Ex acellula esicles as
su oga ed bioma ke s o
P os a e Cance me abolism
A me abolomics app oach o s udy hei
ole in p os a e cance p og ession
Guille mo Bo danaba Flo i
2024
Ex acellula esicles as su oga ed
bioma ke s o P os a e Cance me abolism
A me abolomics app oach o s udy hei ole in p os a e
cance p og ession
Guille mo Bo danaba Flo i
Supe ised by: Juan Manuel Falcón Pé ez
Félix Royo López
2019 - 2024
(cc) 2024 Guille mo Bo danaba Flo i (cc by 4.0)
Po D’Addaia, Cala Molí
acone meu es ima ;
em sen més al eu cos a
com més lluny es às de mi
I eco d s’es iu passa
que sembla a sense i
i que em a e di en un escla :
S’any que e, queda é aquí.
V
Acknowledgemen s
Cada e apa y p oyec o pe sonal es ini o, en cada luga donde nos encon amos es especial y siemp e
dejamos a pe sonas que han sido y se án, di ec a o indi ec amen e esenciales en u camino. Es as
e apas y pe sonas apa ecen en un momen o y, así sin más, e cambian la ida. De es a misma mane a
me sen í yo el día que comencé mi a en u a con el p oEVLi eCycle en el Labo a o io de Exosomas en
Bilbao. Po es o quie o empeza dándole las g acias a JuanMa, po la opo unidad que me b indó sin
conoce me de nada y po el g an ejemplo de je e humano que he enido y que he dis u ado. G acias
ambién po la anquilidad y ayuda que me has dado en odos los p oyec os que hemos ealizado.
Seguimos. A Félix po da me siemp e en odo momen o buenos consejos sean ( amos a llama le)
“polí icamen e” co ec os o no; siemp e con esa c ea i idad y ganas de ayuda que e ca ac e izan.
Insis o en es o po que siemp e me has ayudado a saca las cas añas del uego aún yendo yo un poco
a mi bola. Al es o de g upo de Exosomas, los que es án y los que se ue on, po odo el apoyo y
especialmen e a Cla a (hubiese pues o Cla i a, pe o no e gus a ), Jone, Elisabe h, Pa i (el Alzhei-
me ’s lab). Mención especial a la Pla a o ma con Sebas iaan, Diana y Oihane, un emanso de paz y
b o es a pa es iguales; e a pasa la pue ecilla con la xa ela y pensa : ya es á, casa. Ma ieAzpa-
enMa i ún que e quie o mucho y que e es la mejo ; siemp e has es ado ahí y ya ni ecue do mi
ida sin conoce e.
A odas las pe sonas de CICbioGUNE que de una o ma u o a han pues o su g ani o de a ena pa a
que es a esis enga algo de sen ido. Sob e odo, pa a es pe sonas que han pues o algo más que un
g ani o de a ena en di e en es momen os de o ma desin e esada, g acias a A kai z, Jani e y A iane
po ues a ayuda en el desa ollo de es a esis en cánce de p ós a a.
P aisewo hy people ule all li e-changing e en s and in his case, he p oEVLi eCycle conso ia is an
example o big and collabo a i e p ojec s being also un and inspi ing. I would like o specially men ion
Guido, he coo dina o ac ion, and his eam who a e he s anda d bea e s o good science, c ea i i y
and eam wo k. To Elena who ac ually became a good iend. Big hanks o e e yone in his conso ia,
ESRs, PIs, collabo a o s, lab membe s, ex e nal aine s; you all made his PhD a memo able jou ney.
To Jason and Aled o all hei help o push o wa d pa o his hesis’ wo k. And inally, o Guillaume
and his eam. They hos ed me in Pa is and boos ed his PhD p ojec . I I g ow old in science I’d like o
esemble he ibes o you g oup .
Esas pe sonas que se uel en impo an es son aquellas que e acili an las cosas y e hacen sen i
como en casa. Cuando llegué a Bilbo, la bien enida no pudo se mejo en Ramón y Cajal 42. La e dad
que me sen í como en una segunda casa. Especialmen e a Ma a y al incansable T enco, po odas
las bajadas al Izeki a con a nos cualquie on e ía; po es a ahí y po ene un co azón an g ande
como el que ienes. En las al u as en Blas de O e o ambién se ha quedado un oci o de mí. La casa
es un luga habi ado po su eños (1/8 ya e da el ce i icado) si uado a las a ue as de Deus o y que lo
sien o como un hoga . Con mi segunda casa en BilbaoDeus o es oy muy con o me. La Isa es un odo-
e eno. Te admi o po la capacidad de i a siemp e pa a adelan e y ambién po an epone la gen e
VI
que de e dad e impo a. La e dad has sido mi so p esa y ya no es solo que e quie a muchísimo
sino lo que con ío en i. Luego es á mi Agu, mi medio oli o aquí. Uno más de la amilia y así me he
sen ido yo ambién siemp e con igo. Admi o lo empá ico y gene oso que e es en odas las ace as de
la ida, no e haces idea de lo mucho que me has ayudado. Todo iene su in, pe o yo no quie o que la
casa e mine nunca.
Todo el mundo que se c uza en u ida puede o ma pa e de ella de o ma ugaz, empo al o queda se
pa a siemp e. Todos ellos pueden, en un solo momen o, cambia e la ida o u pe cepción de ella pa a
siemp e. Desconocidos cuando llegas y que luego pa ece que hayan es ado siemp e ahí. Muchas
g acias a odos los que me habéis hecho sen i así en Bilbo, en cua o años he compa ido g andes
momen os con odos y sabéis que siemp e os lle a é conmigo. Y a Ma ía, sabes odo de mí y has
es ado siemp e ahí, e es mi Augens e n.
Dicen los sabios que somos cada momen o y cada pe sona que nos haya dejado un pedaci o den o.
Ag adezco a odas y cada uno de mis amigos que han o mado y que o man pa e de mí. Els dijous
de Mundo, les bi es a la Cí ica, Fes ònomes, Colònies bio ec, indes de ma a -nos a la biblio, San
Joans, bàsicamen o es les p ime es egades de an es coses i qualse ol plan que acabi en – ec. Also,
o all de Bonge d d inks, libe a ion days, D oe endaal pa ies, Du ch wei d e en s, Panda poin s, ICA
club, lib a y days, TT wi h he G eeks; basically o my Wageningen amily. A Na alia, po que hicis e una
mejo e sión de mí y po que básicamen e si no uese po i nunca jamás hubiese hecho un doc o ado
y menos en España. To he c azies lab I ha e e e been and I will e e be, he AG Ba -E ens. Nico,
my supe iso back hen, he one who made me lo e science again.
Po úl imo, a mi amilia. No és ni la amilia més ex ensa del món ni ampoc la més no mal del món.
Pe ò ens es imem, els es imo i m’es imen. Sabe que semp e hi són, de o ma incondicional és el majo
ac e d’amo que mai he pogu sen i . Tinc so de eni - os. Y ol e é al cas ellano pa a mi amilia
a agonesa. La de sang e, que me ae ecue dos inol idables y son mi mesa edonda donde oy a
busca opinión. La amilia es á c eciendo y no puedo sen i me más o gulloso de pode o ma pa e de
ello. Luego es á mi o a amilia a agonesa, mis p imos. No ha ía al a ni menciona os, siemp e habéis
es ado en mi ida y siemp e lo ais a es a . A s’à ia, s’única que enc i sa que semp e a mi a de e -
me eliç i cuida -me. Bé, u i l’a i. Ad ián José, qué oy a deci de i si es que es ás siendo aho a mismo
pa a mí un g an ejemplo en odo. Sa mamà y papá, lo que soy es únicamen e po oso os.
VII
Table o Con en
ACKNOWLEDGEMENTS ......................................................................................................................... V
TABLE OF CONTENT .......................................................................................................................... VII
ABSTRACT .......................................................................................................................................... X
RESUMEN ........................................................................................................................................ XV
INTRODUCTION .................................................................................................................................... 1
I. PROSTATE CANCER DISEASE ............................................................................................................ 2
P os a e gland ................................................................................................................................. 2
P os a e cance p og ession ........................................................................................................... 4
PCa me abolic ewi ing ................................................................................................................... 6
S e oid ho mones me abolism .............................................................................................................. 9
II. EXTRACELLULAR VESICLES ............................................................................................................ 11
EVs as bioma ke s ........................................................................................................................ 12
Role o Lipids in EVs ..................................................................................................................... 13
EVs in me abolism ........................................................................................................................ 15
III. METABOLOMICS ......................................................................................................................... 17
Biological p oblem and expe imen al design ................................................................................ 19
Sample p epa a ion ....................................................................................................................... 19
Da a acquisi ion ............................................................................................................................. 20
Da a p ocessing ............................................................................................................................ 20
S a is ical analysis ......................................................................................................................... 22
Me abolic pa hways associa ion.................................................................................................... 22
IV. MODELS IN RESEARCH ................................................................................................................. 23
V. HYPOTHESIS AND OBJECTIVES ........................................................................................................ 27
RESULTS ................................................................................................................... 29
CHAPTER 1. CURRENT APPROACHES TO STUDY EXTRACELLULAR VESICLES IN CELL PHYSIOLOGY AND ITS APPLICATIONS ...... 31
1.1. In oduc ion ........................................................................................................................ 31
1.1.1. EV he e ogenei y and bioma ke disco e y. ........................................................................ 33
1.1.2. Single esicle analysis echniques o biological cha ac e isa ion o EVs. ............................ 34
1.2. Single- esicle echniques .................................................................................................. 34
1.2.1. Label- ee me hodologies. .................................................................................................. 34
1.2.2. Label-based me hodologies. .............................................................................................. 39
1.3. Recen ad ances in he EV ield due o single esicle analysis ....................................... 42
1.3.1. EV cha ac e isa ion. ........................................................................................................... 42
1.3.2. EV a icking and signalling mechanisms. .......................................................................... 45
1.3.3. EV bioma ke s. .................................................................................................................. 49
1.4. Fu u e ou look ................................................................................................................... 52
1.5. Concluding ema ks .......................................................................................................... 55
CHAPTER 2. EVALUATION OF STEROID HORMONE TRANSCRIPTS ASSOCIATED TO URINARY EXTRACELLULAR VESICLES IN
PROSTATE CANCER PROGRESSION .......................................................................................................... 57
2.1. Backg ound ....................................................................................................................... 57
2.2. Ma e ials and me hods ...................................................................................................... 57
2.2.1. Da abase esou ces. .......................................................................................................... 57
EV bioma ke da ase s ................................................................................................................... 57
PCa bioma ke da ase s ................................................................................................................. 58
2.2.2. Real ime quan i a i e PCR analyses. ................................................................................. 59
VIII
Sample p epa a ion ........................................................................................................................ 59
Real- ime quan i a i e PCR me hodology ........................................................................................ 60
Design o p ime s o SYBR G een and TaqMan qPCR ................................................................... 60
Housekeeping genes analysis ........................................................................................................ 60
2.3. Resul s .............................................................................................................................. 61
2.3.1. Selec ion o a ge s. ........................................................................................................... 61
S udy o s e oid ho mones mRNA ela ed o EVs ............................................................................ 61
Bioma ke po en ial o s e oid ho mone enzymes exp ession ........................................................... 62
2.3.2. Selec ion o coho s. .......................................................................................................... 63
2.3.3. No malisa ion and Housekeeping genes. ............................................................................ 65
2.3.4. E alua ion o mRNA in EVs as a ge s. ............................................................................... 67
2.4. Discussion and Conclusions ............................................................................................. 69
CHAPTER 3. LIQUID CHROMATOGRAPHY COUPLED TO MASS SPECTROMETRY ASSAY TO MEASURE STEROID HORMONE ......... 71
BIOSYNTHESIS PATHWAY ...................................................................................................................... 71
3.1. De elopmen o a Ta ge ed Me abolomics Assay in Endoc ine Tissues o Male Ra s and ................................
Human Samples ..................................................................................................................... 71
3.1.1. In oduc ion ....................................................................................................................... 72
3.1.2. Ma e ials and Me hods ....................................................................................................... 76
3.1.2.1. Tissue and bio luid samples ........................................................................................... 76
3.1.2.2. Wes e n blo Analysis .................................................................................................... 77
3.1.2.3. Chemicals and s anda ds .............................................................................................. 78
3.1.2.4. LC-MS sample p epa a ion ............................................................................................ 78
3.1.2.5. Ul a-high pe o mance liquid ch oma og aphy ................................................................ 78
3.1.2.6. Mass spec ome y ........................................................................................................ 79
3.1.2.7. S a is ical Analysis ......................................................................................................... 80
3.1.3. Resul s .............................................................................................................................. 81
3.1.3.1. Liquid ch oma og aphy and mass spec ome y me hod .................................................. 81
3.1.3.2. Analy e eco e y op imiza ion ........................................................................................ 82
3.1.3.3. Ma ix e ec .................................................................................................................. 85
3.1.3.4. Semi-quan i a ion o s e oids in animal issues ................................................................ 87
3.1.3.5. Quan i a ion o s e oid ho mones in human u ina y samples ........................................... 89
3.1.4. Discussion ......................................................................................................................... 91
3.2. Clinical E alua ion o Me abolic Signa u es as Bioma ke s o P os a e Cance P og ession in .........................
Pa ien U ines ........................................................................................................................ 94
3.2.1. Backg ound ....................................................................................................................... 94
3.2.2. Resul s and discussion ...................................................................................................... 94
3.2.2.1. Clinical coho cha ac e is ics ......................................................................................... 94
3.2.2.2. Signa u e esul ............................................................................................................. 95
3.2.2.3. App oaches o gi e an ou come ..................................................................................... 98
3.2.3. Conclusions ...................................................................................................................... 99
CHAPTER 4. TRANSFERENCE OF BIOLOGICAL COMPONENTS AND FUNCTIONALITIES DRIVEN BY EXTRACELLULAR VESICLES ... 101
4.1. Labelled Choles e ol Demons a es E ec i e EV-Media ed Me aboli e T ans e in P os a e Cance ........ 101
4.1.1. In oduc ion ..................................................................................................................... 102
4.1.2. Ma e ials and me hods ..................................................................................................... 104
4.1.2.1. Cell cul u e, EV p oduc ion and d ug ea men s. .......................................................... 104
4.1.2.2. Isola ion o EVs. .......................................................................................................... 104
4.1.2.3. Cha ac e isa ion o EVs. .............................................................................................. 105
4.1.2.4. EV up ake expe imen s. ............................................................................................... 105
4.1.3. Resul s ............................................................................................................................ 107
4.1.3.1. Choles e ol is quan i iable in cell models and ex acellula esicles. .............................. 107
4.1.3.2. Ex acellula esicles can ans e choles e ol o ecipien cells. .................................... 110
XV
Resumen
El cánce de p ós a a es una en e medad que únicamen e padecen los homb es y una de
las más amenazan es con la edad. Es a a ección causa un g a e p oblema socio-económico
debido a la al a de sensibilidad y especi icidad en el diagnós ico du an e ases iniciales de su
desa ollo. El cánce de p ós a a es mul i ocal y ex emadamen e di e so an o a ni el mole-
cula como his opa ológico. También posee una g an a iedad de mecanismos de p og esión,
pe o las consecuencias isiológicas de i adas y mane a de e oluciona son oda ía una in-
cógni a. El c ecimien o umo al descon olado y me ás asis a ejidos ce canos gene a una
g an di e sidad de pe iles me abólicos, p o eómicos y ansc ip ómicos. Es a he e ogeneidad
sus di e en es ases p opo ciona lexibilidad y adap abilidad a los a amien os y, po lo an o,
ambién complica su diagnós ico.
La comunicación celula es imp escindible pa a el p og eso de cualquie en e medad. Fue
en 1967 cuándo Pe e Wol desc ibió una nue a es uc u a subcelula : esículas expulsadas
po células. Ac ualmen e, se ha demos ado que es as esículas juegan un papel impo an e
en la in e acción celula . Es as esículas ex acelula es son es uc u as delimi adas po bica-
pas lipídicas expulsadas po la g an mayo ía de células. Su amaño oscila en e nanóme os
y mic óme os de diáme o y, pueden con ene lípidos, p o eínas, me aboli os y ácidos nuclei-
cos, incluyendo ARN y ADN. Se a a de es uc u as ex emadamen e he e ogéneas en a-
maño y composición, que es á de e minada en g an medida po las u as de biogénesis que
las o men. Tan o las moléculas de la memb ana como las encapsuladas en el in e io pueden
ac i a cascadas de señalización que pueden p oduci espues as isiológicas como la madu-
ación celula , e en os de coagulación o el es ablecimien o de nichos p eme as á icos, en e
o os.
La ep og amación me abólica mediada po esículas ex acelula es se ha obse ado en
muchos sis emas biológicos donde di e en es células, que pueden ene dis in as unciones y
o ígenes, in e cambian ecu sos me abólicos. Du an e la oncogénesis, las esículas ex ace-
lula es de los ejidos cance osos o los ejidos adyacen es pueden induci cambios me abóli-
cos al p opo ciona ecu sos me abólicos o desencadenando señales necesa ias pa a su p o-
g esión. De hecho, las esículas ex acelula es se desc iben como mediado es ele an es en
XVI
los p ocesos de p og esión del cánce . Has a la apa ición de las ecnologías “omicas”, se
u iliza on en oques de biología molecula y isiología pa a ob ene da os que pudie an desc i-
bi la in e acción en e componen es y unciones biológicas especí icas. La me abolómica
iene como obje i o medi , iden i ica y cuan i ica un g an núme o de me aboli os en un sis-
ema biológico de e minado. Una es a egia de me abolómica di igida u ilizando c oma og a-
ía líquida acoplada a un espec óme o de masas es el más adecuado pa a los obje i os de
es a esis.
La p esen e esis se cen a en el es udio del con enido de las esículas ex acelula es y la
mane a en que in e ac úa con las células ecep o as. Con es e p opósi o, se ha di idido según
paque es de abajo especí icos que incluye on:
Un p ime capí ulo dedicado a la iden i icación bibliog á ica y e isión de las me odologías
ac uales pa a ca ac e iza las esículas ex acelula es. La he e ogeneidad de su es uc u a y
composición di icul a su aplicación en diagnós icos médicos y e apias. Dicha di e sidad am-
bién di icul a el es ablecimien o de oles isiológicos, así como las unciones y composición
de di e en es (sub)poblaciones de esículas ex acelula es. Los mé odos que p omedian el
con enido de las mues as, como es el caso de las “ómicas”, ienden a dilui es as he e oge-
neidades. Los desa ollos ecien es en el análisis indi idual de esículas han e elado nue as
pe spec i as en la comunicación en e células, así como la iden i icación y alidación de bio-
ma cado es. Sin emba go, hay que ene en cuen a que los en oques 'ómicos' siguen siendo
ele an es pa a es udia p ocesos isiológicos asociados a esículas ex acelula es a escala
más global den o de un modelo biológico.
La in es igación más de allada de la ca ga de esículas ex acelula es es de g an u ilidad
pa a de e mina posibles mecanismos de p og esión del cánce en e apas emp anas de su
desa ollo. En es e sen ido, las ho monas es e oides desempeñan un papel i al en la egu-
lación de p ocesos celula es, y la des egulación de es os me aboli os puede p o oca o ag a-
a p oblemas pa ológicos, como en e medades au oinmunes y cánce . En el cánce de p ós-
a a, son los p incipales impulso es de la oncogenicidad en las e apas muy emp anas de la
en e medad. Po lo an o, la cuan i icación de ARNm especí icos en mues as biológicas e-
le an es, como la o ina, es un ecu so pode oso pa a diagnos ica la en e medad. En el capí-
ulo 2, se desa olla on ensayos cuan i a i os de ADN pa a es udia la exp esión de ARNm
especí icos. P ime o, se iden i ica on 32 posibles ma cado es de p og esión del cánce de
XVII
p ós a a u ilizando CANCERTOOL. Es os candida os disc iminan en e mues as de pacien-
es con cánce de p ós a a e hipe plasia benigna, ya sea incluyendo mues as con p esencia
de me ás asis o ambas. Además, di e en es es udios cien í icos los han desc i o en esículas
ex acelula es. Un panel de dichos candida os, se cuan i icó en una coho e de o inas de
pacien es humanos. Lamen ablemen e, no ue posible desc ibi a ningún candida o como un
bioma cado álido en mues as de o ina pa a se p obado más a ondo.
Focalizando en es udios uncionales, el es ado oncogénico de las células cance osas
puede e alua se midiendo la exp esión de genes asociados al c ecimien o dependien e de
and ógenos (Capí ulo 4.2.). La asa p oli e a i a de las células ambién es un indicado com-
plemen a io de la oncogenicidad. La línea celula LNCaP ue el modelo más ap opiado pa a
es udia la dependencia de and ógenos y la exp esión de KLK3 el mejo indicado del a a-
mien o de depleción de and ógenos. T a ando el modelo dependien e de and ógenos LNCaP
con esículas ex acelula es, se pe seguía desc ibi si las esículas ex acelula es podían
man ene el eno ipo oncogénico en las células ecep o as. El es ado oncogénico se e aluó
midiendo la exp esión de KLK3, que es á asociado a la cascada de señalización de and óge-
nos. También se conside ó el es ado p oli e a i o de las células de cánce de p ós a a pa a
e alua los a amien os con esículas ex acelula es pe o es e no p opo cionó esul ados
signi ica i os. T as la depleción de es e oides, la exp esión de KLK3 disminuyó d ás icamen e
con el iempo. Es a pé dida de eno ipo oncogénico ue neu alizada con la p esencia de e-
sículas ex acelula es aisladas de cul i os celula es con c ecimien o oncogénico. Es o indica
la pa icipación de esículas en la ans e encia de eno ipo oncogénico aunque no explica
las implicaciones en la isiología del cánce .
Es os esul ados sugie en una ans e encia de uncionalidades en e las esículas ex a-
celula es y las células ecep o as. Es a ans e encia puede da se a a és de p o eínas que
ac i en cascadas de señalización o con di e en es ecu sos me abólicos. En el capí ulo 3,
pa a es udia la p esencia de me aboli os elacionados con ho monas es e oides en es os
e en os de ans e encia, se desa olló un ensayo de me abolómica. La ex acción y cuan i i-
cación de 11 miemb os cla e en la ed me abólica de ho monas es e oideas, incluyendo an-
d ógenos, es ógenos, p oges ágenos y co icoides, se desc ibe en el capí ulo 3 de es a esis.
Una ca ac e ís ica impo an e de es e ipo de ensayos es que se pueden implemen a me a-
boli os adicionales a pos e io i. El ensayo consis e en una ex acción bi ásica líquido/líquido
y la subsiguien e cuan i icación median e c oma og a ía líquida de al a esolución acoplada a
XVIII
espec ome ía de masas de iempo de uelo. Las e iciencias de ecupe ación oscilan en e
el 74.2% y el 126.9%, y el 54.9% y el 110.7%, pa a los compues os apola es y pola es, es-
pec i amen e. En gene al, la pé dida de in ensidad de señal debido al e ec o de ma iz no
supe a el 30%. El mé odo se ha p obado en di e sas ma ices, como ejidos de a as, o inas
humanas, líneas celula es humanas y esículas ex acelula es. En esumen, es e ensayo
puede medi simul áneamen e me aboli os elacionados con ho monas es e oideas en un
iempo de ejecución de 6 minu os el cual puede inc emen a se a 9 min pa a inclui el coles e ol
en el análisis. Además de su uso en es udios de ans e encia, o o p opósi o del mé odo e a
el de alida una i ma me abólica de ho monas es e oides como bioma cado pa a el cánce
de p ós a a.
Los lípidos y especí icamen e el coles e ol desempeñan un papel undamen al en la p o-
g esión del cánce de p ós a a. El coles e ol es impo an e en ías me abólicas p o umo ales
ya que es el p ecu so p incipal del me abolismo de ho monas es e oideas. Además, las e-
sículas ex acelula es es án en iquecidas con coles e ol, cons i uyendo ap oximadamen e del
50 a 60% de su con enido lipídico. La ans e encia e ec i a de me aboli os asociados a esí-
culas ex acelula es oda ía no se ha explo ado. En el capí ulo 4.1. se a a on cul i os de
células de cánce de p ós a a con coles e ol ma cado en medios desp o is os de lípidos.
Luego, las esículas ex acelula es pu i icadas se suminis a on a células ecep o as no ma -
cadas pa a as ea la in e nalización de coles e ol median e mic oscopía con ocal y su pos-
e io me abolización median e el ensayo di igido UPLC-MS desc i o an e io men e. En es e
abajo, se de ec ó coles e ol asociado a esículas ex acelula es en células ecep o as no
ma cadas. Además, al ma ca las células de cánce de p ós a a con un isó opo es able de
coles e ol, se de ec a on esículas ex acelula es ma cadas. En esumen, se ha demos ado
que un ensayo de me abolómica di igida combinado con un en oque de biología molecula es
ú il pa a es udia la in e nalización mediada po esículas ex acelula es y la ans e encia
e ec i a de me aboli os ele an es como el coles e ol.
Finalmen e, se u ilizó una es a egia me abolómica semidi igida pa a es udia la in e acción
en e di e en es compa imen os de la glándula p os á ica (Capí ulo 5). La glándula p os á ica
es un ó gano complejo y he e ogéneo compues o po el epi elio y el es oma. El es oma
p os á ico es complejo y, en conjun o con la p og esión del umo , expe imen a al e aciones
que incluyen la apa ición de un eno ipo de ib oblas o asociado al cánce . Es a población
celula an he e ogénea a menudo incluye células con un eno ipo simila al mio ib oblas o
XIX
que no es án p esen es en ejido p os á ico sano. Al u iliza biopsias de aguja de ejidos u-
mo ales y no males asociados al mismo pacien e, se pudo es udia la econ igu ación me a-
bólica de los ib oblas os es omales que expe imen an una di e enciación hacia mio ib oblas-
os. Se de e minó que los ib oblas os asociados al cánce son me abólicamen e más ac i os
y, po lo an o, se en aumen adas las ías me abólicas de p oducción de ene gía. Además,
los ib oblas os asociados al cánce mues an un me abolismo lipogénico ele ado an o po
la p esencia de compues os de ese a lipídica como de e ien e es uc u al. Así como en
es a egias an e io es, se incuba on esículas ex acelula es de i adas de líneas celula es de
cánce de p ós a a con ib oblas os p ima ios sanos. Se de ec ó un aumen o en la espi ación
basal de los ib oblas os, e lejando el eno ipo simila a la en e medad. Con es os esul ados,
se puede p opone que el cambio en el pe il me abolómico de los ib oblas os es omales
asociados ejido umo al es á impulsado po ías me abólicas dependien es de oxígeno; sin
emba go, los mecanismos especí icos aún no es án cla os.
INTRODUCTION
2
I. P os a e Cance Disease
The Wo ld Heal h O ganisa ion desc ibes p os a e cance as one o he mos equen ly diagnosed
and deadly ypes o ca cinomas. I is a global heal hca e p oblem wi h an incidence o app oxima ely a
qua e o all cance diagnoses in Eu ope. P os a e cance (PCa) is an exclusi e disease o men ha
misses sensi i e and speci ic diagnos ic ools o su eil ea ly s ages o i s p og ession. This causes a
huge socio-economic p oblem o amilies, pa ne s and ca egi e s, h ough loss o li e o p o ac ed
pallia i e incapaci y. In ac , many men ini ially a end clinic wi h a umou ha is al eady in an ad anced
and incu able s a e. P os a e cance is o en a mul i ocal disease and e en wi hin a single umou ,
se e al molecula and his o-pa hological a angemen s a e obse ed. Many o he unde lying mecha-
nisms o p og ession ha e been p oposed as possible a ge s o diagnos ics and he apeu ics; how-
e e , he physiological d i e s and consequences in i s e olu ion a e no ully unde s ood.
Nowadays, p os a e-speci ic an igen (PSA) blood sc eening es s a e he co ne s one o PCa su -
eillance a ea ly s ages. Up o 40% o men unde go unnecessa y biopsies due o i s poo speci ici y.
Upon biopsy o he pa ien , he se um PSA es is combined wi h he clinical T-s age and Gleason sco e
as s anda d es s o disc imina e pa ien s wi h low, in e media e o high isk o su e PCa1. The p o-
g ession o PCa wi hin p os a ic issue is e y di e se, so i is a me as a ic s ages. Me as ases p inci-
pally occu in bone and lymph node issues bu hey a e also epo ed in li e , lung and b ain issues2.
This di e si y causes a ia ion in he apy esponse and esis ance mechanisms. A high p opo ion o
men exhibi slow g owing umou s ha a e unlikely o p og ess o a li e- h ea ening s age. O en, ini ial
ea men s o and ogen-abla ion eliably igge eg ession bu his is empo a y, and wi hin 2-3 yea s,
cas a e esis an PCa may ecu , becoming a pa icula ly in ac able and agg essi e disease. Ye , dis-
c imina ion o indolen om agg essi e disease is di icul . Fo hese men, he iden i ica ion o no el
a ge able pa hways o be conside ed du ing decision-making p ocess o he apeu ic in e en ion. Fu -
he mo e, a be e unde s anding o PCa disease will also help o design imp o ed ea men s a egies.
Abo e all, unde s anding he molecula and physiological mechanisms o PCa disease hold an eno -
mous po en ial o ans o m disease diagnosis, pa ien s a i ica ion and no el ea men modali ies;
p o iding supe io in o ma ion o weal h. Resea che s need o deciphe he sec e s o PCa, om he
spa k ha unleashes cance de elopmen o he su i al s a egies imp o ed by umou cells.
P os a e gland
The p os a e is he main accesso y gland o he male ep oduc i e sys em. The human p os a e is a
walnu -sized o gan whose base is loca ed a he u ina y bladde neck and he apex a he u ogeni al
diaph agm (Figu e 1). As a back as he mid-six een h cen u y, when And eas Vesalius published his
obse a ions o he male accesso y glands, p os a e ana omy was o medical in e es 3. In e es ingly,
he mac oscopic ana omy o his o gan di e s conside ably be ween species. Roden model o ganisms
such as a s o mice u ilised a e o model human diseases; he e o e, i is impo an o acknowledge he
di e ences in he s uc u e o human and oden p os a es. Unlike human, a o mouse p os a e is no
me ged in o a unique ana omical s uc u e wi h h ee glandula egions bu i is composed o ou dis inc
3
lobula s uc u es (Figu e 1). None heless, he e is no exis ing e idence ha suppo s a di ec ela ion-
ship be ween he speci ic mouse p os a e lobes and he speci ic zones in he human p os a e4.
Figu e 1. Rep esen a ion o a human and a mouse p os a e. On he le , a diag am o an adul human p os a e
whe e he u e h a, he bladde and he h ee majo egions o a p os a e a e indica ed. On he igh , a mouse
p os a e is depic ed. I is s uc u ed in ou lobes (simila o a o ganiza ion): la e al p os a e, do sal p os a e, en al
p os a e and an e io p os a e. 5Rep in ed om U ologic Clinics o No h Ame ica, 282, LaTayia Aa on, Oma E.
F anco, Simon W. Haywa d, Re iew o P os a e Ana omy and Emb yology and he E iology o Benign P os a ic
Hype plasia, Volume 43, Issue 3, Copy igh 2016, wi h pe mission om Else ie .
In humans, he p os a e gland con ains h ee majo glandula egions, which di e bo h his ologically
and biologically: he pe iphe al zone, he cen al zone, and he ansi ion zone. The la e is he main
o igin o p os a e hype plasia6 and ca cinoma is mo e common in he pe iphe al zone7. A a his ological
le el, he p os a e is a b anched duc o gan o ganized in glandula acinis cons uc ed by he epi helium
and sus ained by he s oma7. The wo compa men s in luence each o he ia signalling pa hways o
p omo e p os a e de elopmen and co ec unc ioning8. Bo h issues a e ex emely he e ogeneous wi h
many ypes o cells pa icipa ing in di e en physiological unc ions. The epi helium con ains sec e o y
epi helial cells a ound he glandula lumen, whe e hey sec e e he p os a ic luid (Figu e 2A). These
sec e o y cells, also called luminal cells, a e su ounded by basal cells and spa se neu oendoc ine cells,
bo h a ached o a basal lamina9,10. Basal cells a e associa ed o duc al in eg i y and sec e o y cell
suppo i e unc ions while neu oendoc ine cells ole is a he unclea . Ye , i is known hey sec e e
speci ic ho mones and lack he exp ession o and ogen ecep o . Fundamen ally, he p os a e epi helial
compa men holds he man glandula unc ion: The sec e ion o p os a ic luid o he lumen (Figu e 2A).
The p os a ic luid is he majo con ibu o o he ejacula e olume and con ains ac o s o con ol ma -
u a ion and mo ili y o spe m o he ejacula e’s luidi y, among o he s.
4
Beyond he basal lamina, a p ominen ib omuscula s oma o e s physical suppo and con ac ion
o he gland (Figu e 2A). A majo unc ion o he s omal compa men is o ensu e he app op ia e
mic oen i onmen o he epi helial compa men , which is p o ided p incipally by su ounding ib o-
blas s. In addi ion, i pa icipa es in he ascula isa ion o he p os a e bu also e acua es p os a ic luid
owa ds he u e h a by con ac ion. Rema kably, he s omal compa men p o ides many suppo i e
signals o e ain o es o e gland homeos asis in heal hy condi ions o du ing egene a ion p ocesses6.
P os a e cance p og ession
The s uc u e and well- unc ioning o he p os a e is o en impai ed wi h aging and, inding he mo-
lecula o physiological mechanism ha igge s PCa o spa k is he one-billion dolla ques ion. In gen-
e al, i is accep ed ha umou ini ia ing cells unde go speci ic biological modi ica ions such as he in-
ac i a ion o ce ain pa hways o he exp ession o speci ic genes. The p os a e gland equen ly su e s
om in lamma ion due o an o e g ow h o s omal cells (and epi helial cells) a ansi ion zone and
pe iu e h al a eas, which is usually associa ed o benign p os a ic hype plasia (BPH). Howe e , when
he g ow h is localised in speci ic loci o he p os a e and associa ed o he epi helium i is p one o be
an ini ia ing umou . The iden i ica ion on g oss inspec ion o PCa by palpa ion o he gland is o en
challenging. Acco ding o expe s, he oci should be a leas 5 mm in diame e o eliable diagnosis as
umou s end o be mul i ocal6. Besides, much la ge a eas can nei he be accu a ely iden i ied. As
men ioned, umou s a e mainly ound in he pe iphe al zone11,12, ollowed by he ansi ion zone and
hen cen al zone.
Figu e 2. His ological sec ion o a human p os a e issue slice. Hema oxylin plus eosin p o ides a comp e-
hensi e pic u e o mic oana omy o issues by p ecisely s aining nuclea (hema oxylin) and cy oplasmic (eosin)
componen s. A. Heal hy p os a e issue wi h glandula acini is depic ed; a do ed ed line delimi a es he s uc u e.
The h ee di e en compa men s a e indica ed wi h black a ows. B. P os a e cance issue in an ad anced
s age, wi h Gleason sco e 7 (3+4) and mino componen s o c ib i o m glands. Images a e adap ed
om h p://commons.wikimedia.o g, and ep oduced wi h pe mission unde C ea i e Commons A ibu ion 4.0
In e na ional License h p://c ea i ecommons.o g/licenses/by/4.0/.
5
Table 1. Summa y o he cu en TNM s aging classi ica ion based on AJCC’s classi ica ion.
Adap ed om 7,13,14.
S age
De ini ion
P ima y umou , clinical (T)
TX
P ima y umou canno be assessed.
T0
No e idence o p ima y umou
T1
Clinically inappa en umou no palpable o isible by imaging. Slow g owing and PSA le -
els a e low.
T1a: Tumou inciden al his ologic inding in 5 % o less o issue esec ed.
T1b: Tumou inciden al his ologic inding in mo e han 5 % o issue esec ed.
T1c: Tumou iden i ied by needle biopsy (i.e. because o ele a ed PSA le els.
T2
Tumou con ined wi hin he p os a ea. PSA le els a e medium o low.
T2a: Unila e al. Tumou in ol es hal o one lobe o less.
T2b: Unila e al. Tumou in ol es mo e han hal o one lobe bu no bo h. I can be la ge
enough o be el by DRE.
T2c: Bila e al. Tumou in ol es bo h lobes.
T3
Tumou ex ends h ough he p os a e capsuleb.Locally ad anced umou wi h high le els o
PSA.
T3a: Ex acapsula ex ension (unila e al o bila e al).
T3b: Tumou in ades seminal esicle(s).
T3c: Tumou cells a e poo ly di e en ia ed. Pe haps, in asion o bladde o ec um.
T4
Tumou is ixed o in ades adjacen s uc u es o he han he seminal esicle(s): bladde
neck, ex e nal sphinc e , ec um, le a o muscles, pel ic wall, o all he abo e.
T4a: Tumou has sp ead o egional lymph nodes.
T4b: Tumou has in aded dis an lymph nodes o o he issues such as bones (i.e. pel ic
wall), le a o muscles, ex e nal sphinc e and o he s.
Regional lymph nodes (N)
NX
Regional lymph nodes canno be assessed.
N0
No egional lymph node me as asis.
N1
Me as asis in egional lymph node o nodes.
Dis an me as asesd (M)
MX
Dis an me as asis canno be assessed.
M0
No dis an me as asis.
M1
Dis an me as asis.
M1a: Non- egional lymph node(s).
M1b: Bone(s).
M1c: O he si e(s).
a Tumou ha is ound in one o bo h lobes by needle biopsy, bu no palpable o eliably isible by imaging, is clas-
si ied as T1c.
b In asion in o he p os a ic apex o in o (bu no beyond) he p os a ic capsule is classi ied as T2, no T3.
c The e is no pa hologic T1 classi ica ion.
d When mo e han one si e o me as asis is p esen , he mos ad anced ca ego y is used; pM1c is mos ad anced.
12
ca ego isa ion includes no only esicles bu also lipop o eins, i uses, p o ein agg ega es, ibonucleo-
p o ein complexes and exome es76–78, which p o ides an addi ional laye o di e si y. This is because
such s uc u es can pa icipa e in simila (pa o)physiological p ocesses along wi h EVs.
Figu e 5. Summa y o EV biogenesis, EV-media ed cell- o-cell in e ac ion and in acellula a icking. (a)
Ca go o EVs includes di e en ypes o p o eins nucleic acids and me aboli es. (b) Plasma memb ane in agina ion
endocy ose ex acellula cons i uen s and cell su ace p o eins. This s uc u e can use wi h cons i uen s o he ER,
ans-Golgi ne wo k and mi ochond ia, which leads o he o ma ion o ea ly so ing endosomes (ESEs). Then,
ESEs ma u a e o la e so ing endosomes (LSEs) o ming in aluminal esicles (ILVs) by a second in agina ion o
he memb ane. Finally, mul i esicula bodies (MVBs) a e gene a ed and hey can be so ed o lysosome, unde go-
ing deg ada ion; o anspo ed o plasma memb ane, docking on luminal side o cells. Exocy osis o MVBs e-
leases ILVs as EVs o he ex acellula space. (c) EVs can be in e nalised by cells using di e en pa hways: usion
wi h cell memb anes, a ecep o -media ed en y, cla h in-coa ed in agina ions o lipid a s in e ac ions, among
o he s. Rep oduced om 79 wi h pe mission unde C ea i e Commons A ibu ion 4.0 In e na ional License
h p://c ea i ecommons.o g/licenses/by/4.0/.
EVs as bioma ke s
In 1989, a ask g oup on Bioma ke s and Risk Assessmen p oposed a de ini ion o bioma ke s,
which a e chemicals, me aboli es o chemicals, enzymes and o he biochemical subs ances o docu-
men hei in e ac ion wi h biological sys ems. In his line, bioma ke s can be de ined a b oade sense
by including chemical, physical and biological in e ac ions be ween hese sys ems. The use o bi-
oma ke s in esea ch has g own exponen ially du ing he las decades seeking o es ablish di ec meas-
u emen s o disease causes and a ec ions80,81. One o he mos impo an c i e ia is hei accessibili y
and biological ele ance. A bioma ke should be p esen in a minimally in asi e sou ce and i should
be as much sensi i e as possible. Mo eo e , i has o be meaning ul in a biological amewo k and o en
p o ide a be e unde s anding o he disease’s mechanism. This will de ini ely lead o desc ibe as
13
esponse bioma ke s upon disease p og ession o ea men and hence, p o ide isk s a i ica ion and
p ognosis. Cu en ly, bioma ke s o PCa do no gua an ee an e ec i e and eliable diagnosis due o
he high he e ogenei y and plas ici y o he disease. On op o ha , i s di e si y is usually assessed
elying on issue biopsies. This s a egy is a he in asi e and does no p o ide a eliable p ognos ic
ou pu . Fo his eason, and conside ing ha cu en diagnos ic ools p esen se e al laws, inding o he
bioma ke sou ces is undamen al in he diagnosis ield.
Nea ly all cell ypes expel EVs o he ex acellula milleu wi h signalling o disposal pu poses65,82.
These physiologically ele an cell goods a e exchanged sa ely hanks o he p o ec ion o e ed by he
lipid memb ane. As cellula componen s a e no easily deg aded, EVs ep esen a new sou ce o bi-
oma ke s ha could desc ibe physiological p ocesses o cellula s ages in disease p og ession. De-
pending on he in o ma ion p o ided, bioma ke s can be used as p edic o s when hey p o ide in o -
ma ion on he biological esponse o he apeu ic in e en ions, p ognos ic asse s when hey p o ide
in o ma ion on disease p og ession and/o ecu ence o diagnos ic asse s i hey can dis inguish
heal hy and disease samples83–85. Rema kably, EVs ha e been iden i ied in wide a ie y o human is-
sues o luids, including blood, u ine, sali a, syno ial luid, ce eb ospinal luid, u e ine luid, bile, b eas
milk o aeces, among o he s65. In his line, liquid biopsy may be highly in o ma i e as i is low-in asi e
and could e lec he s a us o a disease be e han a con en ional biopsy. The e o e, no el bioma ke s
can be desc ibed om EVs and hei concomi an compounds o in o m abou he p og ession o a
disease o a physiological esponse.
Role o Lipids in EVs
The biogenesis o EVs commences wi h p ocesses o mic oau ophagy in la e endosomes o ou wa d
budding a he plasma memb ane86. His o ically, he esul ing esicles ha e been called exosomes and
mic o esicles, espec i ely. The biological e en s go e ning hei o ma ion a e di e en ; howe e , hey
a e all somehow d i en by hei lipid composi ion. Fo ins ance, he ansloca ion o acid sphingomye-
linase gene a es ce amides in he ou e lea le o he plasma memb ane, inducing a cu a u e in he
plasma memb ane ha igge s mic o esicle budding87. Ac ually, he dis up ion o lipid o ganiza ion
appea s o be c i ical; no only ce amides a ec hei o ma ion bu also choles e ol/sphingomyelin a io,
and phospha idylse ine asymme y o he ou e lea le . A e mic oau ophagy in la e endosomes, mul-
i esicula bodies (MVBs) con aining in aluminal esicles (ILVs) can each he plasma memb ane in a
dynamic p ocess egula ed by choles e ol. Upon usion wi h plasma memb ane, ILVs a e eleased o
he ex acellula space whe e hey a e called exosomes. Exosomes a e en iched in disa u a ed molec-
ula species o phospholipids, which accoun s o hei inc eased memb ane igidi y87. In summa y,
he e a e many lipid- ela ed pa hways in ol ed in he biogenesis o EVs.
The elease o esicles implies mo e s eps p e ious o budding e en s in which he composi ion o
memb anes plays a c i ical ole. S udies wo king wi h cells in i o show a ema kable en ichmen o
choles e ol, sphingomyelin, glycosphingolipids and phospha idylse ine in EV memb anes88. Mo eo e ,
he p esence o lipids wi h small head g oups as ce amide, diacylglyce ol and phospha idic acid has
been associa ed wi h he o ma ion and elease o EVs89. Conside ing he ole o lipids in EVs one
14
should conside bo h hei s uc u al ole in memb anes and he manne hey modi y memb ane luidi y
and cu a u e, bu also hei po en ial ole as signalling molecules89.
Figu e 6. Classes o lipids. Di e en g oups a e displayed acco ding o hei s uc u e and syn hesis
pa hways.
15
EVs in me abolism
The memb ane-enclosed en i ies ha we bap ised as EVs ca y p o eins, nuclea acids, lipids and
me aboli es. Indeed, EVs a e o en conside ed me abolic machine ies since hey can con ain ac i e
enzymes, me abolic subs a es o ligands o ca y ou enzyma ic eac ions o egula e enzyme ac i i ies.
The e a e a ious mechanisms by which me abolic ca goes a e loaded in o EVs: iden i ying speci ic
miRNA90, p o eins91 o pe o ming pos ansla ional modi ica ions, such as ace yla ion, sumoyla ion o
phospho yla ion92,93 on p o eins and RNA o inc ease he likely hood o ac i ely o passi ely loading
EVs wi h speci ic compounds. In addi ion, EVs a e en i ies wi h a high con en o lipids, a wide amily o
me aboli es (Figu e 6) wi h a e y di e se chemical s uc u e and hence, se e al p ope ies physiologi-
cal unc ion. Al hough EV memb anes a e simila o dono cells hey a e en iched in speci ic lipids as
choles e ol, sphingomyelins, ce amides and glycosphingolipids94–96. This may explain why p o eins wi h
a ini y o ce ain lipids a e en iched in EVs, i.e. e aspanins o lo illin. While some o he mechanisms
o ca go ec ui men ha e been desc ibed, he in ol emen o me abolic asse s in ac ual physiological
p ocesses is o en o e looked97. I is known ha me abolic esou ces a e anspo ed by EVs ye , he
manne hei ca go ( eac ion subs a e -me aboli es- o ac i i y -enzymes-) in e ac s wi h ecipien cell
is no unde s ood nei he comp ehensibly s udied.
Table 2. Glycoly ic enzymes and occu ence in exosomes and PCa-de i ed EVs. Enzymes in he glycoly ic
pa hway and hei equen appea ance in EVs a e lis ed. The numbe s e e o hei placemen in he op 100 mos
equen ly iden i ied p o eins.
H = Human; D = Dog; S = S allion; B = Bull.
P o eomic da a was e ie ed on 2018-10-16 and he o iginal able is ex ac ed om98.
Enzyme
EVpedia
ExoCa a
P o asomal lipid a
Specie
Hexokinase
-
-
No
H, D, S, B
Glucose-6-phospha e isome ase
63
-
Yes
H, D, S, B
6-Phospho uc okinase
-
-
No
H, S
F usc ose-biphospha e aldolase
12
18
Yes
H, D, S, B
T iosephospha e isome ase
20
27
Yes
H, D, S, B
Glyce aldehyde-3-phospha e dehyd ogenase
1
4
Yes
H, D, S, B
Phosphoglyce a e kinase
16
16
Yes
H, D, S, B
Phosphoglyce a e mu ase
87
-
Yes
H, D, S, B
Enolase
2
9
Yes
H, D, S, B
Py u a e kinase
3
12
Yes
H, D, S, B
Lac a e dehyd ogenase-A
7
13
Yes
H, D, S, B
Glycoly ic enzymes equen ly appea in EVs (Table 2) and p os asomes – EVs p oduced by p os-
a ic cells – om li ing cells98. Se e al mechanisms can explain he p esence o hese enzymes in EVs
such as he ac hey a e ancho ed o lipid a s o e aspanin mic odomains99. Indeed, a majo i y o he
glycoly ic enzymes a e ela ed o lipid a s (Table 2). Up- o-da e, he ull se o glycoly ic enzymes has
been iden i ied in EVs100 h ough he e o s o mul iple ini ia i es as EVpedia101, Vesiclepedia102 o Ex-
oca a103. These da abases ha e been compiling iden i ied p o eins in EVs by means o p o eomics
analysis. In iguingly, lac a e dehyd ogenase-A, he enzyme esponsible o con e ing py u a e o lac-
a e by oxida ion o NADH o NAD+, is among he mos equen ly iden i ied p o eins in EVs (Table 2).
16
None heless, EVs do no only con ey componen s bu hey can ans e hei ac i i y. Ex acellula ATP
p oduc ion was obse ed upon addi ion o uc ose o pu i ied p os asomes104. O he s udies desc ibed
a ginase ac i i y o hepa ocy e-de i ed EVs105 o L-aspa aginase ac i i y in EVs de i ed om neu al
s em/p ogeni o cell106. This sugges s EVs may unc ion as independen me abolic uni s wi h a po en ial
impac o he composi ion o he ex acellula mic oen i onmen .
Me abolic ep og aming media ed ia EVs is p esen in many biological sys ems whe e cell ypes
wi h di e en o he same unc ionali y exchange me abolic asse s107. Besides me aboli es and en-
zymes, EVs can modi y me abolic pa hways by he ans e o nucleic acids, which can u he egula es
glycolysis108,109 o OXPHOS110, among o he s. Du ing oncogenesis, no only cance cells in luence he
en i onmen by means o EV elease bu also s omal cells can d i e me abolic changes in cance cells
by p o iding me abolic esou ces equi ed o cance p og ession111. Cance -associa ed ib oblas s
(CAFs) a e de ined by speci ic mo phological ea u es o exp ession o ma ke s including α-smoo h
muscle ac in (α-SMA), ib oblas -speci ic p o ein-1 (FSP1/S100A4), and ib oblas ac i a ion p o ein
(FAP)112. Recen s udies showed he c oss alk be ween cance cells and CAFs plays a i al ole in
umou g ow h by egula ing hei me abolism. Cance cells p omo e an enhanced glycolysis o CAFs
which, in u n, p o ide TCA- and OXPHOS- ela ed me aboli es113. This phenomenon was ecognized
as he “Re e se Wa bu g E ec ”. In e es ingly, EVs de i ed om CAFs ha e been desc ibed as pa ially
esponsible o umou cell su i al in he hos ile, nu ien -dep i ed, and hypoxic en i onmen o PCa.
These EVs con ain se e al me aboli es, including lac a e, ace a e, aminoacids, lipids, and TCA cycle
in e media es, ha u he a ou s glycolysis, educ i e glu amine me abolism, and p oli e a ion o can-
ce cells67. Reduc i e ca boxyla ion o glu amine eplenishes TCA in e media es h ough glu ama e and
ci a e, which a e also p ecu so s o majo mac omolecules ela ed o anabolism and ca abolism. In
addi ion, lac a e acili a es umou cell su i al unde hypoxic and nu ien -de icien condi ions114. These
esul s u he demons a e ha he exis ing me abolic symbiosis be ween PCa cells and CAFs.
Inc easing e idence a e piled up epo ing EVs as ele an media o s be ween CAFs and cance
cells. PCa cells eleasing EVs can igge no mal ib oblas di e en ia ion owa ds myo ib oblas s o
CAFs. In 2010, Webbe e al. desc ibed ha TGF-β1 con ained in EVs could ac i a e ib oblas s as
soluble TGF-β1 does115. Howe e , in u he s udies hey showed PCa-de i ed EVs igge ed TGF-β1-
dependen ib oblas di e en ia ion ha esembles cance ous s omal cells while soluble TGF-β1 did
no deli e p o-angiogenic o umou -p omo ing pheno ype116. O he s udies pe o med in neona al i-
b oblas cells no ed ha colo ec al cance -de i ed EVs could d i e he exp ession o he ac i a ed i-
b oblas ma ke α-SMA. In he same s udy, he au ho s desc ibed me abolic modi ica ions such as ami-
noacid biosyn hesis, enhanced glycolysis ia glucose and lac a e anspo o up egula ion o glycogen
me abolism, upon EV ea men 117. In b eas cance models, a me abolic emodelling o ib oblas s and
inc eased glycolysis h ough cance - eleased EVs was desc ibed117,118. These esul s con ibu e o he
idea o an EV-media ed signalling ha sus ains umou g ow h and he ac i a ion o ib oblas . I u he
sugges s ha in speci ic sys ems o condi ions, EVs could be a he ele an con ibu o s o cance
p og ession.
17
III. Me abolomics
Un il he ise o he omics e a, molecula biology and physiology app oaches we e u ilised o acqui e
da a ha could desc ibe biological componen s and unc ions, sepa a ely. Nowadays, he in e ac ion
be ween hose is an essen ial ocus in he sys ems biology pa adigm, which aims o explo e physiolog-
ical p ocesses o an o ganism as a whole in a holis ic manne . Omics s a egies hold he po en ial o
iden i y he en i e se o biomolecules (Figu e 7) con ained in biological samples. This gene a es a bas
amoun o da a ha can be co ela ed o desc ibe a biological sys em in a comple e and in eg a ed way.
Me abolomics is de ined as he comp ehensi e and quan i a i e analysis o small molecules wi hin
a biological sys em including cells, bio luids, issues o en i e o ganisms, commonly known as me abo-
li es. In li e a u e, he e m me abolome i s appea ed in 1998 e e ing o he en i e se o me aboli es
p esen in an o ganism o any kind. Me aboli es a e biologically ac i e compounds - smalle han 1500
Da - which pa icipa e in all biological p ocesses o a li ing cell as i.e. building blocks o mac omole-
cules, ene gy ca ie s, signal e ec o s o inhibi o s85,119. Dis inc classes o compounds cons i u e he
me abolome: lipids, aminoacids, ino ganic species o nucleo ides, among o he s. Me aboli es a e di i-
cul o s udy due o hei ex eme a ie y o chemical and physical p ope ies, such as molecula weigh ,
pola i y, solubili y and ola ili y. Mo eo e , he numbe o me aboli es conside ed in a me abolome is
highly a iable and igh ly dependen on he s udied o ganism. I anges om 600 me aboli es es ima ed
in Saccha omyces ce e isiae o app oxima ely 200,000 me aboli es anno a ed in he plan kingdom. I
is accep ed human’s me abolome is smalle han plan ’s one bu mo e han 4,000 compounds ha e
al eady been anno a ed120. The e a e wo g oups o me aboli es ega ding hei implica ion in he bio-
logical sys em. P ima y me aboli es a e hose in ol ed in biological p ocesses, hus making hem es-
sen ial o li e. This is he case o amino acids, o ganic acids, lipids, e c. Ins ead, seconda y me aboli es
a e hose me aboli es no essen ial o he cell o li e. This is because hey do no ha e a ole in any
essen ial biological p ocess. The e o e, hey a e es ic ed o a selec ed se o cells, which syn he ize
hem o speci ic biological unc ions.
Me abolomics aims o iden i y and quan i y a la ge numbe o me aboli es in a biological sys em o
esol e speci ic scien i ic hypo heses121. In gene al, he me hods o choice a e Nuclea Magne ic Res-
onance spec oscopy (NMR) o Mass Spec ome y (MS). The mos widely used mass spec ome e s
a e O bi ap and Time-O -Fligh (TOF) based sys ems because hey p o ide he ad an age o analysing
a comple e mass ange wi h ela i ely high acquisi ion a es122. T iple quad upole (QQQ) o Quad u-
pole-TOF (Q-TOF) p o ide a highe sensi i i y and he oppo uni y o analyse agmen a ion pa e ns,
which is e y use ul o iden i y known analy es in ce ain me abolomics app oaches. MS is usually cou-
pled o a sepa a ion sys em such as Gas Ch oma og aphy (GC-MS), Liquid Ch oma og aphy (LC-MS)
o Capilla y Elec opho esis (CE-MS) o deli e he iden i ica ion o nay me aboli e. To inc ease he
sepa a ion powe , he combina ion o se e al sepa a ion app oaches in o mul idimensional sys ems is
becoming common119. All hese analy ical pla o ms and me hodologies gene a e la ge amoun s o high-
dimensional and complex expe imen al aw da a when used in a me abolomics con ex . The amoun o
18
da a, he need o ep oducible esea ch, and he complexi ies o he biological p oblem unde in es i-
ga ion necessi a e a high deg ee o au oma ion and s anda dise analy ical wo k lows. Many ools and
me hods ha e been de eloped o acili a e he p ocessing and analysis o me abolomics da a; mos
seek o pe o m ep oducible da a analysis and o wo k wi h di e en ypes o aw da a.
Figu e 7. Scheme o he main omics s a egies u ilised in sys ems biology app oach. The scheme
ep esen s he co ela ion o each omic app oach o geno ype o pheno ype in any biological sys ems.
Cu en ly, he e is no ac ual consensus ega ding he classi ica ion o me abolomics s udies. Fo his
eason, we p e e o use a classi ica ion based on whe he he esea che knows a p io i which me ab-
oli es will be quan i ied119. A gene al me abolomics wo k low is ep esen ed in Figu e 8 conside ing his
classi ica ion. A a ge ed me abolomics app oach is he quan i a i e analysis – concen a ions in he
samples a e de e mined – o semiquan i a i e analysis – ela i e in ensi ies a e egis e ed – o ew
up on -known me aboli es ha a e associa ed o common chemical classes o linked o selec ed me -
abolic pa hways119. This app oach p o ides a me abolic p o iling o speci ic biochemical pa hways wi h
biological ele ance o he hypo hesis. The use o iso ope-labelled me aboli es (wi h 13C, 15N o deu e -
ium) in expe imen s pe mi s o ace me abolic eac ions and pa hways. I also allows assessing he
u ilisa ion o a subs a e by a me abolic ne wo k. This ype o analysis can also be pe o med in an
un a ge ed manne . The e m un a ge ed me abolomics was coined o de ine he quali a i e o semi-
quan i a i e analysis o he la ges possible numbe o me aboli es om di e en chemical and biological
classes which a e con ained in a biological specimen119. This app oach enables a apid classi ica ion
o samples acco ding o hei o igin o s a us bu also he analysis o di e en compa men s o a sys em,
o ins ance, cell con en ( inge p in ) and sec e ome ( oo p in ) unde con olled condi ions. In his way,
an al e a ion o a small subse o me aboli es can be pinpoin ed in a b oade con ex o analy es and
desc ibe he po en ial in e ac ions wi h o he me aboli es and/o pleio opic e ec s conside ing se e al
pa hways (o modules) o he en i e me abolomics ne wo k. In con as , iden i ica ion eliabili y is loss.
19
Ideally, one would s a wi h an un a ge ed analysis o p o ide a po en ial answe o he hypo hesis and
inally, con i m i by using a a ge ed app oach.
In his hesis, we will wo k wi h liquid ch oma og aphy coupled o mass spec ome y wi h a a ge ed
me abolomics manne . In his app oach, sample p epa a ion is adap ed o he chemical p ope ies o
he selec ed compounds and he sample ma ix hence, educing signal al e a ion o a ge ed analy es
by non-desi ed compounds123,124. As men ioned, some hypo heses may equi e a b oade se o me-
aboli es o p ope ly desc ibe he impac o he biological sys em. As he inclusion o he whole me ab-
olome has echnical limi a ion and complica ions, di e en ex ac ion and sample p epa a ions ha e
been combined in so-called analy ical pla o ms. These pla o ms enable he ex ac ion o chemically
simila me aboli es in di e en se s o analyse hem sepa a ely. The e o e, we can analyse pola and
apola compounds sepa a ely ye main aining a s ong esol ing and iden i ica ion powe wi hou com-
p omising he selec i i y and sensi i i y o he assay.
Fo a ge ed me abolomics, he ypical me abolomics wo k low (Figu e 8) is o ganised in six gene al
s eps: es ablish he biological p oblem and expe imen al design (1), ideally wi hin he inpu om a s a -
is ician, sample p epa a ion (2), u he da a acquisi ion (3), da a p ocessing (4), s a is ical analysis (5)
and pa hway unc ional analyses (6).
Biological p oblem and expe imen al design
A clea o mula ion o he biological p oblem is c ucial because i will go e n u he decisions du ing
he expe imen design. The in eg a ion o all subjec s ela ed o he wo k low is essen ial o bo h con-
side all he po en ial con ounding a iables and ob ain eadily in e p e able esul s. A his poin , he
ype o me abolomics app oach is de ined conside ing he sample size and ype – cells, luids, EVs,
issues o in ac o ganisms -, he expe imen al condi ions, equency o collec ion, s o age condi ions,
me abolic quenching o in e up me abolism and, he analy ical pla o ms and p epa a ion s a egies.
Sample p epa a ion
Once he esea che s o he ound able uled he mos app op ia e expe imen design, he app o-
p ia e sample p epa a ion mus be de ined. Because o he wide a ie y o physicochemical p ope ies
o me aboli es, he ex ac ion p ocedu e is usually op imised o a speci ic se o compounds o chemical
classes in he analy ical pla o ms. I usually in ol es clean-up s eps o emo e sample ma ix in e -
e en s (i.e. p o ein p ecipi a ion wi h me hanol), p econcen a ion s a egies o boos de ec abili y and
he selec ion o app op ia e ex ac ion sol en s125. An impo an conside a ion o make is ha me abo-
lism is a cons an lux; hus, s opping any po en ial me abolic eac ion is impe a i e in sample collec ion,
p ese a ion and manipula ion o a oid any po en ial loss o me aboli e signals.
Me aboli e ex ac ion me hods om he collec ed samples ha e been de eloped o be e ec i e o
speci ic compound classes. This implies each me hod will lead o he loss o me aboli es no speci ic o
he assay. Conside ing all po en ial issues in ex ac ion me hods, an ideal p epa a ion should: (i) inco -
po a e a p ese a i e so ha he me aboli e composi ion e lec s he o iginal one a he sampling mo-
men ; (ii) be as non-selec i e as possible in o de o inco po a e he b oade ange o me aboli es. To
20
his aim, a combina ion o dis inc ex ac ion p o ocols may be conside ed; (iii) be simple and as , o
a oid me aboli e loss; (i ) be ep oducible.
Da a acquisi ion
The chemical di e si y o he me abolome, as well as i s wide dynamic ange, is a d awback o
achie e a comp ehensi e iden i ica ion and quan i ica ion o an en i e me aboli e se in a biological
sys em. I is impo an o emphasize ha no only dis inc chemical p ope ies, s uc u al a angemen s
and unc ionali ies di e en ia e me abolic compounds bu also hei concen a ion le els. Fo ins ance,
suga s and concomi an in e media es ange be ween mic omola o millimola depending on he ma-
ix126,127 bu s e oid ho mones a e a he nanomola ange.
Typically, he analy ical echniques employed o da a acquisi ion a e NMR and MS. Thei s eng hs
and limi a ions mus be conside ed o decide he bes app oach. While NMR spec oscopy is conside ed
as a uni e sal me aboli e de ec ion echnique, wi h a low sample manipula ion and a wide a ie y o
chemical classes de ec ed simul aneously, MS is much mo e sensi i e and speci ic, howe e , i usually
equi es a sepa a ion echnique o educe sample complexi y and ion supp ession128–130. Nowadays,
MS is usually coupled o high-p essu e liquid ch oma og aphy (HPLC) and ul a-high p essu e liquid
ch oma og aphy (UPLC). The mass spec ome e is composed o an ion sou ce, which is he en y poin
o he ionized me aboli es in o he equipmen , a mass analyse o sepa a e he analy es by mass- o-
cha ge a io (m/z) and he de ec o . The m/z abb e ia ion s ands om he alue esul ing om di iding
he mass numbe (m) o an ion by he co esponding cha ge numbe (z) and i is cha ac e is ic o each
chemical o mula131. Analy ical pla o ms o en exhibi s abili y issues o e ime, which can be ci cum-
en wi h he use o a quali y con ol (QC). I is p epa ed by mixing small olumes o all unning samples.
QC is no mally un se e al imes up on samples o check o ins umen al s abili y and in e cala ed
du ing samples un o ensu e da a eliabili y.
Da a p ocessing
A co ec analy e quan i a ion o semiquan i a ion is impe a i e in a ge ed me abolomics. Usually,
an assay is de eloped unde speci ic and op imised condi ions o a speci ic se o me aboli es. The e-
o e, u he alida ion o he pa ame e s speci ici y/selec i i y, accu acy, linea i y, esolu ion, limi s o
de ec ion and quan i ica ion and, ep oducibili y is equi ed o es ablish a me hodology119.
Raw da a collec ed om he mass spec ome e is colossal and unmanageable. Hence o h, he
aim o his s ep is o gene a e a 2D da a able o ea u es o me aboli es de ined as m/z - RT pai s whe e
da a has been al eady co ec ed acco ding o QC, no malised and, i equi ed, ans o med, cen e ed
and/o scaled. Fo mo e de ailed in o ma ion ela e o Chap e 6 o he book 119.
21
Figu e 8. Analy ical wo k low o s udies in me abolomics. This me abolomics wo k low com-
p ises he sequen ial s eps ha unde line bo h a ge ed and un a ge ed analyses. S a ing om a
p oblem o hypo hesis o mula ion i p o ides me abolic pa hway ela ionships. Rep in ed om 119.
RESULTS
31
Chap e 1. Cu en App oaches o S udy Ex acellula
Vesicles in Cell Physiology and I s Applica ions
This chap e ocusses in assessing cu en app oaches in he EV ield including ecen ad ances de-
i ed om he access o new echnologies. Thei applica ion in cance physiology and bioma ke dis-
co e y is also discussed. I has been published as a Re iew A icle in Na u e P o ocols jou nal and i is
appended in he supplemen a y ma e ial.
Bo danaba-Flo i , G. e al. Using single- esicle echnologies o un a el he he e ogenei y o ex acel-
lula esicles. Na P o oc 16, 3163–3185 (2021). DOI: 10.1038/s41596-021-00551-z
1.1. In oduc ion
Since he desc ip ion o minu e bodies ound in a piece o co k by Robe Hooke in 166561, bo h ou
scien i ic knowledge and echnical abili ies ha e inc eased eno mously. As echniques ha e become
mo e accu a e and in ica e, so has ou unde s anding o biological p ocesses and s uc u es. Techno-
logical ad ances in he ield o imaging ha e esul ed in he iden i ica ion o cell s uc u es, such as he
mi ochond ia62 and nuclei63, and he disco e y o di e en le els o cellula complexi y. In 1967, Pe e
Wol isualised pla ele dus in esh pla ele - ee blood plasma using an elec on mic oscope64; hus, a
mammalian esicle-like s uc u e was desc ibed o he i s ime. G adually, hese esicles we e cha -
ac e ised in mo e de ail. I has been es ablished ha hey a e eleased by all kinds o cells (p oka yo ic
and euka yo ic) in o he ex acellula milleu172–175. The p ocess o esicle sec e ion is conse ed
h oughou e olu ion, sugges ing ha such esicles a e likely o ha e speci ic oles in he cellula and
o ganismal de elopmen and su i al176. Indeed, la e disco e ies showed ha sec e ed esicles pa -
icipa e ac i ely in many physiological p ocesses in mammals, o example coagula ion, in lamma o y
esponse, cell ma u a ion, adap i e immune esponse, bone calci ica ion and neu al cell communica-
ion, among o he s65,68. In addi ion o hei c i ical unc ions in no mal physiology69,70, sec e ed esicles
media e in se e al pa hological p ocesses71,72, such as he es ablishmen o p e-me as a ic niche du ing
cance p og ession73,74.
Nowadays, hese sec e ed esicles a e ex ensi ely epo ed and widely known as ex acellula es-
icles (EVs). EVs a e he e ogeneous, nano- o mic ome e-sized, bilaye lipid con aine s sec e ed by
mos cell ypes. They a e mul i-pu pose ca ie s ha can con ain a wide a ie y o ca gos such as lipids,
p o eins, me aboli es, suga s, RNA (mRNA, miRNA, siRNA) and e en DNA65. When hey a e aken up
by ecipien cells, hey igge in acellula signalling h ough EV su ace molecules o by he elease o
ca go in o cell compa men s ia endocy ic pa hways. These p ocesses can u he ac i a e down-
s eam gene ic o me abolic pa hways in he ecipien cell67,68. In mammals, EVs ha e been ound in
body luids like plasma, u ine, sali a, b eas milk and seminal luid, among o he s. They a e classi ied
in o h ee g oups acco ding o hei biogenesis mechanisms and biophysical p ope ies:
exosomes: ypically 30–150 nm in diame e , de i ed om in acellula endosomal com-
pa men s,
32
mic o esicles: 100–1000 nm in diame e , p oduced by ou wa d budding and pinching-o
he plasma memb ane, and
apop o ic bodies: 50–5000 nm in diame e , eleased as blebs by cells unde going apop-
osis64,65.
In a sys ema ic e iew o guidelines o his ield, he In e na ional Socie y o Ex acellula Vesicles
(ISEV)75 endo sed a ca ego isa ion o EVs isola ed using ul acen i uga ion in o la ge, medium and
small EVs. Howe e , i is impo an o no e ha ul acen i uga ion p ecipi a es no only esicles bu also
lipop o eins, i uses, p o ein agg ega es, ibonucleop o ein complexes and exome es76–78, which p o-
ides an addi ional laye o di e si y bu also a bias upon analysis o EV samples. Fu he mo e, he e
is e idence ha exosomes, mic o esicles and apop o ic bodies con ain subpopula ions wi h unique
oles in biological p ocesses177,178. These subpopula ions a e igh ly in eg a ed wi h a b oad ange o
biological p ocesses and exhibi a wide ange o unc ionali ies, which makes hem an ou s anding
sou ce o po en ial bioma ke s o ea ly diagnosis, d ug deli e y sys ems o he apeu ics, o accine
p oduc ion sys ems72,179–181.
Du ing he las ew decades, he in e es in EVs and hei applica ions has g own conside ably. Many
a icles and e iews ha e ocused on he unc ional ole o EV he e ogenei y. Thei ole in speci ic bio-
logical p ocesses, such as ca go a icking o egula ion o signalling pa hways, and hei po en ial as
bioma ke s ha e also been examined68,72,178,182,183. None heless, mos o hese s udies examine he
esicles in bulk and use ensemble-a e aging assays. Al hough such me hods ha e been p o en use ul
in speci ic cases, i is impo an o ealise ha he ex ensi e he e ogenei y o s uc u e, composi ion and
unc ion o single esicles a e masked in such assays177,182–184. Fo example, he inabili y o de ec he
he e ogenei y o molecula s a es o eac ion pa hways, indi idual p o eins o nucleic acids may lead o
a misin e p e a ion o ensemble measu emen s185,186. Recen de elopmen s in single- esicle analysis
(SVA) ha e opened new oppo uni ies o he examina ion o he e ogenei y wi hin EV (sub)popula ions
a he indi idual EV le el and hei cha ac e isa ion on he nanome e scale176,182. This new in o ma ion
is pa amoun o unde s anding he biological unc ions o EVs and o hei po en ial clinical use.
Di e en EV popula ions and subpopula ions can be isola ed acco ding o hei physicochemical
p ope ies, ye he exis ing isola ion echnologies a e in ica e and s ill need o be u he de el-
oped177,182. The e a e i e main g oups o echniques o so ing EV popula ions and subpopula ions,
based on ul acen i uga ion, size, immunoa ini y cap u e, polyme p ecipi a ion and mic o luidics65.
Since each echnique ype so s EVs using a di e en p inciple, each me hod can yield di e en EV
subpopula ions om he same sample187,188. Mo eo e , he highly concen a ed EV p epa a ions may
con ain con aminan s, such as la ge p o ein agg ega es and lipop o eins, le behind by some o he
isola ion echniques189,190. In e es ingly, di e en app oaches can also a ec he physicochemical su -
ace cha ac e is ics o EVs191.
In consequence, some echniques end o en ich o disc imina e agains speci ic EV popula ions. As
so ing EVs in o popula ions is usually based on physical p ope ies only, we ha e o assume ha such
classi ica ion is la gely a bi a y wi h espec o he composi ion o unc ion o EVs. Vesicle isola ion and
33
en ichmen echniques can help o yield mo e homogeneous EV subpopula ions, albei only o pa ic-
ula echnique-speci ic pa ame e s. In conclusion, al hough a ious isola ion me hods could help d i ing
EV analysis owa ds a single- esicle app oach176, many di e en composi ions and unc ionali ies a e
s ill expec ed o be ound wi hin such EV popula ions.
In his e iew, we desc ibe he cu en me hods used o s udy single- esicles , and hei con ibu ions
o he unde s anding o EV biology and bioma ke disco e y. Single- esicle expe imen s can deli e
di ec in o ma ion on he he e ogeneous composi ion o EVs. They e eal mul iple molecula s a es ha
go e n EV unc ionali y and anspo and p o ide s a is ically alid in o ma ion o en los in la ge en-
semble expe imen s66,182,192.
1.1.1. EV he e ogenei y and bioma ke disco e y.
The e a e many echnological challenges o be me in he de elopmen o EV-based diagnos ics.
The ele an esicles mus be iden i ied and isola ed om complex bio luids, and a speci ic disease-
ela ed EV popula ion o popula ion mix has o be de ec ed. Biomedical s udies o EVs o en ocus on
seeking sui able bioma ke s o he diagnosis o a ious diseases193,194. Fo example, he unc ional ole
o EVs in a ious ypes o cance has been ex ensi ely s udied71,73,74,180. In his e iew, we concen a e
on p os a e cance (PCa) diagnos ics as an example applica ion o which he SVA o EVs has made
impo an con ibu ions. Acco ding o he Wo ld Heal h O ganiza ion, PCa is among he mos equen ly
diagnosed ypes o cance , accoun ing o app oxima ely a qua e o all cance diagnoses in Eu ope195,
ye he lack o sensi i e diagnos ic ools and insu icien knowledge o he mechanisms o cance eme -
gence and p og ession a e o majo conce n. PCa is a he e ogeneous pa hological s a e, bo h in he
p ima y umou in he p os a e issue and a he me as a ic s age. I is un o una ely no ecommendable
o examine PCa di e si y elying solely on issue biopsies, since hese a e highly in asi e p ocedu es
and do no gua an ee an e ec i e and eliable diagnosis196–199. The se um p os a e-speci ic an igen
(PSA) es —s ill he co ne s one o PCa sc eening—is pa icula ly ques ionable. Up o 40% o men
unde go unnecessa y biopsies as a esul o poo speci ici y o he assay.
Rema kably, liquid biopsy has a po en ial o ma ke iden i ica ion and p o ides be e e idence o
PCa di e si y han he con en ional solid issue biopsy. In pa icula , p os a e- and PCa-de i ed EVs
and concomi an ma ke s a e highly abundan in u ine, blood and ejacula e samples60,200. Hence, hese
body luids could be used o de ec ing and measu ing he p og ession o he disease. Fo example, he
EVs eleased by PCa cells ca y unique p os a e-speci ic memb ane p o eins (e.g. TMPRSS2,
STEAP2, PSMA, PPAP2A, e c.) ha enable he de ec ion o pa hogenic p os a e EVs and hei cap u e
o ex i o cha ac e isa ion201. Al hough hese da a show ha liquid biopsies may be highly in o ma i e
and minimally in asi e p ocedu es, he me hods o esicle isola ion, cha ac e isa ion and iden i ica ion
o disease diagnos ics emain challenging. Up o da e, he e a e no s anda dised ope a ing p ocedu es
o esicle isola ion and cha ac e iza ion o di e en ypes o samples and diseases, which complica es
he employmen o liquid biopsies as a clinical sou ce o EV bioma ke s.
34
1.1.2. Single esicle analysis echniques o biological cha ac e isa ion o EVs.
Mos o he s udies e iewed in his a icle examine he h ee p oblems ha can be add essed by
aking ad an age o SVA echniques: (1) cha ac e isa ion o EV he e ogenei y including subpopula ions,
su ace (memb ane p o ein and lipid) composi ion and esicle con en , (2) s uc u al s udies o EV mem-
b ane and soluble p o eins and he assays o p obe he me abolic ac i i y o hese p o eins in a na i e-
like en i onmen and (3) cha ac e isa ion o he EV con en and unc ion depending on he cells o o igin.
The las ask p esen s an in e es ing dicho omy: do he EVs e lec he p ope ies o hei cells o o igin,
o a e hey comple ely independen communica ion asse s? On he one hand, i has been epo ed ha
EV su ace and con en depend on he pa en al cells202–204. On he o he hand, some ecen epo s
desc ibe se e al EV subpopula ions, wi h a ange o di e en unc ionali ies, o igina ing om he same
cell ype66,67,176,182,205,206. In iguingly, ano he ecen a icle using SVA echniques demons a es ha he
T2SS-like amily o p o eins is, in ac , esponsible o selec i e ca go loading in o EVs gene a ed by
he mic oo ganism Shewanella esiculosa207.
1.2. Single- esicle echniques
As inc easing numbe s o esea che s ha e highligh ed he impo ance o accu a e EV (sub)popu-
la ion so ing and pheno yping, so a , mo e han 20 new SVA echniques ha e been de eloped66,176,182.
Many o hese use mic o luidic de ices designed o in eg a e a ious echnologies o imp o e EV so ing
and de ec ion. Mo eo e , se e al o hese me hods ha e been used o cha ac e ising EVs a he single-
esicle le el208–219. Some o hese echniques can di ec ly p o ide in o ma ion on esicle su ace, con-
en , size and shape while o he may equi e an ups eam physicochemical cha ac e isa ion o he se-
lec ed EV subpopula ions o conduc su ace p o iling, moni o he exp ession o bioma ke s and quan-
i y hem in body luids. These echnological ad ances should help o design new diagnos ic de ices
o small sample sizes, using minimally in asi e me hods.
Twel e di e en me hods a e p esen ed in Table 5 and discussed in de ail in he ollowing sec ions.
Some o hese me hods u ilise labelling echniques (such as luo escence o nanopa icle coa ing) o
isualise he EVs, and o he s wo k as label- ee sys ems (Figu e 9). I is impo an o no e ha in some
cases label- ee app oaches may hinde he de ec ion o EVs because hey o en p oduce weak signals,
which can be enhanced using a suppo ing labelling echnique.
1.2.1. Label- ee me hodologies.
Nanopa icle acking analysis (NTA) is a echnique based on he B ownian mo ion o mic opa icles
in suspension and i is used o de e mine he size dis ibu ion in pa icle popula ions208,220. In his ap-
p oach, mic opa icles a e de ec ed by sca e ing he ligh o a lase beam, which is acked and ec-
o ded a ideo ame a es. Howe e , his app oach has some disad an ages and limi a ions. Fo in-
s ance, he accu a e assessmen o pa icle size dis ibu ion equi es speci ic ack leng hs, a s eady
empe a u e and a la ge numbe o eplica es o p o ide obus esul s. Ca e should be aken when
compa ing di e en samples because a ia ions in bu e iscosi y and mic opa icle concen a ion in-
oduce s a is ical e o s. Mo eo e , he close p oximi y o wo pa icles can esul in o e lap o he
35
sca e ing signals. Accu a e de ec ion o pa icles wi h a diame e below 60 nm is challenging, ega d-
less o he NTA machine used221. Fu he mo e, esicles canno be disc imina ed om o he pa icles,
such as p o ein agg ega es o i us pa icles. The esicles can be p obed speci ically, and undesi ed
pa icles excluded om he analysis only by employing luo escen ma ke s, ye only a ac ion o EVs
may ca y known ma ke s ha can be used o labelling a speci ic subpopula ion. Gene al luo escen
labels (such as lipophilic ca bocyanines DiO o DiI) can be used ins ead. Howe e , i is impo an any
non-a ached label is emo ed since his can mask he luo escence signal emi ed by labelled EVs66.
Figu e 9. Schema ic o e iew o he main SVA echniques discussed in his e iew. Da a isualisa ion and single-
esicle in e p e a ion using each SVA me hodology a e depic ed. In he cen e o he igu e, a ( umo igenic) cell
eleasing EVs is shown. The echniques can be di ided in o wo g oups: label- ee (a-e) and label-based ( -i) me h-
odologies. The me hods used he e a e: a) C yo-elec on mic oscopy222,223, b) AFM, c) NTA176,224, d) RTM223, e) SP-
IRIS225, ) h FC226, g) ddPCR227, h) SRM228 and i) luo escence mic oscopy (TIRF image o synap ic esicles is
depic ed)229. Re e ences show he sou ce o he images.
Raman weeze s mic ospec oscopy (RTM), also known as lase weeze s Raman spec oscopy
(LTRS), can be employed o examine he chemical con en o EVs. This app oach can be used o
36
in es iga e bo h he su ace and he in e nal olume o single EVs, e ealing speci ic biomolecula sig-
na u es o p o eins, lipids, nucleic acids and ca o enoids as majo con ibu o s204–206,216,225–231. RTM is
an inelas ic sca e ing-based me hod. I employs a igh ly ocused lase beam o bo h op ical apping
o single (o e y ew) esicles in aqueous medium and exci a ion o subsequen Raman sca e ing,
which p o ides a ib a ional inge p in om he apped cons i uen biomolecules. The main inhe en
ad an age o RTM lies in he signal linea i y, which allows bo h quali a i e and quan i a i e biochemical
cha ac e isa ion o single EVs. This me hod is also label- ee and p o ides da a wi h high in o ma ion
con en 209,223,230. The main disad an age is ha he sca e ing e iciency is usually e y low and hus
p o ides a a he low le el o in o ma i e Raman signal. As a esul , an ex ended da a collec ion ime is
equi ed. The e o e, RTM, wi h a ypical p ocessing capaci y o 0.2 pa icles pe min, is no conside ed
a high- h oughpu me hodology230. RTM can, howe e , be used o ob ain in e es ing, unique in o ma ion
no only o EVs209–211,223,230–236 bu also o many o he biopa icles like liposomes, lipid laye s on syn-
he ic nanopa icles and o he s237–242.
Se e al me hods ha e been de eloped o compensa e o he low Raman signal s eng h in RTM.
Fo example, he esicle concen a ion can be inc eased by d op-coa ing deposi ion o he sample,
ollowed by d ying243–247. Un o una ely, his app oach esul s in loss o in o ma ion abou indi idual EVs,
as does any o he analy ical s udy o a bulk sample. Ano he s a egy o inc ease he Raman signal is
o use su ace-enhanced Raman spec oscopy (SERS). In his me hod, EVs can be exposed o a ious
signal-enhancing nanopa icles and/o subs a es o ob ain a s eng hened biomolecula signal212,248–
254. The main p oblem o label- ee SERS is ha he enhancemen e ec depends s ongly on he dis-
ance be ween he biomolecule and he nanopa icle/subs a e, and anishes a dis ances longe han
a ew nanome e s248. The e o e, his me hod is mainly sui able o cha ac e isa ion o biomolecules on
he ou e su ace o EVs. In addi ion, Raman modes co esponding o molecula ib a ions pe pendic-
ula o he SERS su ace a e p e e ably enhanced248. As a esul , he o e all SERS ib a ional spec um
is usually dis o ed, lacks ep oducibili y, and is o en di icul o in e p e .
In elec on mic oscopy, a beam o elec ons is emi ed on o a sample in a acuum en i onmen . The
wa eleng h o elec ons is sho e han he isible ligh used in op ical mic oscopy; hus, he me hod
gi es images o much highe esolu ion, ypically below 1 nm66. C yogenic ansmission elec on mi-
c oscopy (c yo-TEM) is among he elec on mic oscopy me hods mos commonly u ilised o EV cha -
ac e isa ion. In con as o he leng hy sample p epa a ion needed o o he TEM me hods (usually ak-
ing hou s), no hea y me als o ixa i es a e added, and no dehyd a ion s eps a e equi ed. This also
limi s sample damage and a e ac e ec s, bu yields lowe con as images255. In c yo-TEM, he sam-
ples a e p epa ed by apid eezing, ypically wi h liquid e hane209,256. In his p ocess, he wa e i i ies,
ins ead o o ming o de ed c ys als, and he na i e s uc u e o EVs is p ese ed257. The i s exosome
isualisa ion was achie ed using c yo-EM in 2008258. Since hen, his echnique has success ully e-
ealed EV polymo phism by imaging he memb ane bilaye s, EV s uc u es and in e nal ea u es o
indi idual EVs209,213,255,259. E en hough c yo-TEM is an ex emely use ul echnique o high- esolu ion
isualisa ion o EVs, his app oach is ela i ely low- h oughpu . C yo-TEM images ypically only con ain
a ew EVs (al hough he h oughpu could be enhanced by using au oma ed sea ch). In addi ion, c yo-
37
Table 5. Summa y o main SVA echniques o indi idual EV cha ac e isa ion.
Technique
De ec ion p inciple
In o ma ion ob ained
Th oughpu
Time pe
analysis (a)
Sample p epa a ion (b)
Is sample
eusable?
Loading ol-
ume
Wo king con-
cen a ion
(mL-1)
High- esolu ion low
cy ome y
Elas ic ligh sca e ing. Au o- luo-
escence o luo escence om
ex e nal labels.
P o iling(c) o EVs in a he e ogene-
ous luid mix u e. Func ionalised lu-
o escen labels cha ac e ise spe-
ci ic popula ions.
High
~1 min
Immuno luo escence o lu-
o escen conjuga e s aining
p o ocol.
No (d)
20–100 µL
107–1010 (e)
Nanopa icle ack-
ing analysis
Imaging o B ownian mo ion pa h-
ways o EVs, using elas ic ligh
sca e ing o luo escen labels.
Pa icle concen a ion, size dis ibu-
ion.
Mode a e
~1 min
Dilu ion o concen a ion o
EVs o he ange op imal o
he me hod.
Yes
0.3–1mL( )
108–109
Raman weeze s
mic ospec oscopy
Raman sca e ing om op ically
apped single EVs.
Biomolecula (g) composi ion o he
su ace and he in e io o single
EVs.
Low
~1-5 min(h) pe
one(i) apping
e en
Concen a ion o EVs o he
ange op imal o he
me hod.
Yes
~100 µL
107–1011(j)
Su ace-enhanced
Raman spec os-
copy (SERS)
Raman sca e ing enhanced by
ex e nal ac i e su ace/coa ing.
Pa ial(k) biomolecula (g) composi ion
o he memb ane o single EVs.
Mode a e
~1-10 s pe
one(i) EV
Fixa ion and coa ing p o o-
col.
No
50–100 µL
109 –1011
SERS wi h ex e nal
labels
Enhanced Raman signal om
SERS nano ags.
Numbe o EVs wi h speci ic unc-
ionalised SERS nano ags a -
ached.
High
~10 s
Fixa ion and SERS-label
s aining.
No
10–100 µL
10 –107(l)
C yo- ansmission
elec on mic oscopy
(C yo-TEM)
T ansmission elec on mic os-
copy imaging.
Mo phological EV cha ac e isa-
ion(m). Di ec isualisa ion o single
EVs and examina ion o con ami-
nan s.
Low
~1 hou (h)
Vi i ica ion o wa e in EVs
dispe sed on ca bon g id,
using as -plunge eezing.
No
2–10 µL
1010–1012
A omic o ce mi-
c oscopy (AFM)
Imaging ( as e scanning) exploi -
ing in e ac ion o ce be ween he
p obing ip and immobilised EV.
P ecise(q) mo phological, mechani-
cal and biochemical (o) cha ac e isa-
ion o he EV su ace.
Low
~1 min pe im-
age(s)
Immobilisa ion p o ocol.(p)
No
5–25 µL
Rela i e(q)
Table con inued
44
Table 6. Summa y o EV cha ac e isa ion s udies using SVA echniques.
Technique
Main conclusion
Re
AFM
The i s ime ha plan exosomes ha e been isualised in hei na i e s a e. They ha e been obse ed on he in e nal
laye s o he cell walls and hei ca go assessed.
313
Ma ix esicles ini ia e changes du ing he mine alisa ion o he ex acellula ma ix. In he cou se o his p ocess, he
ma ix esicles inc ease hei size and c ys allini y and change shape.
314
AFM-IR
The i s a emp o p obe he di e ences be ween he molecula cons i uen s (p o eins, lipids and DNA) and s uc u es
o indi idual esicles in wo sub ypes o placen a s em cells. In his wo k, p o ein agg ega es ha e been success ully
di e en ia ed om esicle s uc u es.
266
RTM
Vesicle shape and size depend on he lipid composi ion o he memb ane. A dec ease in choles e ol concen a ion
inc eases he local memb ane cu a u e and s e ches he esicle.
241
Cance esea ch
h FC (immuno-
luo escence)
Mic oen i onmen acidi y o he p os a e umou inc eases he elease o p os a e-speci ic EVs.
331
EVs om PCa cells a e e y he e ogeneous, bu speci ic popula ions a e no associa ed wi h di e en cance s ages.
Mainly mic o esicles and se e al exosome subpopula ions a e ound, acco ding o he su ace signa u e.
332
NTA
Inc ease in he numbe o eleased exosomes unde acid pH (6.5), independen o umou his o ype.
330
Tumou cells elease mo e EVs han non- umo igenic cells (mos likely due o he acidic en i onmen in umou cells).
327
He e ogenei y- ela ed esea ch
NTA
Desc ip ion o di e en EV popula ions in human glioblas oma cells.
322
NTA (wi h luo-
escence)
The i s a emp o use his echnique o de e mine he concen a ion and pa icle dis ibu ion in speci ic EV subpopu-
la ions, acco ding o su ace ma ke s such as CD9, CD63, imen in and LAMP-1.
328
Elec on mi-
c oscopy
(c yo-TEM)
La ge di e si y o exosome mo phology e ealed, ega dless o he cell ype and o igin, sugges ing ha di e en exo-
some subpopula ions om he same cell line pe o m di e en unc ions.
302
Visualisa ion o a la ge spec um o EVs om ce eb ospinal luid, including mul ilaye esicles, single, double and dou-
ble-memb ane esicles, and in e nal esicula s uc u es. These speci ic subpopula ions a e sugges ed o se e as
po en ial bioma ke s o Pa kinson’s disease.
321
Two di e en s imuli a ec ing EV elease. Lipopolysaccha ides and s a a ion condi ions in human leukaemia mono-
cy ic cell line (THP-1) a ec he ype o EVs shed and, p obably, he shedding p ocess.
222
AFM
EVs isola ed om di e en species a e imaged. Using he apping mode, he mechanical p ope ies o EVs a e as-
sessed, concluding ha he a EVs a e mo e agile han he mouse esicles.
326
RTM
Du ing cell g ow h and s a a ion-induced agg ega ion, Dic yos elium discoideum p oduces EVs ha di e d as ically
in hei biomolecula composi ion (nucleic acids, p o eins, lipids, ca o enoids).
209
De ec ion o ou EV popula ions wi h speci ic p o ein, phospholipid and choles e ol su ace signa u es. Thei unc ion-
ali y de e mines hei dis ibu ion; he popula ions a e sha ed among se e al cell ypes.
230
In EV p epa a ions om a hepa ocy es and human u ine, single EVs om he same sample ha e di e en biochemi-
cal p ope ies, well beyond he “usual” a ia ions o EVs om a hepa ocy es o human u ine. A quan i a i e me hod
de eloped o measu e he concen a ion o nucleic acids in a single EV.
223
Combined mo pho-chemical p o iling o indi idual EVs is p oposed based on Raman-enabled nanopa icle apping
analysis.
233
45
SVAs ha e been ex ensi ely u ilised in cance esea ch, o example, o cha ac e ise he EVs in
PCa. NTA-based esea ch has es ablished cance cells p oduce la ge amoun s o EVs han non- u-
mo igenic cells194,217,327 and ha low ex acellula pH inc eases he elease o EVs om cance
cells328,329. In e es ingly, NTA s udies also suppo he idea ha umo igenic cells up egula e EV p o-
duc ion due o he acidi ica ion o he immedia e mic oen i onmen 327–330. Logozzi e al. ha e employed
a combina ion o h FC and immuno luo escence o disco e ha acidic mic oen i onmen o p os a e
umou s induces he elease o PCa-speci ic EVs330. These EVs a e e y he e ogeneous, and se e al
popula ions can be iden i ied, acco ding o hei su ace composi ion. Howe e , i is impo an o no e
ha his he e ogenei y is no ela ed di ec ly o di e en cance s ages333.
In summa y, he highligh ed epo s o single-EV cha ac e isa ion demons a e ha EV mo phology
and composi ion a e la gely independen o cell o igin and ha ce ain (sub)popula ions a e in ol ed in
a ious diseases. These disco e ies indica e ha mo phologically di e en EV popula ions may be dis-
ibu ed acco ding o speci ic unc ions and biogenesis pa hway, a he han hei o iginal cell- ype.
1.3.2. EV a icking and signalling mechanisms.
SVA app oaches ha e ad anced EV esea ch by acking pa icula molecules and examining he
changes in cells unde di e en condi ions. Ea ly in 2020, FRET s udies in T cells showed ha he
concen a ion o ee zinc in cells is a majo egula o o he ma u a ion p ocess in insulin-s o ing esi-
cles334. SVA has also con ibu ed o disco e ies epo ing an inc ease in in acellula Ca2+ and/o p o ein
C unde ce ain s imula ing and ac i a ing condi ions in ed blood cells. These condi ions al e cell mo -
phology and cause an inc ease in he elease o mic o esicles335. Table 7 summa ies mo e examples
o ecen ad ances in esea ch in (a) esicle–cell communica ion and a icking and (b) esicle usion
and endocy ic pa hways.
EVs a e se e as in o ma ion ca ie s o cell pa hways and may igge some diseases due o his
ac i i y. Neu odegene a i e diseases like Alzheime ’s a e caused by he mis olding and agg ega ion o
au p o ein. Polanco e al. ha e desc ibed a p ion-like sp ead o au p o ein seeds b ough abou by
EVs, employing c yo-TEM336 and SRM (PALM and STORM)228. The con ibu ion o EVs o ans-syn-
ap ic au ansmission has been con i med using c yo-TEM in ano he s udy337. This single- esicle
me hodology has also been u ilised ecen ly o s udy he nea -na i e 3-D a chi ec u e o EVs sec e ed
a e in ec ion wi h polio i us. C yo-TEM omog aphy has gene a ed images o i ions and i al s uc-
u es con ained in EVs be o e cell lysis338. Mo eo e , FRET mic oscopy has been used o ack he iad
p o ein VOR (VAP-A, ORP3, Rab7), pa amoun o he ans e o EV-de i ed componen s o he nu-
cleus339. This ype o esea ch has he apeu ic po en ial o diminishing he p og ession o neu odegen-
e a i e diseases (in he case o he VOR complex, by inhibi ing EV-media ed in e cellula communica-
ion).
Cance mechanisms ha e also been analysed using single-molecule echniques. Manna ola e al.
ha e pe o med a ddPCR expe imen using os eo opic melanoma cells and obse ed ha EVs could
induce he up egula ion o genes such as CXCR7340. Thus, EVs may ac as chemo axis agen s and,
46
hence, pa icipa e in he p og ession o cance ; howe e , addi ional esea ch in his ield is s ill equi ed
o achie e be e unde s anding o how EVs con ibu e o cance 340.
Table 7. Mechanis ic s udies o esicles using SVA echniques ocusing on esicle–cell communica ion, a ick-
ing and esicle usion and endocy ic pa hways.
Technique
Main conclusion
Re
Vesicle–cell communica ion and a icking esea ch
FRET mic oscopy
Tau p o ein mis olding and monome agg ega es sp ead in a p ion-like manne causing neu odegene a i e dis-
eases such as Alzheime ’s. Ex acellula esicles play a ole in he ansmission o pa hological au seeds.
336
The iad p o ein VOR is essen ial in he egula ion o he endocy ic nuclea ans e o EV-de i ed componen s. Ta -
ge ing VOR migh ha e he apeu ic po en ial by inhibi ing EV-media ed in acellula communica ion.
339
Fluo escence mi-
c oscopy (SRM)
In i o isualisa ion o EVs in he zeb a ish emb yo. Up ake o EVs by endo helial cells and blood pa olling mac o-
phages is shown. The s udy demons a es umou EVs ac i a ing mac ophages and p omo ing me as asis.
218
Elec on mic oscopy
(c yo-TEM)
Mul iple i ions and unique mo phological componen s o ming a ma -like s uc u e a e anspo ed ia in ec ious
EVs o 100–1000 nm in diame e . These esicle-con aining en e o i us a e sec e ed om hos cells p io lysis.
338
ddPCR
EVs om os eo opic melanoma cells induce chemo axis and cance p og ession ia up egula ion o CXCR7 o non-
os eo opic melanoma cells.
340
Vesicle usion and endocy ic pa hway esea ch
AFM
Desc ip ion o mechanical p ope ies o e y h ocy e-de i ed EVs acco ding o hei p o ein–lipid a io. While a high
a io is associa ed wi h so esicles, a low a io co esponds o s i EVs. These mechanical di e ences a e linked o
se e al esicula ion and budding mechanisms.
341
Elec on mic oscopy
(c yo-TEM)
Visualisa ion o he SNARE-media ed memb ane usion-by-hemi usion o small esicles wi h cellsshows usion in-
e media es whe e lipid monolaye s pa ially mix en ou e o comple e bilaye me ge .
342
FRET mic oscopy
Desc ip ion o he molecula mechanism o SNAREs du ing di e en memb ane usion s ages, (docking, hemi usion
and ull usion) by acking he lipid-mixing p ocess a he single- esicle le el.
343
Fluo escence mi-
c oscopy
The usion pa hways a e he e ogeneous wi h an a es ed hemi usion s a e p edomina ing. The usion o wo lipid
bilaye s occu s spon aneously in a single s ep when hey a e b ough in o close p oximi y.
344
Fluo escence mi-
c oscopy (TIRF)
He e ogenei y o endocy ic esicle beha iou s upon in e nalisa ion. P io o scission, esicles emain p oximal o he
plasma memb ane o a iable pe iods. Cla h in uncoa ing is also a iable.
345
Calcium ac i a es synap o agmin-1, esul ing in SNARE-media ed usion o syn he ic esicles used as an exocy ic
model o synap ic e en s. An op imal dis ance o he usion is 5 nm.
346
Al e a ions in memb ane choles e ol con en shi hemi usion in e media es o ull usion memb ane and a ec he
s abili y o usion po es. A la ge inc ease in choles e ol le els boos s indi idual SNARE-media ed usion e en s.
347
Disassembly o he cla h in la ice su ounding coa ed esicles is he obliga o y las s age in hei li e cycle. The
s udy isualises he ec ui men o auxilin and Hsc70 which essen ial o his well-s udied endocy ic p ocess.
348
h FC ( luo escen
conjuga es)
Cha ac e isa ion o mic o esicles om ed blood cells unde s imula ion condi ions. An inc ease in in acellula Ca2+
o p o ein kinase C le els leads o al e a ions in cell mo phology and inc eased elease o mic o esicles.
335
FRET mic oscopy
F ee zinc concen a ion in insulin-s o ing esicles is quan i ied using a no el FRET-based zinc senso . The concen-
a ion o ee zinc is impo an o insulin-s o ing esicle ma u i y, hence i al e s insulin a icking.
334
47
Vesicle budding and shedding and he mechanical p ope ies o he esicles a e poo ly unde s ood.
Rema kably, a ecen compa a i e e iew sugges s ha biomechanical analysis o single EVs p o ides
key insigh s in o hei biological s uc u e, bioma ke unc ions, and po en ial he apeu ic unc ions325.
So kin e al. ha e used AFM o s udy hese p ope ies in e y h ocy e and EV memb anes unde di e en
condi ions342. They ha e es ablished ha s i ness is in e sely p opo ional o he p o ein–lipid a io and
linked i o se e al di e en budding mechanisms341. On he one hand, budding o p o ein- ich so es-
icles is possibly d i en by p o ein agg ega ion, and on he o he , budding o s i esicles wi h low mem-
b ane-p o ein con en is likely o be d i en by cy oskele on-induced buckling341. A u he in es iga ion
compa ing EVs o igina ing om heal hy e y h ocy es and om hose wi h he edi a y sphe ocy osis has
suppo ed hese obse a ions. I also unco e ed mechanical and esicula ion di e ences be ween
hese wo EV popula ions wi h po en ial use as diagnos ic pa ame e s349.
Vesicle endocy ic pa hways ha e been in es iga ed p ima ily using mic oscopy-based echniques.
One majo EV endocy osis pa hway is media ed by he o ma ion o cla h in-coa ed esicles (Figu e
10b). In his p ocess, in acellula cla h in in e ac s wi h he memb ane, p oducing a memb ane in agi-
na ion ha will o m an endosome h ough which he EVs can be in e nalised. The disassembly o he
cla h in la ice su ounding coa ed endosomes is a manda o y las s ep in hei li e cycle. The ec ui -
men o auxilin and Hsc70 ( luo escen ly labelled) was di ec ly isualized using an in e ed luo escence
mic oscope equipped wi h TIRF ha dwa e and desc ibed as essen ial o cla h in-based in e nalisa ion
e en s348. The cla h in-d i en uncoa ing is a a iable p ocess in which he endocy osing cla h in-coa ed
esicles emain p oximal o he memb ane o di e en pe iods p io o he scission o plasma mem-
b ane. The dynamics o cla h in media ed endocy osis we e assayed using luo escen ly agged p o-
eins and TIRF mic oscopy345.
Fusion s a es, dynamics and mechanisms o esicle in e nalisa ion du ing single- esicle usion
e en s ha e been di ec ly examined using c yo-TEM, FRET and TIRF mic oscopy. Cha ac e is ics and
kine ics o indi idual usion e en s can be quan i ied o he lipids o DNA-lipid complexes in ol ed in
he p ocess. Di e en usion pa hways exis : esicles and cell memb ane me ge ia a di ec usion o
memb anes (Figu e 10a) o using p o ein-media ed mechanisms (Figu e 10c). These mechanisms a e
in ol ed in bo h endocy osis and exocy osis e en s. Examining indi idual gian unilamella esicles by
luo escence mic oscopy, i has been shown ha du ing a di ec usion e en , he hemi usion s a e
p edomina es, and he usion o wo bilipid laye s occu s in a single s ep when hey a e su icien ly
close344.
Du ing he las decade, he usion mechanism based upon SNARE-media ed in e nalisa ion pa h-
ways (Figu e 10c) has been in es iga ed employing SVA. A he molecula le el, SNARE p o eins me-
dia e esicle usion wi h he a ge memb ane and wi h memb ane-bound compa men s. Recen ly,
Ma ie e al. ha e isualised SNARE single- usion e en s using c yo-TEM342. The sequence o usion
in e media es om lipid monolaye s o a comple e bilaye me ge has been epo ed in homo ypic ac-
uoles342. Hu e al. ha e co ela ed he memb ane usion s ages wi h a molecula mechanism using
econs i u ed esicles343. A a single- esicle le el, hey ha e aced he lipid-mixing p ocess using FRET
mic oscopy and co ela ed i wi h he docking, hemi usion and ull usion s ages343. They also epo
48
ha an op imal dis ance o a SNARE-media ed usion is 5 nm. In e es ingly, some egula o s o usion
pa hways we e iden i ied in ela ed s udies. Calcium ac s as an ac i a o o synap o agmin-1, leading
o he usion o synap ic esicles wi h he p esynap ic memb ane346. S a on e al. ha e iden i ied cho-
les e ol as an impo an egula o o usion dynamics, shi ing he p ocess om hemi usion in e medi-
a es owa ds ull usion memb anes347. La ge amoun s o choles e ol p eclus e -SNAREs, which se e
as unc ional docking and usion pla o ms. These clus e s subs an ially a ec he s abili y o po es by
inc easing he ac ion o ully open po es and accele a ing usion e en s. Consequen ly, high choles-
e ol con en igge s as and indi idual SNARE-media ed usion e en s347.
Figu e 10. Schema ic ep esen a ion o endocy ic and usion pa hways ecen ly in es iga ed using SVA ech-
niques. A lipid bilaye om a ecipien cell is shown in do ed o ange and EVs lipid bilaye in do ed ed; he in a-
cellula con en is shaded in blue and esicle con en , in ed. (a) di ec usion o EVs wi h he plasma memb ane348.
(b) Cla h in-based in e nalisa ion349,352. TIRF mic oscopy allows he examina ion o he cla h in uncoa ing p ocess.
In (c), SNARE-media ed memb ane usion is shown346. FRET mic oscopy acili a es he analysis o he h ee main
s ages o endocy ic and usion pa hways: docking, hemi usion and ull usion. Memb ane composi ion enhances
his usion pa hway h ough -SNARE-machine y ec ui men and en ichmen 351.
Du ing he las decade, mul imodal imaging pla o ms ha e been es ed in i o in a numbe o di -
e en cellula models o disease 350–352. These pla o ms ha e conside able po en ial o be used in i o,
o ins ance, in mice353–356. Howe e , hese sys ems usually ail o pe o m single-EV acking and ha e
been success ul only in ex i o cul u es350. Ne e heless, he e is an animal model wo h men ioning
due o i s physiological cha ac e is ics and anspa ency. Zeb a ish emb yo has eme ged ecen ly as a
p ospec i e model o acking EVs and assessing hei dissemina ion and up ake in i o357,358. In 2019,
Hyenne e al. epo ed an app oach o acking indi idual ci cula ing umou EVs in he zeb a ish em-
b yo218 using con ocal mic oscopy and a combina ion o chemical and gene ically encoded p obes o
image EVs in i o. The au ho s desc ibed, o he i s ime, he hemodynamic beha iou o umou EVs
and hei in a ascula a es . The s udy shows ha he endo helial cells and blood mac ophages apidly
49
ake up ci cula ing umou EVs. These EVs ac i a e blood pa olling mac ophages and p omo e me a-
s a ic ou g ow h218. A back- o-back s udy pe o med by Ve weij e al. combined he gene ic labelling
(using a CD63-pHluo in exosomal epo e ) o speci ic issues wi h elec on mic oscopy o ack endog-
enous EVs in blood and u he un a el hei mechanisms o biogenesis, biodis ibu ion, and a ge cells
h oughou he zeb a ish emb io357. In iguingly, Sung e al. ha e ecen ly epo ed a CD63-pHluo in-
mSca le usion p o ein ha can be used o image se e al s ages o he exosome li ecycle in i o359.
This epo e likely can be used o isualize exosomes in i o and is a p ospec i e ool o unde s anding
he physiological oles o exosomes.
1.3.3. EV bioma ke s.
SVA echniques hold he capaci y o disco e new, speci ic and e ec i e bioma ke s in EVs ha
ha e been missed by ensemble me hods and can be used in disease diagnos ics. While he esolu ion
and sensi i i y o SVA echniques s ill needs imp o ing, hey p o ide an accu a e cha ac e isa ion o EV
subpopula ions and assessmen o bioma ke s. Ex ensi e esea ch has been ca ied ou seeking he
biological bioma ke s o se e al diseases such as p os a e cance 60,224,308, ib omyalgia360, endome ial
cance 361, colo ec al cance 362,363 and li e -associa ed diseases364, among o he s. Figu e 3 summa ises
ecen ad ances in cance esea ch a he single- esicle le el. Cu en ly, luo escen assays, which a e
o en used in cance sc eening365, can de ec miRNAs and co ela e hei p esence wi h indi idual EVs
and EV popula ions. Exosome-localised miRNA-21 migh be used o di e en ia e be ween cance pa-
ien s and assess umou p og ession and esponse o ea men 365. Mo eo e , a speci ic lipid-p o ein
signa u e may iden i y umou -de i ed EVs as Raman spec a wi hin he ange o 2800 o 3100 cm-1
appea o be a dis inguishing ea u e o genuine cance EVs234. These indings open he way o ea ly
cance de ec ion. Ne e heless, he bioma ke s discussed he e a e ei he gene ic (canno disc imina e
be ween di e en cance s) o come om 2-D cance model esea ch and may no adequa ely diagnose
clinical cance s.
The de elopmen o nex -gene a ion sequencing SVA me hods has op imised he iden i ica ion o
cance bioma ke s. Speci ically, a ge ed sequencing using cance gene panels has allowed he s udy
o EV-de i ed and ci cula ing ee DNA300, esul ing in he disco e y o gene ic bioma ke s o se e al
diseases. Fo ins ance, nine miRNAs ha e been p o iled in se um EVs. Using hese p o iles, ch onic
hepa i is C pa ien s can be dis inguished om heal hy indi iduals wi h accu acy abo e 95%366. Diag-
nos ic oppo uni ies p esen ed by EV-speci ic gene ic bioma ke s ha e been widely e iewed367–370. I
has been shown ha cance -de i ed EVs ewi e and modi y he p e-me as a ic mic oen i onmen , sup-
po ing umou g ow h and me as asis du ing cance p og ession67,71,74,295. This esea ch desc ibes a
se o po en ial ma ke a ge s o be used as ea ly diagnosis o PCa. The p omo ion and p oli e a ion o
PCa igge ed by EVs p oduced as a esul o DIAPH3 loss o g ow h ac o s imula ion ha e also been
epo ed295. O he s udies ha e shown ha miRNA quan i ica ion in issues can iden i y PCa by de ec -
ing he exp ession o RNU24371 o miR-130b372.
Recen ly, se e al s udies ha e epo ed speci ic and sensi i e bioma ke s o cance de ec-
ion227,251,296,298,373–375. ddPCR has de ec ed and quan i ied he IDH1 ansc ip in ce eb ospinal luid-
50
de i ed EVs o pa ien s wi h glioma umou s in he b ain296. In lung cance , ele en cance -speci ic SERS
signals ha e been ob ained, allowing o di e en ia e be ween EV popula ions de i ed om heal hy and
lung cance cells wi h high sensi i i y251. Addi ionally, he CD147 p o ein ound in EVs has been iden i-
ied as a bioma ke o colo ec al cance (CRC) diagnosis362. Fu he s udies ha e iden i ied BRAF and
KRAS soma ic mu a ions in plasma-de i ed EV popula ions om CRC pa ien s227. In iguingly, Melo e
al. ha e desc ibed an explici bioma ke , glypican 1 (GPC1), ound in EV popula ions om panc ea ic
cells con aining di e en KRAS oncogenic isome s374. Hence, GPC1-EV iden i ica ion could acili a e
ea ly panc ea ic cance diagnosis374. In 2020, he EV- anspo ed HULC lncRNA (long non-coding RNA
highly up egula ed in li e cance ) was sugges ed as a chemo axis agen o cell in asion and mig a ion.
This encapsula ed HULC is a po en ial bioma ke o human panc ea ic adenoca cinoma diagnosis298.
Figu e 11. Schema ic o e iew o e iewed bioma ke s disco e ed using SVA ech-
niques. The me hods shown he e a e: (A) h FC226,362,374, (B) RTM211,232,235,243,251, (C)
TIRF361 and (D) ddPCR227,296,298,370. Mo e in o ma ion o speci ic bioma ke s disco -
e ed using each me hod can be ound in Table 8.
The ecen ad ances in cance bioma ke esea ch a e a p oo -o -concep o non-in asi e diagnos ic
ools based on EV inge p in ing in combina ion wi h mul i a ia e s a is ical analysis212. The in es iga-
ions in he ield o non-in asi e diagnos ics o PCa ha e been s imula ed by he disco e y o in insic
bioma ke s de ec ed in u ina y-de i ed EVs. As a esul o SVA esea ch, se e al bioma ke s ha e been
associa ed wi h di e en s ages o PCa. Biggs e al. ha e measu ed he le els o ci cula ing p os a e
mic opa icles in plasma om PCa pa ien s226 and used hese mic opa icles in a liquid biopsy pla o m
o iden i y and cha ac e ise pa ien s. This s udy ound ha subjec s wi h an ad anced and agg essi e
umo (in Gleason scale, sco ing 8 o highe ) can be iden i ied independen ly o hei PSA alue226. The
51
lipid and su ace p o ein signa u es o p os a e-de i ed EVs ha e been desc ibed using
RTM210,232,235,243. Se e al cha ac e is ics ha e been highligh ed, indica ing po en ial PCa bioma ke s.
Fo example, a shi in he s uc u e o su ace p o eins om alpha-helix- ich in p os a e EVs o be a-
shee - ich p o eins in PCa-speci ic EVs (isola ed om blood samples) has been obse ed243. Mo eo e ,
O o e al. ha e de ec ed chemical signa u es in he Raman and Rayleigh sca e ing da a232 ha e i-
cien ly di e en ia e be ween he EV popula ions om no mal p os a e and PCa cells211,234. The au ho s
ha e ained a con olu ional neu al ne wo k o p edic p ecisely he cellula o igin o EVs o an au o-
ma ed diagnosis o PCa235. In ano he s udy, using a ious SVA echniques such as nanoscale low
cy ome y and ddPCR, Joncas e al. ha e explo ed cas a ion- esis an PCa370, iden i ying KLK3 and
he and ogen ecep o a ian 7 (AR-V7) as speci ic bioma ke s o his cance 370.
Table 8. Summa y o ecen EV bioma ke - ela ed disco e ies achie ed using SVA echniques, ocused on can-
ce diagnos ic bioma ke s.
Technique
Main conclusion
Re
Flow cy ome y (immuno luo es-
cence)
De ec ion o CD147 as EV bioma ke o CRC diagnosis.
362
De ec ion o GPC1 as EV bioma ke o panc ea ic cance diagnosis. Cance -cell
EVs con ain oncogenic K asG12D.
374
Measu es ci cula ing p os a e mic opa icles (PMPs) le els in plasma. This PCa "liq-
uid biopsy" can iden i y pa ien s wi h Gleason sco e ≥ 8, i espec i e o hei PSA.
226
Fluo escence mic oscopy (TIRF)
Employs a luo escen assay o de ec exosome miRNA-21 (miR-21-EX) as a can-
ce -sc eening assay. miR-21-EX can be used o dis inguish be ween cance pa-
ien s, umou p og ession s ages and ea men esponses.
365
Raman (d y EVs)
The su ace p o ein signa u e shi s om alpha-helix- ich p o eins o be a-shee - ich
p o eins in p os a e cance -speci ic blood EVs.
243
RTM
Chemical signa u es om Raman spec a can be used o di e en ia e be ween EV
popula ions de i ed om heal hy and p os a e cance cells. A ained con olu ional
neu al ne wo k can iden i y he cellula o igin o EVs.
232,2
35
Raman analysis coupled wi h Rayleigh sca e ing dis ibu ion is used o de ec spe-
ci ic lipid and p o ein signa u es o umou -de i ed EVs.
234
Label- ee SERS
Repo o eal- ime and label- ee diagnosis o lung cance by de ec ing 11 cance -
speci ic SERS signals (p o eins and lipids) ha dis inguish EV popula ions om
heal hy and lung cance cells wi h high sensi i i y.
251
ddPCR
De ec ion and quan i ica ion o mu an and wild- ype IDH1 RNA ansc ip s in EVs
om ce eb ospinal luid o pa ien s wi h glioma umou s.
296
Iden i ies soma ic BRAF and KRAS mu a ions in plasma-de i ed EVs om CRC pa-
ien s. P obes a wide ange o cance cells and he de i ed EV popula ions.
227
Iden i ies KLK3 and AR-V7 (and ogen ecep o a ian ) as bioma ke s o cas a-
ion- esis an p os a e cance (which p og esses e en unde s e oid dep i a ion he -
apy).
370
Ex acellula esicle- anspo ed HULC p omo es cell in asion and mig a ion. This
encapsula ed HULC is iden i ied as a bioma ke o human panc ea ic duc al adeno-
ca cinoma (PDAC).
298
52
1.4. Fu u e ou look
Indi idually analysed EVs p o ide excellen p ospec s o u u e basic and p ac ical esea ch wi h a
iew o hal disease p og ession and con ol cell– o–cell communica ion p ocesses. To exploi he ull
po en ial o SVA echniques, biological alida ion and ep oducibili y mus mee he demands o clinical
applica ions. Each echnique has i s speci ic ad an ages and disad an ages, and he exac choice o
he me hod o analysis depends on he esea ch ques ion, he na u e o he samples and EV cha ac-
e is ics. These echniques s ill need o imp o e hei quan i a i e de ec ion powe , lowe hei cos and
inc ease hei eliabili y, esolu ion and h oughpu . In addi ion o echnologies in use o SVA de ec ion,
we highligh se e al p omising app oaches ha ha e ye o p o e hei po en ial in SVA.
Con en ional me hodologies ha e he po en ial o be applied in SVA, and low cy ome y is a good
example. I s implemen a ion using inno a i e app oaches can p o ide new ea u es and capabili ies,
as shown by esicle impac elec ochemical cy ome y (VIEC). This elec ochemis y-based low cy om-
e y echnique uses single up u ed esicles whose con en is de ec ed and quan i ied based on Fa a-
day’s law exploi ing he p oduced oxida ion cu en 376,377. Ex ensi e s udies o he egula ion o neu o-
ansmi e a icking by Ewing and colleagues, ocusing on ca echolamine exocy osis377–384 ha e
demons a ed he p ospec i e applica ion o his app oach in he EV ield, highligh ing elec ochemical
low cy ome y as p ospec i e asse in he s udies o EV unc ions and biology in he nea u u e. The
VIEC-based expe imen s ha e examined neu o ansmi e con en a a single- esicle le el in a pheo-
ch omocy oma cell line. Se e al s udies ha e es ablished ha he neu o ansmi e ca echolamine is
only pa ially eleased om he esicles du ing an exocy osis e en 377. Mo eo e , ca echolamine con-
cen a ion is a key ac o in egula ing esicle size since esicula ansmi e con en is ela i ely con-
s an and independen o esicle size381. None heless, many di e en agen s may egula e exocy osis
e en s. Using VIEC, he g oup has la e e i ied ha he zinc and cispla in con en se e as majo
egula o s in hese p ocesses382,385.
Video mic oscopy is ano he common echnique ha is po en ially use ul in EV analysis. In 2010,
Zupanc e al. de eloped an e icien algo i hm o ans o m ideo sequences in o quan i a i e da a383.
Thei wo k was a c ucial s ep owa ds he c ea ion o au oma ed compu e analysis and led o he
de elopmen o ano he , mo e popula , me hodology, NTA. In 2008, he- hen eme ging luo escen a-
iome ic image analysis (FRIA) me hod was used o de e mine he pos -endocy ic a e and anspo
kine ics o in e nalised ca go384. FRIA p esen ed a b eak h ough in his ield a he ime and i s applica-
ion led o colocaliza ion o EV ca go wi h o ganelle ma ke s. Howe e , he echnique has no been e y
success ul in u he EV esea ch possibly due o he eme gence o o he mic oscopy app oaches such
as TIRF o SRM ha enable he s udy o EV in e naliza ion and a e wi h a be e esolu ion in luo es-
cence images and a mo e s aigh o wa d analysis.
The las decade has seen he de elopmen o se e al echniques wi h p ima y applica ions in ana-
ly ical ields o he han EV analysis. Howe e , some, mos no ably adio- equency analysis and SP-
IRIS, a e applicable o single- esicle esea ch and could play an impo an ole in scanning and e alu-
a ion o speci ic EV popula ions o cha ac e ising se e al EV pa ame e s in a single expe imen .
53
Radio- equency analysis, also known as elec ically con olled uneable b oadband in e e ome ic
dielec ic spec oscopy, has only been p esen ed in se e al con e ence pape s386–389 a e i s i s pub-
lica ion. In his me hod, speci ic senso s a e used o pe o m a highly sensi i e and uneable b oadband
adio- equency analysis. One s udy applying his me hod o EVs showed ha he highly concen a ed
adio- equency ields s imula es s ong in e ac ions be ween esicles, which can be de ec ed and
quan i ied390. Speci ically, he au ho s could de ec and scan a ype o EVs, gian unilamella esicles,
a mul iple equency poin s and de e mine hei molecula composi ion. In 2017, Wu e al. epo ed a
sepa a ion me hod based on acous o luidics and c ea ed a pla o m employing hese acous ic apping
o weeze s phenomenon391. This echnique isola es EVs om whole blood in a label- ee and con ac -
ee manne 391. An acous ic wa e alls upon a esicle, and i s sca e ing ac s as a d i ing o ce o e ain
i . A yea la e , Ku e al. demons a ed isola ion and en ichmen o EVs using a simila acous ic ap
echnology392. In ollow-up esea ch, he me hod was used o isola e RNA and sequence miRNAs om
EVs. Since hen, an acous ic-based mic o luidic pla o m has been eleased, coupling EV apping ech-
nology wi h nex -gene a ion sequencing echniques. Toge he , hese pla o ms o m a obus and au o-
ma ed s a egy o bioma ke disco e y in small sample olumes393.
SP-IRIS, has so a ound limi ed use in EV esea ch, bu has he po en ial o ill a unique expe i-
men al niche. SP-IRIS can cha ac e ise he size and pheno ype (su ace bioma ke s) o EVs wi h no
need o co ela e wo sepa a e measu emen s214,394. This ea u e p o ides SP-IRIS wi h a high h ough-
pu and subs an ially educes he amoun o alse nega i es and posi i es compa ed o echniques ha
assess wo cha ac e is ics in indi idual measu emen s. Due o he la e al esolu ion o mic oscopy
(340–435 nm), highly concen a ed samples cause signal o e lap and a subsequen shi in he appa -
en esicle size. S ikingly, despi e he mic oscope esolu ion d awback indi idual Fla i i idae pa icles
o app oxima ely 40 nm ha e been iden i ied and cha ac e ised using SP-IRIS269. Besides he examples
p esen ed in Table 9, he e is a comme cialised pla o m o EV pheno yping de eloped by Nano iew
Biosciences using SP-IRIS, highligh ing i s po en ial applica ion in cha ac e ising limi ed inpu EV sam-
ples. Se e al pape s ha e p oposed o he highly-p omising au oma ed on-chip pla o ms using SP-IRIS
o EV cha ac e iza ion214,269,395–397. These pla o ms ha e been combined wi h immunoblo ing o so
and cha ac e ise EV popula ions om a sample and can de ec size and pheno ype a a single-pa icle
le el, isualising and quan i ying ei he i uses o single EVs o bo h, in an uncha ac e ised sample.
One o he g ea es ad an ages o such mic o luidic pla o ms is ha hey only need e y small
sample olume (~20 µL) o an e ec i e analysis396,398. O he echniques di e en om SP-IRIS ha e
been implemen ed in such de ices o cha ac e ise EVs om EV- egula ed diseases and examine hei
u u e use as diagnos ic ools. EVs de i ed om ans usion- ela ed acu e lung inju y (TRALI) ha e
been in es iga ed using SP-IRIS coupled wi h AFM mechanics. Obeid e al. used his app oach o de-
e mine ha ce ain ypes o EVs igge neu ophil ex acellula aps (NETs) and ha hese NETs a e
likely o media e in TRALI399.
Fluo escence mic oscopy coupled wi h on-chip nano low cy ome y enables au oma ed quan i a i e
SVA o body luid samples398. Acco ding o Yoko a e al., he mo phology and de o mabili y o EVs om
di e en cell lines can be in es iga ed using nanopa e ned e he ing o EVs in combina ion wi h AFM400.
60
U ina y EVs (uEVs) we e ob ained by ul acen i uging u ine samples as desc ibed elsewhe e59. In
b ie , app oxima ely 50 o 100 mL o u ine we e collec ed o which aliquo s o 50 mL we e cen i uged
a 2,000g o 10 min, il e s e ilised (0.22 μm po e size) and immedia ely ozen a -80 ºC o u he
p ocessing. When collec ion was highe han 50 mL, aliquo s o ei he 1 mL o 10 mL we e gene a ed
p io eezing. In hese expe imen s, u ine samples o 1 mL o 10 mL we e hawed a oom empe a u e
and cen i uged o 5 min a 2,000g o emo e any p ecipi a e. Then, RNA associa ed o uEVs was
isola ed by using a U ine Exosome RNA Isola ion Ki (P oduc #47200, No gen Bio ek Co po a ion,
Canada). P o ocol is ex ensi ely desc ibed elsewhe e188. The isola ed RNA was quan i ied in nucleic
acid concen a ion using a NanoD opTM (ND-1000 Spec opho ome e , The mo Fishe Scien i ic, MA,
USA) de ice in o de o op imize he e o ansc ip ion o cDNA by means o Supe Sc ip TM VILOTM pol-
yme ase (#11754-050, In i ogenTM, MA, USA). An app oxima ely 100% cDNA con e sion e iciency
was conside ed o p oceed wi h u he assays.
Real- ime quan i a i e PCR me hodology
Real- ime quan i a i e PCR (qPCR) was u ilised o quan i y he exp ession o selec ed a ge genes
in uEV-de i ed RNA. RNA de i ed om PCa cell lines (PC-3, DU145, 22R 1, LNCaP, BPH-1) and a
uni e sal human RNA empla e (QS0639, The moFishe Scien i ic, MA, USA)) we e u ilised o op imise
qPCR me hodology. To pe o m qPCR assays, SYBR® G een Selec Mas e Mix was pu chased om
Applied Biosys ems (#4472897, MA, USA). Reac ion mix was p epa ed ollowing manu ac u e ’s ins uc-
ions and qPCR was un as speci ied in Table 10. Mel ing cu es o each amplicon we e de e mined o
e alua e he e iciency and speci ici y o he ampli ica ion. A Quan S udio™ 5 Real-Time PCR Ins umen
(The mo Fishe Scien i ic, MA, USA) was u ilised o un he eac ion and Quan S udio™ Real-Time PCR
So wa e 1.3 o he analysis. Unless speci ied, all expe imen s we e un wi h 1 ng o DNA pe eac ion.
Design o p ime s o SYBR G een and TaqMan qPCR
P ime s o he selec ed a ge genes we e designed ollowing a p o ocol sha ed by ExosomeDx o
clinical ials in u ine. The speci ic p ocedu e and pa ame e s canno be speci ied as i is hei indus ial
p ope y. Rema kably, he design was pe o med conside ing a u u e applica ion in a clinical ial. This
means he sc eening o po en ial bioma ke a ge s was designed o wo k wi h wo lanking p ime s
when using SYBR G een polyme ase – ha equi es less op imisa ion – wi h a TaqMan p obe in case
we could mo e o wa d o a clinical ial, which will equi e a highe sensi i i y. The a ge genes es ed
in his chap e a e: SRD5A2, CYP1A2, CYP3A5, CYP3A7, CYP3A4, SULT1A, STS, SULT2B1,
SRD5A1, SRD5A3, AKR1C2 and CYP11B1. As housekeeping genes (HKG) GAPDH, ACTB, 18s RNA,
SPEDF, EEA1F1, RPL6 and KLK3 we e es ed.
Housekeeping genes analysis
To ensu e an adequa e no malisa ion o quan i a i e RNA con en , app op ia e HKG a e equi ed. In
gene al, cell lines and o he well-es ablished ma ices al eady ha e HKG desc ibed in li e a u e. Due o
he he e ogenei y o human samples, he es ablishmen o a s eadily exp essed ansc ip is equi ed.
To pe o m such analysis he e a e many no malisa ion algo i hms a ailable405. As in e e y modelling
s a egy, when many algo i hms a e a ailable he use o se e al app oaches p o ides a mo e obus
61
and us able ou come. Fo his eason, se e al e e ence genes we e es ed in hese u ine samples
wi h No mFinde 406 and Bes Keepe 407 algo i hms. Bo h wi h hei limi a ions and s eng hs ound ou he
mos obus HKG in PCa cell lines was GADPH combined wi h ACTB (da a no shown). EE1AF1 is also
a op candida e as a HKG bu in some samples i does no ampli y, he e o e, should no be conside ed
as a HKG a p io i. They we e also applied o u ine samples (Figu e 16).
Table 10. Real- ime qPCR condi ions. These qPCR condi-
ions we e op imised o housekeeping genes and a ge
genes in uEVs.
#
Tempe a u e
Time
Tempe a u e g adien
Hold s age
1
50.0 ºC
120 s
1.6 ºC pe s
2
95.0 ºC
120 s
1.6 ºC pe s
PCR s age (40 cycles)
1
95.0 ºC
15 s
1.6 ºC pe s
2
60.0 ºC
60 s
1.6 ºC pe s
Mel Cu e S age
1
95.0 ºC
15 s
1.6 ºC pe s
2
60.0 ºC
60 s
1.6 ºC pe s
3
95.0 ºC
15 s
0.5 ºC pe s
2.3. Resul s
2.3.1. Selec ion o a ge s.
S udy o s e oid ho mones mRNA ela ed o EVs
The luminous da kness o PCa wo ks in mys e ious ways in ad anced s ages, howe e , i is well-
de ined s e oid ho mones a e c ucial in ea ly PCa p og ession. Fo his eason, gene ansc ip s ela ed
o his pa hway associa ed o EVs we e he objec i e in his sec ion. O e he decades, mRNA encap-
sula ed inside he EV lipid bilaye ha e been inc easingly u ilised as p og ession ma ke s o se e al
diseases. In ac , mRNA associa ed o EVs eme ged as an ou s anding sou ce o in o ma ion o cha -
ac e ise biological samples and hence, o desc ibe speci ic signa u es o hei composi ion as bi-
oma ke s. A web-based da abase con aining o e 500 da ase s ela ed o EV s udies – Vesiclepedia –
was used o disco e po en ial ma ke s o ea ly s age p og ession o PCa associa ed o EVs.
Vesiclepedia compiles mo e 18,000 di e en p o eins associa ed o EVs in mo e han 500 di e en
s udies (Re ie al: No embe , 2021). App oxima ely 8,000 p o eins ha e been linked o p os a e e-
sea ch wi h s udies using ei he cell lines o u ine samples. In e es ingly, he e a e unique se s o p o-
eins ela ed o cell lines, which a e also speci ic o di e en ypes o cells. In his unmanageable da a-
base, he e is a se o 61 enzymes pa icipa ing in he biosyn hesis o s e oid ho mones (Ex ac ed om
KEGG) and i conce ns his chap e . Figu e 13 shows a o al o 43 mRNAs associa ed o EVs conside -
ing only da ase s uploaded o Vesiclepedia; his also means 18 enzyme- ela ed p o ein o mRNA ha e
no been e e desc ibed in EVs. Rema kably, 24 o hese mRNAs ha e been linked o EVs bu none
was iden i ied in p os a e ela ed esea ch. The e a e 10 mRNAs – AKR1C3, AKR1C2, AKR1C1,
62
CYP17A1, SULT2B1, SULT1E1, HSD17B8, HSD17B12, DHRS11 and COMT- ha a e associa ed o
bo h cell line- and u ine- ela ed esea ch while 9 mRNAs - HSD17B2, HSD17B6, CYP1A1, UGT1A6,
UGT1A9, UGT2B7, UGT1A8, HSD11B2 and CYP7B1 - a e unique o s udies pe o med wi h uEVs.
Figu e 13. Venn Diag am including he enzymes o s e oid ho mone biosyn he-
sis de ec ed in EVs and in di e en da ase s in silico. Da ase s included we e all
il e ed o all enzymes ela ed o s e oid ho mones biosyn hesis. Mo eo e , i epo s
he names o he a ge genes we selec ed o es in his chap e .
Bioma ke po en ial o s e oid ho mone enzymes exp ession
The nex s ep was o e alua e which o he 61 ho mone s e oid- ela ed genes could be ele an in
ca ego ising pa ien samples. The s a is ical appliance CANCERTOOL was u ilised o epo he exp es-
sion o hese genes o in e es along g oups o samples in di e en s udy cases. In he Y-axis a Log2-
no malized gene exp ession is ep esen ed and depending on he g oups o samples compa ed i in-
o ms abou he s a us o he gene:
A. In p os a e cance : compa ing non- umo al s PCa specimens.
B. By p og ession: compa ing non- umo al, p ima y umo and me as a ic PCa specimens.
C. By Gleason G ade: i is indica ed as GS6, GS7, GS8, GS8+9, GS9 and GS10 and; i in o ms
abou he p esence o a ansc ip in a speci ic Gleason a chi ec u e.
D. Disease F ee Su i al: Kaplan-Meie cu es ep esen ing he disease- ee su i al (DFS) o
pa ien g oups acco ding o he exp ession o a ge genes. I indica es whe he he gene p og-
noses PCa agg essi eness.
The ool p o ides a epo o each single gene in e e y de ini ion abo e. These epo s we e checked
manually o assess whe he each speci ic ansc ip exp ession could de ine cance p og ession. The
disc imina ion be ween no mal and PCa specimens (S a us in PCa) and, including me as a ic s ages
(S a us by p og ession) we e conside ed he mos ele an aspec s o aim o ea ly s age bioma ke s.
63
In Table 11, a summa y o his analysis is epo ed. Only he ansc ip s able o signi ican ly disc imina e
pa ien s acco ding o de ini ions A and B a e shown. The plus sign (+) indica es a signi icancy close o
0.05 and double sign (++) close o o e 0.01; unsu e means ha he e missing esul s o ha hey a e
no conclusi e and a minus sign (-) indica es no signi icance in disc imina ion. The s a i ica ion by GS
was no obus and in many cases exp ession da a was sp ead ou due o a sho numbe o samples
pe g oup. This ca ego isa ion is o impo ance because including e y agg essi e PCa samples o com-
pu e s a is ical di e ences may hinde ele an in o ma ion o ea ly s ages o p og ession. Finally, DFS
indica es agg essi eness o he cance ollowed o e ime and conside ing dea h by cance as a meas-
u e. This indica o is no much ele an o conside disease ecu ences a his s age.
Among he 32 candida es we ha e il e ed using CANCERTOOL (Table 11), he i s 12 mos ele-
an en ies in his classi ica ion we e selec ed o be es ed as mRNA bioma ke s associa ed o uEVs.
Figu e 13 summa ises he analysis and u he indica es whe he candida es ha e been al eady iden i-
ied. Acco ding o esiclepedia, 10 o hem ha e no been e e de ec ed in u ine samples. The e a e
wo in e es ing candida es, AKR1C2 and SULT2B1, epo ed in EVs and u ine samples ha a e also
ele an as a pa ien classi ie . Anyway, he 12 candida es will be assessed once he coho s a e de-
signed.
2.3.2. Selec ion o coho s.
One o he mos impo an hallma ks o PCa is i s poo p ognosis. Al hough he classi ica ion o PCa
pa ien s is becoming mo e speci ic, he ools o pa ame e s u ilised o ca ego ise hem a e no backing
up. The diagnos ic ools a e highly in asi e, ime consuming and equi e high expe ise in examina ion.
Nowadays, he gold s anda d bioma ke be o e unde going a biopsy is PSA concen a ion in blood. I s
poo p ognosis a ea ly s ages o de elopmen has al eady been discussed in his hesis (see in
INTRODUCTION), demons a ing he exis ence o a esea ch niche. The e o e, bioma ke s wi h a su-
pe io capaci y o s a i y pa ien samples claim he spo ligh o cance diagnosis.
Indeed, an analysis wi h a bina y classi ie model demons a ed PSA is no a good classi ie o PCa
pa ien g oups in his u ina y coho o 646 pa ien s (Figu e 14). A ecei e ope a ing cha ac e is ic
(ROC) cu e illus a es he ue posi i e ac ion agains he alse posi i e ac ion o a sample popula ion
a di e en h eshold alues. Thus, by inc emen ing he alue o he classi ie he success ulness in
classi ica ion is assessed. The a ea unde he cu e (AUC) es ima es how well he classi ie disc imi-
na es be ween wo g oups o samples. In Figu e 14, ROC cu es and AUC alues o each compa ison
be ween g oups o pa ien s a e p esen ed. To no e, an AUC alue close o 0.5 o lowe indica es an-
dom-d i en classi ica ion by he analysed pa ame e ; he highe he alue, he be e he classi ie is.
F om his da a, one can conclude PSA is no a good classi ie o pa ien g oups in his coho . Only when
compa ing a he ad anced PCa (GG4 and GG5) o e y low de eloped PCa (GG1) he ROC cu es
disc imina e g oups easonably. This means PSA could be app op ia e o classi y low de eloped cance
and ad anced cance pa ien s. In his ega d, he lack o an app op ia e classi ie a ea ly s ages o
de elopmen highligh s he impo ance on pa ien s a i ica ion esea ch.
64
Figu e 14. ROC cu es compa ing all g oups o pa ien s in he sample coho (n=646). The
AUC a e indica ed o assess PSA pe o mance as a bioma ke .
Table 11. Summa y o CANCERTOOL esul s o signi ican genes o in e es in disc imina ing PCa speci-
mens. The columns indica e disc imina ion o s a us: A. in PCa B. by p og ession C. by Gleason g ade D. Disease
ee su i al. Repo s o all genes a e included in he Supplemen a y ma e ial. A plus sign (+) indica es signi icancy
65
close o 0.05 and double sign (++) close o o e 0.01; unsu e means ha he e a e no conclusi e and a minus sign
(-) indica es no signi icance in disc imina ion.
Gene
Desc ip ion
A
B
C
D
SRD5A2
S e oid 5 alpha- educ ase 2
++
++
+
+
CYP1A2
Cy och ome P450 amily 1 sub amily A membe 2
++
++
+
-
CYP3A5
Cy och ome P450 amily 3 sub amily A membe 5
++
++
+
+
CYP3A7
Cy och ome P450 amily 3 sub amily A membe 7
++
+
-
-
CYP3A4
Cy och ome P450 amily 3 sub amily A membe 4
++
++
-
Unsu e
SULT1A1
a yl sul o ans e ase
++
++
-
-
STS
S e oid Sul a ase
+
+
+
-
SULT2B1
Sul o ans e ase amily 2B membe 1
+
+
Unsu e
+
SRD5A1
S e oid 5 alpha- educ ase 1
+
+
-
Unsu e
SRD5A3
S e oid 5 alpha- educ ase 3
+
Unsu e
-
Unsu e
AKR1C2
Aldo-ke o educ ase amily 1 membe C2
+
+
-
Unsu e
CYP11B1
Cy och ome P450 amily 11 sub amily B membe 1
+
+
-
-
AKR1D1
Aldo-ke o educ ase amily 1 membe D1
+
-
Unsu e
-
CYP7B1
Cy och ome P450 amily 7 sub amily B membe 1
+
+
-
Unsu e
HSD17B6
Hyd oxys e oid 17-be a dehyd ogenase 6
+
++
-
-
HSD17B7
Hyd oxys e oid 17-be a dehyd ogenase 7
+
+
-
Unsu e
HSD17B8
Hyd oxys e oid 17-be a dehyd ogenase 8
+
+
-
+
DHRS11
Dehyd ogenase/Reduc ase 11
+
+
-
-
CYP2E1
Cy och ome P450 amily 2 sub amily E membe 1
+
Unsu e
-
Unsu e
CYP1B1
Cy och ome P450 amily 1 sub amily B membe 1
+
+
-
Unsu e
CYP19A1
Cy och ome P450 amily 19 sub amily A membe 1
+
Unsu e
-
+
UGT2B11
UDP glucu onosyl ans e ase amily 2 membe B11
+
Unsu e
+
Unsu e
UGT2B28
UDP glucu onosyl ans e ase amily 2 membe B28
+
Unsu e
Unsu e
-
UGT2B10
UDP glucu onosyl ans e ase amily 2 membe B10
+
-
-
Unsu e
UGT2B7
UDP glucu onosyl ans e ase amily 2 membe B7
+
+
-
-
UGT2B4
UDP glucu onosyl ans e ase amily 2 membe B4
+
Unsu e
-
-
COMT
Ca echol-O-me hyl ans e ase
+
+
Unsu e
-
LRTOMT
Leucine- ich ansmemb ane and O-me hyl ans e ase domain con aining
+
+
-
-
HSD11B1L
Hyd oxys e oid 11-be a dehyd ogenase 1 like
-
+
-
-
HSD11B1
Hyd oxys e oid 11-be a dehyd ogenase 1
Unsu e
+
-
Unsu e
SULT1E1
Sul o ans e ase amily 1E membe 1
Unsu e
+
+
-
CYP1A1
Cy och ome P450 amily 1 sub amily A membe 1
Unsu e
++
Unsu e
Unsu e
As a consequence, small coho s mimicking demog aphic pa ame e s o he whole popula ion we e
designed o es he p oposed bioma ke s. Two coho s o 15 u ine samples (o ei he 1 mL olume o
66
10 mL olume) wi h BPH, low agg essi e and highly agg essi e PCa samples we e es ablished. The
objec i e was o ind good HKG o be used in hypo he ical clinical ials as well as assessing which
speci ic bioma ke s a e associa ed o EVs and s ill hold po en ial o disc imina e be ween pa ien g oups.
2.3.3. No malisa ion and Housekeeping genes.
The genes 18s RNA, ACTB, EE1AF1, GAPDH, KLK3, RPL6 and SPDEF we e examined as HKG o
no malise u ine coho s in upcoming assays. Once p ime s we e designed, a qPCR assay was un using
RNA ex ac ions om he 1 mL olume coho . Two con ol RNA ex ac ions om wa e and PBS we e
included. None o he HKG ampli ied using hese samples in excep ion o 18s RNA ha showed qui e
con incing ampli ica ion cu es wi h ampli ica ion cycle (C ) alues a ound 34 cycles (da a no shown in
Figu e 15). Ye , he HKG could s ill be used as i s C alues using sample RNA ex ac ion ange om
17 o 22 cycles, app oxima ely. Figu e 15 depic s he mean (± SD) o C alues o each HKG in all RNA
samples o he coho . The qPCR assay de e mined SPDEF and RPL6 a e no ampli iable in all samples
and hey exhibi a high a iabili y in C alues along samples. Hence, hey may in oduce mo e unce -
ain y a he han no malisa ion capaci y.
Figu e 15. Rep esen a ion o mean (± SD) ampli ica ion cycle (C ) alues o HKG es es
in 1 mL olume coho . I is exp essed as an a e age o echnical eplica es (n=3) and he
e o ba s co espond o s anda d de ia ion o hese echnical eplica es. In his ype o assays,
C alues a e a quan i ica ion measu emen . The bigge he C is, he lowe he concen a ion
o DNA/RNA.
In gene al, C alues o hese HKG candida es a e somewha high (excep o 18s RNA). To de e -
mine he bes candida e, a comp ehensi e analysis o HKG exp ession along samples and be ween
g oups is equi ed. The es ablishmen o a gene o combina ion o genes whose exp ession is ea men -
and g oup-independen is impe a i e o ind a gene ha only p o ides RNA/DNA con en in o ma ion.
Bes Keepe and No mFinde algo i hms we e selec ed o pe o m such e alua ion.
67
Figu e 16. Ou come o he HKG analysis by No mFinde algo i hm. S a-
bili y alues de e mine he likeliness o each gene o be an app op ia e HKG
in hese se o samples.
Bes Keepe desc ibes he gene al a iabili y o samples o he analysed gene. This algo i hm de e -
mined EE1AF1 and GAPDH showed he lowes a iabili y. In addi ion, i desc ibed ha he bes combi-
na ion o genes whe e hose including ACTB and GAPDH. No mFinde esul s a e epo ed in Figu e
16. This algo i hm conside s sample a iabili y bu i also includes he s anda d de ia ion along samples
and a ia ion be ween g oups o epo a s abili y alue. The lowe he s abili y alue is he be e he
HKG. No mFinde poin ed ou ACTB, EE1AF1 and GAPDH as he bes HKG bu also he combina ion
o hem. Fo his eason, we ha e selec ed ACTB, EE1AF1 and GAPDH as HKG o analyse hese co-
ho s and use hem o no malisa ion pu poses upon RNA quan i ica ion.
2.3.4. E alua ion o mRNA in EVs as a ge s.
The in silico app oach issued 12 candida es o which only he exp ession o six could be epo ed in
cell lines (Table 12). A his s age, a PCa cell line se was u ilised o assess p ime s pe o mance. Re-
ma kably, SULT2B1 has been epo ed in PCa cell lines while esiclepedia only desc ibes i in u ine
s udies. The use o cell line based empla es has i s own limi a ions since se e al genes may no be
exp essed in all o hem. The use o a uni e sal RNA de i ed om issues could o e come his limi a ion
and p ime design can be alida ed eliably.
Table 12. Summa y o a ge ho mone s e oid genes es ed in cell line-de i ed RNA. The able epo s
whe he he a ge gene was de ec ed in he wo a ailable empla es. Uni e sal RNA s ands o a comme -
cial empla e composed o a mix u e o 10 di e en human cell lines (The moFishe Scien i ic, MA, USA).
In house PCa cells a e RNA ex ac ed om PCa cell lines cul u ed in ou lab.
Gene a ge
Uni e sal RNA
In house PCa
cells
Gene a ge
Uni e sal RNA
In house PCa
cells
SRD5A2
Non de ec ed
Non de ec ed
STS
De ec ed
De ec ed
CYP1A2
Non de ec ed
Non de ec ed
SULT2B1
De ec ed
De ec ed*
CYP3A5
De ec ed
De ec ed
SRD5A1
Non de ec ed
Non de ec ed
CYP3A7
De ec ed
De ec ed*
SRD5A3
Non de ec ed
Non de ec ed
CYP3A4
Non de ec ed
Non de ec ed
AKR1C2
De ec ed
Non de ec ed
SULT1A1
De ec ed
De ec ed
CYP11B1
Non de ec ed
Non de ec ed
* De ec ion in only one ype o PCa cell line
Ano he limi a ion o he s udy a e he low yields in RNA ob ained in u ine samples. Upon RNA ex-
ac ion, 1 mL olume coho yielded 0.388±0.301 μg o RNA while 10 mL olume coho yielded
0.541±1.100 μg o RNA in o al. These a e, in gene al, low yields excep o ew 10 mL olume samples
o which high concen a ion o RNA was ob ained a e isola ion. The ac ha 10 mL ex ac ion we e
only 2- old he yield o he 1 mL coho al eady indica es a loss e iciency upon olume inc ease o a
poo ex ac ion speci ici y o EV-associa ed RNA om u ine.
68
In Figu e 17, he selec ed HKG and a ge genes ha showed ampli ica ion in ou empla es a e
analysed. The C alues o each gene o in e es pe sample a e ep esen ed. I is obse ed HKG be-
ha e simila o no malisa ion analysis. S ikingly, RNA ex ac ions om 10 mL olume samples meas-
u ed lowe RNA exp ession o no malising genes. This al eady hin s ha ei he he RNA ex ac ion o
he qPCR assay migh ha e been ine icien . To assess whe he a la ge olume o ini ial sample may
ca y sal s o o he con aminan s hampe ing he ex ac ion o qPCR assay, a se ial dilu ion (1:5 and
1:10 o he ex ac ed RNA) and a highe amoun o empla e (4 ng ins ead o 1 ng) we e analysed by
qPCR. Values o C a ied acco ding o se ial dilu ion o empla e in each eac ion: a highe amoun o
empla e imp o ed qPCR ou come by a dec ease o app oxima ely 2 C and he dilu ion inc eased C
alues as expec ed. This sugges s ha he e a e no compounds impai ing qPCR eac ion and hence,
issues may be due o RNA ex ac ion o a low ini ial quan i y o EV-associa ed RNA in samples. Pe haps
a highe amoun o ex ac ed RNA would inc ease eliabili y in he assay o could imp o e he sensi i i y
o quan i ica ion. In ac , quan i ica ion sensi i i y could be imp o ed in u u e wo ks by sc eening a ge
genes di ec ly using TaqMan quenching app oach.
Figu e 17. A e age C alues (± SD) o each RNA sample ex ac ed om u ine in analysed genes. The op
g aph depic s he esul s om 1 mL olume samples and he bo om he esul s om 10 mL olume sample. Each
do co esponds o a single u ina y RNA p epa a ion analysed o one single gene. They a e colou ed as ollows: in
blue, GG5 pa ien RNA samples; in g een, GG2 pa ien RNA samples; in ed, BPH pa ien RNA samples.
Focussing on he six es ed genes wi h con i med wo king p ime s only CYP3A5, SULT2A1 o
SULT2B1 ampli ied in u ine samples. They we e measu ed only occasionally and in ew samples. Also,
C alues we e highe han 38 in gene al. Non-speci ic ampli ica ions s a occu ing om his s anda d
h eshold. Indeed, a mel ing cu e analysis con i med ha mel ing empe a u e o p ime s - a cha ac e -
is ic ha indica es an adequa e and speci ic annealing o he empla e – was inco ec , sugges ing un-
speci ic ampli ica ions.
69
2.4. Discussion and Conclusions
The explo a ion o new bioma ke s in cance esea ch ose in e es due o he limi a ions ha sup-
pose a poo diagnosis and p ognosis o cance disease. The appea ance o no el bioma ke s ca ie s
such as EVs combined wi h cu ing-edge me hodologies as single- esicle app oaches can p o ide al-
e na i es o disco e disease ma ke s. In his line, he bioin o ma ics analysis pe o med in his wo k
aimed o desc ibe po en ial candida es associa ed o EVs o disc imina e be ween pa ien g oups. Web-
based da abases compile se e al p o eins o mRNA ela ed o s e oid ho mones and associa ed o EVs.
These EV da ase s de i e om hund eds o s udies which include cell lines, issues and body samples.
Besides, EV-associa ed candida es we e u he il e ed using CANCERTOOL o conside hei ele-
ance in disc imina ing cance g oups. To no e, CANCERTOOL da abases compile ansc ip omic anal-
yses o p os a e p ima y samples. One could a gue ha speci ic mRNAs ound in PCa p ima y samples
would no necessa y be p esen in EV samples o any kind.
Up- o-da e, concen a ion o PSA in blood is measu ed ou inely o diagnose a pa ien wi h p os a e
issues. I s poo p ognosis a ea ly s ages is well documen ed bu also i s poo pa ien s a i ica ion; his
highligh s he need o disco e no el bioma ke s. In e es ingly, Exosome Dx eleased in 2020 a PSA-
independen ool o diagnose PCa. By combining he gene exp ession o PCA3, ERG and SPDEF in
uEVs hey could p edic he likelihood o high g ade PCa occu ence408. The launch o such es demon-
s a es he po en ial using uEVs con en as bioma ke s. Howe e , I also poin s ou he challenge in
es ablishing bioma ke s o e y ea ly s ages. Acco ding o SCOPUS, o e 18,000 s udies ha e been
eleased desc ibing bioma ke s associa ed o PCa disease; howe e , only 35 conside EVs and s e oid
ho mone enzymes. Joncas e al.370 is he unique wo k one could ind desc ibing an EV-associa ed
mRNA ha could disc imina e plasma samples a di e en s ages o disease p og ession using ddPCR.
Mo eo e , sec ion 1.3.3. EV bioma ke s. o Chap e 1 demons a es he po en ial o SVA echniques o
op imise he iden i ica ion o cance bioma ke s. Speci ically, he use o ddPCR may enable he de ec ion
o ansc ip s ha a e p esen in a small subse o EVs in body luids. This app oach is able o dis ibu e
single EVs in o indi idual d ople s, which allows he iden i ica ion o hei gene ic ca go o in e es .
In his chap e , he collabo a i e wo k wi h Exosome Dx is summa ised. A clinical ial a e alida ion
o assays was p ojec ed, howe e , i u ned ou o no happen. S ill one was able o:
i. Design an in silico s udy om which ma ke s p esen in EVs we e de e mined. Mo eo e , one
analysed which bioma ke s hold a po en ial in ea ly classi ica ion o pa ien s.
ii. Demons a e PSA was no a good classi ie in a la ge u ine coho . PSA could only be accep a-
ble o disc imina e GG1 pa ien s o highly agg essi e PCa pa ien s.
iii. Design a qPCR assay o he sc eening o EV-associa ed mRNA a ge s. This assay ound ou
app op ia e HKG and no malising genes and; could es some o he po en ial bioma ke s.
Sadly, no posi i e esul s we e ob ained bu we included se e al sugges ions in he chap e o
op imise his app oach in u u e wo k o s udies o he same kind.
76
We desc ibe a me hod o he de ec ion o endogenous s e oid ho mones and hei in e media es
using liquid/liquid ex ac ion and ul a-pe o mance liquid ch oma og aphy (UPLC) coupled o high es-
olu ion ime-o - ligh mass spec ome y (h LCMS). UPLC p o ides as cycling imes and a high ch o-
ma og aphic esolu ion. The high mass esolu ion ob ained wi h ime-o - ligh mass spec ome y esul s
in high speci ici y while sensi i i ies a e on pa wi h iple quad upole me hods. This me hod was applied
o me abolically p o ile se e al animal issues and u ina y EVs (uEVs). Di e en biological ma ices
including p os a e, ad enal gland, es icle, b ain and li e o Wis a male a s bu also human u ina y
samples we e es ed in his assay. To ou knowledge, he p esen wo k p esen s o he i s ime a
eliable and op imized h LCMS assay o analyse key endogenous s e oid ho mones in endoc ine issue,
bioliquids and EVs.
3.1.2. Ma e ials and Me hods
3.1.2.1. Tissue and bio luid samples
Tissues and se um we e ob ained om h ee wild- ype (Wis a , RjHan:WI) a s ob ained om Jan ie
Labs, Le Genes -Sain -Isle, F ance. All u ine samples we e ob ained om a heal hy male on ei he he
mo ning o he a e noon. uEVs we e ob ained by ul acen i uging u ine samples as desc ibed else-
whe e60. U ine samples and uEVs we e cha ac e ized in se e al physicochemical pa ame e s and p o-
ein ma ke s, espec i ely.
Tissue and se um sample p epa a ions om a s
Tissues and se um we e ob ained om h ee wild- ype (Wis a , RjHan:WI) male a s which a e 17
weeks old. Ra s we e ed wi h s e ile wa e and a s anda d die ad libi um in a empe a u e- and ligh
cycle-con olled animal acili y ollowing he Spanish Guide o he Ca e and Use o Labo a o y Animals
(RD 53/2013 - BOE-A-2013-1337). All a s unde wen a li e pe usion p ocedu e and we e sac i iced
by bleeding. Immedia ely a e he pe usion, b ain, p os a e, es icles and ad enal gland issues we e
emo ed and di ec ly ozen a -80 ºC in d y ice o u he use. Li e issue was collec ed om animals
in which li e pe usion could no be comple ed. Li e , p os a e and es icle issues we e cu on d y ice
and aliquo ed in o po ions be ween 50 o 80 mg p io me aboli e ex ac ion. Ad enal glands we e s o ed
indi idually as each indi idual weigh ed app oxima ely 80 mg. The exac weigh o all issue samples
was calcula ed and u ilised no malise he de ec ed me aboli es. Blood was collec ed du ing he pe u-
sion p ocedu e in o a BD Mic o aine ® blood collec ion ube wi h sepa a o gel (BD, F anklin Lakes,
NJ). Then, i was cen i uged a 8,000g o 15 min and he se um ac ion ans e ed in o a clean Ep-
pendo ® ube p io s o age a -80 ºC o u he p ocessing.
Human u ine samples
All u ine samples we e ob ained om a heal hy male on ei he he mo ning ( i s u ine o he day
a e as ing) o he a e noon (a e lunch, app oxima ely a 4pm) o obse e ci cadian a ia ions in a-
indi idual. Each sample g oup (mo ning and a e noon samples) has h ee eplica es collec ed in inde-
penden days by spon aneous mic u i ion. App oxima ely 80 mL o u ine we e collec ed o which 50 mL
77
we e cen i uged a 2,000g o 10 min, il e s e ilised (0.22 μm po e size) and immedia ely ozen a -
80 ºC o u he p ocessing. An aliquo o he o iginal u ine was also s o ed a -80ºC o u he analysis.
The six di e en u ine samples we e cha ac e ized ega ding he ollowing pa ame e s: blood and ke-
one bodies p esence in u ine, glucose concen a ion, pH alue and densi y (Table 19). Dip-and- ead
s ipes we e in oduced in o hawed u ines samples and physicochemical pa ame e s we e measu ed
wi h an OneS epTM Plus S ipe U ine Analyse (Hen y Schein Inc., Mel ille, NY).
EV isola ion p ocedu e
U ine samples we e hawed a oom empe a u e and cen i uged o 5 min a 2,000g o emo e any
p ecipi a e . Then, hey we e cen i uged a 10,000 g o 30 min o ob ain a pelle (P10K ac ion) con-
aining EVs o bigge size han in he emaining supe na an . In a nex s ep, he u ine supe na an was
ul acen i uged a 100,000g o 90 min; he esul ing pelle (F ac ion P100K) con ains EVs o smalle
size han P10K. F ac ion P10K and P100K we e bo h washed in 50 mL o phospha e-saline bu e
(PBS) and ul acen i uga ed a 100,000g o 90 min. A e wa ds, bo h ac ions we e esuspended in
50 μL o PBS and s o ed a -80 ºC oge he wi h he u ine supe na an (SN100K) o P100K o u he
analysis.
3.1.2.2. Wes e n blo Analysis
An aliquo o 6 µL o each u ina y EV (uEV) p epa a ion was loaded and sepa a ed unde non-
educing condi ions in 4–12% Bis-T is P o ein gels (In i ogen Inc., Wal ham, MA). Wes e n blo ing
was pe o med o de e mine he p esence and ela i e amoun o uEVs in each sample and ac ion, o
his eason only he app oxima ely 10% o each uEV isola ed ac ion was u ilized. In b ie , he p o eins
we e ans e ed o ni ocellulose memb anes and hen, hey we e blocked o 1 h (in 5% non- a milk
and 0.1% Tween-20 PBS solu ion). Then, he p ima y an ibody was incuba ed o e nigh (app oxima ely
16 h) a 4 ºC, washed and incuba ed o 1 h wi h a seconda y HRP-conjuga ed an ibody a oom em-
pe a u e. The p ima y an ibodies used in his s udy we e: MoαCD63 (clone H5C6) pu chased om
De elopmen al S udies Hyb idoma Bank (Iowa, IA), MoαCD9 (clone 209306) om R&D Sys ems (Min-
neapolis, MN), RbαAQP2 (clone A7310) ob ained om Sigma-Ald ich (S . Louis, MO), RbαCOX-IV
(clone 3E11) om Cell Signaling Technology (Dan e s, MA), MoαCD10 (clone F-4) om San a C uz
Bio echnology Inc. (Dallas, TX), and inally, RbαAnnexin V (ab14196) and RbαLAMP2A (clone
EPR4207(2)) we e bo h pu chased om Abcam (Camb idge, UK). Jackson ImmunoResea ch, Inc. p o-
ided he mouse and abbi HRP-conjuga ed seconda y an ibodies. A Cla i y Wes e n ECL ki om Bio-
Rad (He cules, CA) was u ilized o he chemiluminescence de ec ion o bands. Ei he by scanning
Ame shamTM Hype ilmTM MP pho osensible ilms (Cy i a, Uppsala, Sweden) o using a luminescen
image analyse ImageQuan TM LAS 4000 (GE Heal hca e, Chicago, IL), he p o eins in he ni ocellulose
memb anes coming om di e en u ine samples and ac ions we e iden i ied.
78
3.1.2.3. Chemicals and s anda ds
DHEA, DHT, co isol (in me hanol solu ion) and he sodium sal o and os e one sul a e we e ob-
ained om Ce illian Co po a ion (Round Rock, TX). Supelco (Belle on e, PA) p ocu ed and os enedi-
one. The sodium sal s o DHEAS and p egnenolone we e ob ained om A an i Pola Lipids, Inc. (Ala-
bas e , AL). Tes os e one, aldos e one, co icos e one, es one, p egnenolone 3-sul a e (sodium sal
o m), leucine-enke aline (Leu-Enk), chlo o o m (>99.8% pu e; o ch oma og aphy g ade) and ammonia
solu ion we e pu chased om Sigma-Ald ich (S . Louis, MO). LC-MS g ade wa e , ace oni ile, o mic
acid and me hanol we e pu chased om Fishe chemical (Fai Lawn, NJ).
3.1.2.4. LC-MS sample p epa a ion
S e oid me aboli es we e ex ac ed by liquid-liquid ex ac ion using a me hanol/wa e mix u e and
chlo o o m as ex ac ion liquids. EV ac ions we e sonica ed o 15 min in a o al olume o 400 µL 50%
/ me hanol/wa e mix u e con aining 1 mM ammonia o lysa e EVs. The cell cul u e (DU145 cell line),
ixed on cul u e well pla es, was sc apped a e 5 min incuba ion wi h 500 μL 50% / me hanol/wa e
mix u e con aining 1 mM ammonia. Tissue aliquo s - app oxima ely 50 mg - we e lysed using 1.4 mm
zi conium oxide beads in o s anda d 2 mL homogenize ubes (P ecellys, Mon igny, F ance). Each
sample was homogenized in 500 μL 50% / me hanol/wa e mix u e con aining 1 mM ammonia by
pe o ming wo cycles o 40 s a 6,000 pm in a Fas P ep-24TM 5G bead bea ing g inde (MP Biomed-
icals, Solon, OH). A e lysis, 400 μL o he homogena e – ei he issue, EV ac ion o DU145 cell cul u e
- was ans e ed o a clean Eppendo ® ube. Subsequen ly, 400 μL o LC-MS g ade chlo o o m was
added on op o he 400 μL o any lysa ed sample and shaken o 60 min a 1,400 pm a 4 ºC. Then,
he samples we e cen i uged o 30 min a 14,000 pm a 4 ºC in o de o p ecipi a e p o eins and o
sepa a e he o ganic om he aqueous phases.
The aqueous ( op) and o ganic (bo om) phases we e sepa a ed. The p o ein ac ion p ecipi a ed
on he meniscus be ween hese wo immiscible phases. Then, 250 μL o each ac ion was ans e ed
o clean Eppendo ® ubes and e apo a ed using a cen i ugal acuum concen a o . The pelle s om
he o ganic ac ion we e dissol ed in 100 μL pu e me hanol and he pelle s om he aqueous ac ions
we e dissol ed in 50% / me hanol/wa e . All esuspended pelle s we e cen i uged o 30 min a
13,000 pm and 4 ºC. Finally, 80 μL o he esuspended pelle s we e ans e ed o deac i a ed glass
ials o 96-wells pla es o injec ion in o he h LCMS sys em.
3.1.2.5. Ul a-high pe o mance liquid ch oma og aphy
The ch oma og aphic sepa a ion o he analy es was pe o med wi h an ACQUITY UPLC I-Class
PLUS Sys em (Wa e s Inc.). This sys em was equipped wi h a cooled (10 ºC) P ocess Sample Manage
wi h a sample loop o 10 µL and a Sample O ganize , a Bina y Sol en Manage and a High Tempe a-
u e Column Hea e . A e e sed-phased 1.0 mm x 100 mm BEH C18 column (Wa e s Inc.), he mo-
s a ed a 40ºC, was used o sepa a ing he analy es. Samples we e injec ed om ei he 2 mL deac-
i a ed glass ials o 700 µL Round 96-well polyp opylene pla es.
79
Ch oma og aphic beha iou was op imized wi h espec o peak in ensi y and an adequa e sepa a-
ion o he 11 analy es along he un. G adien elu ion was accomplished wi h an aqueous mobile phase
(eluen A) consis ing o 99.9% wa e wi h 0.1% o mic acid and an o ganic mobile phase (eluen B)
consis ing o 99.9% ace oni ile wi h 0.1% o mic acid. The low a e was 140 µL pe min. Se e al g a-
dien s we e es ed du ing he op imiza ion p ocess (Table 13) in o de o a oid b eak- h ough (elu ion
o analy e in he injec ion peak) and ob ain good peak sepa a ion. The op imal g adien was as ollows:
s a a 30% B, a linea inc ease o 80% B in 3.8 min., a s ep inc ease om 80% o 99%, cons an a
99% o 1.0 min and back o 30% B in 0.2 minu es. The o al cycle ime om injec ion o injec ion was
6 minu es. The injec ion olume o all samples was 2 µL.
Table 13. Summa y o he six g adien u ilised in op imiza ion ounds o es ablish he bes
me hodology wi h espec o peak in ensi y and analy e sepa a ion.
Time (min)
G adien 1
G adien 2
G adien 3
G adien 4
G adien 5
G adien 6
% A
% B
% A
% B
% A
% B
% A
% B
% A
% B
% A
% B
0
95
5
90
10
85
15
80
20
80
20
70
30
3.8
-
-
10
90
10
90
10
90
20
80
20
80
4
1
99
1
99
1
99
1
99
1
99
1
99
4.8
1
99
1
99
1
99
1
99
1
99
1
99
5
95
5
90
10
85
15
80
20
80
20
70
30
3.1.2.6. Mass spec ome y
A Time-O -Fligh mass spec ome e SYNAPT G2-S (Wa e s Inc.) was u ilized o he de ec ion o
analy es. The ins umen was ope a ed in ei he posi i e (ESI+) o nega i e (ESI-) elec osp ay ioniza-
ion mode and in ull-scan mode wi h a scan ange be ween 50 Da and 1200 Da and scan ime o 0.2
seconds.
Table 14. Summa y z-sp ay sou ce pa ame e s op-
imized o m/z 556.2771 in ESI+ and ESI-.
Pa ame e
ESI+
ESI-
Capilla y ol age
1.00 kV
2.00 kV
Sampling cone ol age
25
40
Sou ce O se
80
80
Sou ce empe a u e
120 ºC
120 ºC
Desol a ion empe a-
u e
450 ºC
450 ºC
Desol a ion gas low
5.00 L/h
5.00 L/h
Cone gas low
1,000 L/h
600 L/h
Nebulise
6.00 ba
6.00 ba
The z-sp ay sou ce pa ame e s: empe a u es, gas lows, capilla y posi ion and ol ages we e uned
as de ailed elsewhe e. Op imal sou ce pa ame e s o his assay in ei he ESI+ o ESI- a e summa ised
in Table 14. Ion op ics we e ine- uned by sp aying Leu-Enk (100 ppb), a a a e o 10 µL pe min, o a
80
esolu ion o e 20,000 (FWHM) o m/z 556.2771. The same Leu-Enk solu ion was sp ayed as a lock
mass o co ec o m/z luc ua ions along he assay. The lock mass solu ion was in oduced in o he
sou ce e e y 90 seconds using a second ESI p obe and i was eco ded o 0.5 s. Mass spec ome e
spec a was co ec ed acco ding o luc ua ions de ec ed in he lock mass.
3.1.2.7. S a is ical Analysis
Analy e eco e y s udy
The ex ac ion s ep e iciency was assessed by pe o ming a eco e y assay wi h a ious mix u es
o o ganic sol en s and wa e . Fi e di e en ex ac ion bu e s we e es ed in his assay: 25/75% /
and 50/50% / o me hanol/wa e mix u e, 25/74.9/0.1% / / and 50/49.9/0.1% / / o me hanol/wa-
e / o mic acid mix u e and 50/50% / o me hanol/wa e mix u e wi h 1mM ammonia. To compa e and
calcula e he eco e ies o 10 di e en analy es, a cul u e o a p os a e cance cell line - DU145 - was
spiked wi h analy e s anda ds. Each well con aining 5·105 cells was spiked wi h a mix o s anda ds a 2
µM be o e lysis (p e-spiked) and a he esuspension s age (pos -spiked) wi h a s anda d mix a 10 µM.
Thus, he p e-spikes con ained 1 nmol in 500 µL and pos -spikes (aqueous and o ganic ac ions) con-
ained he same o al amoun in 100 µL, which would be he heo e ical maximum absolu e i no loss
du ing he ex ac ion. In addi ion, o each ex ac ion solu ion, non-spiked samples we e p epa ed in
o de o co ec o endogenous me aboli es in he ma ix. Samples o p e-spiked, pos -spiked and non-
spiked condi ions and he i e di e en ex ac ion bu e s we e p epa ed in biological iplica es.
Only he absolu e peak a eas we e aken in o conside a ion o es ablish he eco e y e iciency in
he ex ac ion s ep. A e age peak a eas we e ob ained by mean smoo hing he aw signals o iplica es.
The eco e y (R) was de e mined by di iding he co ec ed p e-spike a e age by he co ec ed pos -
spike a e age and ep esen ed as a pe cen age (Eq. 1). Bo h p e-spiked and pos -spiked aw signals
ough o be co ec ed by sub ac ing he endogenous analy es signal in DU145 cul u e ma ix (Snon-spike).
Howe e , as Snon-spike o DU145 cul u e ma ix was less han 0.05% o he signal, endogenous co ec ion
was neglec ed du ing calcula ion. Impo an ly, p e-spikes we e co ec ed wi h espec o analy e loss
(α) du ing ex ac ion p ocedu e. Mo eo e , aw signals o each sample did no ha e o be co ec ed by
he amoun o ini ial samples because e e y well con ained he same amoun o cells.
𝑅 (%)=𝛼(𝑆𝑝𝑟𝑒−𝑠𝑝𝑖𝑘𝑒 − 𝑆𝑛𝑜𝑛−𝑠𝑝𝑖𝑘𝑒)
𝑆𝑝𝑜𝑠𝑡−𝑠𝑝𝑖𝑘𝑒 − 𝑆𝑛𝑜𝑛−𝑠𝑝𝑖𝑘𝑒
𝑥100
(1)
S udy o ma ix e ec in analy e quan i ica ion
𝑀𝐸 (%)=𝑆𝑝𝑜𝑠𝑡−𝑠𝑝𝑖𝑘𝑒 − 𝑆𝑛𝑜𝑛−𝑠𝑝𝑖𝑘𝑒
𝑆𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑𝑠
𝑥100
(2)
In o de o assess he ma ix e ec (ME) in he quan i ica ion o he analy es, he pos -spiked aw
signal was compa ed o an equi alen aw signal o a mix u e o analy es (10 µM) in solu ion. Pos -
spiked aw signals we e co ec ed by sub ac ing he endogenous analy es de ec ed in he non-spiked
81
DU145 cul u e samples. Then, he nume a o was di ided by he a e age peak a eas o he s anda ds
and exp essed as a pe cen age (Eq. 2).
Analy e semi-quan i ica ion
In his wo k, a calib a ion cu e was p epa ed in solu ion wi h 50% / me hanol/wa e o he semi-
quan i ica ion o analy es. This calib a ion cu e consis ed o a se ially dilu ed mix u e con aining all he
analy es s a ing a a concen a ion o 10 µM. The ini ial concen a ion was dilu ed o hal concen a ion
wice, esul ing in 5 µM and 2.5 µM concen a ion is he cu e. Then, his se o iple s was dilu ed in
i e decades; i esul ed in he ollowing 15 di e en concen a ions pe analy e: 10, 5, 2.5, 1, 0.5, 0.25,
0.1, 0.05, 0.025, 0.01, 0.005, 0.0025, 0.001, 0.0005 and 0.00025 µM. The calib a ion samples we e
injec ed a he beginning and a he end o each expe imen ; he a e age o hese wo poin s was used
o semi-quan i y o me aboli es in issues.
The limi o de ec ion (LOD) o each analy e was se o be he lowes concen a ion a which he
signal- o-noise (S/N) a io was abo e 3. The LOQ was de ined as he lowes concen a ion a which S/N
a io was abo e 10. The highes quan i iable concen a ion is he highes concen a ion pe analy e ha
i s he calib a ion cu e wi h an accep able accu acy and p ecision (CV ≤ 15%)413.
In gene al, he da a o a calib a ion cu e anges o e se e al o de s o magni ude, i is no linea
and ends o be he e oscedas ic426. Fo his eason, he ela ion be ween he peak a ea and sample
concen a ion was de e mined by powe - i ing427. Powe i ing esul ed in a calib a ion cu e (Eq. 3)
wi h α and b as he i ed pa ame e s. Once he sample concen a ions we e calcula ed using a calib a-
ion me hod in solu ion, he amoun (in nanomole) pe g am o issue weigh was es ima ed.
𝑃𝑒𝑎𝑘 𝑎𝑟𝑒𝑎 = α[𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛]𝑏
(3)
3.1.3. Resul s
3.1.3.1. Liquid ch oma og aphy and mass spec ome y me hod
We ha e compa ed six di e en ch oma og aphic me hodologies (Table 13) o sepa a e he analy es
sa is ac o ily. The g adien 6 (30% B o 80% B in 3.8 min; de ailed s eps in Table 13) showed he bes
peak sepa a ion along his un ime compa ed o o he es ed g adien s (da a a ailable in 430). Due o
he na u e o he s a iona y phase, analy es elu e in o de o inc easing hyd ophobici y. The esul ing
ex ac ed ion cu en (XIC) ch oma og ams o a s anda d mix u e a 10 µM a e depic ed in Figu e 21.
In b ie , aldos e one (m/z 361.2015; ESI+) elu es a 0.99 min, co isol (m/z 363.2171; ESI+) a 1.20 min,
DHEAS (m/z 367.1579; ESI-) a 1.60 min, co icos e one (m/z 347.2222; ESI+) a 1.68 min, and os-
e one sul a e (m/z 369.1736; ESI-) a 1.85 min, p egnenolone sul a e (m/z 395.1892; ESI-) a 2.23 min,
es one (m/z 271.1698; ESI+) a 2.39 min, and os enedione (m/z 287.2011; ESI+) and DHEA (m/z
289.2168; ESI+) coelu e a 2.40 min, DHT (m/z 291.2324; ESI+) a 2.65 min, p egnenolone (m/z
317.2481; ESI+) a 3.25 min.
82
Table 15. Summa y o he i e di e en ex ac ion bu e s es ed in he
eco e y e iciency expe imen s.
Ex ac ion bu e
% me hanol
% H2O
pH modi ie
Bu e 1
50
50
None
Bu e 2
50
49
1% o mic acid
Bu e 3
25
75
None
Bu e 4
25
74
1% o mic acid
Bu e 5
50
49.975
1 mM ammonia
The bu e s di e in hei me hanol con en and he compound u ilised o une he pH o he
bu e (pH modi ie ).
Rega ding he mass spec ome y me hod, Leu-Enk signal (m/z 556.2771) was aimed a a esolu ion
o o e 20,000 (FWHM) and p o ided he necessa y mass accu acy o e alua e assay analy es. Iso ope
pa e n ma ching and he use o chemical s anda ds con i ming elu ion imes u he ensu ed speci ici y.
In gene al, mass accu acies o he analy es in solu ion we e be ween -1 o 1 mDa. No e-wo hy, se e al
analy es we e no adequa ely sepa a ed du ing ch oma og aphic elu ion. Co icos e one and DHEAS
elu e a simila e en ion imes - 1.60 min and 1.68 min -, howe e , he MS could p ope ly dis inguish
hem by hei m/z di e ence and hei agmen a ion pa e n. Mo eo e , DHEAS was no de ec ed wi h
a high in ensi y signal in ESI+ mode. Fo his eason, co icos e one was measu ed in ESI+ and DHEAS
in ESI- mode. Likewise, es one, DHEA and and os enedione elu ed in app oxima ely 2.40 min. In his
case, one could only ely in MS sensi i i y (es one m/z 271.1698, DHEA m/z 289.2168, and os enedi-
one m/z 287.2011) and agmen a ion pa e n ha was sensi i e enough o dis inguish and quan i y
hem sepa a ely.
3.1.3.2. Analy e eco e y op imiza ion
A e wa ds, we e alua ed he eco e y o 11 analy es using a biphasic liquid-liquid me hod and an-
alysing hem wi h he op imized h LCMS me hod. Ex ac ion was pe o med using DU145 cell line as
ma ix. Fi e di e en mix u es o o ganic sol en s and wa e , con aining ei he o mic acid o ammonia
o modi y he pH o he ex ac ion bu e o no pH modi ie we e assessed (Table 15). Addi ion o o mic
acid s i ed o lowe ing he pH app oxima ely o 3 while 1mM ammonia modi ied he ex ac ion bu e
o pH 8-9 in o de o chemically neu alize unc ional g oups o s e oid compounds. F om p e ious ex-
pe imen s in ou me abolomics pla o m, we obse ed ha in liquid-liquid ex ac ion equi es a leas
25% o ganic sol en du ing he ex ac ion s ep o p ecipi a e he p o eins. This is impo an o a oid
clogging he ch oma og aphic sys em427. Mo eo e , he e ec i i y o issue homogeniza ion using beads
has been epo ed as high and does no di e much o he homogeniza ion o o he ma ices such as
u ine o cell cul u es427,428. The e o e, he calcula ed eco e ies a e ul ima ely dependen on he ex ac-
ion bu e u ilized ega dless he homogeniza ion me hodology.
Du ing he op imiza ion p ocess, i was de e mined s e oid sul a e compounds a e eco e ed com-
ple ely in he aqueous ac ion whils s e oids wi hou sul a e g oup a e ound in he o ganic ac ion.
No ably, only co isol was de ec ed sys ema ically in bo h ac ions (Figu e 22); howe e , i was majo ly
eco e ed in he o ganic (80% o highe ) a he han in he aqueous (app oxima ely 20%) ac ion.
83
Figu e 21. Ex ac ed ion cu en ch oma og ams o he analy es om a mix u e a 10 uM concen a ion o
each s anda d. In each ch oma og am, he op imal ES (+ o -), m/z alue and signal in ensi y o de ec ion a e
indica ed. They a e o de ed by dec easing e en ion ime. F om op o bo om p egnenolone, dihyd o es os e one,
DHEA, and os enedione, es one, p egnenolone sulpha e, and os e one sulpha e co icos e one, DHEAS, co isol
84
and aldos e one. In g een, he pa ame e s o he analy es de ec ed in ES- mode; in da k o ange, he pa ame e s
o he analy es de ec ed in ES+ mode.
Mo eo e , he addi ion o o mic acid o he ex ac ion bu e led o a d ama ic decay o eco e ies
in sul a e compounds and a sligh dec ease in he es o s e oid analy es (Figu e 22). One can in e
ha he p esence o p o ons in he bu e do no s abilize s e oid cha ges and se e ely hampe s he
ex ac ion o sul a e s e oids in a pola en i onmen . Supplemen a ion o 1mM ammonia ou pe o med
he ex ac ion in e ms o eco e y and obus ness com-pa ed o he o he ex ac ion liquids. No ably,
eco e y alues using di e en pe cen ages o me hanol in he ex- ac ion bu e do no di e much.
Howe e , he ex ac ion e iciency o sul a e compounds using 25% / me hanol unde pe o ms 50%
/ me hanol wi h a eco e y loss o 40 o 50%.
Figu e 22. Reco e ies (± s anda d de ia ion) o he selec ed panel o s anda d analy es a e shown (n=6).
Fo each analy e, he eco e ies using di e en ex ac ion bu e s a e depic ed. In g een, he esul s o an ex ac ion
using only a mix u e wi h he sol en (50% me hanol da k g een and 25% me hanol ligh g een). In blue, he esul s
wi h ex ac ion bu e s con aining 1% o o mic acid (50% me hanol da k blue and 25% me hanol ligh blue). Re-
co e ies ob ained wi h a 50% me hanol (1mM NH3) ex ac ion a e depic ed in ed. a, analy e eco e ies o he
o ganic ac ion. b, analy e eco e ies o he aqueous ac ion.
In Table 16, he eco e ies o 11 selec ed analy es using a mix u e o 50/50% / me hanol/wa e
wi h 1mM ammonia as ex ac ion bu e a e epo ed. In gene al, he p esen me hodology is able o
eco e and de ec o e 90% o he ini ially spiked analy e. Only DHT was de ec ed in a lowe pe cen -
age, app oxima ely 80% o he ini ially spiked DHT was eco e ed. As expec ed in a biphasic ex ac ion,
ho mone s e oids we e e ie ed in an apola en i onmen and sul a ed s e oids in a pola sol en .
Besides co isol, p egnenolone sul a e was also epo ed in bo h ac ions; i was mainly eco e ed in
he mo e pola sol en and a de iso y amoun in he o ganic ac ion. Using his me hodology, he e-
co e ies o 10 µM o analy e anged om 74.2% o 126.9%. These alues a e accep able o ou ine
mu i-analy e h LCMS analysis since all he esul s a e ep oducible429. Thus, ex ac ion using 50/50%
/ o me hanol/wa e mix u e wi h 1 mM ammonia was selec ed o u he expe imen s in di e en
biological ma ices.
85
Fu he mo e, he pe o mance o he op imized me hodology was es ed using u ine as ma ix since
i has a high in e es o clinical applica ions. Six u ines om a male indi idual we e pooled and aliquo ed
in di e en wo olumes o assess ma ix e ec o eco e y e iciency. Table 17, he eco e ies o 10
analy es a e epo ed; DHEA eco e y has no been e ie ed because i s peak was masked by es os-
e one’s signal. In gene al, o e 85% o he ini ially spiked analy e is eco e ed and de ec ed in 50 µL
u ine ma ix. Impo an ly, sul a ed s e oids a e no eco e ed wi h he same e iciency; DHEAS and
p egnenolone sul a e epo a eco e y e iciency o 75.7% and 54.9%, espec i ely. Reco e ies o he
analy es using 250 µL u ine as ma ix desc ibes a sligh dec ease in non-sul a ed s e oids while he
e iciency decay is d ama ic in sul a ed species.
Table 16. Summa y o he op imized me hod cha ac e is ics. The eco e ies (± s anda d de ia ion) and
ma ix e ec as signal loss (± s anda d de ia ion) o he ex ac ion p ocedu e in wo di e en biological ma-
ices (n=6; biological ma ix: DU145 cell) a e epo ed. In addi ion, LOD and LOQ alues o he analy es in
he adequa e ac ion a e compiled. LOD: Limi o de ec ion; LOQ: Limi o quan i ica ion.
Analy e
F ac ion
Reco e y [%]
Ma ix e ec [%]
LOD [nM]
LOQ [nM]
P egnenolone
O ganic
97.2 (± 1.9)
25.2 (± 3.1)
2.5 nM
10 nM
Aqueous
-
24.0 (± 2.8)
DHEA
O ganic
122.7 (± 2.9)
37.7 (± 5.7)
5.0 nM
50 nM
Aqueous
-
28.0 (± 6.2)
-
-
And os enedione
O ganic
102.2 (± 3.2)
30.8 (± 4.6)
0.25 nM
0.5 nM
Aqueous
-
23.2 (± 4.5)
Es one
O ganic
103.7 (± 3.8)
25.5 (± 4.8)
5.0 nM
10 nM
Aqueous
-
25.7 (± 4.0)
DHT
O ganic
74.2 (± 3.4)
23.1 (± 3.9)
0.25 nM
1.0 nM
Aqueous
-
23.4 (± 2.9)
Co isol
O ganic
114.3 (± 3.8)
25.9 (± 4.2)
0.5 nM
1.0 nM
Aqueous
22.28 (± 4.5)
17.6 (± 4.7)
Aldos e one
O ganic
99.8 (± 1.77)
18.7 (± 4.3)
0.5 nM
2.5 nM
Aqueous
-
17.7 (± 5.1)
Co icos e one
O ganic
109.4 (± 3.1)
25.1 (± 3.6)
0.25 nM
1.0 nM
Aqueous
-
20.2 (± 3.2)
Tes os e one
O ganic
126.9 (± 1.7)
14.3 (± 1.9)
0.25 nM
0.25 nM
Aqueous
-
8.0 (± 2.1)
P egnenolone sul a e
O ganic
6.9 (± 2.7)
25.2 (± 3.1)
0.25 nM
1.0 nM
Aqueous
94.8 (± 1.9)
24.0 (± 2.8)
DHEAS
O ganic
-
42.6 (± 1.1)
0.25 nM
0.5 nM
Aqueous
108.0 (± 1.4)
42.5 (± 0.1)
3.1.3.3. Ma ix e ec
I is well known ha phospholipids and o he lipids ypically en iched in biological ma ices such as
issues, body luids o cell cul u es can cause ion supp ession in mass spec ome y, he eby hampe ing
he analy e signal129,130. This phenomenon nega i ely in luences he de ec ion o analy es and may un-
de es ima e hei quan i ica ion. Fo a speci ic ma ix, he highe he ion supp ession e ec is he highe
92
assay is e y con enien as all me aboli es (excep co isol) a e eco e ed in one ac ion. This pe mi s
a as e measu emen o s e oid ho mones in di e se biological ma ices.
Exis ing quan i a ion me hods o s e oid ho mone compound ha e a wide span o LOQ, anging
om 0.002 o 10 ng pe mL. Howe e , i is highly dependen on he analysed ma ix, i.e. a u ine ma ix
shows a ange om 0.002 o 0.2 ng pe mL415,416, whils cell ma ices display a highe LOQ up o 10 ng
pe mL413. This sugges s he ma ix e ec also depends on he speci ic ma ix whe e he me aboli es
a e con ained. Compa ing hese s udies, cell ma ices epo a lowe sensi i i y compa ed o u ine; his
is impo an when applying his me hod in u u e expe imen s o assays. In ac , his obse a ion spo -
ligh s he majo limi a ion o his s udy: The quan i a ion has been pe o med semiquan i a ely. Ion sup-
p ession in mass spec ome y a ec s nega i ely he analy e signal, and subsequen ly unde es ima es
i s quan i a ion o i simply hampe s i s de ec ion. Mo eo e , ion supp ession may be limi ing he de ec-
ion o ce ain s e oid compounds in se e al ma ices, i.e. EV p epa a ions. In consequence, his me hod
should be u ilised in ma ices ha acili a e s e oids de ec ion. A ma ix-spiked calib a ion is usually he
app op ia e me hod o quan i y absolu e amoun s o analy es in samples427. In his wo k, a calib a ion
cu e o he analy e s anda ds was p epa ed in solu ion wi h 50% / me hanol/wa e as sol en . Such
app oach canno compu e absolu e amoun s o he analy es in issue since he ma ix e ec is no
conside ed, howe e , a semi-quan i a i e app oxima ion o he me aboli es in issues can be calcula ed.
In his assay, he epo ed LOQ ange lays be ween 0.50 and 50 nM (equi alen o 0.14 and 14.42 ng
pe mL) in solu ion, simila ly o p e ious s udies. Howe e , i is ad ised o use ma ix-spiked cu es in
u he expe imen s using his assay.
The ime equi ed o pe o m he ch oma og aphic sepa a ion is ypically long in li e a u e; hey epo
un imes om o e 10 min up o 45 min59,409,416–420,425. Only he wo k o Quanson e al.413 and Indapu ka
e al.414 desc ibed a me hodology wi h a sho un ime (4 o 5 min); howe e , hey es ed and applied
he me hod solely in cell ma ices: PCa and induced plu ipo en s em cell lines, espec i ely. Indapu ka
e al.414 de eloped a me hodology speci ic o es adiol- ela ed me aboli es and Quanson e al.413 meas-
u ed and ogens wi h ul a-pe o mance con e gence ch oma og aphy. In 2012, Maeda e al.418 accom-
plished he sepa a ion, de ec ion and quan i ica ion o a panel o s e oids in a o gans excep in he
li e bu using an HPLC sys em. Fo his eason, sample p epa a ion s a egy demanded high olumes
o ex ac ion bu e – 15 mL o ace oni ile pe sample – and equi ed a o al un ime o 11 min. In his
wo k, he olumes and un ime a e lowe han 1 mL and he 10 min.
In o de o es he pe o mance o ou me hodology, we ha e measu ed s e oid ho mone analy es
om se e al a issues: ad enal glands, es is, p os a e, li e and b ain. The da a shown in Table 18 is
in acco dance wi h he ac ha he pa hway is issue-dependen in egula physiological condi ions.
Two me aboli es ups eam he pa hway, p egnenolone and and os enedione, we e quan i ied in ad enal
glands bu could no be quan i ied in p os a e o b ain. This hin s ha ad enal glands a e in cha ge o
me abolising choles e ol in o s e oid compounds in complex o ganisms such as a s; i is in line wi h
p e ious indings in li e a u e435–437. Likewise, ad enal glands a e known o p oduce co icoid ho mones.
Ou da a con i ms his since co icos e one is quan i ied in a highe amoun – h ee o ou o de s o
93
magni ude – when compa ed o p os a e, b ain and es icles. Ad enal glands also seem o accumula e
and ogens (Table 18); howe e , he p esence o ac i e and ogens (DHT) is 2- old highe in p os a e
compa ed o o he issues. Impo an ly, he a io DHT/ es os e one, which a e he ac i e and non-ac i e
pai ed and ogens, was app oxima ely 11 in p os a e while ad enal gland and es is we e below 1. Be-
cause he p esence o ac i e and ogen plays a physiological ole in p os a e, he a io DHT/ es os e one
was also highe in his issue.
Since he i s u ina y me abolomics a emp s o analyse u ina y samples and o he bio luids, se e al
me hodologies ha e been de eloped du ing he las yea s416–418. Ne e heless, none o he epo ed
me hodologies was op imal o assess s e oids in EV sample p epa a ions, issues o body luids in a
as and simple manne . Up o da e, many s udies ha e shown me abolomics in EVs60,417,438, bu none
o hem has epo ed he de ec ion o s e oid ho mones in a a ge ed app oach. A plausible explana ion
is ha he iden i ica ion and de ec ion o compounds simila in molecula mass – e en he same one in
some cases – hampe s he alloca ion o mass signals wi h he co esponding ch oma og aphic peak.
Fo hose s e oids, i.e. DHEA and es os e one, which sha e empi ical o mula, he iden i ica ion o each
speci ic compound emains challenging using MS and i elies on ch oma og aphic sepa a ion.
Impo an ly, we ha e been able o quan i y s e oid ho mones in u ine samples and de i ed uEV in a
as and simple manne . Howe e , only one DHEAS was de ec ed in uEVS and co isol, and os enedi-
one and DHEAS we e de ec ed in u ine samples. These EVs we e isola ed by ul acen i uga ion in-
cluding a washing s ep o a oid any con amina ion om he soluble ac ion. U ine samples om a
heal hy man we e collec ed in di e en days and in ime collec ion (mo ning and a e noon). Time col-
lec ion was a pa ame e o be assessed om a me abolomics pe spec i e bu we ound ou ha in e -
day a iabili y had also a high impac in he analysis. Mo ning samples a e conside ed o con ain a
highe concen a ion o s e oids coming om he p os a e, possibly due o accumula ion and leakage
owa ds he u ina y ac a nigh . Howe e , his end was no desc ibed in ou mo ning samples. The
eason may be ha u ine sample U003 (Table 19) was no a ailable o me abolomics analysis since
he analysis o he soluble ac ions o u ine (a e uEV isola ion), which includes U003, mo ning samples
had a highe concen a ion o DHEAS. This highligh s he impo ance o analysing a la ge coho o
ob ain signi ican esul s non-dependen o a unique high concen a ed sample.
In he end, his is a as and sensi i e me hod ha was success ully applied o he de ec ion and
quan i ica ion o a panel o s e oid ho mone compounds in biological samples in 6 min un ime pe
sample. The sensi i i y o his me hod makes i ideally sui ed o mul iple in i o applica ions. In his
manusc ip , we explo e he analysis o s e oids in se e al a issues and also human u ine and uEV
samples. This has e iden applica ions in p o iling he me abolic s a us o pa ien s su e ing any ho -
mone-dependen disease. To no e, he assay equi es a longe cleanse s ep o wash he column ou o
lipids and pep ides when unning a long expe imen wi h many issue samples. To ou knowledge, his
is he i s h LCMS-based me hod able o de ec and quan i y s e oid ho mones associa ed o EVs iso-
la ed om body luids in a a ge ed app oach.
94
3.2. Clinical E alua ion o Me abolic Signa u es as
Bioma ke s o P os a e Cance P og ession in
Pa ien U ines
3.2.1. Backg ound
The use o body luids as a sou ce o bioma ke s ha e been in ensi ied o e las yea s. Cu en ly,
u ine samples a e one o he mos impo an sou ces o iden i y PCa bioma ke s since i is nea he
p os a e and hey can p o ide biological in o ma ion. Documen ed s udies ha e shown me aboli es and
me abolic signa u es con ained in u ine samples can disc imina e heal hy and disease pa ien s439–441.
Mo eo e , a me abolomics s udy in uEVs desc ibed se e al al e ed me aboli es o which ew s e oid
ho mones we e heigh ened60. In gene al, hese ype o app oaches aim o desc ibe me abolic bi-
oma ke s in al eady dis inc samples. This p o ides a aluable diagnosis in o ma ion; howe e , hey
o en p esen a poo p ognosis, as hey canno p edic he likeliness o a pa ien o de elop he disease
o how i will p og ess.
In his sec ion, a me hod o he de ec ion and quan i ica ion o s e oid ho mones in biological sam-
ples has been de eloped and op imised as hese ho mones a e he majo d i e s o p og ession a
ea ly s ages. This app oach will be used in upcoming chap e o desc ibe he ans e ence o me abo-
li es ia EVs. None heless, ano he pu pose o he me hod was o alida e a s e oid ho mone me abolic
signa u e as a bioma ke o PCa.
3.2.2. Resul s and discussion
3.2.2.1. Clinical coho cha ac e is ics
In Chap e 2. E alua ion o S e oid Ho mone T ansc ip s Associa ed o U ina y Ex acellula Vesi-
cles in P os a e Cance P og ession a coho o samples ob ained om Basu o’s Hospi al has been
p esen ed. F om ha coho , a clinically ele an coho was designed o seek o speci ic signa u es o
de ine he s a us o disease in pa ien s. In his sec ion, i has been u ilised o assess he ho mone s e oid
me abolic signa u e o pa ien s in u ine. This coho consis s o 85 u ine samples o 1 mL olume se-
lec ed o co e he 5 g oups o PCa pa ien s (GG1, GG2, GG3, GG4 and GG5) and con ol BPH. Rel-
e an in o ma ion abou he pa ien s and samples is compiled in Table 21. To no e, all u ine samples
we e collec ed p io any biopsy hence, me abolic signa u es o pa ien s in o m abou hei s a us a i s
isi o clinician. Diagnosis in e ms o GS, pT, Pn and pN we e epo ed a e unde going a second
biopsy, a so-called su gical specimen biopsy, which was pe o med a e 1 o 3 yea s. The s aging
men ioned in Table 21 is well explained in P os a e Cance Disease sec ion in he INTRODUCTION.
Impo an ly, whe he pe ineu al in asion was obse ed clinically (Pn) o alida ed ana omopa hologi-
cally (pN) is speci ied.
95
Table 21. Summa y o clinical cha ac e is ics o u ina y
samples included in he s udy. Pa ien g oups, pT and Pn
s ages we e de e mined upon su gical specimen biopsy.
3.2.2.2. Signa u e esul
Me abolomics assays equi e an adequa e pe o mance in he speci ic biological ma ices o be an-
alysed. In e ms o accu acy and sensi i i y, he u ilised ma ix may in luence on he ou come; howe e ,
he mos impo an pa ame e o es a e he e iciencies o analy e eco e y. This pa ame e will in o m
whe he he assay is wo king success ully. Simila o sec ion 3.1. De elopmen o a Ta ge ed Me abo-
lomics Assay in Endoc ine Tissues o Male Ra s and Human Samples, eco e ies o he analy es
in di e en olumes o u ine we e calcula ed (Figu e 25). Non-sulpha ed me aboli es we e eco e ed in
Pa ame e
Pa ien s (%, n=85)
Median Age (IQR)
67(63,73)
BMI (IQR)
26.6 (25.3,28.1)
PSA alue, ng/mL
< 4
41 (48)
4-10
35 (41)
> 10
9 (11)
ADT ea men
no a ailable
P io p os a ec omy
Yes
84 (99)
Unknown
1 (1)
Family his o y
PCa
6 (7)
O he cance
39 (46)
None
33 (39)
Unknown
7 (8)
Pa ien g oup (GS)
BPH
35 (41)
GG1
(3+3)
10 (12)
GG2
(3+4)
10 (12)
GG3
(4+3)
10 (12)
GG4
(8)
6 (7)
GG5
(9-10)
14 (16)
pT s age
None
37 (43)
2
26 (31)
3
a
10 (12)
b
12 (14)
Pn s age
No epo ed
38 (44)
Pn0
3 (4)
Pn1
44 (52)
pN s age
No epo ed
44 (52)
pN0
10 (12)
pNx
30 (35)
pN1
1 (1)
ADT, and ogen dep i a ion he apy; GS, Gleason sco e; IQR,
in e qua ile ange; pT, PCa p og ession s age; Pn, Pe ineu-
al in asion (Clinical); pN, posi i e lymph nodes (Ana o-
mopa hological)
96
a ange be ween 70 o 110%, app oxima ely. In con as , sulpha ed me aboli es - DHEAS - ange be-
ween 45 o 75% app oxima ely, exhibi ing a dec eased eco e y alues compa ed o p e ious sec ion.
In gene al, an inc ease in u ine olume leads o a lowe eco e y o analy e; in sulpha ed me aboli es
hey su e a d ama ic decay. This esul indica es ha u ine ma ix nega i ely a ec s he pe o mance
o he assay. In he case o pola analy es, he loss is a he ele an and i is some hing o conside in
he decision making p ocess upon designing an expe imen . Anyway, eco e ies a e s ill adequa e so
we decided o p oceed quan i ying he coho .
Figu e 25. Reco e ies (± s anda d de ia ion) o a selec ed panel o s anda d analy es wi h u ines
as ma ix a e shown (n=6). Fo each analy e, he eco e ies using a di e en olume o u ine o
unde go analy e ex ac ion a e depic ed. Ei he s a ing wi h 250 µL o 50 μL o u ines was es ed. In
blue, he analy es eco e ed in he aqueous o ganic ac ion. In ed, he analy es eco e ed in he
aqueous o ganic ac ion. Ex ac ion wi h 50% me hanol (1mM NH3) was u ilised o u ines.
Thence, a o al o 85 u ine samples we e analysed and quan i ied in s e oid ho mones con en . The
u ine olume u ilised in his app oach was 250 μL since apola ho mones a e p esen in low le els and
one would like o maximise de ec ion e en comp omising analy e e iciency o eco e y. The analy es
aldos e one, and os enedione, es os e one, co isol, choles e ol, and os e one sulpha e and DHEAS
we e success ully de ec ed in his coho . P io analysis, analy e signal was no malised using median
old change442,443 o co ec o me aboli e abundance in insic o he u ine sample. Ho mone s e oid
le els in u ines ailed o disc imina e be ween PCa and BPH pa ien s (Figu e 26). The sco e plo indi-
ca es a endency o sp ead ou bu he wo g oups o samples a e no sepa a ed. Fu he uni a ia e
analysis con i med his esul . In e es ingly, ew signi ican di e ences we e measu ed when PCa sam-
ples we e ca ego ised acco ding o hei disease s age (Figu e 27). Figu e 27 depic s he ou mos
ele an me aboli es ound back in u ine samples. I shows ha and os e one, DHEAS and co isol
le els a e di e en ially quan i ied be ween BPH and some PCa g oups bu also, wi hin PCa g oups.
97
The majo issue o hese esul s is ha no signa u e can de e mine whe he a sample is diagnosed
wi h PCa o BPH. Documen ed epo s using EV p epa a ions desc ibed DHEAS as a po en ial ma ke
o PCa60; howe e , analysed u ine samples do no suppo his hypo hesis. The ac he me aboli e
DHEAS is eadily soluble in bio luids and i is esidual in EV samples de i ed om u ines444 sugges ed
ha i s e alua ion in u ine could ha e been mo e in o ma i e o he s a us o he disease. None heless,
he new da a o e s a be e classi ica ion o PCa sample g oups and shows ew signi ican di e ences
be ween hem. A limi a ion o his subg ouping is he sample size because i is a he small a g oup
le el. Mo eo e , s e oid ho mone le els do no desc ibe a classi ica ion pa e n no desc ibe all he
di e en subg oups. No ably, he classi ica ion o samples is e y ele an in p oceeding wi h hese
app oaches, he e o e di e ences in classi ica ion o building up coho may hinde signi ican esul s.
This makes he da a qui e di icul o in e p e . Hence, p edic i e ools o alida ion models migh be o
in e es o ea his da a.
Figu e 26. Sco e plo co esponding o PCA analysis o PCa and BPH u ine
samples. This PCA model was gene a ed wi h all he samples in o de o di e -
en ia e bo h g oups. In he axes, op wo mos ep esen a i e ea u es o dis in-
guish samples g oups a e plo ed. In pu ple iangles PCa samples and in g een
ci cle, BPH samples. Red a ows indica e he a iables ha explain a iabili y.
98
Figu e 27. No malised quan i ica ion o he ele an analy es de ec ed majo ly in he coho o u ine sam-
ples. The BPH and PCa le els o and os enedione, and os e one sulpha e, co isol and DHEAS a e epo ed as
well as he signi ican di e ences be ween he g oups.
3.2.2.3. App oaches o gi e an ou come
As u ine samples o his coho we e collec ed a i s isi , me abolomics da a ep esen s he s a us o
he pa ien a ha ime. This me abolomics da a is o use i i can disc imina e be ween pa ien g oups.
A logis ic eg ession is a widely used bina y classi ica ion model, which could p o ide a decision sys em
by gi ing a ma ix wi h da a as an inpu . This means ha a ma ix wi h s e oid ho mones da a, a mul i-
a ia e logis ic eg ession can be w i en o decide he ou pu in a bina y manne i.e. PCa o BPH (Eq.
4). Howe e , he decision can also be igge ed be ween PCa g oups. In his app oach, he idea was o
build up a model, a logis ic eg ession wi h diagnosis da a. Then, challenge he model wi h a numbe
o andom samples o es whe he i classi ies he samples success ully. Because no me aboli e was
di e en ially p esen in PCa s BPH, no alid model could be buil . One could nei he apply i success-
ully o classi y PCa g oups.
F(x)= 𝛽0+ 𝛽1𝑥1+ 𝛽2𝑥2+ ⋯ + 𝛽𝑝𝑥𝑝
𝑤ℎ𝑒𝑟𝑒 𝑥 𝑎𝑟𝑒 𝑚𝑒𝑡𝑎𝑏𝑜𝑙𝑖𝑡𝑒𝑠 𝑝𝑎𝑟𝑡𝑖𝑐𝑖𝑝𝑎𝑡𝑖𝑛𝑔 𝑖𝑛 𝑡ℎ𝑒 𝑠𝑖𝑔𝑛𝑎𝑡𝑢𝑟𝑒
(4)
Such bina y app oaches a e no use ul in assessing he classi ica ion o se e al g oups using a
me abolic signa u e. Fo his eason, o he p edic ing model ound in li e a u e a e sugges ed: Gaussian
99
Naï e Bayes, Random Fo es , XGBoos , deep neu al ne wo k o suppo ec o machines. Howe e ,
he de elopmen o a p ope pipeline o e alua e a me abolic signa u e o clinical pu poses lays beyond
he scope o his hesis.
Un il diagnosis ( ia cylinde and/o su gical specimen) is comple ed, a couple o yea s could ha e
elapsed hence, he da a can also be conside ed longi udinal. The app oaches al eady desc ibed do no
conside longi udinal da a and i could also be an in e es ing app oach. One possibili y would be he
usage o a machine lea ning amewo k, which equi es a longi udinal collec ion o da a. This means
he analysis o a u ine sample is equi ed in each ime poin o de e mine he a ia ion o signa u e. The
R package ‘Me abolicSu ’ iden i ies bioma ke signa u es by disco e ing p edic i e me aboli es o clas-
si y he expec ed p og ess in o g oups. These app oaches a e in e es ing bu do no qui e i he da a
ga he ed in his clinical coho . One has o acknowledge his limi a ions and pe haps p opose o build
up a model wi h pa ien diagnosis ou pu s. In his manne , known me abolic signa u es lead o speci ic
ajec o ies o diagnosis o e ime. Hence, a p edic i e model can calcula e p obabili ies o speci ic
ou comes. Ne e heless, one is hypo hesising and p oposing he e a possible ollow up o an in e es ing
esea ch ield o compu ing sciences.
3.2.3. Conclusions
In his sec ion, he me abolomics assay has been u he desc ibed and e alua ed o u ine samples. I
has been u he u ilised wi h clinical pu poses o es whe he p e ious esul s can be ep oduced. One
can conclude:
i. S e oid ho mones can be measu ed and quan i ied in u ines om pa ien s. The e iciencies o
hei eco e ies a e accep able o his ype o analysis.
ii. Signi ican di e ences ha e been epo ed be ween PCa g oups and, be ween BPH and PCa
g oups in co isol, and os enedione and DHEAS le els. Un o una ely, no s e oid ho mone sig-
na u e could dis inguish be ween BPH and PCa u ine samples. This sugges s ha a co ec
e alua ion and classi ica ion o he sample is also equi ed o ind ou bioma ke s and compa e
s udies.
iii. No model could ha e been success ully buil up wi h he p o ided da a. One has sugges ed
ollow up oppo uni ies; howe e , o con inue wi h his ype o esea ch was beyond he scope
o his hesis.
101
Chap e 4. T ans e ence o Biological Componen s and
Func ionali ies D i en by Ex acellula Vesicles
This chap e ocusses in desc ibing wo app oaches o assess he ans e o me aboli es o unc-
ions o ecipien cells by means o EVs. The assay desc ibed in Chap e 3.1 has been u he de eloped
o ace labelled me aboli es in ecipien cells and hence, de e mine whe he EVs may pa icipa e in
eeding me abolic pa hways impo an in PCa ea ly p og ession. This wo k has been compiled in a
manusc ip , which is unde a submission p ocedu e, and has no been accep ed ye . Chap e 4.1 in-
cludes he las e sion o he manusc ip . Fu he mo e, he ans e o oncogenic signalling d i en by
EVs has been explo ed. This app oach consis s in es ablishing a PCa model wi h a eadou esponsi e
o and ogen signalling. Thence, oncogenic ma ke s we e measu ed upon ea men wi h EVs. This pa
has been included in Chap e 4.2.
Bo danaba-Flo i , G., an Liempd, S., Cab e a, D. e al. Labelled-choles e ol demons a es e ec-
i e EV-media ed me aboli e ans e in a p os a e cance model. Unde submission.
4.1. Labelled Choles e ol Demons a es E ec i e EV-
Media ed Me aboli e T ans e in P os a e Cance
Lipids and speci ically choles e ol play a pi o al ole in p os a e cance p og ession. Choles e ol
se es as signal o p o umo igenic me abolic pa hways because i is he main p ecu so o s e oid
ho mone me abolism. Typically, he sou ce o choles e ol is ex acellula (apo)lipopa icles; howe e ,
ex acellula esicles could also p o ide choles e ol. The e ec i e ans e o ex acellula esicle-as-
socia ed me aboli es o ecipien cells has no been explo ed ye .
In his wo k, p os a e cance cell cul u es we e ea ed wi h labelled choles e ol in lipid-deple ed
media. Then, pu i ied ex acellula esicles we e ed o non-labelled ecipien cells o ace he in e nal-
isa ion o choles e ol by con ocal mic oscopy and i s u he me aboliza ion by a ge ed UPLC-MS as-
say. Choles e ol associa ed o ex acellula esicles was de ec ed in unlabelled ecipien cells. Upon
ea men wi h an inhibi o o choles e ol a icking, an accumula ion o choles e ol was obse ed bu
he egula anspo o mi ochond ial compa men was no ully impai ed. Mo eo e , we show s able
iso ope labelling in p os a e cance cells, which p oduce labelled ex acellula esicles. Upon ea men
wi h hese esicles, s able iso ope was de ec ed in p os a e ecipien cells.
In summa y, we epo an op imized and apid UPLC-MS assay o de ec ion o s e oid- ela ed me-
aboli es om cells and ex acellula esicles. Fu he mo e, we demons a ed ha a a ge ed me abo-
lomics assay combined o a molecula biology app oach is use ul o s udy ex acellula esicle-media ed
in e nalisa ion and he e ec i e ans e o ele an me aboli es as choles e ol.
108
TopF up ake. No ably, labelled s e oid- ela ed ho mones we e no de ec ed a e 24 hou incuba ion
wi h D6-choles e ol in hese p os a e cance cells.
Figu e 29. Choles e ol up ake by ecipien LNCaP and hNAF cells when labelled choles e ol is supple-
men ed in LPDS media. (A) Con ocal images o bo h ecipien cells upon ea men wi h 1 µM TopF (in he le )
and he no malised luo escence quan i ica ion o TopF signal o he basal signal de ec ed in ecipien cells no
ea ed wi h TopF (in he igh ). In con ocal panel, om le o igh he columns show: DAPI s aining o nuclei (in
blue channel), TopF signal (in g een channel) and he esul o me ging he wo channels. (B) Rela i e quan i ica-
ion o choles e ol iso opes in LNCaP and hNAF ecipien cells (n=6) upon ea men wi h choles e ol supplemen ed
media, D6-choles e ol supplemen ed media and, D6-choles e ol and U18666A inhibi o supplemen ed media.
Nex , EVs we e isola ed and pu i ied a e incuba ing he p oducing LNCaP and hCAF cells wi h 1
µM BODIPY-choles e ol (TopF) o 70 µM D6-choles e ol (Scheme o p oduc ion in Figu e 31A). EV
p epa a ions we e cha ac e ised and quan i ied o use hem in ollowing ea men s. As an example, in
Figu e 30 we compiled he ypical cha ac e isa ion expe imen s done in any EV p oduc ion. Figu e 30B
shows ha EV ac ions in bo h p oducing cells a e he i s ou ac ions o he SEC p o ocol used in
his wo k acco ding o ypical EV ma ke s. Also, in he i s ac ions i shows no p esence o ApoB100,
a ma ke o VLDL apolipopa icles. The bulk o soluble p o eins a e elu ed in he la e ac ions o he
SEC pu i ica ion (Figu e 30A and B). Impo an ly, choles e ol con en is mo e p ominen in he i s
109
ac ions wi h an app oxima ely 2- o 3- old highe concen a ion o choles e ol in LNCaP-de i ed EVs
compa ed o hCAF EVs (Figu e 30A). EV ac ions we e u he pooled oge he and quan i ied by NTA
p io ea ing ecipien cells. NTA shows ha pa icle popula ions con ained in EV ac ions a e homo-
geneous since hey exhibi a simila dis ibu ion o pa icle diame e (Mode o 157 nm in LNCaP and
177 nm in hCAF) wi h a unique peak (Figu e 30C). The quan i ica ion was used in he ollowing sec ion
o ea ecipien cells wi h a simila numbe o EVs.
Figu e 30. Cha ac e isa ion o choles e ol-con aining EVs (EV p epa a ions used in Figu e 32) in ou p o-
duc ion app oach and expe imen s. (A) Choles e ol con en and p o ein quan i ica ion o he 13 ac ions ob-
ained by SEC sepa a ion o condi ioned media in p oducing LNCaP (in blue) and hCAF (in pu ple) cells. (B) Pon-
ceau and Wes e n blo ing esul s o CD9, CD63 and ApoB an ibodies in he i s 12 ac ions ob ained by SEC
sepa a ion o condi ioned media. (C) Nanopa icle acking p o ile o he pooled i s ou ac ions ob ained in SEC
(EV-en iched ac ions). In blue, he modes in nm o pa icle diame e popula ions a e indica ed.
110
4.1.3.2. Ex acellula esicles can ans e choles e ol o ecipien cells.
Once p oducing cells we e labelled and hei eleased EVs pu i ied, we ob ained choles e ol-labelled
( luo escen ly o iso opically) EVs eady o ea ecipien cells (Figu e 31A). By UPLC-MS we we e able
o iden i y and quan i y choles e ol and D6-choles e ol upon ea men wi h EV p epa a ions.
Figu e 31. T ans e o iso ope labelled choles e ol media ed by EVs using a UPLC-MS assay o de ec ion.
(A) Schema ic o e iew o he p oduc ion and pu i ica ion o EVs om choles e ol-labelled LNCaP and hCAF cells
ha a e used o ea ecipien LNCaP and hNAF cells. (B) D6-choles e ol de ec ed in ecipien LNCaP cell as a
pe cen age o o al de ec ed choles e ol upon ea men wi h: EVs p oduced in choles e ol supplemen ed media
(g ey), EVs p oduced in D6-choles e ol supplemen ed media (ligh g een) o EVs p oduced in D6-choles e ol sup-
plemen ed media and U18666A supplemen a ion (da k g een). EVs om p oducing LNCaP and hCAF cells we e
used in his expe imen . (C) D6-choles e ol de ec ed in ecipien LNCaP and hNAF cells as a pe cen age o o al
de ec ed choles e ol upon ea men wi h: EVs p oduced in choles e ol supplemen ed media (g ey), EVs p oduced
in D6-choles e ol supplemen ed media (ligh g een) o EVs p oduced in D6-choles e ol supplemen ed media and
U18666A supplemen a ion(da k g een). Only EVs p oduced by LNCaP cells we e used in his expe imen .
In Figu e 31BC, we show he esul s o wo independen expe imen s in which ecipien cells we e
ea ed wi h EVs ca ying ei he choles e ol o D6-choles e ol. Recipien LNCaP cells incuba ed wi h
simila numbe o EVs p oduced by choles e ol and D6-choles e ol ea ed hCAF o LNCaP e ained
D6-choles e ol associa ed o EVs (Figu e 31B). I is obse ed ha D6-choles e ol associa ed o EVs was
111
up ake by ecipien hNAF and LNCaP cells in minu e ela i e amoun s, ep esen ing less han 0.1% o
o al choles e ol in ecipien cells. Impo an ly, D6-choles e ol up ake was consis en in bo h expe imen s
and speci ic o labelled EVs because he e was no ace o he iso ope in ecipien cells ea ed wi h
choles e ol-EVs. Figu e 31B desc ibes a highe up ake o D6-choles e ol by ecipien LNCaP cells when
ea ed wi h LNCaP-de i ed EVs a he han hCAF-de i ed EVs. Simila ly, when ea ing ecipien hNAF
and LNCaP cells wi h app oxima ely hal he numbe o LNCaP-de i ed EVs (Figu e 31C) compa ed o
Figu e 31B, hNAF showed a highe up ake o choles e ol. To no e, he obse ed ela i e quan i ica ions
a e e y small so esul s may be highly a iable om e e y o he expe imen . Ul ima ely, one can con-
clude D6-choles e ol associa ed o EVs was ans e ed o (non-labelled) ecipien cells.
4.1.3.3. Choles e ol associa ed o ex acellula esicles is anspo ed o mi-
ochond ial compa men .
T acing labelled me aboli es demons a ed he abili y o EVs o ans e hei associa ed choles e ol
o he ecipien cells LNCaP and hNAF. Howe e , such a bulk ensemble app oach canno image he
up ake o EVs o di e en cellula loca ions. To image he up ake o TopF associa ed o EVs by ecipien
LNCaP and hNAF cells, we used a dual agging wo k low o load EVs om p oducing cells wi h TopF
and label hem wi h he memb ane dye MemB igh 472. Then, unlabelled ecipien cells unde wen di -
e en ea men s while ed wi h hese labelled EV p epa a ions.
We i s con i med he up ake o choles e ol was associa ed o EVs by moni o ing he choles e ol as
TopF signal and EVs associa ed o MemB igh signal (Figu e 35). Recipien LNCaP ea ed wi h dual
agged EVs showed pa ial colocalisa ion o bo h signals (Figu e 35A) while he ea men wi h ee
choles e ol ollowed by a MemB igh s aining o cells did no show a simila colocalisa ion pheno ype
(Figu e 32B). Co- acking o choles e ol and EV-associa ed MemB igh in simila compa men s sug-
ges s choles e ol signal is associa ed o EVs.
Then, we ea ed ecipien cells wi h he NPC1 inhibi o U18666A, which hampe s in acellula a -
icking o choles e ol in endosomal compa men 473. The ea men did no a ec ecipien LNCaP up-
ake o choles e ol (Figu e 32B) bu i induced an accumula ion pheno ype o choles e ol no obse ed
in absence o he inhibi o (Figu e 32A). Mi oT acke s aining and colocalisa ion analysis con i med ha
he in acellula anspo o choles e ol was sligh ly impai ed (Figu e 32C) sugges ing choles e ol om
EVs is anspo ed by he endocy ic pa hway o o he cellula compa men s. Impo an ly, choles e ol
shows a high deg ee o colocalisa ion in mi ochond ial compa men (Figu e 32C), anging om 50% o
70% app oxima ely and depending on he image analysis app oach, deno ing a high p esence o cho-
les e ol in mi ochond ial memb anes. The ac ha a well cha ac e ised choles e ol a icking inhibi o
as U18666A did no d ama ically hampe ed choles e ol colocalisa ion o mi ochond ia (s ill up o 50%)
indica es ha he e migh be o he pa hways in ecipien LNCaP o e aking he anspo o choles e ol
o make i a ailable in mi ochond ia.
112
Iden ical expe imen s in ecipien hNAF - p os a ic s oma ib oblas – did no show a simila accu-
mula ion pheno ype by ea ing wi h U18666A inhibi o (Figu e 33A). Co- acking o TopF and Mi o-
T acke con i ms ha he anspo owa ds mi ochond ial compa men was no signi ican ly impai ed
(Figu e 33C). None heless, he ea men a ec ed choles e ol up ake om EVs (Figu e 33B).
Figu e 32. EV-associa ed choles e ol is anspo ed o mi ochond ial compa men in LNCaP ecipien
cells. (A) Con ocal images o LNCaP ecipien cells upon ea men wi h LNCaP EVs (Mock), LNCaP-TopF EVs
(BODIPY-choles e ol loaded EVs) and LNCaP-TopF EVs and U18866A inhibi o . All ea men s we e incuba ed
wi h Mi oT acke . F om le o igh he columns show: DAPI s aining o nuclei (in blue channel), TopF signal (in
g een channel), Mi o acke signal (in ed channel) and he esul o me ging he h ee channels. (B) No malised
luo escence quan i ica ion o TopF signal o he basal signal de ec ed in Mock. (C) Co- acking quan i ica ion o
TopF signal o Mi oT acke signal using a co ec ed Mande ’s coe icien and objec -based colocalisa ion.
113
Figu e 33. EV-associa ed choles e ol is anspo ed o mi ochond ial compa men in hNAF ecipien cells.
(A) Con ocal images o hNAF ecipien cells upon ea men wi h LNCaP EVs (Mock), LNCaP-TopF EVs (BODIPY-
choles e ol loaded EVs) and LNCaP-TopF EVs and U18866A inhibi o . All ea men s we e incuba ed wi h Mi o-
T acke . F om le o igh he columns show: DAPI s aining o nuclei (in blue channel), TopF signal (in g een
channel), Mi o acke signal (in ed channel) and he esul o me ging he h ee channels. (B) No malised luo es-
cence quan i ica ion o TopF signal o he basal signal de ec ed in Mock. (C) Co- acking quan i ica ion o TopF
signal o Mi oT acke signal using a co ec ed Mande ’s coe icien and objec -based colocalisa ion.
In line wi h desc ibing he ans e o me aboli es associa ed o EVs om PCa models o o he PCa
models, we pe o med expe imen s wi h a simila layou bu using p oducing LNCaP and hCAF cells.
Bo h EV p epa a ions we e gi en as a ea men – wi h simila numbe o EVs – o ecipien LNCaP
cells. In Figu e 34A one can obse e he ans e o choles e ol associa ed o EVs, showing a simila
pheno ype e en wi h di e en EV p epa a ions. Mo eo e , co- acking o TopF and Mi oT acke shows
no signi ican quan i a i e di e ences in he localisa ion o choles e ol in he mi ochond ial compa men
114
(Figu e 34C). In e es ingly, he ea men wi h di e en EV p epa a ions a ec ed signi ican ly he up ake
o choles e ol by ecipien LNCaP cells. Up ake a ia ions a e simila o he di e ences in choles e ol
con en desc ibed in Figu e 29B, he e o e, he ac ha EV-associa ed ac ions om LNCaP ca y
h ee imes mo e choles e ol han hCAF EVs (Figu e 30A.) may explain hese up ake di e ences. This
suppo s he idea ha he ans e ed choles e ol is associa ed o EVs since up ake quan i ica ions a e
dose dependen on EV p epa a ions.
Figu e 34. EV-associa ed choles e ol om LNCaP and hCAF p oducing cells is anspo ed o mi ochon-
d ial compa men in LNCaP ecipien cells. (A) Con ocal images o LNCaP ecipien cells upon ea men wi h
LPDS media (Mock), LNCaP-TopF EVs (BODIPY-choles e ol EVs) and hCAF-TopF EVs. All ea men s we e in-
cuba ed wi h Mi oT acke . F om le o igh he columns show: DAPI s aining o nuclei (in blue channel), TopF
signal (in g een channel), Mi o acke signal (in ed channel) and he esul o me ging he h ee channels. (B)
No malised luo escence quan i ica ion o TopF signal o he basal signal de ec ed in Mock. (C) Co- acking quan-
i ica ion o TopF signal o Mi oT acke signal using a co ec ed Mande ’s coe icien and objec -based colocalisa-
ion.
115
Figu e 35. LNCaP ecipien cells up ake TopF and MemB igh signals associa ed o EVs. (A) Con ocal im-
ages o LNCaP ecipien cells upon ea men wi h TopF supplemen ed media and no MemB igh , LNCaP-TopF
EVs labelled wi h MemB igh o LNCaP-TopF EVs labelled wi h MemB igh and U18666A inhibi o . (B) Con ocal
images o LNCaP ecipien cells ea ed wi h TopF and MemB igh supplemen ed media and TopF, MemB igh and
U18666A inhibi o supplemen ed media. In con ocal panels, om le o igh he columns show: Mi o acke signal
(in ed channel), TopF signal (in g een channel) and he esul o me ging he wo channels.
116
4.1.4. Discussion
The ele ance o me abolomics app oaches beyond he disco e y o bioma ke s and he me abolic
p o iling o issues in di e en disease s ages is in he spo ligh . Many e o s ha e been done in de-
sc ibing me abolic di e ences be ween he epi helium and s oma in PCa bu also be ween hose is-
sues in di e en p og ession s ages o he disease. Fu he o ha , his wo k desc ibes a me abolomics
app oach o s udy he ans e o me aboli es in cell- o-cell communica ion e en s media ed by EVs.
The de ec ion o D6-choles e ol ecipien cells ea ed wi h EV p epa a ions con aining labelled choles-
e ol demons a ed he ans e o choles e ol be ween physiologically ele an cells in p os a e cance
disease.
On one hand, luo escen ly-labelled choles e ol showed i s anspo o mi ochond ia whe e i can
be u he me abolised in o p egnenolone, he i s p ecu so o p oduce o he s e oid ho mones. This
sugges s ha choles e ol ca ied by EVs can po en ially be me abolised in o o he s e oid ho mones
ele an o PCa p og ession. On he o he hand, iso ope labelled choles e ol associa ed o EVs was
up ake by ecipien hNAF and LNCaP cells. The ac only a small pe cen age o choles e ol was labelled
– less han 0.1% o o al choles e ol in ecipien cells – indica es he ea men wi h EVs was no su i-
cien . Fo me abolomics, we equi e en imes mo e cells pe ea men compa ed o luo escence as-
says due o ep oducibili y and, he p oduc ion o labelled EVs is limi ing. E en so, D6-choles e ol was
quan i ied consis en ly h ough expe imen s and i s de ec ion is speci ic o labelled EV p epa a ions
since i could no be de ec ed upon ea men wi h unlabelled EV p epa a ions. Besides he de ec ion
o labelled choles e ol in ecipien cells, i s usage o me abolise o he s e oid could no be demons a ed
ye .
In his s udy we showed he ans e ed choles e ol is ela ed o ex acellula ma e ial de i ed om
labelled (p oducing) cell cul u es. The esul s o co- agging EVs wi h TopF and MemB igh and, expe -
imen s using EV p epa a ions wi h di e en choles e ol con en con i med his hypo hesis. Fu he mo e,
expe imen s inhibi ing he anspo o choles e ol ia endocy ic pa hway s eng hened he hypo hesis
labelled choles e ol came om an ex e nal sou ce and i unde goes in e naliza ion owa ds lysosomes
p io dis ibu ion o o he compa men s. By using he well-known endosomal a icking inhibi o
U18666A, we would ha e expec ed an accumula ion o choles e ol in he endocy ic compa men and
he e o e, a d ama ic decay o choles e ol in he mi ochond ial compa men . E en hough colocalisa ion
expe imen s showed a dec ease o choles e ol up ake o a dec ease o choles e ol in mi ochond ial
compa men and, he cha ac e ising in acellula accumula ion in con ocal images, choles e ol was s ill
anspo ed o mi ochond ia. U18666A impai s NPC1 ac i i y, in cha ge o ansloca ing choles e ol cap-
u ed by NPC2 o esicula memb anes. Thus, his inding indica es ha ecipien cells may be using
o he pa hways di e en o NPC complex o o e ake choles e ol a icking owa ds mi ochond ial com-
pa men . Mi ochond ia a e known o no pa icipa e in esicula a icking as o he o ganelles and, i is
STARD3 he p o ein in cha ge o ansloca ing choles e ol om esicula o mi ochond ial compa men
upon gene a ion o con ac si es474,475. The e o e, o he p o eins han NPC1 and NPC2 migh be ans-
117
loca ing choles e ol owa ds endocy ic compa men memb anes p io anspo o mi ochond ia. Pe -
haps o he p o ein anspo e s such as LIMP2 and LAMP2 can make choles e ol a ailable o be ans-
po ed o mi ochond ia46.
Choles e ol is essen ially anspo ed and deli e ed by (apo)lipopa icles ia bloods eam. Lipids
(and choles e ol) a e p ocessed in he li e , he o gan in cha ge o manu ac u ing hese lipid-con aining
pa icles o u he deli e ing o o he issues. The e o e, one would expec ha only li e -de i ed mod-
els exhibi lipid ese oi s and majo ly expel lipid-con aining pa icles ins ead o EVs. Indeed, he main
limi a ion o his s udy is he possible p esence o lipid-con aining pa icles in he EV p epa a ions be-
cause EV isola ion echniques a e mainly based on size o p ecipi a ion pa ame e s and he ones based
on p o ein ma ke s o o he pa ame e s a e no well-s anda dised ye . Fo his eason, we pu i ied EVs
wi h SEC because i is cleane han o he echniques such as ul acen i uga ion and he main con am-
ina ion we could ind is o VLDL because hey ha e a simila size compa ed o EVs. None heless, he
sou ce o LNCaP, hCAF and hNAF a e human excised p os a e issues and hey ha e no been e-
po ed o p oduce any lipopa icles. Also, apolipop o eins cha ac e is ic o VLDL pa icles – ApoB100
and ApoE (da a no shown) - we e no ound in EV p epa a ions. The ac we obse e a deg ada ion
pa e n o ApoB100 in soluble p o ein ac ions by Wes e n blo ing indica es he cells a e no using
apolipop o eins o manu ac u e pa icles and a e elimina ing hem ins ead.
4.1.5. Conclusions
In conclusion, he UHPLC-MS assay de eloped in his wo k demons a ed o be sui able o acing
iso ope labelled me aboli es o he ho mone s e oids pa hway. We ha e been able o de ec D6-choles-
e ol in cell cul u es and hence, o p oduce EVs ca ying D6-choles e ol. Fu he mo e, EVs we e able
o ans e choles e ol o ecipien cells. Al hough his ans e was con i med, no p oduc ion o o he
s e oid ho mones could be epo ed using LNCaP and hNAF as e e ence cell lines.
In addi ion, ea men o ecipien cells wi h EVs labelled wi h luo escen choles e ol demons a ed
he in e nalisa ion o choles e ol o cellula compa men s. Also, he quan i ica ion o TopF was dose
dependen upon choles e ol concen a ion in EV p epa a ions and; he quan i ica ion o D6-choles e ol
in ecipien cells dec eased wi h a ea men o a mino numbe o EVs pe cell. This sugges s ha EV
p epa a ions a e he sou ce o D6-choles e ol de ec ed in ecipien cells. Finally, co- acing choles e ol
and Mi oT acke showed EV-associa ed choles e ol is anspo ed pa ially o mi ochond ial compa -
men .
124
mRNA and i usually leads o a e y high a iabili y because o he loss in sensi i i y. Ac ually, he e o
ba s a e no included in he g aph because hey a e la ge and lay beyond he g aph ange, which
makes meaningless any analysis The e o e, he assay would no con iden ly p o ide an ou pu o he
and ogen deple ion ea men .
Oncogenici y ma ke s
Al hough KLK3 is desc ibed in li e a u e as he majo ma ke o oncogenici y in and ogen-dependen
p og ession476, o he ma ke s we e also es ed in LNCaP model o s eng hen he e alua ion o he
oncogenic pheno ype. The selec ed genes ha e been p esen ed in documen ed epo s as bioma ke s
in PCa diagnos ic es s: SPDEF478, HOXC6477, DLX1477 and PCA3439. SPDEF is a ansac i a o o PSA
p omo e and is exp essed in men wi h ad anced PCa. HOXC6 is a homebox gene in ol ed in o gan
de elopmen and cell p oli e a ion. PCA3 is long non-coding RNA highly o e exp essed in PCa cells.
DLX1 may unc ion as a ansc ip ional egula o o mul iple TGF supe amily membe s.
Figu e 39. Fold change exp ession o a ge genes (mean ± SD;
n=3) o e ime (in days) in ecipien LNCaP cell line. Ta ge gene
exp ession is calcula ed as 2-ΔΔC compa ed o exp ession a day 0.
ACTB and GAPDH exp ession le els we e conside ed o
no malisa ion. In da k ed, ea men wi h and ogen-con aining media
and, in ligh ed, and ogen deple ed media. Solid lines do no ep esen
expe imen al da a, hey a e added jus as a guide o he eye.
Using a simila app oach, Figu e 38 ep esen s he old change o e ime o 5 di e en po en ial
oncogenic ma ke s no malised wi h 3 HKG upon and ogen signalling deple ion. Ta ge SPDEF, DLX1
and PCA3 gene exp essions we e no esponsi e o and ogen deple ion. One could expec his esul
because hose ansc ip s we e desc ibed in highly g ade PCa, usually and ogen independen . How-
e e , HOXC6 appea ed as an adequa e ma ke o oncogenici y when no malised wi h ACTB and
18s RNA exp ession. No e o ba s a e shown un il now because expe imen s we e no ye pe o med
in biological iplica es, only echnical iplica es we e included.
125
Acco ding o he esul s, LNCaP was a sui able model o ollow and ogen-dependen oncogenici y
and, he ansc ip s KLK3 and HOXC6 he oncogenic ma ke s esponding o his ho mone signalling.
Thence, a old change exp ession was analysed o e ime including app op ia e biological eplica es
and he wo bes no malise s o p e ious expe imen s in e ms o ep oducibili y and s abili y (Figu e
39). This expe imen con i med KLK3 exp ession was dependen on and ogen signalling, howe e ;
HOXC6 exp ession was a he a iable and, al hough i s exp ession o e ime displays a simila en-
dency as in Figu e 38, he po en ial esponse o and ogen signalling was no signi ican . The e o e,
LNCaP was selec ed as he cell model o be ea ed wi h EVs and KLK3 exp ession he ma ke o ollow
oncogenici y o e ime.
EV-media ed ans e o oncogenici y
Figu e 40. Summa y o oncogenic ma ke s o e ime (in days) unde p esence o absence o s e oid
ho mones and upon ea men wi h EV p epa a ions. A. Fold change exp ession o a ge KLK3 gene (mean ±
SD; n=3) o e ime (in days) in ecipien LNCaP cell line. Ta ge gene exp ession is calcula ed as 2-ΔΔC compa ed
o exp ession a day 0. ACTB and GAPDH exp ession le els we e conside ed o no malisa ion. B. Posi i e e en s
o nuclea KI-67 (%) o e ime (in days) in ecipien LNCaP cell line. Posi i e e en s we e coun ed using a ci ome e .
The colou s co espond o di e en ea men s as indica ed in he legend. Addi ion o deple ion o and ogens a e
depic ed in da k o ligh ed (+/- DHT); BPH-1 de i ed EVs (C l EVs) in g een, LNCaP de i ed EVs (AD EVs) in
da k o ligh blue and, in yellow, PC-3 de i ed EVs (AI EVs).
Once he model was e alua ed, i was challenged wi h EV ea men s in absence o and ogens. EVs
we e isola ed om h ee ecognised PCa models ha we e selec ed acco ding o hei s a us and e-
126
sponse o and ogen signalling. Con ol (C l) s ands o he non- umo al cell line BPH-1, and ogen de-
penden (AD) LNCaP line equi es and ogen signalling o sus ain umo al g ow h and and ogen inde-
penden (AI) PC-3 uses non-and ogen signalling o p omo e ou g ow h. In o de o gene a e EV sam-
ples, cells we e cul u ed in comple e media and LNCaP was also s imula ed wi h 10 mM DHT. P o ein
i e o EV p epa a ions was simila in all ea men s and hose we e calcula ed as 3 p oducing cells
p o iding EVs o 1 ecipien cell.
Exp ession o KLK3 (as old change) was calcula ed using di e en no malise s o e ime (Figu e
40). As in p e ious expe imen s, a highe old change o KLK3 desc ibes oncogenic g ow h o he cell
cul u e. In Figu e 40, he old change o KLK3 using wo di e en pai s o p ime s and wo di e en
no malise s a days 3, 6 and 9 is ep esen ed. Simila o p e ious sec ions, LNCaP esponded wi h a
dec ease in oncogenic g ow h upon deple ion o and ogens and, when and ogens we e supplied, on-
cogenic ma ke s inc eased hei ansc ip ion le els. This con i ms an app op ia e pe o mance o he
model o u he analyse EV ea men s depic ed in he same igu e. In gene al, he impac o EV ea -
men s in KLK3 exp ession is a he low; and in some cases, i is negligible. E en hough, he ea men
wi h AI-de i ed EVs ends o show a lowe KLK3 exp ession compa ed o AD-de i ed EVs he di e -
ences a e no qui e ele an . Mo eo e , he exp ession o KLK3 inc eased in LNCaP cells g owing in
and ogen-deple ed media a ea men days 6 and 9. This inding sugges s ha ea men s wi h EVs
ha e no long- e m e ec in sus aining oncogenic g ow h. Undoub edly, he layou o his expe imen
does no ep esen a eliable pic u e o a physiological con ex . EVs a e con inuously eleased by p o-
ducing cells and so hey a e p o ided o ecipien cells. Pe haps, a con inuous supply o EVs would
esul in an oncogenic pheno ype sus ained o e ime. The limi a ion u ns in o echnical because EV
p oduc ion is highly a iable, and i equi es ime and a high olume o condi ioned media o ou line
such app oach.
None heless, Figu e 40 depic ed a ele an e ec o EV ea men s a day 3 (sho - e m). Al hough
EV ea men s canno mimic esul s obse ed wi h and ogen-supplemen ed media, he d ama ic de-
c ease in KLK3 exp ession is sligh ly neu alised. While and ogen deple ion dec eases i s exp ession
by app oxima ely 75%, EV ea men s neu alise his decay o 50%. In o he wo ds, he old change
exp ession o KLK3 a day 3 is app oxima ely 2- o 2.5- old he exp ession in and ogen-deple ed media.
As al eady sugges ed, he supply o EVs o e ime is ele an o assess whe he hey can suppo he
oncogenic g ow h obse ed in p e ious expe imen s wi h and ogen ligands in he media. Howe e , EV
samples and hei p oduc ion is highly a iable and so a e EV ea men s; hence, one would expec a
high a iabili y in he oncogenic ma ke exp ession caused by he ea men s in ecipien LNCaP cells.
An EV mig a ion assay, using T answell® mig a ion chambe s o 400 nm po e size, is o in e es o
sol e issues in EV p oduc ion and o assess his unc ional ans e in a mo e physiological app oach.
127
4.2.4. Conclusions
In he p e ious sec ion o he chap e , he ans e o me aboli es con ained in EVs has been e alua ed.
In he p esen sec ion, he ans e o biologically ele an unc ionali ies media ed by EVs is shown.
The main pu pose was o es ablish a unc ional in e ac ion o EVs wi h ecipien cells and, s udy he
capabili y o EVs o sus ain and ogen-dependen oncogenici y. Since he ac ual composi ion o EVs
d i ing oncogenic pheno ype ha e no been e alua ed, a mechanis ic explana ion is missing. Howe e ,
one can conclude ha :
i. LNCaP is an app op ia e model o s udy and ogen-dependen signalling in PCa. Mo eo e , i
was he only cell line wi h oncogenic ma ke s esponding o and ogen ea men s; acco ding o
he oncogenic ma ke s es ed, 22R 1 and BPH-1 did no show and ogen-dependency o sus-
ain g ow h.
ii. No all he oncogenic epo ed genes we e app op ia e ma ke s o in o m abou and ogen de-
penden p og ession. Measu ing he exp ession o he genes SPDEF, DXL1 and PCA3 could
no in o m abou oncogenic g ow h o PCa cells. The exp ession o ansc ip s HOXC6 and
KLK3 was al e ed upon and ogen deple ion, sugges ing hem as ma ke s o oncogenici y. How-
e e , HOXC6 was a he a iable and hence, only KLK3 was u ilised in his wo k.
iii. EV ea men p o oked an e ec ega dless he ype o p oducing cells om whe e hey we e
isola ed. A day 3, he old change exp ession o KLK3 was app oxima ely 2- o 2.5- old he
exp ession in and ogen-deple ed media.
i . EVs showed a neu alisa ion in he loss o oncogenic pheno ype. Howe e , a one sho ea -
men wi h EVs could no sus ain his oncogenic pheno ype o e ime.
129
Chap e 5. Me abolic Al e a ions o No mal and Cance -
Associa ed Fib oblas s om Human S oma Samples
This chap e ocusses in s udying he in e ac ion be ween he wo main compa men s o p os a e.
The me abolic ewi ing o s omal-de i ed ib oblas s om a no mal egion o he p os a e compa ed o
a egion wi h he p esence o a umou was e alua ed. I is well-known ha epi helium in e ac s wi h
s oma and ice e sa. Documen ed epo s show EVs a e one o he playe s o his communica ion
d i ing di e en ia ion o ib oblas s o cance -associ ed ib oblas s. In his chap e , we also discuss me -
abolic al e a ions in no mal ib oblas s caused by PCa-de i ed EVs. The wo k has been published as
an O iginal A icle in Biochimica e Biophysica Ac a (BBA) - Molecula Basis o Disease Jou nal and i
is appended in he supplemen a y ma e ial. The o iginal manusc ip u ilised in he publica ion is included
and o ma ed in his sec ion.
Bo danaba-Flo i , G., Royo, F., Albóniga, O.E. e al. In eg a ion o p o eomics and me abolomics e-
eals me abolic al e a ions o p os a e cance ib oblas s om pa ien ’s s oma samples. Biochim. Bio-
phys. Ac a. Mol. Basis. Dis. Unde e ision
Figu e 41. G aphical abs ac o Chap e 5. Me abolic Al e a ions o No mal and Cance - Associa ed
Fib oblas s om Human S oma Samples. Ma ched-needle biopsies om PCa pa ien s we e ob ained and
associa ed ib oblas s we e ob ained. Me abolomics analysis was pe o med o de e mine he dis inc me abolic
p o ile o no mal and umou ib oblas s. Seaho se analysis was u ilised o de e mine any cen al me abolism
changes ela ed o TGF-β and EVs.
130
The p os a e gland is a complex and he e ogeneous o gan composed o epi helium and s oma.
P os a e cance is mos commonly seen eme ging om luminal epi helial cells and equi es he assis-
ance o adjacen s oma. P os a ic s oma is composi ionally complex, and in conce wi h disease
p og ession unde goes many al e a ions which include he eme gence o cance associa ed ib oblas s
(CAFs). This he e ogeneous cell popula ion o en con ains cells wi h a myo ib oblas -like pheno ype
ha a e no no mally p esen in heal hy p os a e issue. In his wo k, we s udied he me abolic ewi ing
o s omal ib oblas s ollowing myo ib oblas di e en ia ion. Fi s , he me abolic abundances o no mal-
and cance -associa ed ib oblas s de i ed om needle biopsies o he same pa ien was analysed using
UPLC-MS. I was de e mined CAFs we e me abolically mo e ac i e and, he e o e, ene gy p oducing
me abolic pa hways we e enhanced. Also, CAFs showed a heigh ened lipogenic me abolism as bo h
ese oi species and building block compounds. In e es ingly, lipid me abolism a ec s mi ochond ia
unc ioning ye he mechanisms o lipid-media ed unc ions a e unclea . The ac oxidised a y acids
and glu a hione sys em a e ele a ed in CAFs s eng hens he hypo hesis ha inc eased me abolic ac-
i i y is ela ed o mi ochond ia. In u he expe imen s measu ing he me abolic lux wi h a Seaho se
bioanalyse , we s udied whe he TGF-β1 and ex acellula esicles (EVs) could s imula e myo ib oblas
di e en ia ion in no mal ib oblas s. An inc ease o basal espi a ion in no mal ib oblas s was epo ed,
mi o ing he disease-like pheno ype. This indica es an al e ed me abolism associa ed o mi ochond ia
in CAFs and ea ed ib oblas s. Hence, one p oposes ha he change in he me abolomics p o ile o
umou -associa ed s omal ib oblas s is d i en by oxygen-dependen me abolism, possibly associa ed
o mi ochond ia; howe e , he speci ic mechanisms a e s ill unclea .
5.1. In oduc ion
The p os a e gland is globally composed o epi helium and s oma, which a e ex emely he e oge-
neous issues. The epi helium is o ganized as glandula acini and i con ains cuboidal o columna se-
c e o y epi helial cells wi h apical junc ion complexes, a con inuous laye o basal cells10, and spa se
neu oendoc ine cells, each a ached o a basal lamina9. Beyond he basal lamina, a p ominen ib o-
muscula s oma composed o smoo h muscle, ib oblas s, blood essels, au onomic ne e ib es, in-
lamma o y cells, and ex acellula ma ix componen s o e s physical suppo and con ac ion o he
gland479. In p os a e ca cinoma (PCa), he in e s i ial s oma is o en abno mally ich in myo ib oblas ic
cells467,468, capable o suppo ing umou g ow h, ascula iza ion, angiogenesis, and me as asis in
i o115. T ans o ming g ow h ac o β1, TGF-β1, emains among he mos c i ical ac o s o myo ib o-
blas ic di e en ia ion and he gene a ion o a umou eac i e s oma. In iguingly, we ha e p e iously
shown ha cance ex acellula esicles (EVs) can igge ib oblas o myo ib oblas di e en ia ion in
an EV-TGF-β1 dependen manne 115. In u he s udies, he essen ial ole o EVs in di ec ing his s omal
cell di e en ia ion owa ds cance -associa ed myo ib oblas -like pheno ype was desc ibed116. Howe e ,
he mechanisms by which EV-ac i a ed s omal cells suppo umou g ow h emain unclea .
Ou p e ious s udies wi h biopsy ma e ial om pa ien s whe e umo al g ow h was loca ed in one
hal o he p os a e and no he o he , e ealed clea di e ences be ween no mal and disease egions.
His ological examina ion showed a smoo h muscle s omal a chi ec u e a ound glandula s uc u es in
131
no mal issue while he glands we e diso ganised and showed an al e ed, ib osis-like in e s i ial s oma
obse ed in disease-associa ed issue480. A panel o an ibody ma ke s con i med he highe abundance
o α-Smoo h Muscle Ac in (α-SMA)-posi i e myo ib oblas cells in umou -associa ed issue. S ikingly,
EVs isola ed om p os a e cance epi helial cell lines gene a ed a myo ib oblas -like pheno ype in no -
mal ib oblas s116. In ha wo k, we demons a ed he essen ial ole o EVs in di ec ing s omal di e en-
ia ion o a p o- umo igenic pheno ype, exhibi ing p o-angiogenic p ope ies and enhancemen o u-
mou g ow h in xenog a models. Fu he unc ional assays and p o eomics p o iling wo k highligh ed
ha s oma ac i a ion media ed by EV s imula ion mi o s he na u ally occu ing ib oblas di e en ia-
ion du ing disease480.
Al e ed me abolism is a hallma k o PCa and se e al me aboli es and me abolic pa hways a e al-
eady dis inc i e in di e en p os a e ypes o issue481,482. In his line, Ande sen e al. epo ed highe
le els o ene gy- ela ed pa hway me aboli es, such as ADP, ATP, and glucose as well as highe le els
o he an ioxidan au ine in s omal issue compa ed o cance and non-cance epi helium483. Besides,
inc eased le els o c ucial me aboli es o a y acid oxida ion and building blocks in lipid syn hesis we e
desc ibed in cance issue. O he s udies also epo ed me abolic ewi ing o eac i e s oma, showing
di e en le els o ce ain me aboli es be ween highly di e en ia ed s oma compa ed o poo ly di e en-
ia ed s oma484. The s omal-epi helial in e ac ions ha e a dominan ole in umou g ow h, in asion
and me as asis. Ac ually, many epo s o e he las decades showed he in e ac ion o eac i e s oma
wi h PCa9,469,484–488 and EVs in luencing aspec s o cance biology such as angiogenesis116,480,489,490 and
umou p og ession488,491–493. Ye , ew s udies ha e in es iga ed he ole o umou EVs in al e ing me -
abolic p ocesses in s omal cell compa men s.
Wi h he ise o omics e a, en i e se s o biomolecules – genes, p o eins o me aboli es - con ained
in a biological issue, cell, luid, o o ganism can be iden i ied. A p o eomics analysis o he same
ma ched- no mal and disease s oma issues was able o dis inguish bo h pheno ypes and desc ibe a
disease-like pheno ype480. Simila ly, a ansc ip omic analysis o de i ed EVs om he same ype o
samples disc imina ed no mal om disease s oma samples494. This manusc ip p esen s a b oad sem-
i a ge ed me abolomics app oach, which analyses he me abolome o no mal and disease s oma is-
sues, highligh ing he me abolic di e ences o ma ched no mal ib oblas s and cance -associa ed ib o-
blas s wi hin indi idual PCa pa ien s. Fu he mo e, we show an al e ed me abolism o no mal ib oblas s
ea ed wi h EVs om a PCa cell line.
5.2. Ma e ial and me hods
5.2.1. S omal p ima y cell cul u es.
Six pa ien -ma ched no mal and umou -associa ed needle biopsies we e isola ed om adical p os-
a ec omy. These we e aken om si es o palpable disease and also om appa en ly no mal issue
om he opposi e side o he same p os a e. Tissue collec ion and consen ing was managed h ough
he Wales Cance Bank. Co es we e manually dissec ed in o 1mm3 pieces and subjec ed o mechanical
homogeniza ion ollowed by 200 U pe mL collagenase-I diges ion o 15 o 20 hou s a 37 °C. Cells
132
we e cul u ed in S omal Cell Basal Medium (SCBM) supplemen ed wi h human ib oblas g ow h ac o -
B, insulin, e al bo ine se um (FBS) and GA-1000 (Lonza, Wokingham, UK). o a ound wo weeks un il
only s omal cells we e e ained. 8,23Subsequen cul u es we e main ained in DMEM/F12 media (Lonza)
wi h 10% FBS deple ed o bo ine EVs. Cul u es we e con i med ee o epi helial cells by immuno-
luo escence s aining o cy oke a in p io seeding s omal cells in 96 well pla es 480,494. A sample o each
pa ien -ma ched p os a ic s omal cell cul u e was collec ed and ozen o me abolomics analysis using
liquid ch oma og aphy coupled o mass spec ome y.
5.2.2. Me aboli e ex ac ion.
In me abolomics, he e is no single pla o m o me hod able o analyse he en i e me abolome o a
biological sample. The e o e, me aboli es we e ex ac ed by ac iona ing he cell samples in o pools o
species wi h simila physicochemical p ope ies. In b ie , p o eins we e p ecipi a ed by adding me hanol
o he cell lysa e. Chlo o o m sol en was added o he me hanol ex ac ion mix u e and his biphasic
mix u e was incuba ed a -20 ºC o 30 min. Then, h ee di e en ac ions we e collec ed: (1) a y acyls,
bile acids, s e oids and lysoglyce ophospholipids, we e ob ained a e cen i uging he supe na an a
16,000g o 15 min, d ying and econs i u ing in me hanol, (2) o aminoacids, aliquo s o 5 μL om he
i s ac ion we e de i a ised and d ied, and (3) glyce olipids, choles e yl es e s, sphingolipids and glyc-
e ophospholipids we e ob ained by mixing he chlo o o m ex ac ion mix u e wi h H2O (pH 9) and incu-
ba ing a -20 ºC o 60 min. A e cen i uging a 16,000g o 15 min, he o ganic phase om his hi d
ac ion was collec ed hen, d ied and econs i u ed in 50/50% / ace oni ile/isop opanol. The aque-
ous phase ha con ains pola me aboli es, including cen al ca bon me abolism, was collec ed, d ied
and econs i u ed in H2O.
Quali y con ol (QC) sample o calib a ion and alida ion we e included in his wo k low o co ec
o esponse ac o s be ween and wi hin ba ches; and o assess he quali y o da a.
5.2.3. LC-MS analysis.
An app op ia e UPLC-MS me hod was used o each pla o m. The ins umen s and he condi ions
o he ch oma og aphic sepa a ion and mass spec ome ic de ec ion a e summa ized in Table 23. A
es mix u e o s anda ds was analyzed be o e and a e he en i e se o andomized, duplica ed sample
injec ions o check o e en ion ime s abili y, mass accu acy and sensi i i y. All da a we e p ocessed
using he Ta ge Lynx applica ion manage o MassLynx 4.1 so wa e (Wa e s Co p., Mil o d, USA). A
se o p ede ined ea u es, de ined as e en ion ime - mass- o-cha ge a io pai s, R -m/z, co esponding
o me aboli es included in he analysis a e ed in o he p og am. Associa ed ex ac ed ion ch oma o-
g ams (mass ole ance window = 0.05 Da) a e hen peak-de ec ed and noise- educed in bo h he LC
and MS domains such ha only ue me aboli e ela ed ea u es a e p ocessed by he so wa e. Then,
a lis o ch oma og aphic peak a eas is gene a ed o each sample injec ion.
133
Table 23. UPLC-MS Analysis pla o ms o me abolomics analysis.
Pla o m 1
Pla o m 2
Pla o m 3
Pla o m 4
Column ype
UPLC BEH C18, 1.0 x
100 mm, 1.7 μm
UPLC BEH C18m 2.1 x
100 mm, 1.7 μm
UPLC BEH C18, 1.0 x
100 mm, 1.7 μm
UPLC HSS T3, 1.0 x
150 mm, 1.8 μm
Flow a e
0.14 mL pe min
0.40 mL pe min
0.14 mL pe min
0.10 mL pe min
Sol en A
H2O + 0.05% Fo mic
acid
H2O + ACN + 10 mM
Ammonium Fo ma e
10 mM Ammonium Bi-
ca bona e (pH = 8.8)
10 mM T ibu ylamine +
15 mM Ace ic Acid + 2%
Me hanol (pH = 5.0)
Sol en B
ACN + 0.05% Fo mic
acid
ACN + Isop opanol + 10
mM Ammonium Fo ma e
ACN
Me hanol
(%B), ime
0%, 0 min
40%, 0 min
2%, 0 min
0%, 0 min
(%B), ime
50%, 2 min
100%, 10 min
8%, 6.5 min
4%, 1.5 min
(%B), ime
100%, 13 min
40%, 15 min
20%, 10 min
20%, 3 min
(%B), ime
0%, 18 min
40%, 17 min
30%, 11 min
25%, 8 min
(%B), ime
-
-
100%, 12 min
50%, 10 min
(%B), ime
-
-
2%, 14 min
45%, 15 min
(%B), ime
-
-
-
100%, 16-20 min
(%B), ime
-
-
-
0%, 21-25 min
Column empe a u e
40 ºC
60 ºC
40 ºC
40 ºC
Injec ion olume
2 μL
3 μL
2 μL
2 μL
Sou ce empe a u e
120 ºC
120 ºC
120 ºC
120 ºC
Nebulisa ion N2 low
600 L pe hou
1000 L pe hou
600 L pe hou
600 L pe hou
Nebulisa ion N2 em-
pe a u e
350 ºC
500 ºC
350 ºC
300 ºC
Cone N2 low
30 L pe hou
30 L pe hou
10 L pe hou
50 L pe hou
Capilla y ol age
2.8 kV
3.2 kV
3.2 kV
2.8 kV
Cone ol age
50 V
30 V
30 V
100
5.2.4. Da a analysis.
Da a no malisa ion and quali y con ol
A e da a inspec ion in e ms o ep oducibili y and peak in eg a ion, each me aboli e was co ec ed
and no malized using he in ensi y o an app op ia e in e nal s anda d included in he analysis and
ollowing he p ocedu e ully desc ibed by an de Kloe e al.495. Finally, any emaining ze o alues in
he co ec ed da ase we e eplaced wi h missing alues p io a e aging o ob ain a da ase u he used
o s a is ical analyses. A inal no maliza ion p ocedu e was applied by di iding e e y sample by i s
p o ein con en .
Mul i a ia e and uni a ia e analysis
Once da a was no malized and p epa e o s a is ical analysis, a i s app oach based on mul i a i-
a e analysis was pe o med wi h SIMCA-P ( e sion 13.0). Fi s ly, a non-supe ised p incipal componen
analysis (PCA) was u ilised o educe dimensionali y and o s udy da a quali y, assess ep oducibili y o
Me aboli es 2022,12, 714 2 o 17
5-
α
dihyd o es os e one (DHT), a e he majo ligands in his molecula pa hway and cause
he p og ession o PCa a ea ly s ages [4,6].
Figu e 1.
Schema ic ep esen a ion o he s e oid ho mone biosyn hesis pa hway in ele an o gans
and i s egula ion. CRH s imula es he elease o ACTH om he pi ui a y gland. ACTH s imula es
he p oduc ion o co isol (exe s nega i e eedback on CRH and ACTH) and DHEAS in ad enal
glands. Pulses o GnRH om hypo halamic neu ons s imula e pulses o LH as well as FSH. LH s im-
ula es es os e one p oduc ion in es is. Li e main ains pa hway’s homeos asis and se e al p ocesses
may happen: sul desul a ion makes me aboli es a ailable o eed he pa hway while p ocesses indi-
ca ed wi h a la end a ow inac i a e me aboli es ha a e in ci cula ion. Bold a ows indica e a highe
ac i i y o he speci ic eac ion. In bold, he me aboli es ha a e majo ly p oduced in each speci ic o -
gan a e ep esen ed. ACTH: ad enoco ico opin; CRH: co ico opin- eleasing ho mone; FSH: ollicle
s imula ing ho mone; GnRH: gonado opin- eleasing ho mone; LH: lu einizing ho mone; CYP17A1:
S e oid 17-alpha-monooxygenase; CYP19A1: a oma ase; SULT: hyd oxys e oid sul o ans e ase;
STS: s e oid sul a ase; 3
β
-HSD: 3
β
-Hyd oxys e oid dehyd ogenase; 17
β
-HSD: 17
β
-Hyd oxys e oid
dehyd ogenase; DHEA: dehyd oepiand os e one; DHEAS: DHEA sul a e.
Me aboli es 2022,12, 714 3 o 17
In mammals, he p ecu so o s e ol biosyn hesis is choles e ol, which is u he u ilized
in he ad enal glands, gonads and sexual-de i ed issues o p oduce s e oid ho mones.
The e a e 99 me aboli es in ol ed in he s e oid ho mone biosyn hesis pa hway and o e
100 eac ions a e ca alyzed by 61 di e en enzymes [
7
,
8
]. All o he s e oid compounds sha e
a s e ane backbone s uc u e. The physiological ole o each indi idual s e oid ho mone is
p ima ily de ined by he layou o double bonds, hyd oxyl and ke o g oups a ound his
basic s e ane backbone s uc u e [
1
]. The main s uc u al di e ence be ween he classes
is he ca bon a om a angemen i.e., he and ogens a e C-19, he es ogens a e C-18, he
p oges ogens a e C-20 and he co icoids a e C-21.
In he i s s ep o he s e oid ho mone biosyn hesis, choles e ol is in e nalized in o he
mi ochond ia whe e i is ed as a subs a e o p oduce p egnenolone (Figu e S1, Supplemen-
a y Ma e ials). This is he main p ecu so o s e oid ho mones p oduced de no o [
4
] inside
he mi ochond ia. P egnenolone can be con e ed o p oges e one o dehyd oepiand os-
e one (DHEA), which can be u he me abolized o glucoco icoids and mine aloco icoids
(C-21) o o and ogens (C-19), such as es os e one, DHT o and os e one and es ogens
(C-18), espec i ely (Figu e S1, Supplemen a y Ma e ials). In e es ingly, his me abolic
ne wo k is issue-dependen . Di e en o gans a e specialized on pa icula modules o
he pa hway ha a e physiologically ele an o pe o m hei unc ion. Fo ins ance, he
ad enal glands a e he p oduce s o C-21 ho mones, while p os a e shows a high SRD5A
ac i i y, which ca alyzes he con e sion o es os e one o DHT (Figu e 1).
Indeed, his is an in ica e ne wo k o me aboli es. Many o hese me aboli es pa -
icipa e as ligands in a wide span o signaling cascades and biological p ocesses, and
hei le els a y s ongly be ween di e en biological compa men s. While choles e ol
is he unique de no o p ecu so in s e oid ho mone biosyn hesis, he e exis s an in e -
change be ween cells and issues ha anaple o ically eeds he pa hway a he in e media e
s eps [
9
]. This means ha he compounds ups eam o he pa hway can be p o ided by
he cell en i onmen . In his line, sul a ed s e oids a e o in e es since hey a e, unlike
hei unsul a ed coun e pa s, eadily soluble in he cy oplasm and in bio luids, such as
blood o u ine. No ably, he sul a es o s e oids a e conside ed endogenous and ac i e
neu os e oids [
9
,
10
]. O e he pas ew decades, i has been es ablished ha sul ona ion
is no only a p ocess o inac i a e and exc e e s e oid ho mones; i also ac s as a sys emic
ese oi o pe iphe al o local s e oidogenesis in non-s e oidogenic issues, i.e., he b ain
o p os a e [
9
,
11
]. In addi ion, i has been epo ed ha he sec e ed esicles, also known
as ex acellula esicles (EVs), pa icipa e in many o he physiological p ocesses [
12
,
13
]
and hey can con ain a wide a ie y o ca gos, such as lipids, p o eins, me aboli es, suga s
and e en DNA [
12
–
15
]. The ho mone s e oids and ela ed ca gos a e anspo ed by he
blood and o he body luids as sul a ed species, bu hey could also be anspo ed by EVs
o each he a ge issues.
The s e oid ho mone me abolism and he consequences o dys egula ion ha e gained
in e es wi hin he biomedical communi y o unde s and and diagnose ho mone-dependen
diseases, a he han he his o ic usage o s e oid ho mones in he apeu ics. Indeed, a num-
be o me hods o de ec and quan i y s e oid ho mones ha e been epo ed du ing he las
wo decades. Many o he s udies desc ibe me hodologies o de ec s e oids om se e al bio-
logical sou ces: cell cul u es [
3
,
16
,
17
]; u ine samples [
18
–
20
]; animal issues [
21
–
23
]; human
se um [
24
–
26
]; human hai [
27
] and was e wa e [
28
,
29
]. In gene al, s e oid me abolomics
me hodologies ocus on p o iling a speci ic se o me aboli es o in e es in a ge ed issues
(o in ci cula ion) a he han analyzing s e oidogenesis s a us in a sys em o o gans and
ela ed luids. The me hods a e usually de eloped o simila non-sul a ed s e oids ha
e icien ly ionize in he same mode, a oiding he explo a ion o he de ec ion and quan-
i ica ion o many di e en s e oids simul aneously [
16
,
23
,
25
,
26
]. Me hodologically, hese
s udies desc ibe a a ie y o ex ac ion, sepa a ion and de ec ion me hods. In pa icula , he
solid phase ex ac ion (SPE) and e e sed phase liquid ch oma og aphic-based me hods
a e deployed in he isola ion and sepa a ion o hese compounds. The de ec ion is mos ly
pe o med wi h iple quad upole ins umen s. In addi ion, gas ch oma og aphy-coupled
Me aboli es 2022,12, 714 4 o 17
MS me hods was also u ilized in a ew o he s udies. All o hese me hods ha e hei
ad an ages and disad an ages.
We desc ibe a me hod o he de ec ion o endogenous s e oid ho mones and hei
in e media es, using liquid/liquid ex ac ion and ul a-pe o mance liquid ch oma og a-
phy (UPLC), coupled wi h high esolu ion ime-o - ligh mass spec ome y (h LCMS).
UPLC p o ides as cycling imes and a high ch oma og aphic esolu ion. The high mass
esolu ion ob ained wi h ime-o - ligh mass spec ome y esul s in high speci ici y, while
he sensi i i ies a e on pa wi h iple quad upole me hods. This me hod was applied o
me abolically p o ile se e al animal issues and u ina y EVs (uEVs). Di e en biological
ma ices, including p os a e, ad enal gland, es icles, b ain and li e o Wis a male a s
bu also human u ina y samples, we e es ed in his assay. To ou knowledge, he p esen
wo k p esen s o he i s ime a eliable and op imized h LCMS assay o analyze he key
endogenous s e oid ho mones in endoc ine issue, bioliquids and EVs.
2. Ma e ials and Me hods
2.1. Tissue and Bio luid Samples
The issues and se um we e ob ained om h ee wild- ype (Wis a , RjHan:WI) a s
ob ained om Jan ie Labs, Le Genes -Sain -Isle, F ance. All o he u ine samples we e
ob ained om a heal hy male on ei he he mo ning o he a e noon. uEVs we e ob ained
by ul acen i uging u ine samples as desc ibed elsewhe e [
5
]. U ine samples and uEVs
we e cha ac e ized in se e al physicochemical pa ame e s and p o ein ma ke s, espec i ely.
Fo a mo e de ailed in o ma ion on sample collec ion, p epa a ion and cha ac e iza ion
e e o Figu e S1 (Supplemen a y Ma e ials).
2.2. Chemicals and S anda ds
The DHEA, DHT, co isol (in me hanol solu ion) and he sodium sal o and os e one
sul a e we e ob ained om Ce illian Co po a ion (Round Rock, TX, USA). Supelco (Belle-
on e, PA, USA) p ocu ed and os enedione. The sodium sal s o DHEAS and p egnenolone
we e ob ained om A an i Pola Lipids, Inc. (Alabas e , AL, USA). The es os e one,
aldos e one, co icos e one, es one, p egnenolone 3-sul a e (sodium sal o m), leucine-
enkephalin (Leu-Enk), chlo o o m (>99.8% pu e; o ch oma og aphy g ade) and ammonia
solu ion we e pu chased om Sigma-Ald ich (S . Louis, MO, USA). The LC-MS g ade
wa e , ace oni ile, o mic acid and me hanol we e pu chased om Fishe Chemical (Fai
Lawn, NJ, USA).
2.3. LCMS Sample P epa a ion
The s e oid me aboli es we e ex ac ed by liquid–liquid ex ac ion using a me hanol/
wa e mix u e and chlo o o m as ex ac ion liquids. The EV ac ions we e sonica ed
o 15 min in a o al olume o 400
µ
L 50% / me hanol/wa e mix u e con aining
1 mM ammonia o lysa e EVs. The cell cul u e (DU145 cell line), ixed on cul u e well
pla es, was sc apped a e 5 min incuba ion wi h 500
µ
L 50% / me hanol/wa e mix u e
con aining 1 mM ammonia. Tissue aliquo s—app oxima ely 50 mg—we e lysed, using
1.4 mm zi conium oxide beads in o s anda d 2 mL homogenize ubes (P ecellys, Mon igny,
F ance). Each sample was homogenized in 500
µ
L 50% / me hanol/wa e mix u e
con aining 1 mM ammonia by pe o ming wo cycles o 40 s a 6000 pm in a Fas P ep-
24TM 5G bead bea ing g inde (MP Biomedicals, Solon, OH, USA). A e lysis, 400
µ
L o
he homogena e—ei he issue, EV ac ion o DU145 cell cul u e—was ans e ed o a
clean Eppendo
®
ube. Subsequen ly, 400
µ
L o LCMS g ade chlo o o m was added on
op o he 400
µ
L o any lysa ed sample and shaken o 60 min a 1400 pm a 4
◦
C. Then,
he samples we e cen i uged o 30 min a 14,000 pm a 4
◦
C in o de o p ecipi a e he
p o eins and o sepa a e he o ganic om he aqueous phases.
The aqueous ( op) and o ganic (bo om) phases we e sepa a ed. The p o ein ac ion
was p ecipi a ed on he meniscus be ween hese wo immiscible phases. Then, 250
µ
L
o each ac ion was ans e ed o he clean Eppendo
®
ubes and e apo a ed using a
Me aboli es 2022,12, 714 5 o 17
cen i ugal acuum concen a o . The pelle s om he o ganic ac ion we e dissol ed in
100
µ
L pu e me hanol and he pelle s om he aqueous ac ions we e dissol ed in 50% /
me hanol/wa e . All o he esuspended pelle s we e cen i uged o 30 min a 13,000 pm
and 4
◦
C. Finally, 80
µ
L o he esuspended pelle s we e ans e ed o deac i a ed glass
ials o 96-well pla es o injec ion in o he h LCMS sys em.
2.4. Ul a-High Pe o mance Liquid Ch oma og aphy (UPLC)
The ch oma og aphic sepa a ion o he analy es was pe o med wi h an ACQUITY
UPLC I-Class PLUS Sys em (Wa e s Inc., Mil o d, MA, USA). This sys em was equipped
wi h a cooled (10
◦
C) P ocess Sample Manage wi h a sample loop o 10
µ
L and a Sample
O ganize , a Bina y Sol en Manage and a High Tempe a u e Column Hea e . A e e sed-
phased 1.0 mm
×
100 mm BEH C18 column (Wa e s Inc., Mil o d, MA, USA), he mos a ed
a 40
◦
C, was used o sepa a ing he analy es. The samples we e injec ed om ei he 2 mL
deac i a ed glass ials o 700 µL ound 96-well polyp opylene pla es.
The ch oma og aphic beha io was op imized wi h espec o he peak in ensi y
and an adequa e sepa a ion o he 11 analy es along he un. The g adien elu ion was
accomplished wi h an aqueous mobile phase (eluen A) consis ing o 99.9% wa e wi h
0.1% o mic acid and an o ganic mobile phase (eluen B) consis ing o 99.9% ace oni ile
wi h 0.1% o mic acid. The low a e was 140
µ
L pe min. Se e al g adien s we e es ed
du ing he op imiza ion p ocess (Table S1, Supplemen a y Ma e ials) in o de o a oid
b eak- h ough (elu ion o analy e in he injec ion peak) and o ob ain a good peak sepa a ion.
The op imal g adien was as ollows: s a a 30% B; a linea inc ease o 80% B in 3.8 min.; a
s ep inc ease om 80% o 99%; cons an a 99% o 1.0 min and back o 30% B in 0.2 min.
The o al cycle ime om injec ion o injec ion was 6 min. The injec ion olume o all o
he samples was 2 µL.
2.5. Mass Spec ome y
A ime-o - ligh mass spec ome e SYNAPT G2-S (Wa e s Inc.) was u ilized o he
de ec ion o he analy es. The ins umen was ope a ed in ei he posi i e (ESI+) o nega i e
(ESI-) elec osp ay ioniza ion mode and in ull-scan mode wi h a scan ange be ween 50 Da
and 1200 Da and scan ime o 0.2 s.
The z-sp ay sou ce pa ame e s: empe a u es; gas lows; capilla y posi ion and ol -
ages we e uned, as de ailed elsewhe e [
30
]. The op imal sou ce pa ame e s o his assay
in ei he ESI+ o ESI
−
a e summa ized in Table S2 (Supplemen a y Ma e ials). The ion
op ics we e ine- uned by sp aying Leu-Enk (100 ppb), a a a e o 10
µ
L pe min, o a
esolu ion o e 20,000 (FWHM) o m/z556.2771. The same Leu-Enk solu ion was sp ayed
as a lock mass o co ec o m/z luc ua ions along he assay. The lock mass solu ion was
in oduced in o he sou ce e e y 90 s using a second ESI p obe and i was eco ded o
0.5 s. Mass spec ome e spec a was co ec ed acco ding o luc ua ions de ec ed in he
lock mass.
2.6. S a is ical Analysis
2.6.1. Analy e Reco e y S udy
The ex ac ion s ep e iciency was assessed by pe o ming a eco e y assay wi h a i-
ous mix u es o o ganic sol en s and wa e . Fi e di e en ex ac ion bu e s we e es ed in
his assay: 25/75% / and 50/50% / o me hanol/wa e mix u e;
25/74.9/0.1% / /
and 50/49.9/0.1% / / o me hanol/wa e / o mic acid mix u e and 50/50% / o
me hanol/wa e mix u e wi h 1mM ammonia. To compa e and calcula e he eco e -
ies o 10 di e en analy es, a cul u e o a p os a e cance cell line-DU145-was spiked wi h
he analy e s anda ds. Each well con aining 5
·×
10
5
cells was spiked wi h a mix o s an-
da ds a 2
µ
M be o e lysis (p e-spiked) and a he esuspension s age (pos -spiked) wi h a
s anda d mix a 10
µ
M. Thus, he p e-spikes con ained 1 nmol in 500
µ
L and pos -spikes
(aqueous and o ganic ac ions) con ained he same o al amoun in 100
µ
L, which would
be he heo e ical maximum absolu e i he e was no loss du ing he ex ac ion. In ad-
Me aboli es 2022,12, 714 6 o 17
di ion, o each ex ac ion solu ion, he non-spiked samples we e p epa ed in o de o
co ec o endogenous me aboli es in he ma ix. The samples o he p e-spiked, pos -
spiked and non-spiked condi ions and he i e di e en ex ac ion bu e s we e p epa ed
in biological iplica es.
Only he absolu e peak a eas we e aken in o conside a ion o es ablish he eco e y
e iciency in he ex ac ion s ep. The a e age peak a eas we e ob ained by mean smoo hing
he aw signals o iplica es. The eco e y (R) was de e mined by di iding he co ec ed
p e-spike a e age by he co ec ed pos -spike a e age and ep esen ed as a pe cen age
(Equa ion (1)). Bo h he p e-spiked and pos -spiked aw signals ough o be co ec ed by
sub ac ing he endogenous analy es signal in he DU145 cul u e ma ix (S
non-spike
). How-
e e , as he S
non-spike
o DU145 cul u e ma ix was less han 0.05% o he signal, endogenous
co ec ion was neglec ed du ing he calcula ion. Impo an ly, he p e-spikes we e co ec ed
wi h espec o analy e loss (
α
) du ing he ex ac ion p ocedu e. Mo eo e , he aw signals
o each sample did no ha e o be co ec ed by he amoun o ini ial samples, because e e y
well con ained he same amoun o cells.
R(%) =
αSp e−spike −Snon−spike
Spos −spike −Snon−spike
×100 (1)
2.6.2. S udy o Ma ix E ec in Analy e Quan i ica ion
In o de o assess he ma ix e ec (ME) in he quan i ica ion o he analy es, he
pos -spiked aw signal was compa ed o an equi alen aw signal o a mix u e o analy es
(10
µ
M) in solu ion. The pos -spiked aw signals we e co ec ed by sub ac ing he endoge-
nous analy es de ec ed in he non-spiked DU145 cul u e samples. Then, he nume a o
was di ided by he a e age peak a eas o he s anda ds and exp essed as a pe cen age
(Equa ion (2)):
ME (%) = Spos −spike −Snon−spike
Ss anda ds
×100 (2)
2.6.3. Analy e Semi-Quan i ica ion
In his wo k, a calib a ion cu e was p epa ed in solu ion wi h 50% / me hanol/wa e
o he semi-quan i ica ion o he analy es. This calib a ion cu e consis ed o a se ially
dilu ed mix u e con aining all o he analy es, s a ing a a concen a ion o 10
µ
M. The
ini ial concen a ion was dilu ed o hal concen a ion wice, esul ing in 5
µ
M and 2.5
µ
M
concen a ion in he cu e. Then, his se o iple s was dilu ed in i e decades; i esul ed
in he ollowing 15 di e en concen a ions pe analy e: 10; 5; 2.5; 1; 0.5; 0.25; 0.1; 0.05; 0.025;
0.01; 0.005; 0.0025; 0.001; 0.0005 and 0.00025
µ
M. The calib a ion samples we e injec ed a
he beginning and a he end o each expe imen ; he a e age o hese wo poin s was used
o semi-quan i y he me aboli es in he issues.
The limi o de ec ion (LOD) o each analy e was se o be he lowes concen a ion a
which he signal- o-noise (S/N) a io was abo e h ee. The LOQ was de ined as he lowes
concen a ion a which he S/N a io was abo e 10. The highes quan i iable concen a ion
was he highes concen a ion pe analy e ha i s he calib a ion cu e wi h an accep able
accu acy and p ecision (CV ≤15%) [16].
In gene al, he da a o a calib a ion cu e ange o e se e al o de s o magni ude,
he da a a e no linea and end o be he e oscedas ic [
31
]. Fo his eason, he ela ion
be ween he peak a ea and he sample concen a ion was de e mined by powe - i ing [
30
].
The powe i ing esul ed in a calib a ion cu e (Equa ion (3)) wi h
α
and bas he i ed
pa ame e s. Once he sample concen a ions we e calcula ed using a calib a ion me hod in
solu ion, he amoun (in nanomole) pe g am o issue weigh was es ima ed:
Peak a ea =α[concen a ion]b(3)
Me aboli es 2022,12, 714 7 o 17
3. Resul s
3.1. Liquid Ch oma og aphy and Mass Spec ome y Me hod
We compa ed six di e en ch oma og aphic me hodologies (Table S1, Supplemen a y
Ma e ials) o sa is ac o ily sepa a e he analy es. The g adien 6 (30% B o 80% B in 3.8 min;
de ailed s eps in Table S2, Supplemen a y Ma e ials) showed he bes peak sepa a ion along
his un ime compa ed o o he es ed g adien s (da a a ailable in [
32
]). Due o he na u e
o he s a iona y phase, analy es elu e in o de o inc easing hyd ophobici y. The esul ing
ex ac ed ion cu en (XIC) ch oma og ams o a s anda d mix u e a 10
µ
M a e depic ed in
Figu e S2 (Supplemen a y Ma e ials). In b ie , aldos e one (m/z361.2015; ESI+) elu es a
0.99 min, co isol (m/z363.2171; ESI+) a 1.20 min, DHEAS (m/z367.1579; ESI
−
) a 1.60 min,
co icos e one (m/z347.2222; ESI+) a 1.68 min, and os e one sul a e (m/z369.1736; ESI
−
)
a 1.85 min, p egnenolone sul a e (m/z395.1892; ESI
−
) a 2.23 min, es one (m/z271.1698;
ESI+) a 2.39 min, and os enedione (m/z287.2011; ESI+) and DHEA
(m/z289.2168
; ESI+)
co-elu e a 2.40 min, DHT (m/z291.2324; ESI+) a 2.65 min, p egnenolone (m/z317.2481;
ESI+) a 3.25 min.
Rega ding he mass spec ome y me hod, he Leu-Enk signal (m/z556.2771) was
aimed a a esolu ion o o e 20,000 (FWHM) and p o ided he necessa y mass accu acy
o e alua e assay analy es. Iso ope pa e n ma ching and he use o chemical s anda ds
con i ming elu ion imes u he ensu ed he speci ici y. In gene al, he mass accu acies o
he analy es in solu ion we e be ween −1 o 1 mDa. I is no ewo hy ha se e al analy es
we e no adequa ely sepa a ed du ing he ch oma og aphic elu ion. The co icos e one
and DHEAS elu e a simila e en ion imes—1.60 min and 1.68 min-, howe e , he MS
could p ope ly dis inguish hem by hei m/zdi e ence and hei agmen a ion pa e n.
Mo eo e , he DHEAS was no de ec ed wi h a high in ensi y signal in ESI+ mode. Fo
his eason, he co icos e one was measu ed in ESI+ and he DHEAS in ESI
−
mode.
Likewise, es one, DHEA and and os enedione elu ed in app oxima ely 2.40 min. In his
case, one could only ely on he MS sensi i i y (es one m/z271.1698, DHEA m/z289.2168,
and os enedione m/z287.2011) and on a agmen a ion pa e n ha was sensi i e enough
o dis inguish and quan i y hem sepa a ely.
3.2. Analy e Reco e y Op imiza ion
A e wa ds, we e alua ed he eco e y o 11 analy es using a biphasic liquid–liquid
me hod and analyzed hem wi h he op imized h LCMS me hod. The ex ac ion was
pe o med, using he DU145 cell line as a ma ix. Fi e di e en mix u es o o ganic
sol en s and wa e , con aining ei he o mic acid o ammonia o modi y he pH o he
ex ac ion bu e o no pH modi ie , we e assessed (Table S3, Supplemen a y Ma e ials).
The addi ion o o mic acid s i ed o lowe ing he pH app oxima ely o h ee, while
1mM ammonia modi ied he ex ac ion bu e o pH 8–9 in o de o chemically neu alize
he unc ional g oups o he s e oid compounds. F om he p e ious expe imen s in ou
me abolomics pla o m, we obse ed ha in liquid–liquid ex ac ion equi es a leas 25%
o ganic sol en du ing he ex ac ion s ep o p ecipi a e he p o eins. This is impo an
o a oid clogging he ch oma og aphic sys em [
30
]. Mo eo e , he e ec i i y o issue
homogeniza ion using beads has been epo ed as high and does no di e much om
he homogeniza ion o o he ma ices, such as u ine o cell cul u es [
30
,
33
]. The e o e, he
calcula ed eco e ies a e ul ima ely dependen on he ex ac ion bu e u ilized, ega dless
o he homogeniza ion me hodology.
Du ing he op imiza ion p ocess, i was de e mined ha he s e oid sul a e compounds
we e eco e ed comple ely in he aqueous ac ion, whils s e oids wi hou sul a e g oup
we e ound in he o ganic ac ion. No ably, only co isol was de ec ed sys ema ically
in bo h o he ac ions (Figu e S3, Supplemen a y Ma e ials); howe e , i was majo ly
eco e ed in he o ganic (80% o highe ) a he han in he aqueous (app oxima ely 20%)
ac ion. Mo eo e , he addi ion o o mic acid o he ex ac ion bu e led o a d ama ic
dec ease in he eco e ies o he sul a e compounds and a sligh dec ease in he es
o he s e oid analy es (Figu e S3, Supplemen a y Ma e ials). One can in e ha he
Me aboli es 2022,12, 714 8 o 17
p esence o p o ons in he bu e do no s abilize s e oid cha ges and se e ely hampe s
he ex ac ion o sul a e s e oids in a pola en i onmen . The supplemen a ion o 1mM
ammonia ou pe o med he ex ac ion in e ms o eco e y and obus ness, compa ed o
he o he ex ac ion liquids. No ably, he eco e y alues using di e en pe cen ages o
me hanol in he ex ac ion bu e do no di e much. Howe e , he ex ac ion e iciency o
he sul a e compounds using 25% / me hanol unde pe o ms 50% / me hanol, wi h a
eco e y loss o 40 o 50%.
In Table 1, he eco e ies o he 11 selec ed analy es, using a mix u e o 50/50% /
me hanol/wa e wi h 1mM ammonia as he ex ac ion bu e , a e epo ed. In gene al, he
p esen me hodology is able o eco e and de ec o e 90% o he ini ially spiked analy e.
Only DHT was de ec ed in a lowe pe cen age; app oxima ely 80% o he ini ially spiked
DHT was eco e ed. As expec ed in a biphasic ex ac ion, he ho mone s e oids we e
e ie ed in an apola en i onmen and he sul a ed s e oids in a pola sol en . Besides
co isol, p egnenolone sul a e was also epo ed in bo h o he ac ions; i was mainly
eco e ed in he mo e pola sol en and a de iso y amoun in he o ganic ac ion. Using
his me hodology, he eco e ies o 10
µ
M o analy e anged om 74.2% o 126.9%. These
alues a e accep able o ou ine mu i-analy e h LCMS analysis since all o he esul s a e
ep oducible [
34
]. Thus, ex ac ion using 50/50% / o me hanol/wa e mix u e wi h
1 mM ammonia was selec ed o u he expe imen s in di e en biological ma ices.
Table 1.
Summa y o he op imized me hod cha ac e is ics. The eco e ies (
±
s anda d de ia ion)
and ma ix e ec as signal loss (
±
s anda d de ia ion) o he ex ac ion p ocedu e in wo di e en
biological ma ices (n= 6; biological ma ix: DU145 cell) a e epo ed. In addi ion, LOD and LOQ
alues o he analy es in he adequa e ac ion a e compiled. LOD: Limi o de ec ion; LOQ: Limi
o quan i ica ion.
Analy e F ac ion Reco e y (%) Ma ix E ec (%) LOD (nM) LOQ (nM)
P egnenolone O ganic 97.2 (±1.9) 25.2 (±3.1) 2.5 nM 10 nM
Aqueous - 24.0 (±2.8)
DHEA O ganic 122.7 (±2.9) 37.7 (±5.7) 5.0 nM 50 nM
Aqueous - 28.0 (±6.2) - -
And os enedione O ganic 102.2 (±3.2) 30.8 (±4.6) 0.25 nM 0.5 nM
Aqueous - 23.2 (±4.5)
Es one O ganic 103.7 (±3.8) 25.5 (±4.8) 5.0 nM 10 nM
Aqueous - 25.7 (±4.0)
DHT O ganic 74.2 (±3.4) 23.1 (±3.9) 0.25 nM 1.0 nM
Aqueous - 23.4 (±2.9)
Co isol O ganic 114.3 (±3.8) 25.9 (±4.2) 0.5 nM 1.0 nM
Aqueous 22.28 (±4.5) 17.6 (±4.7)
Aldos e one O ganic 99.8 (±1.77) 18.7 (±4.3) 0.5 nM 2.5 nM
Aqueous - 17.7 (±5.1)
Co icos e one O ganic 109.4 (±3.1) 25.1 (±3.6) 0.25 nM 1.0 nM
Aqueous - 20.2 (±3.2)
Tes os e one O ganic 126.9 (±1.7) 14.3 (±1.9) 0.25 nM 0.25 nM
Aqueous - 8.0 (±2.1)
P egnenolone sul a e O ganic 6.9 (±2.7) 25.2 (±3.1) 0.25 nM 1.0 nM
Aqueous 94.8 (±1.9) 24.0 (±2.8)
DHEAS O ganic - 42.6 (±1.1) 0.25 nM 0.5 nM
Aqueous 108.0 (±1.4) 42.5 (±0.1)
Fu he mo e, he pe o mance o he op imized me hodology was es ed, using u ine
as he ma ix since i has a high in e es o clinical applica ions. Six samples o u ine om
a male indi idual we e pooled and aliquo ed in di e en wo olumes o assess he ma ix
Me aboli es 2022,12, 714 9 o 17
e ec on he eco e y e iciency. In Table 2, he eco e ies o he 10 analy es a e epo ed;
DHEA eco e y has no been e ie ed, because i s peak was masked by es os e one’s
signal. In gene al, o e 85% o he ini ially spiked analy e is eco e ed and de ec ed in
50
µ
L u ine ma ix. Impo an ly, he sul a ed s e oids a e no eco e ed wi h he same
e iciency; DHEAS and p egnenolone sul a e epo a eco e y e iciency o 75.7% and
54.9%, espec i ely. The eco e ies o he analy es using 250
µ
L u ine as ma ix desc ibes a
sligh dec ease in he non-sul a ed s e oids while he e iciency decay is d ama ic in he
sul a ed species.
Table 2.
Summa y o he eco e ies using he op imized me hodology in u ine ma ix. The eco e ies
(
±
s anda d de ia ion) o wo di e en olumes (50
µ
L and 250
µ
L) o p e-pooled u ine a e epo ed
(n= 3).
Analy e U ine Volume Reco e y (%)
P egnenolone 50 µL 92.4 (±3.6)
250 µL 99.3 (±4.8)
And os enedione 50 µL 93.0 (±3.9)
250 µL 79.3 (±3.8)
Es one 50 µL 94.2 (±3.3)
250 µL 84.8 (±4.8)
DHT 50 µL 76.3 (±4.1)
250 µL 71.2 (±3.76)
Co isol 50 µL 87.0 (±3.0)
250 µL 72.4 (±3.6)
Aldos e one 50 µL 110.7 (±2.9)
250 µL 103.1 (±3.2)
Co icos e one 50 µL 96.2 (±2.8)
250 µL 84.3 (±3.6)
Tes os e one 50 µL 104.1 (±2.1)
250 µL 96.3 (±5.1)
P egnenolone sul a e 50 µL 54.9 (±1.5)
250 µL 25.5 (±1.2)
DHEAS 50 µL 75.7 (±2.5)
250 µL 44.0 (±4.2)
3.3. Ma ix E ec
I is well known ha he phospholipids and o he lipids, ypically en iched in biologi-
cal ma ices, such as issues, body luids o cell cul u es, can cause ion supp ession in mass
spec ome y, he eby hampe ing he analy e signal [
35
,
36
]. This phenomenon nega i ely
in luences he de ec ion o he analy es and may unde es ima e hei quan i ica ion. Fo a
speci ic ma ix, he highe he ion supp ession e ec is, he highe he signal loss. The e o e,
he conclusions d awn by de ec ing and quan i ying he analy es unde hese condi ions
could be misleading.
The ma ix e ec o each analy e was de ined as he signal loss measu ed a he esus-
pension s ep (sample spiked wi h 10
µ
M analy e mix) compa ed o 10
µ
M o each analy e in
solu ion. The signal loss was calcula ed in i e di e en ex ac ion p ocedu es, because hey
can in luence ion supp ession. The ma ix e ec epo ed in his wo k was es ima ed o a
p os a e cance cell line (DU145) cul u e and u ine samples. To no e, signal loss is speci ic
o each ma ix and each independen expe imen . In u he expe imen s, in which quan-
i ica ion is equi ed, he ma ix e ec should be calcula ed in e e y pa icula assay. F om
ou op imiza ion expe imen s, one can in e ha he ma ix e ec is ac ion-dependen ,
because he e is a signi ican di e ence be ween signal loss compa ing o ganic and aqueous
ac ions (Figu e S4, Supplemen a y Ma e ials). This phenomenon is likely obse ed due
Me aboli es 2022,12, 714 10 o 17
o a di e en ial ex ac ion o he phospha idylcholine (o o he lipid) compounds [
30
,
35
].
S ikingly, his ac ion dependency was no obse ed upon he addi ion o ammonia o
he ex ac ion liquid. Mo eo e , he p esence o ammonia esul ed in a signal loss o up o
hal - old compa ed o ex ac ion liquids wi h acidic modi ie o no pH modi ie addi ion.
This sugges s ha he ammonia impai s he ex ac ion o he lipidic compounds om he
biological ma ix, hence, dec easing he ion supp ession phenomenon in mass spec ome y.
In Table 1, he ma ix e ec (exp essed as signal loss (%)) o a DU145 cul u e o
11 selec ed analy es, using a 50/50% / o me hanol/wa e mix u e wi h 1mM ammonia
o ex ac ion, is epo ed. In gene al, he p esen me hodology loses app oxima ely 15
o 40% o he signal o non-sul a ed analy es bu i mainly lays be ween 20 o 30% loss.
On he o he hand, he sul a ed s e oids display a 40 o 50% loss o signal, ega dless o
he ex ac ion ac ion. The signal loss o he 10
µ
M analy es spiked in DU145 cell line
we e: 25.2% o p egnenolone, 37.7% o DHEA, 30.8% o and os enedione, 25.5% o
es one, 23.1% o DHT, 25.9% and 20.2 % o co isol in he o ganic and aqueous ac ion,
espec i ely, 18.6% o aldos e one, 25.0% o co icos e one, 46.1% o p egnenolone sul a e
and 42.5% o DHEAS. All o he analy es a e majo ly eco e ed back in a pa icula ac ion
o he ex ac ion p ocedu e, which is he one selec ed o epo he ma ix e ec . Signal loss
o sul a e compounds e e o aqueous ac ion measu emen and he o he s e oids e e o
signal loss in o ganic ac ion.
3.4. Semi-Quan i a ion o S e oids in Animal Tissues
The h LCMS me hod was mos sensi i e in de ec ing and os enedione, DHT, co icos-
e one, p egnenolone sul a e and DHEAS wi h a LOD (S/N > 3) o 250 pM in a
50/50% /
me hanol/wa e solu ion. The de ec ion limi o co isol and aldos e one was 0.5 nM,
and a LOD o 2.5 nM was de e mined o p egnenolone. The leas esponsi e ions we e
hose o DHEA and es one wi h a LOD o 5.0 nM. Wi h ega ds o he quan i ica ion
limi s, and os enedione and DHEAS we e he mos sensi i e compounds, wi h a LOQ
(S/N > 10) o 0.5 nM in solu ion. The co isol, co icos e one, p egnenolone sul a e and
DHT we e in he second g oup o he mos quan i iable ions showing a LOQ o 1.0 nM. The
quan i a ion limi o aldos e one was 2.5 nM, while a LOQ o 0.01
µ
M was es ima ed o
p egnenolone and es one. The DHEA was he compound wi h he highes quan i a ion
h eshold (0.05 µM).
We ound ha he concen a ion ange o he s e oid ho mones is ypically low in
issues, anging om pico- o nanomole pe g am o issue, and canno be de ec ed in some
issues (Table 3). Only p egnenolone, and os enedione, DHT, co icos e one, co isol and
es os e one we e de ec ed in he issues o se um o Wis a a s. P egnenolone and co isol
a e only quan i ied in he ad enal gland issue, howe e , p egnenolone is also de ec ed
in he b ain and es icles. Ad enal gland and es icles epo ed picomole amoun s o
and os enedione pe g am o issue. Mo eo e , DHT was quan i ied in he p os a e, ad enal
gland and es icles. In p os a e, he amoun o DHT was wo- old he quan i a ion in he
o he issues. The es os e one and co icos e one we e quan i ied in all o he measu ed
a samples. In gene al, hey we e epo ed in he picomole pe g am ange in issues. In
se um, hey we e quan i ied in he nM ange. In e es ingly, he ad enal gland desc ibed
nanomole pe g am concen a ions o co icos e one. Fu he mo e, es os e one was ound
in a one o de o magni ude highe amoun in he ad enal gland and es icles compa ed o
p os a e and b ain.
Me aboli es 2022,12, 714 11 o 17
Table 3.
Quan i a ion o h ee independen Wis a a issues: ad enal gland, p os a e and b ain.
Ad enal glands o he same animal we e i e ed independen ly, also, he p os a e lobes o each a .
The a e ages in nmol pe g am o issue, s anda d de ia ions and coe icien s o a ia ion (%) o he
h ee g oups o samples a e epo ed.
Analy e Quan i ica ion
(nmol/g Tissue) Ad enal Gland P os a e B ain Tes icle Se um (nM)
P egnenolone Amoun 7.04 - De ec ed De ec ed -
S . de . 3.74
%c 53
And os enedione
Amoun 5.97 ×10−3- De ec ed 1.45 ×10−3De ec ed
S . de . 3.35 ×10−31.38 ×10−3
%c 56 95
DHT Amoun 3.47 ×10−37.57 ×10−3De ec ed 2.70 ×10−3De ec ed
S . de . 1.02 ×10−32.40 ×10−37.92 ×10−4
%c 29 31 29
Co icos e one Amoun 18.89 4.01 ×10−32.42 ×10−21.25 ×10−328.01
S . de . 10.05 5.15 ×10−37.04 ×10−37.98 ×10−43.31
%c 53 128 29 63 12
Co isol Amoun 0.45 - - - -
S . de . 0.19
%c 43
Tes os e one Amoun 4.53 ×10−36.92 ×10−47.02 ×10−49.18 ×10−30.20
S . de . 1.47 ×10−32.36 ×10−44.29 ×10−44.53 ×10−30.02
%c 32 34 60 49
The s anda d de ia ions and coe icien s o he a ia ion a e a he la ge, indica ing an
impo an a iabili y among he samples ob ained om he same s ain bu independen
animals. One could expec his biological a ia ion and i sugges s ha ea men s, s ess o any
p ocedu e applied o animals can po en ially in luence he ou come in u he expe imen s.
3.5. Quan i a ion o S e oid Ho mones in Human U ina y Samples
Six di e en u ine samples we e cha ac e ized in se e al physicochemical pa ame e s
(Table S4, Supplemen a y Ma e ials) o examine whe he he sample collec ion esul ed in
homogenous sample g oups, ega dless o he me abolomics’ analysis. No blood, ke one
bodies o glucose we e de ec ed in he u ine sample, and he pH alue and densi y o he
u ine we e simila in all o he samples. The u ine samples we e cen i uged in wo se ial
s eps a 10,000
×
g o 30 min o isola e he so-called P10K ac ion— ypically con aining
esicles o 150 o 200 nm diame e and abo e— ollowed by a 100,000
×
gcen i uga ion
o 90 min o isola e he so-called P100K— ypically con aining esicles o 100 o 150 nm
diame e and below (up o 50 nm) [
37
]. The supe na an o he second cen i uga ion was
also analyzed and e e ed o as SN100K.
In his se o u ine samples, he cu en me hodology is able o de ec and quan i y
and os enedione, co isol and DHEAS (Table 4). The o he s e oids o he panel we e below
he LOQ and, in gene al, also below he LOD. The and os enedione and co isol we e
de ec ed only in he u ine and SN100K. I was no possible o de ec hem associa ed wi h
he EVs, and hey a e majo ly solubilized in he u ine. The and os enedione was ound
in lowe concen a ions compa ed o co isol and he a iabili y be ween he collec ion
days was high (40 o 60%) ega dless o he collec ion ime. Conce ning co isol, he
a iabili y was ex emely high be ween he mo ning collec ion days (app oxima ely 50 o
85%) whils he concen a ion o he a e noon collec ed samples was s able (app oxima ely
2% a ia ion). DHEAS was he compound de ec ed in he highes concen a ion (
µ
M
ange) soluble in u ine, compa ed o and os enedione and co isol (nM ange). Simila
o and os enedione, he DHEAS showed a high a iabili y o e independen collec ion
Ti le: In eg a ion o p o eomic and me abolomic analysis e eal dis inc me abolic al e a ions o
1
p os a e cance -associa ed ib oblas s compa ed o no mal ib oblas s om pa ien ’s s oma samples
2
Au ho names: Guille mo Bo danaba-Flo i 1,†, Félix Royo1,2, Oihane E. Albóniga3, Aled Clay on4,
3
Juan Manuel Falcón-Pé ez1,2,3 and Jason Webbe 5,†
4
A ilia ions:
5
1Exosomes Labo a o y, Cen e o Coope a i e Resea ch in Biosciences (CIC bioGUNE), De io, Spain;
6
2Cen o de In es igación Biomédica en Red de En e medades Hepá icas y Diges i as (Cibe ehd),
7
28029 Mad id, Spain;
8
3Me abolomics Pla o m, Cen e o Coope a i e Resea ch in Biosciences (CIC bioGUNE), De io,
9
Spain;
10
4Di ision o Cance and Gene ics, School o Medicine, Ca di Uni e si y, Ca di , UK;
11
5Ins i u e o Li e Science, Swansea Uni e si y Medical School, Swansea Uni e si y, Swansea, UK.
12
†Co esponding au ho s: gbo [email protected] (G. B.-F.); j.p.webbe @swansea.ac.uk (J. W.)
13
Highligh s:
14
Lipogenic me abolic pa hways a e mo e p e alen in CAFs.
15
Ene gy p oducing pa hways al e na i e o glycolysis suppo CAFs me abolic ac i i y.
16
Mi ochond ial me abolism is al e ed in he i s s eps o ib oblas di e en ia ion.
17
Ex acellula esicles p o oke a highe basal espi a ion associa ed o mi ochond ia in no mal
18
ib oblas s.
19
Abs ac :
20
The p os a e gland is a complex and he e ogeneous o gan composed o epi helium and s oma.
21
P os a e cance is mos commonly seen eme ging om luminal epi helial cells and equi es he
22
assis ance o adjacen s oma. P os a ic s oma is composi ionally complex, and in conce wi h
23
disease p og ession unde goes many al e a ions which include he eme gence o cance associa ed
24
ib oblas s (CAFs). This he e ogeneous cell popula ion o en con ains cells wi h a myo ib oblas -like
25
pheno ype ha a e no no mally p esen in heal hy p os a e issue. In his wo k, we s udied he
26
me abolic ewi ing o s omal ib oblas s ollowing myo ib oblas di e en ia ion. Fi s , he me abolic
27
abundances o no mal- and cance -associa ed ib oblas s de i ed om needle biopsies o he same
28
pa ien was analysed using UPLC-MS. I was de e mined CAFs we e me abolically mo e ac i e and,
29
he e o e, ene gy p oducing me abolic pa hways we e enhanced. Also, CAFs showed a heigh ened
30
lipogenic me abolism as bo h ese oi species and building block compounds. In e es ingly, lipid
31
me abolism a ec s mi ochond ia unc ioning ye he mechanisms o lipid-media ed unc ions a e
32
unclea . The ac oxidised a y acids and glu a hione sys em a e ele a ed in CAFs s eng hens he
33
hypo hesis ha inc eased me abolic ac i i y is ela ed o mi ochond ia. In u he expe imen s
34
measu ing he me abolic lux wi h a Seaho se bioanalyse , we s udied whe he TGF-β1 and
35
ex acellula esicles (EVs) could s imula e myo ib oblas di e en ia ion in no mal ib oblas s. An
36
inc ease o basal espi a ion in no mal ib oblas s was epo ed, mi o ing he disease-like pheno ype.
37
This indica es an al e ed me abolism associa ed o mi ochond ia in CAFs and ea ed ib oblas s.
38
Hence, one p oposes ha he change in he me abolomics p o ile o umou -associa ed s omal
39
ib oblas s is d i en by oxygen-dependen me abolism, possibly associa ed o mi ochond ia; howe e ,
40
he speci ic mechanisms a e s ill unclea .
41
Keywo ds:
42
Mass spec ome y; P os a e cance ; Me abolism; Ex acellula esicles; Human p ima y ib oblas s
43
Abb e ia ions:
44
2-deoxy-d-glucose, 2-DG; α smoo h muscle ac in, α-SMA; Adenosine iphospha e, ATP; Cance -
45
associa ed ib oblas , CAF; Choles e yl es e , CE; Ce amides, Ce ; Ex acellula acidi ica ion a e,
46
ECAR; Ex acellula esicle, EV; Fa y acid, FA; Ca bonyl cyanide p- i luo o me hoxyphenylhyd azone,
47
FCCP; Oxydised a y acis, FFAox; Glu a hione, GSH; Hyd oxyeicosapen anoic acid, HEPE; Liquid
48
ch oma og aphy coupled o mass spec ome y, LC-MS; Lysophospha idylcholines, LPC;
49
Glyce ophosphocholines, LPE; Mass spec ome y imaging, MSI; Nico inamide adenine
50
dinucleo ide, NAD; N-acyl e hanolamines, NAE; Oxygen consump ion a e, OCR; P os a e cance ,
51
PCa; P incipal componen analysis, PCA; Phospha e bu e ed saline, PBS; Reac i e oxygen species,
52
ROS; S anda d e o o he mean, SEM; T ica boxylic acid, TCA; T ans o ming g ow h ac o be a 1,
53
TGF-β1; Ul a-high pe o mance liquid ch oma og aphy coupled o mass spec ome y, UPLC-MS.
54
G aphical abs ac :
55
56
57
In oduc ion:
58
The p os a e gland is globally composed o epi helium and s oma, which a e ex emely he e ogeneous
59
issues. The epi helium is o ganized as glandula acini and i con ains cuboidal o columna sec e o y
60
epi helial cells wi h apical junc ion complexes, a con inuous laye o basal cells1, and spa se
61
neu oendoc ine cells, each a ached o a basal lamina2. Beyond he basal lamina, a p ominen
62
ib omuscula s oma composed o smoo h muscle, ib oblas s, blood essels, au onomic ne e ib es,
63
in lamma o y cells, and ex acellula ma ix componen s o e s physical suppo and con ac ion o he
64
gland3. In p os a e ca cinoma (PCa), he in e s i ial s oma is o en abno mally ich in myo ib oblas ic
65
cells4,5, capable o suppo ing umou g ow h, ascula iza ion, angiogenesis, and me as asis in i o6.
66
T ans o ming g ow h ac o β1, TGF-β1, emains among he mos c i ical ac o s o myo ib oblas ic
67
di e en ia ion and he gene a ion o a umou eac i e s oma. In iguingly, we ha e p e iously shown
68
ha cance ex acellula esicles (EVs) can igge ib oblas o myo ib oblas di e en ia ion in an EV-
69
TGF-β1 dependen manne 6. In u he s udies, he essen ial ole o EVs in di ec ing his s omal cell
70
di e en ia ion owa ds cance -associa ed myo ib oblas -like pheno ype was desc ibed7. Howe e , he
71
mechanisms by which EV-ac i a ed s omal cells suppo umou g ow h emain unclea .
72
Ou p e ious s udies wi h biopsy ma e ial om pa ien s whe e umo al g ow h was loca ed in one hal
73
o he p os a e and no he o he , e ealed clea di e ences be ween no mal and disease egions.
74
His ological examina ion showed a smoo h muscle s omal a chi ec u e a ound glandula s uc u es in
75
no mal issue while he glands we e diso ganised and showed an al e ed, ib osis-like in e s i ial s oma
76
obse ed in disease-associa ed issue8. A panel o an ibody ma ke s con i med he highe abundance
77
o α-Smoo h Muscle Ac in (α-SMA)-posi i e myo ib oblas cells in umou -associa ed issue. S ikingly,
78
EVs isola ed om p os a e cance epi helial cell lines gene a ed a myo ib oblas -like pheno ype in
79
no mal ib oblas s7. In ha wo k, we demons a ed he essen ial ole o EVs in di ec ing s omal
80
di e en ia ion o a p o- umo igenic pheno ype, exhibi ing p o-angiogenic p ope ies and enhancemen
81
o umou g ow h in xenog a models. Fu he unc ional assays and p o eomics p o iling wo k
82
highligh ed ha s oma ac i a ion media ed by EV s imula ion mi o s he na u ally occu ing ib oblas
83
di e en ia ion du ing disease8.
84
Al e ed me abolism is a hallma k o PCa and se e al me aboli es and me abolic pa hways a e al eady
85
dis inc i e in di e en p os a e ypes o issue9,10. In his line, Ande sen e al. epo ed highe le els o
86
ene gy- ela ed pa hway me aboli es, such as ADP, ATP, and glucose as well as highe le els o he
87
an ioxidan au ine in s omal issue compa ed o cance and non-cance epi helium11. Besides,
88
inc eased le els o c ucial me aboli es o a y acid oxida ion and building blocks in lipid syn hesis
89
we e desc ibed in cance issue. O he s udies also epo ed me abolic ewi ing o eac i e s oma,
90
showing di e en le els o ce ain me aboli es be ween highly di e en ia ed s oma compa ed o poo ly
91
di e en ia ed s oma12. The s omal-epi helial in e ac ions ha e a dominan ole in umou g ow h,
92
in asion and me as asis. Ac ually, many epo s o e he las decades showed he in e ac ion o
93
eac i e s oma wi h PCa2,12–17 and EVs in luencing aspec s o cance biology such as
94
angiogenesis7,8,18,19 and umou p og ession17,20–22. Ye , ew s udies ha e in es iga ed he ole o
95
umou EVs in al e ing me abolic p ocesses in s omal cell compa men s.
96
Wi h he ise o omics e a, en i e se s o biomolecules – genes, p o eins o me aboli es - con ained in a
97
biological issue, cell, luid, o o ganism can be iden i ied. A p o eomics analysis o he same ma ched-
98
no mal and disease s oma issues was able o dis inguish bo h pheno ypes and desc ibe a disease-
99
like pheno ype8. Simila ly, a ansc ip omic analysis o de i ed EVs om he same ype o samples
100
disc imina ed no mal om disease s oma samples23. This manusc ip p esen s a b oad semi a ge ed
101
me abolomics app oach, which analyses he me abolome o no mal and disease s oma issues,
102
highligh ing he me abolic di e ences o ma ched no mal ib oblas s and cance -associa ed ib oblas s
103
wi hin indi idual PCa pa ien s. Fu he mo e, we show an al e ed me abolism o no mal ib oblas s
104
ea ed wi h EVs om a PCa cell line.
105
Ma e ials and me hods:
106
S omal p ima y cell cul u es
107
Six pa ien -ma ched no mal and umou -associa ed needle biopsies we e isola ed om adical
108
p os a ec omy. These we e aken om si es o palpable disease and also om appa en ly no mal
109
issue om he opposi e side o he same p os a e. Tissue collec ion and consen ing was managed
110
h ough he Wales Cance Bank. Co es we e manually dissec ed in o 1mm3 pieces and subjec ed o
111
mechanical homogeniza ion ollowed by 200 U pe mL collagenase-I diges ion o 15 o 20 hou s a 37
112
°C. Cells we e cul u ed in S omal Cell Basal Medium (SCBM) supplemen ed wi h human ib oblas
113
g ow h ac o -B, insulin, e al bo ine se um (FBS) and GA-1000 (Lonza, Wokingham, UK). o a ound
114
wo weeks un il only s omal cells we e e ained. 8,23Subsequen cul u es we e main ained in
115
DMEM/F12 media (Lonza) wi h 10% FBS deple ed o bo ine EVs. Cul u es we e con i med ee o
116
epi helial cells by immuno- luo escence s aining o cy oke a in p io seeding s omal cells in 96 well
117
pla es 8,23. A sample o each pa ien -ma ched p os a ic s omal cell cul u e was collec ed and ozen o
118
me abolomics analysis using liquid ch oma og aphy coupled o mass spec ome y.
119
Me aboli e ex ac ion
120
In me abolomics, he e is no single pla o m o me hod able o analyse he en i e me abolome o a
121
biological sample. The e o e, me aboli es we e ex ac ed by ac iona ing he cell samples in o pools o
122
species wi h simila physicochemical p ope ies. In b ie , p o eins we e p ecipi a ed by adding me hanol
123
o he cell lysa e. Chlo o o m sol en was added o he me hanol ex ac ion mix u e and his biphasic
124
mix u e was incuba ed a -20 ºC o 30 min. Then, h ee di e en ac ions we e collec ed: (1) a y
125
acyls, bile acids, s e oids and lysoglyce ophospholipids, we e ob ained a e cen i uging he
126
supe na an a 16,000g o 15 min, d ying and econs i u ing in me hanol, (2) o aminoacids, aliquo s o
127
5 μL om he i s ac ion we e de i a ised and d ied, and (3) glyce olipids, choles e yl es e s,
128
sphingolipids and glyce ophospholipids we e ob ained by mixing he chlo o o m ex ac ion mix u e wi h
129
H2O (pH 9) and incuba ing a -20 ºC o 60 min. A e cen i uging a 16,000g o 15 min, he o ganic
130
phase om his hi d ac ion was collec ed hen, d ied and econs i u ed in 50/50% /
131
ace oni ile/isop opanol. The aqueous phase ha con ains pola me aboli es, including cen al ca bon
132
me abolism, was collec ed, d ied and econs i u ed in H2O.
133
Quali y con ol (QC) sample o calib a ion and alida ion we e included in his wo k low o co ec o
134
esponse ac o s be ween and wi hin ba ches; and o assess he quali y o da a.
135
LC-MS analysis
136
An app op ia e UPLC-MS me hod was used o each pla o m. The ins umen s and he condi ions o
137
he ch oma og aphic sepa a ion and mass spec ome ic de ec ion a e summa ized in Table S1. A es
138
mix u e o s anda ds was analyzed be o e and a e he en i e se o andomized, duplica ed sample
139
injec ions o check o e en ion ime s abili y, mass accu acy and sensi i i y. All da a we e p ocessed
140
using he Ta ge Lynx applica ion manage o MassLynx 4.1 so wa e (Wa e s Co p., Mil o d, USA). A
141
se o p ede ined ea u es, de ined as e en ion ime - mass- o-cha ge a io pai s, R -m/z,
142
co esponding o me aboli es included in he analysis a e ed in o he p og am. Associa ed ex ac ed
143
ion ch oma og ams (mass ole ance window = 0.05 Da) a e hen peak-de ec ed and noise- educed in
144
bo h he LC and MS domains such ha only ue me aboli e ela ed ea u es a e p ocessed by he
145
so wa e. Then, a lis o ch oma og aphic peak a eas is gene a ed o each sample injec ion.
146
Da a analysis
147
Da a No maliza ion and quali y con ol
148
A e da a inspec ion in e ms o ep oducibili y and peak in eg a ion, each me aboli e was co ec ed
149
and no malized using he in ensi y o an app op ia e in e nal s anda d included in he analysis and
150
ollowing he p ocedu e ully desc ibed by an de Kloe e al.25. Finally, any emaining ze o alues in
151
he co ec ed da ase we e eplaced wi h missing alues p io a e aging o ob ain a da ase u he
152
used o s a is ical analyses. A inal no maliza ion p ocedu e was applied by di iding e e y sample by
153
i s p o ein con en .
154
Mul i a ia e and uni a ia e analysis
155
Once da a was no malized and p epa e o s a is ical analysis, a i s app oach based on mul i a ia e
156
analysis was pe o med wi h SIMCA-P ( e sion 13.0). Fi s ly, a non-supe ised p incipal componen
157
analysis (PCA) was u ilised o educe dimensionali y and o s udy da a quali y, assess ep oducibili y o
158
he analy ical p ocedu e, isualize endencies be ween g oups and de e mine he p esence o ou lie s.
159
A e wa ds, supe ised pa ial leas squa es disc iminan analysis (PLS-DA) and o hogonal PLS-DA
160
(OPLS-DA) we e pe o med ollowed by a sui able alida ion me hod, a c oss- alida ion analysis o
161
a iance (CV-ANOVA), in eg a ed in SIMCA-P so wa e. Supe ised models ha we e alida ed we e
162
u he used o a iable selec ion. To his end, a a iable impo ance on p ojec ion (VIP) sco e and
163
absolu e alue o p(co ) g ea e han 1 and 0.8, espec i ely, we e used as cu -o poin s o a iable
164
selec ion.
165
Finally, and as a complemen a y s a is ical analysis, uni a ia e analysis was pe omed. In o de o es
166
no mali y, Shaphi o es was used; hence ei he pai ed s uden ’s es 26 o Wilcoxon signed- ank es
167
was applied o assess compa isons signi icance. A e ha , he da ase was exp essed as a me aboli e
168
old-change and signi icance (p- alue) and u he depic ed in a olcano plo .
169
Pa hway analysis and pa hway en ichmen
170
A p ope biological in e p e a ion is c ucial in any me abolomics s udy o deli e a comp ehensi e
171
assessmen o expe imen al condi ions. To e alua e he p ominence o ce ain me abolic pa hways, a
172
Pa hway analysis was compu ed using Me aboAnalys 5.0 and inpu ing a da ase o samples and
173
quan i ied me aboli es. I pe o ms an o- ep esen a ion analysis ha in eg a es en ichmen and
174
pa hway opology analysis o isualise speci ic al e ed pa hways in he human me abolic ne wo k27.
175
Then, p o eomics da a was included in a Join -pa hway analysis o e alua e me abolic al e a ions
176
conside ing wo se s o physiologically ele an molecules in ib oblas samples. This o e -
177
ep esen a ion analysis module pe o ms an in eg a ed me abolic pa hway analysis by combining
178
me abolomics and p o eomics da a collec ed om he exac same samples and me hodology27. The
179
Me aboAnalys 5.0 web-based ool includes he no malisa ion, ans o ma ion and scaling o da a o
180
comple e da a in eg a ion.
181
Fu he mo e, a lipid me abolic ne wo k analysis28 pe o med wi h LINEX 2.4.1 webapp o obse e
182
unc ional associa ions o lipid classes. I compu es speci ic lipid ne wo ks based on compounds and
183
lipid classes connec ions using a lipidomics da ase .
184
Ex acellula esicle isola ion
185
EVs we e pu i ied om condi ioned media o DU145 p os a e cance cell (ATCC, Tedding on, UK)
186
g own in In eg a bio eac o lasks (In eg a Biosciences Co p, Hudson, NH, USA) 24. EV samples we e
187
collec ed using he suc ose cushion me hod and esuspended in PBS. Thence, samples we e
188
quan i ied using he BCA-p o ein assay (Pie ce/The mo, No humbe land, UK), and s o ed a -80°C.
189
Fo ea men s o s omal cell cul u es, EV we e used a 200 µg pe mL (app oxima ely equi alen o
190
1.5 ng pe mL o EV-associa ed TGF-β1) o 72 hou .
191
Seaho se assay
192
Oxygen consump ion a e and glycoly ic ac i i y we e assessed using a XF24 Ex acellula Flux
193
Analyse (Seaho se Biosciences) o p obe O2 and pH, espec i ely. Fib oblas s we e equilib a ed in
194
unbu e ed media (60 min a 37 ºC in a CO2- ee incuba o ) p io ans e o he XF24 analyse . Fo
195
mi ochond ial espi a ion, he oxygen consump ion was measu ed o e he assay. Fi s , basal oxygen
196
consump ion (OCR) was de e mined, and hen oligomycin (1 µg pe ml), FCCP (0.3 µM), FCCP (0.6
197
µM), and 2 µM o enone we e sequen ially injec ed o assess maximal oxida i e capaci y, ATP
198
p oduc ion, coupling e iciency (OCR pe cen age dedica ed o p oduce ATP) and basal espi a ion. To
199
analyse glycoly ic ac i i y, he ex acellula pH was measu ed o e he assay. Fi s , base-line (non-
200
glycoly ic) ex acellula acidi ica ion (ECAR) was de e mined, and hen media alone ollowed by
201
glucose (10 mM), oligomycin (1 µg pe ml), and 2-Deoxyglucose (0.1 M) we e sequen ially injec ed o
202
assess maximal glycoly ic capaci y and glycolysis a e.
203
S a is ical analyses we e pe o med by pai ed S uden ’s - es using G aphPad PRISM 9.5 so wa e
204
(G aph Pad, San Diego, CA, USA). Each expe imen was analysed indi idually hen, he mean ± SEM
205
was ep esen ed. All p- alues lowe han 0.05 a e conside ed signi ican as: * P>0.05, ** P>0.01, ***
206
P>0.001.
207
Resul s:
208
CAFs exhibi a di e en ial p o eomic, ansc ip omic and me abolomics landscapes
209
In PCa, he eme gence o myo ib oblas s wi hin he in e s i ial s oma is desc ibed as he majo
210
di e ence be ween no mal and umou eac i e s oma12,29. This eac i e s oma coe ol es wi h
211
p os a e cance in i is capable o suppo ing i s g ow h. In p e ious s udies, we ha e cha ac e ised
212
no mal ib oblas s and CAFs de i ed om he same pa ien ’s needle biopsies o he ypical ma ke s o
213
eac i e s oma, α-Smoo h Muscle Ac in (α-SMA), Cy oke a ine, Desmin and Vimen in7,8,23. CAFs we e
214
no a homogeneous popula ion o myo ib oblas s bu a he e ogenous mix u e o ib oblas s a dis inc
215
di e en ia ion s ages, including a p opo ion o cells which a e α-SMA posi i e7. The no mal ib oblas s
216
lacked α-SMA. Howe e , hey could be induced o exp ess α-SMA when ea ed wi h ei he soluble o
217
esicle-associa ed TGF-β18.
218
In 2016, Webbe e al.8 desc ibed a se o p o eins ha could disc imina e CAFs and no mal ib oblas s
219
isola ed om he pa ien —ma ched needle biopsies desc ibed in his s udy. A mo e ecen s udy
220
explo ed he oppo uni y o use EV-de i ed RNA om hese ib oblas s as indica o s o al e ed umou
221
en i onmen 23. In his wo k, Shepha d e al. iden i ied 19 di e en ially exp essed ansc ip s ha
222
disc imina e disease om no mal s omal EVs, indica ing ansc ip ional di e ences be ween pa ien
223
samples. The me abolomics s udy included in his wo k adds se e al indings o ou published da a
224
ob ained by p o eomics and ansc ip omics. A comp ehensi e e alua ion o da a showed a clea
225
endency o enhanced abundances in mos o he me aboli es in sample 1161-no mal du ing da a
226
analysis and no maliza ion compa ed o o he pa ien -ma ched issues. The beha iou o his sample
227
was also di e en om he emaining samples as demons a ed by PCA (Fig. S1). Mo eo e , i is on
228
he line ha desc ibe he con idence ellipse based on Ho elling’s T2 (signi icance le el = 0.05). Fo
229
hese easons, sample 1161-no mal was excluded om u he s a is ical analysis. A e wa ds, PLS-DA
230
and OPLS-DA models we e buil and he sco es plo s a e included in Fig. 1A and 1B. A clea
231
sepa a ion endency was obse ed in bo h models mainly h ough PC1. E en wi h his sepa a ion
232
endency, none o he models was alida ed (CV-ANOVA p- alue >0.05). Howe e , hose a iables
233
ha in luence on he mos in g oup sepa a ion we e selec ed based on hei VIP and p(co ) alues. In
234
o al 64 and 12 me aboli es ul il VIP g ea e han 1 and ǀp(co )ǀ g ea e han 0.8 in PLS-DA and
235
OPLS-DA, espec i ely. In o de o selec hose me aboli es o ele an impo ance, a Venn Diag am
236
including hese me aboli es was buil o selec only common me aboli es in PLS-DA and OPLS-DA
237
models (Fig. 1C). Rele an me aboli es include ce amides (Ce ), phospha idylcholines (PC) and
238
choles e yl es e s (CE) (Table S3).
239
240
Figu e 1. Summa y o mul i a ia e me abolomics analysis o no mal ib oblas s and CAFs. A. Sco e sca e
241
plo o he PLS-DA model o ib oblas s ob ained om no mal and cance needle biopsies. Model diagnos ics (A =
242
2; R2X = 0.682; R2Y = 0.729; Q2 = 0.337; CV-ANOVA = 0.581). In g een, CAF samples and, in pu ple, no mal
243
ib oblas s. B. Sco e sca e plo o he OPLS-DA model o ib oblas s ob ained om no mal and cance needle
244
biopsies. Model diagnos ics (A = 2; R2X = 0.682; R2Y = 0.721; Q2 = 0.241; CV-ANOVA = 0.799). In g een, CAF
245
samples and, in pu ple, no mal ib oblas s. In g een, CAF samples and, in pu ple, no mal ib oblas s. C. Venn
246
diag am o ea u es (me aboli es) ha in luence o he sepa a ion o CAF and no mal ib oblas s g oups in PLS-DA
247
and OPLS-DA models. Resul s a e compiled in Table S3.
248
Disease-associa ed ib oblas s show lipogenic and ene gy-p oducing me abolic al e a ions
249
Reac i e s oma ele ance g ows upon p og ession and in asion o umou cells o neighbou loca ions
250
and issues since i suppo s s uc u al g ow h and nu ien a ailabili y. In his line, cell popula ions in
251
no mal s oma unde go a myo ib oblas u no e cons i u ing he so-called CAFs. In o de o s udy he
252
me abolic p o ile o his wo issue sub ypes, ou pla o ms based on UPLC-MS we e u ilised o
253
analyse me aboli es. They a e ac iona ed in pools o species wi h simila physicochemical p ope ies.
254
The pla o ms include: (1) Fa y acyls, bile acids, s e oids and lysoglyce ophospholipids; (2)
255
Glyce olipids, glyce ophospholipids, s e ol lipids and sphingolipids; (3) Amino acids; (4) Pola
256
me aboli es p o iling, including cen al ca bon me abolism.
257
258
Figu e 2. Summa y o me abolomics s udy (conside ing indi idual me aboli es) compa ing no mal and
259
cance -associa ed ib oblas s isola ed om needle biopsies o adical p os a ec omy specimens. A.
260
Volcano plo compa ing CAFs and no mal ib oblas s as -log10(p- alue) agains log2( old change). The di e en
261
me aboli e classes a e depic ed in di e en shapes and colou s; ele an me aboli es a e labelled. B. Pa hway
262
o e ep esen a ion analysis depic ed as –log10 (p- alue) agains pa hway impac . Pa hway impac s ands o he
263
ela i e impo ance o he speci ic module in he analysed me aboli e se . I combines pa hway o e ep esen a ion
264
esul s and cen ali y measu es. Rep esen a i e pa hways we e labelled, size o nodes ep esen s pa hway impac
265
and hei signi icance anges om high (in ed) owa ds o ange, yellow and whi e indica ing a lowe signi icance.
266
He e, all indi idual me aboli es and hei old changes we e conside ed o ank he en ichmen and signi icance o
267
each pa hway. AA: aminoacids; TCA: ica boxylic acid cycle ela ed me aboli es; CHD: Ca bohyd a es de i a i es;
268
N : Nucleo ides; Redox: elec on dono and accep o s; Ns: Nucleosides; Vi : Vi amins; HexCe : Hexosylce amides;
269
LPE: Lysophospha idyle hanolamines; PC: Phospha idylcholines; PE: Phospha idyle hanolamines; PI:
270
Phospha idylinosi ols; SM: Sphingomyelins; LPC: Lysophospha idylcholines; Ce : Ce amides; NAE: N-
271
o p ocesses and al e a ions occu ing in each pa ien . E en so, he ou come o his s udy desc ibed
447
me abolic di e ences wi h spa se impac in he en i e me abolome. Once combined wi h p o eomics
448
da a om he exac same se o samples, he ou come acqui ed a highe ele ance in de e mining
449
impo an me abolic p ocesses d i ing myo ib oblas di e en ia ion. The in eg a ion o signi ican
450
p o eins and me aboli es di e en ially measu ed in no mal and cance ib oblas s con i med a majo
451
al e a ion in lipid me abolism. A a ia ion o glyce ophospolipids - i.e. PC(O-22:1/20:4) and PC(P-
452
18:0/20:4) - indica es an en ichmen o he majo memb ane ype o lipids. In e es ingly, his class o
453
lipids ha e been associa ed o mi ochond ia dynamics and well- unc ioning32,33. Al hough lipids can
454
pa icipa e in signalling p ocesses o be used as building blocks, in his s udy mos me abolic
455
al e a ions poin ou uelling o ene gy s o age p ocesses. Majo ee a y acid pa hways – linoleic and
456
a achidonic - a e al e ed oge he wi h an accumula ion o choles e yl es e s and ew oxidised a y
457
acids. This indica es a mobilisa ion o lipids o p oduce ene gy bu also he p oduc ion o lipid
458
ese oi s eadily a ailable o anspo . Mo eo e , cen al me abolism pa hways –
459
glycolysis/gluconeogenesis and pen ose phospha e - a e posi i ely al e ed in CAFs only including he
460
p o eome o he analysis. This sugges s ha as p o ein que ies we e he mos p ominen and
461
o e lapped a highe numbe o pa hways compa ed o me aboli es, he ou pu o his in eg a i e
462
analysis could be de e mined majo ly by di e ences in he p o eome.
463
Back in 2016, his p o eomics s udy desc ibed many p o eins al e ed in no mal and cance ib oblas s.
464
Howe e , se e al o hose we e unique o speci ic ea men s wi h ei he sTGF-β1 o EV-associa ed
465
TGF-β1. Fo ins ance, hey desc ibed a highe p esence o Annexin-I, which has a ole in egula ing
466
VEGF unc ion, in CAFs as well as al e ed mi ochond ial p o eins, linking CAFs o mi ochond ial
467
ea angemen . In his manusc ip , we app oached he assessmen o me abolic al e a ions upon
468
sTGF-β1 o EV-associa ed TGF-β1 ea men s by Seaho se s ess assays. E en conside ing he
469
limi a ion o such assays, whe e O2 consump ion and pH acidi ica ion a e eco ded o e ime, he
470
glycoly ic a e and oxida i e mi ochond ial ac i i y o cells was assessed. ECAR in o ms ha glycoly ic
471
ac i i y associa ed o CAFs was no inc eased. This, oge he wi h he ac ha he in eg a ion o
472
p o eomics and me abolomics indica es an al e a ion in cen al me abolism, sugges s ha hese
473
pa hways we e no used in he ca abolic bu in he anabolic di ec ion. Only basal espi a ion, in he
474
mi ochond ial s ess assay, was dis inc i e when compa ing CAFs and no mal ib oblas s. Mo eo e ,
475
he ea men wi h sTGF-β1 and EVs con aining TGF-β1 showed a simila ou come. This esul links
476
he dis inc i e me abolic abundances o mi ochond ial me abolism. Ou me abolomics da a o e s a
477
plausible explana ion because ele a ed le els o GSH a e usually co ela ed o he p esence o ROS
478
due o mi ochond ial ac i i y while al e ed le els o FFAox may indica e he bu ning o lipids in
479
mi ochond ia seeking o ene gy and coun e ac ing ROS. Pe haps a longe s imula ion o a highe
480
dose o sTGF-β1 o EVs is equi ed o obse e u he me abolic al e a ions associa ed o cance in
481
no mal ib oblas s. None heless, his da a demons a es ha me abolic al e a ions a e ela ed o a
482
highe ac i i y o mi ochond ia, ye he speci ic ole o cance -associa ed EVs and he me abolic
483
mechanisms in s omal di e en ia ion and shi owa ds i s eac i e s a e emain unclea .
484
Conclusions:
485
The pheno ype o cance -associa ed s oma is cha ac e is ic and di e ge om heal hy s oma a bo h
486
ansc ip ional and p o eomic le el. In his manusc ip , we discussed me abolic di e ences measu ed
487
in pa ien -ma ched no mal and umou ib oblas s om p os a e adjacen egions. This app oach has
488
no p eceden s in li e a u e. A mul ipla o m me abolomics analysis o hese ib oblas s de e mined an
489
inc eased me abolic ac i i y o CAFs. Also, many lipid classes we e al e ed, indica ing an enhanced
490
lipogenic me abolism. Lipid ese oi species as choles e yl es e we e measu ed in CAFs oge he
491
wi h building block compounds as lysophospha idylcholines. Fu he mo e, al e a ions in GSH sys em
492
and oxidised a y acids sugges s he in ol emen o mi ochond ia in CAF dis inc me abolism.
493
He ewi h, his in eg a ed s udy poin s ou se e al pa hways ha may be ele an du ing ib oblas
494
di e en ia ion.
495
P e ious s udies demons a ed he ea men wi h TGF-β1 o TGF-β1-EVs p o oke a esponse in
496
no mal ib oblas s owa ds myo ib oblas s. Thence, we assessed whe he EVs con aining TGF-β1 o
497
soluble TGF-β1 could igge a me abolic esponse in no mal ib oblas s. O en, in such di e en ia ion
498
p ocesses, cells despe a ely look o new ene gy sou ces and building blocks in o de o sus ain
499
g ow h. Acco ding o ou me abolomics and Seaho se da a, he al e na i e pa hway o ob ain ene gy is
500
no glycolysis. Mo eo e , Seaho se da a shows ha basal espi a ion associa ed o mi ochond ia was
501
inc eased in CAFs so as no mal ib oblas s upon TGF-β1 o EV s imula ion. In conclusion, one can
502
p opose he al e a ion in me abolic abundances o umou -associa ed ib oblas s is d i en by an
503
oxygen-dependen me abolism associa ed o mi ochond ia. The me abolic s a us o no mal ib oblas s
504
is al e ed upon EV (and TGF-β1) s imula ion and i is associa ed o mi ochond ia me abolism.
505
Howe e , he speci ic mechanisms u ilised o igge di e en me abolic esponses emains unde ined.
506
Acknowledgemen s:
507
We hank Exosomes lab and Me abolomics Pla o m s a a CIC bioGUNE o expe imen assis ance
508
and guidance. We would like o hank OWL me abolomics o pe o ming he me abolomics assay and
509
s a is ical analysis o p os a ic human ib oblas samples.
510
Funding sou ces:
511
The au ho s o his s udy we e suppo ed by unds om he Eu opean Union’s Ho izon 2020 esea ch
512
and inno a ion p og amme unde g an ag eemen no. 860303. The s udy was also suppo ed om a
513
Cance Resea ch Wales P og amme G an . The Eu opean Ne wo k on Mic o esicles and Exosomes in
514
Heal h and Disease (ME-HAD) wi h he cos ac ion STSM-BM1202 unde g an numbe 190514-
515
044080 inanced a Sho -Te m Scien i ic Mission o J. W.
516
Au ho con ibu ions:
517
This s udy was concep ualised and designed by J. W., A. C., F. R. and J.M.F.-P. Expe imen s we e
518
pe o med by J. W. and F. R. wi h he supe ision o A. C. and J.M.F.-P. Da a analysis and o iginal
519
d a w i ing was done by G. B.-F. wi h close supe ision o O. E. A. All au ho s c i ically e iewed,
520
edi ed and discussed he manusc ip ; eading and ag eeing o he published e sion o he a icle.
521
Con lic s o in e es :
522
The au ho s decla e no con lic o in e es .
523
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524
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eman a zabal zazu
Uni e sidad
del País Vasco
Euskal He iko
Unibe si a ea
