REVIEW ARTICLE
published: 04 Ma ch 2014
doi: 10.3389/ onc.2014.00039
Bio enseg i y o he ex acellula ma ix: physiology,
dynamic mechanical balance, and implica ions in oncology
and mechano he apy
I eneTadeo1,Ana P. Be begall1,2, Luis M. Escude o3,Tomás Ál a o4and Rosa Nogue a2*
1Founda ion INCLIVA, Hospi al Clínico de Valencia, Valencia, Spain
2Depa men o Pa hology, Medical School, Uni e si y o Valencia, Valencia, Spain
3Ins i u o de Biomedicina de Se illa, Hospi al Uni e si a io Vi gen del Rocío/CSIC/Depa amen o de Biología Celula de la Uni e sidad de Se illa, Se ille, Spain
4Depa men o Pa hology, Hospi al deTo osa, Ve ge de la Cin a, IISPV, URV,To osa, Spain
Edi ed by:
Jozse Dudas, Medical Uni e si y
Innsb uck, Aus ia
Re iewed by:
Xose S. Puen e, Uni e sidad de
O iedo, Spain
Ch is ian Oli e P i z, Medical
Uni e si y Innsb uck, Aus ia
*Co espondence:
Rosa Nogue a, Depa men o
Pa hology, Medical School, Uni e si y
o Valencia, A da. Blasco Ibañez 15,
Valencia 46015, Spain
e-mail: nogue a@u .es
Cells ha e he capaci y o con e mechanical s imuli in o chemical changes.This p ocess is
based on he enseg i y p inciple, a mechanism o ensional in eg i y.To da e, his p inciple
has been demons a ed o ac in physiological p ocesses such as mechano ansduc ion
and mechanosensing a di e en scales ( om cell sensing h ough in eg ins o molecu-
la mechanical in e en ions o e en localized massage). The p ocess in ol es in a- and
ex acellula componen s, including he pa icipa ion o ex acellula ma ix (ECM) and
mic o ubules ha ac as comp ession s uc u es, and ac in ilamen s which ac as en-
sion s uc u es.The nucleus i sel has i s own enseg i y sys em which is implica ed in cell
p oli e a ion, di e en ia ion, and apop osis. Despi e p esen ad ances, only he ip o he
icebe g has so a been unco e ed ega ding he ole o ECM compounds in in luencing
bio enseg i y in pa hological p ocesses. G oups o cells, oge he wi h he su ounding
g ound subs ance, a e subjec o di e en and speci ic o ces ha ce ainly in luence bio-
logical p ocesses. In his pape , we e iew he cu en knowledge on he ole o ECM
elemen s in de e mining bio enseg i y in malignan p ocesses and desc ibe hei implica-
ion in he apeu ic esponse, esis ance o chemo- and adio he apy, and subsequen umo
p og ession. O iginal da a based on he s udy o neu oblas ic umo s will be p o ided.
Keywo ds: bio enseg i y, cance , ex acellula ma ix, mechano he apy, neu oblas oma
INTRODUCTION
The s udy o spa ial and empo al esponses o mechanical o ces
o issue s uc u es o biological o ganisms is a g owing ield in
heal h sciences. Such esponses can be modi ied by mechano he -
apeu ic in e en ions, anging om he molecula le el o whole
body sys ems, and in ol ing a b oad spec um o a ge molecules
belonging o hemic oen i onmen . Ino de oca y ou mechan-
o he apy e ec i ely,we should conside he s abilizing elemen s o
ension and comp ession, o bio enseg i y sys ems, exis ing a all
s uc u al le els in he body. Tenseg i y is an a chi ec u al p inci-
ple pu o h by Buckmins e Fulle in he 1960s (1,2). Acco ding
o he enseg i y p inciple, s uc u es o enseg i y sys ems a e
s abilized by con inuous ension wi h discon inuous comp ession
(3). Coming unde he e m bio enseg i y, he enseg i y p inci-
ple applies o essen ially all de ec able scales in he body, om he
musculoskele al sys em o p o eins o DNA (4,5).
In his e iew,we highligh he cu en challenges and on-going
issues o dissec ing he mechanisms o umo ex acellula ma ix
(ECM) bio enseg i y and discuss how hese concep s may be ans-
la ed in o ea men and p ognosis o cance . To illus a e he
bio enseg i y p inciple,we p esen some p elimina y esul s on he
ma hema ical in eg a ion o mul imodal da a, combining imag-
ing and non-imaging umo issue da a, acqui ed in he con ex
o neu oblas oma (NB) s udies, sugges ing es able hypo heses o
making p ognos ic p edic ions and he apeu ic esponse ela ed
wi h his p inciple.
CELL AND TISSUE BIOTENSEGRAL PHYSIOLOGY
Se e al s udies ha e demons a ed ha cells can unc ion as inde-
penden p e-s essed enseg i y s uc u es h ough hei cy oskele-
on a chi ec u e. Ingbe de ined he p e-s essed enseg i y model
as a s uc u al suppo on biological sys ems. I is cons i u ed by
a numbe o con inuous elemen s o ension and a numbe o
discon inuouselemen s esis an ocomp essionp o idingas abi-
lizeds uc u e(6–14).Asa enseg i yne wo k,asinglecellp esen s
such con inuous ension (media ed by cy oskele on elemen s such
as mic o ilamen s and in e media e ilamen s) and local discon-
inuous comp ession (media ed by ECM and o he cy oskele on
elemen s such as mic o ubules). The indi idual p e-s essed cells
a e poised and eady o ecei e mechanical signals and con e
hem in o biochemical changes (15). The e o e, cell memb ane,
nucleus, and all he o ganelles a e ha d-wi ed by he cy oskele al
sca old. When he mechanical cue is ecei ed, sensed mainly by
ocal adhesion complexes induced by in eg ins, he signal modi ies
hecy oskele al sca old.Thus, helocalmechanicalsignalisampli-
ied and p opaga ed h ough a se ies o o ce-dependen biochem-
ical eac ions, whe eby in a-cellula signaling pa hways become
sequen ially ac i a ed h ough mechano ansduc ion (16). A he
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Tadeo e al. Bio enseg i y o he ex acellula ma ix
molecula le el, se e al elemen s esis comp ession, such as all
s uc u es con aining alpha-helix, be a-shee , o e en DNA back-
bone s uc u es, while o he s, such as a ac ion and epulsion
o ceso molecula ,a omic,andionicbonds (suchasVan de Waals
o ces, co alen bonds, e c.), esis con inuous ension. Many mol-
ecules display such s uc u es and a e subjec o hese wo o ces
a di e en s ages along he mechanical in a-cellula signal pa h-
ways. Among hese abundan and in e mingled pa hways, many
emain unknown. We belie e ha knowledge on how we could
po en ially in e e e wi h hese signaling pa hways o cascades may
p o ide new he apeu ic a ge s. Ne e heless, as many molecules
play mul iple oles in di e en pa hways, molecula he apy based
on mechano ansduc ion,should be ca ied ou on speci ic a ge s
o a oid ad e se e ec s.
The enseg i y a chi ec u e o he cy oskele on and signal pa h-
ways is linked o he enseg i y elemen s o he ou e and inne
nuclea memb ane h ough KASH–SUN b idges, whe e KASH
p o eins a e loca ed in he ou e nuclea memb ane (Nesp in-1
and Nesp in-2 link nuclei wi h ac in ilamen s,Nesp in-3 in e ac s
wi h in e media e ilamen s,andNesp in-4binds omic o ubules)
(17–19) and SUN p o eins in he inne nuclea memb ane (Samp1
and lamin). This connec ion is c i ical o in a-cellula o ce
ansmission in physiological homeos asis (20–22) and migh
ensu e ha ch oma in o ganiza ion is no pe u bed when is-
sues expe ience s ess, and may be undamen al o no mal
de elopmen (23).
The sel -balanced mechanical s abili y o he cy oskele on
enables he mac o-mechanical o ces o be con e ed in o molec-
ula changes. Since cells a e connec ed o each o he h ough cell
junc ions, media ed mainly by cadhe ins, hese changes no only
a ec he cell ha ecei es he signal,bu a e also ans e ed o he
neighbo ing cells. Indeed, ecen biophysical s udies ha e e ealed
ha he size o cell–cell con ac s can be egula ed in esponse o
he mechanical o ces exe ed on hose junc ions and ha cells a e
also able o egula e he o ces exe ed on hei junc ions (24–26).
I is known ha cell–cell junc ions a e ancho ed o neighbo -
ing cells and ocal adhesions o ECM, and all a e connec ed o
he in a-cellula cy oskele al ne wo k, he e o e he o ces ha
c oss hese s uc u es luc ua e s ongly when issue is emodeled.
I is becoming appa en ha hese s uc u es do no jus ans-
mi o ces while main aining issue cohesion, bu also espond o
luc ua ions in o ce by ac i ely in luencing cell mo phology and
beha io (27). The biological signi icance o his mechano ans-
duc ion is o p omo e coo dina ed cy oskele al eo ganiza ions
ha can de ine changes in shape ac oss he whole issue. The basal
lamina plays a cen al ole in his p ocess. I p o ides physical
suppo o epi helial cells, su ounds muscle cells, a cells, and
Schwann cells, and is he en i onmen whe e cells and ECM bind
h ough ocal adhesions and in eg ins. Acco dingly, he shape o
issue cells ( ound o la ened) and he h ee-dimensional s uc-
u e o he issue pa e ns ha cons i u e glands,al eoli,duc s,and
papillae (among o he s),depend on he s i ness and lexibili y and
on he coo dina ed mo emen o he basal lamina (28).
ROLE OF ECM IN BIOTENSEGRITY
Du ing he las decade, cell-ma ix con ac s based on he ans-
memb ane adhesion ecep o s om he in eg in amily o ocal
adhesions ha e eme ged as he majo mechanosensi i e s uc-
u al elemen s ha connec , collec , p ocess, and in eg a e he
in o ma ion o he ECM. Recen p o eomic s udies ha e no
only ound many mo e componen s, bu also ha e e ealed ha
many o hese elemen s a e ec ui ed o ocal adhesions in a
o ce-dependen manne , suppo ing he iew ha ocal adhesions
ha bo a ne wo k o mechanosensi i e p ocesses (29). In eg ins
a e ansmemb ane αβ he e odime ecep o s ha unc ion as
s uc u al and unc ional b idges be ween he cy oskele on and
ECM molecules. Speci ically, α8β1 o enseg in can bind o se -
e al ECM molecules and has been shown o be associa ed wi h
ocal adhesion poin s, whe e i pa icipa es in he egula ion o
sp eading, adhesion, g ow h, and su i al in di e en neu onal
and mesenchymal-de i ed cell ypes (30,31).
Va ious in e connec ed cells bind o hei mic oen i onmen ,
o ming a mechanical enseg al sys em, which implies he exis-
ence o a mechanical balance be ween comp ession (ECM) and
ension (cell) o ces. The ECM is made up o a mix u e o g ound
subs ance [glycosaminoglycans (GAGs) – mos ly hyalu onan,p o-
eoglycans, and glycop o eins] si ua ed in close ela ionship wi h
a ib ous sca old [ e iculin (Re F) – elas in and collagen ibe s
(Col F)], and supplies much o he s uc u al suppo a ailable o
pa enchymal cellsin issues,byadding ensile s eng hand lexibil-
i y (32). The ECM is a dynamic and mul i unc ional egula o and
has i s own bio enseg i y wi h Re F and elas in ibe s ac ing as en-
sional elemen s, and g ound subs ance and Col F as comp ession-
esis ance elemen s. This enseg al ne wo k is conside ed o be a
solid-s a e egula o y sys em o all cell unc ions, esponsible o
changes in genes and p o eins, as well as al e a ions in cell shape
and mo emen (33–35). One esul o cell–ECM bio enseg i y is
subs a e igidi y, which can con ol nuclea unc ion and hence
cell unc ion (36). Cells can use his subs a e igidi y o exe
ac ion o ces, hus al e ing he ECM. Indeed, in a s a e o ec-
ip ocal isome ic mechanical ension, a dynamic balance exis s
be ween cell ac ion o ces and poin s o esis ance wi hin he
ECM. This dynamic bio enseg al sys em wi h i s mechano ans-
duc ion phases (Figu e 1) enables ou cells o mechanosense,
modi ying hei mic oen i onmen , hus p omo ing ECM emod-
eling in homeos asis and in issue diso de s (37). Manipula ion o
his mechanical balance could be used o p omo e issue egen-
e a ion. In ac , a ious s udies ha e demons a ed ha di e en
elas ici ies o he ECM d i e mesenchymal s em cell di e en ia ion
in a e y speci ic way. Neu ogenic, myogenic, o os eogenic di e -
en ia ions a e induced unde iden ical ma ix se um condi ions,
wi h a ia ions in ECM so ness, s eng h, and s i ness (38). Fu -
he mo e, ECM s i ness guides cell mig a ion. I has been shown
ha ib oblas s p e e igid subs a es and when placed on lexible
shee s o polyac ylamide, hey mig a e om he so o he s i
a eas (39). Unde homeos a ic condi ions, collagen ib ils ha e
a minimal u no e . Howe e , his u no e is accele a ed du -
ing issue emodeling and umo de elopmen , as e idenced by
he se um le els o i s deg ada ion p oduc s (40). S udies o he
ECM ha e e ealed ha he componen s o he umo mic oen-
i onmen a e undamen al, no only o he egula ion o umo
p og ession (41,42), bu also a e essen ial e en be o e he umo
appea s. The s omal cells a e able o ans o m he adjacen cells
h ough an al e a ion in he homeos a ic egula ion o he issue,
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Tadeo e al. Bio enseg i y o he ex acellula ma ix
FIGURE 1 | Mechano ansduc ion phases.(A) When o ces a e exe ed on
a issue a e p oli e a ion o umo and s oma cells, a cell wi hin he cell
mass ( o example, he one ma ked in g een) is subjec o mechanical
de o ma ion in a phase called mechanocoupling. Following he p inciple o
enseg i y a he issue le el, ECM elemen s apply comp ession o ces o he
issue cells and issue cells exe ension o ces be ween hemsel es and o
he ECM. Bio enseg al elemen s o he boxed a ea a e de ailed in (B–D).
(B) This pa icula umo cell ac i a es a biochemical coupling phase in which
he mechanical signal is ans o med in o in a-cellula biochemical signals
h ough he in eg in–cy oskele on–nuclea ma ix s uc u e o
s e ch-ac i a ed ca ion channels wi hin he cell memb ane, among o he
mechanisms. Some essen ial playe s in mechanosensing a e shown (44–47).
(C) The biochemical signal is ansmi ed o neighbo cells (ma ked in pu ple)
h ough cell–cell junc ions. Following he p inciple o enseg i y a he cell
le el, he comp ession elemen s a e he mic o ubules, whe eas he ension
elemen s a e he in e media e ilamen s and he ac in ilamen s.
(D) A ib oblas neighbo cell can p oduce a s i ECM which in u n enables
he i s cell o la en and mig a e h ough his s i ened ECM. The e ec o
cell esponse phase a ises om he bio enseg i y p inciple: a simple
cy oskele on when he ECM comp ession is low swi ches o a complex
cy oskele on when he ECM exe s high comp ession on he cells and he
cells exe high ension on he ECM.
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Tadeo e al. Bio enseg i y o he ex acellula ma ix
including he con ol o a chi ec u e, adhesion, cell dea h, and
p oli e a ion (43).
CELL AND TISSUE BIOTENSEGRITY IN CANCER
Cance can be unde s ood as a disease o he de elopmen al
p ocesses ha go e n how cells o ganize in o issues (48). The
umo mic oen i onmen is comp ised o a a ie y o cell ypes
lying among a ne wo k o a ious ECM ibe s me ged wi hin he
in e s i ial luid and g adien s o se e al chemical compounds,
which cons an ly in e play wi h malignan cells (34). The e o e,
we can in e ha he p e iously desc ibed bio enseg al sys ems
also exis wi hin umo issue. In ac , he dynamic mechanical
balance achie ed h ough mechanosenso s,cy oskele al enseg i y,
molecula bio enseg al in a-cellula pa hways, ECM wi h com-
p essi eand esis an elemen s,suppo i e cells(suchas ib oblas s
and mul iple umo -associa ed immune cells), and ascula and
lympha ic essels ensional s uc u es, can be as impo an as he
gene ic ins abili y o umo cells in he pa hogenesis and e olu ion
o he malignan p ocess (42,49). In his ega d, a ious s udies
ha e demons a ed he impo ance o cell–ECM bio enseg i y in
cance (34,48,50). Indeed, a desmoplas ic eac ion is equen in
manysolid umo s,suchasb eas ,p os a e,colon,o lung,inwhich
high le els o TGF-b and PDGF a e ound. These g ow h ac o s
a e p oduced by he mesenchymal cells o he umo s oma and
induceimmunopheno ypicchanges. These changesa eobse able
by s udying ac in-alpha, myosin, imen in, desmin, and he al e ed
p oduc ion o se e al ECM p o eins, such as collagens, laminin,
enascin, ECM me allop o einases (MMP), and MMP-inhibi o s
(43). Addi ionally, ECM s i ness modula es cance p og ession;
cance cells p omo e s i ening o hei en i onmen , which in
u n eeds back o inc ease malignan beha io s such as loss o
issue a chi ec u e and in asion (51). Fo ins ance, he speed o
malignan cells in i o is a ec ed by he geome y o he ECM.
Human glioma cells mo e as e h ough na ow channels han
h ough wide channels o in non-s e ched 2D su aces. This is
hough o be igge ed by an inc ease in he pola i y o he ac-
ion o ces be ween cell and ECM (52). Indeed, ecen publica ions
desc ibe ha no only neoplas ic ECM s i ness, bu also he i m-
ness o umo cells play a signi ican ole in umo p og ession.
The i mness o umo cells, especially he me as a ic cells, has
been ound o be lowe han ha o he no mal cells o he same
sample, and is caused by he loss o ac in ilamen s and/o mic o-
ubules and he subsequen lowe densi y o sca old (53,54). In
his ega d, i has been shown ha he ans o ma ion om a
benign p oli e a i e cell in o a malignan cell can be p oduced by
a peculia pheno ypic change, known as epi helial–mesenchymal
ansi ion (55). This ans o ma ion in ol es b eaking con ac
wi h sis e cells and inc easing mo ili y, as a esul o a change in
he epi helial cy oskele on, wi h i s co esponding p op ie ies o
a pseudomesenchymal pheno ype, which enables mig a ion, in a-
sion, and dissemina ion (56). While no mal cells adhe e o hei
en i onmen h ough in eg ins, and hei body has a p ope con-
sis ency, umo cells lose ha consis ency and enseg i y,becoming
easily de o mable elemen s (causing pleomo phism), wi h high
elas ici y (enhancing in il a ion) and wi h an inc eased deg ee o
mobili y (enabling me as asis) (57). In b eas cance , he genomic
p o ile exp essing mainly mesenchymal ea u es is ac ually ound
in he mos in asi e cell lines (58). Mo eo e , i has been published
ha ch onic g ow h s imula ion, ECM emodeling, al e a ion o
cell mechanics, and dis up ion o issue a chi ec u e a e non-
gene ic in luences on cance p og ession (42,49). These ideas
no only ag ee wi h basic p edic ions o cellula enseg i y, bu
also suppo he idea ha he apy based on he manipula ion o
he bio enseg i y p inciple cues should be conside ed as a way o
e e he malignan pheno ype (59,60).
In cance esea ch, he hallma k which includes he physical
aspec s o issue has been less in es iga ed, bu i is known ha
his hallma k is one o he mos basic mechanisms in enhancing
umo p oli e a ion and c ea ing esis ance o cance ea men ,
among o he p ocesses (61). A p e ious s udy by ou g oup
akes in accoun some s uc u al elemen s o ECM ha ha e
he capaci y o in luence physical condi ions and sugges s ha
Schwannian s oma cells a e no he only impo an ac o in he
his opa hologic analysis o neu oblas ic umo s (50). Speci ically,
mul i-pa ame ic analysis o o he umo s oma componen s
(Re F, Col F, GAG, and immune cells) de ec ed by classic his-
ochemis y (HC) and immunohis ochemis y (IHC) echniques
andinco po a edin oaquan i a i emo phologicalanalysiswould
imp o e he alue o he In e na ional NB Pa hology Classi ica-
ion (62). As we will show la e , chemo he apy and adio he apy
a e known o ac on umo cells as well as on s omal cells and
ECM elemen s (63,64). As a consequence, inju y o he ECM can
con ibu e di ec ly o ea men esis ance, c ea ing niches o esis-
an umo cells (64,65). Fu he mo e, damage o DNA induces
he p oduc ion o cy okines and g ow h ac o s by s omal cells,
his igge s in lamma ion, cell su i al, and umo p og ession,
hus he e ec o he apy on ECM may be o p omo e elapse o
chemo esis ance (66,67). We hypo hesize ha s udying he di -
e en elemen s o he ECM, as one o he main con ibu o s o
bio enseg i y, h ough objec i e mo phome ic analysis and he
c ea ion o ma hema ical ne wo ks o his ologic sec ions s ained
wi h HC and IHC, can shed ligh on how bio enseg i y in luences
umo mic oen i onmen and could p o ide clues o i s ac ion
mechanism.
EVIDENCES OF ECM BIOTENSEGRITY CHANGES IN
MALIGNANT TISSUE
I is known ha umo cells al e he mechanical p ope ies o
he mic oen i onmen in o de o c ea e a o able condi ions o
hei p oli e a ion and/o dissemina ion (68). In addi ion, adhe-
sion molecules such as E-cadhe in a e in ol ed in he p ocesses o
issue di e en ia ion and mo phogenesis and play an impo an
ole in modula ing he in asi eness o umo cells in b eas cance
and o he epi helial umo s (69). Fo ins ance, he ecip ocal com-
munica ion be ween he s omal cells and he issue pa enchyma
di ec s gene exp ession, and in p os a e ca cinoma and b eas ca -
cinoma, he oncogenic po en iali y a ises om s oma-associa ed
ib oblas s, immune esponse, and he al e a ions o bio enseg-
i y (49,70). De egula ion and diso ganiza ion o he composi-
ion, s uc u e, and s i ness o he ECM elemen s p og essi ely
inc ease in e s i ial luid p essu e, leading o limi ed pene a ion
and dissemina ion o he apeu ic agen s wi hin solid umo s, hus
enabling he c ea ion o niches wi hin issues and o gans ha
o e sanc ua y o umo s andac i a e he apy esis ance p og ams
F on ie s in Oncology | Molecula and Cellula Oncology Ma ch 2014 | Volume 4 | A icle 39 | 4
Tadeo e al. Bio enseg i y o he ex acellula ma ix
(11–13,64,65). Tumo cells a e no he only cells ha change he
mechanical p ope ies o he mic oen i onmen . Despi e all he
e o s o umo cells o make ECM elemen s wo k o hei su -
i al and p oli e a ion, umo s omal cells, speci ically, immune
sys em cells, y o e e se he pa hological condi ion. Indeed,
wo lymphop oli e a i e synd omes ( ollicula lymphoma and
Hodgkin lymphoma) a e good examples o he ac ha a umo
can be conside ed as unc ional issue, connec ed and dependen
on he mic oen i onmen ,which sends and ecei es signals o and
om he umo issue i sel . In such synd omes, umo mic oen-
i onmen s omal cells, including immune esponse, de e mine
he mo phology, clinical s a i ica ion, agg essi eness, p ognosis,
and esponse o ea men o he umo (71).
In he nex wo sub-sec ions, we desc ibe he me hods de el-
oped o he s udy o bio enseg i y in neu oblas ic umo s.
MORPHOMETRIC ANALYSIS OF ECM ELEMENTS – AN EXAMPLE IN NB
Accu a e quan i ica ion o pa hology specimens using imaging
echnology o analyze he a ia ions in s uc u al issue ha a ise
om in e ac ions be ween umo and s oma cells and ECM ele-
men s is p o iding impo an in o ma ion. This app oach would
allow bio enseg al pa e ns o be included in compu a ional o -
mula ions o isk s a i ica ion sys ems and aid in designing be e
an i-cance ea men s a egies (29). Howe e , he alidi y o
he model depends on he quali y o he da a. This quan i i-
ca ion depends on he s aining, scanning, image analysis, and
s a is ical e alua ion. Fo ha pu pose, au oma ic s ained sec ions
mus be digi ized using mic oscopic p epa a ion scanne s such as
Ape io Scanscope XT (Ape io echnologies) o Pano amic Midi
(3Dhis ech) o wi h a pho omic oscope i a scanne is no a ail-
able. Di e en image analysis sys ems can be used, such as Image
P o-plus so wa e (Media cybe ne ics), ImageScope (Ape io ech-
nologies),Pano amic iewe (3DHis ech), eeso wa e (ImageJo
he NIH), o sel -designed so wa e o ob ain cus omized mac os
o algo i hms (in o ma ic p o ocols) o de ec and cha ac e ize
he quan i y (numbe o objec s and a ea occupied), size (a ea,
wid h, leng h), shape (aspec , oundness, pe ime e a io, ac-
al dimension), and o ien a ion (angle), among o he pa ame e s,
o he ECM elemen s o in e es . All sys ems p o ide ma k-up
images o masks, which ep esen he ecognized and measu ed
elemen in whi e upon a black backg ound. The use o issue
mic oa ays is ad ised o s anda diza ion pu pose o backg ound
sub ac ion and colo segmen a ion, gi en ha hese echniques
end o be dependen o he in ensi y o he s aining and algo-
i hms mus be ecalib a ed wi h e e y change o in ensi y/g ound
noise/con as s aining, hus losing objec i eness. Fu he de ails
ega ding objec i e quan i ica ion o di e en cell and ECM ele-
men s and a lowcha o he analysis used by ou g oup, a e
desc ibed elsewhe e (50).
Neu oblas ic cells a e known o be commi ed in a complex
in e ac ion wi h he su ounding umo mic oen i onmen and
webelie e ha pa ien swi hneu oblas ic umo s,speci ically hose
s ill subjec o he apeu ic ailu e despi e cu en knowledge, could
bene i om no el he apeu ic s a egies which could o igina e
om he s udy o ECM bio enseg i y. To in es iga e such new
he apeu ic a ge s, we ha e objec i ely quan i ied Re F, Col I
F, GAGs (Gomo i, Masson’s ich ome, and Alcian blue pH 2.5
HC, espec i ely), blood essels (CD31 IHC, Dako), lymph essels
(D2-40 IHC, Dako), and cell ma ke s, including leukocy e lin-
eage (CD45/LC IHC, Dako) and NB cells, in p ima y NB. A i s
app oach o hee alua iono he oleo ECMbio enseg i yinneu-
oblas ic umo s is he obse a ion o he ma k-up images o issue
mic oa ays cylinde s comp ising a mix u e o umo and no mal
issue ei he in he p ima y and/o me as a ic loca ion. In he pa -
icula case p esen ed in Figu e 2,included o illus a i e pu pose,
a clea dis up ion in he o ganiza ion o he ECM elemen s can be
obse ed when passing om he no mal issue a ea o he neoplas-
ic issue. In he umo a ea, Re F becomes diso ganized, Col I F
is sligh ly inc eased (al hough minimal), GAGs almos disappea ,
CD45 eac i e cells accumula e, and blood essels a y in size and
cha ac e is ics.
S a is ical analysis o he quan i a i e da a o ibe s, GAGs,
umo cells, and immune sys em ma ke s compa ed wi h he
cu en pa ame e s used o p edic isk o elapse (s age, age,
his opa hology, s a e o MYCN oncogene, s a e o 11q egion,
o e all genomic p o ile, and ploidy) (72–74) and o he gene ic
ma ke s o p ognos ic in e es in a subse o 78 p ima y neu-
oblas ic umo s has al eady been published by ou g oup, and
highligh s he in e es o s udying ECM in neu oblas ic umo s
(50). The ac ha ECM elemen s di e depending on he cha -
ac e is ics o he umo s and, mo e in e es ingly, he ac ha he
cha ac e is ics o ECM elemen s a e ela ed o p ognosis ( elapse
o o e all su i al) ad oca es on behal o he egula o y ole o
ECM bio enseg i y in umo p og ession.
DEVELOPMENT OF MATHEMATICAL TOPOLOGY OF ECM
ELEMENTS – AN EXAMPLE IN NB
The combina ion o mul idisciplina y e o s by clinicians, biol-
ogis s, pa hologis , bioenginee s, and bios a is icians could eluci-
da e how ECM elemen s in e ac wi h umo and s omal cells. In
his espec , a new and in e es ing app oach is o analyze biopsies
by con e ing he issue in o a ma hema ical ne wo k o cell- o-cell
con ac s (75–78). Using g aph heo y concep s, hese ne wo ks can
p o ide o ganiza ional in o ma ion ha seems ele an in emb y-
ologic de elopmen and disease. Fo example, his me hod has
been applied o he analysis o neu omuscula diseases, se ing
as a diagnos ic ool able o quan i y he se e i y o he pa hology
in a muscle biopsy (77). We p opose ha his echnology can be
adap ed o he analysis o umo biopsies. I is al eady possible o
compa e di e en ma k-up images ob ained om he analysis o
se e al ma ke s which ha e been assessed on se ial hin sec ions
wi h p ese ed his ology. These o e lapping images enable se -
e al ma ke s o be conside ed a he same ime and allow he
co-loca ion and s udy o he in e ac ion be ween con inuous en-
sional elemen s and discon inuous comp ession elemen s. In his
ega d,we ha e analyzed he ela ionship be ween di e en biopsy
componen s aking he cell nuclei as a e e ence. The p ocedu e
is based in he iden i ica ion o he cell nuclei and he calcula-
ion o hei espec i e cen oids. These cen oids se e as seeds
o pe o m a Vo onoi diag am o Vo onoi cells (79,80). A new
pa i ioned image is p oduced in which each nuclei is associa ed
wi h i s co esponding Vo onoi cell. In his way, i is possible o
cons uc a ne wo k based on he neighbo ingVo onoi cells. Topo-
logical app oaches and he use o Vo onoi cells need o be able
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Tadeo e al. Bio enseg i y o he ex acellula ma ix
FIGURE 2 |Tissue mic oa ay cylinde s ep esen ing an ad enal
gland in il a ion by a poo ly di e en ia ed neu oblas oma.
Hema oxylin and eosin s aining o (A) ad enal gland and (A’) umo
issue. (B–F) Se e al his ochemical and immunohis ochemical
s ainings o ex acellula ma ix elemen s and (B’–F’) hei
co esponding ma k-up images a e image analysis a e shown. The
cylinde issue was s ained wi h (B) Gomo i o Re F, (C) Masson’s
ich ome o Col I F, and (D) Alcian blue pH 2.5 o GAGs; and
immunos ained wi h (E) an i-CD45 o leukocy e-common an igen and
(F) an i-CD31 o Blood essels. Tumo s end o dis up he opology o
he ECM o he o gan in which hey se le, p oducing changes in
bio enseg i y. AG, an igen.
o cap u e he p esence and ela i e disposi ion o issue he e o-
genei ies de i ed om, o example, he luminal space o glands,
blood and lymph essels, o la ge ex acellula spaces. They will
be e lec ed h ough di e en cha ac e is ics and will be aken in o
accoun o he s udy, es ing i hey can be pa o he ele an ea-
u es ha de ine a speci ic condi ion. The selec ion o egions o
in e es in each biopsy will allow s udying only umo issue a eas
wi hou a i ac s ha could bias he s udy, leading o w ong con-
clusions. The combina ion o g aph- ela ed pa ame e s wi h he
mo phome ic in o ma ion will enable a comp ehensi e analysis
o he changes a ising om di e en comp ession o ces in ela-
ion o he di e en ypes o ECM (s i /so , o ganized/chao ic)
in combina ion wi h he umo s oma cells such as immune cell
in il a es. We ha e pe o med p elimina y compa isons based
F on ie s in Oncology | Molecula and Cellula Oncology Ma ch 2014 | Volume 4 | A icle 39 | 6
Tadeo e al. Bio enseg i y o he ex acellula ma ix
FIGURE 3 | Ne wo k cons uc ion om a NB biopsy image.(A) Gomo i
s aining o isualize Re F and nuclei om he NB biopsy. (B) Combina ion
o nuclei (g een) and Re F ma k-up images ob ained om (A). The whi e
ci cum e ence ep esen s he egion o in e es p ocessed in (C).(C) Each
nucleus (g een) is aken as a seed o build a Vo onoi diag am ( ed) o
Vo onoi cells. This allows he c ea ion o a ne wo k o cell- o-cell con ac s
(black lines) ha can be used o ex ac opological cha ac e is ics o he
issue.
on he gene ic ea u es o NB using Re F and blood essels (in
addi ion o he nuclei) as he e e ence ea u es o p o iding he
biological clues. This p ocedu e has shown some hin s o dis-
c imina ion ega ding he o ganiza ion and co-loca ion o hese
elemen s (Figu e 3). We ound ha some ne wo k cha ac e is ics
we e ele an o pe o m his ini ial sepa a ion. This sugges s ha
di e se backg ounds can espond di e en ly o he pa hological
p ocess depending on he o ganiza ion o he umo . Following
he same app oach, we will use o he ma k-up images o he posi-
i e cells s ained wi h he di e en monoclonal an ibodies agains
he di e en cells o he leukocy e lineage. We hope ha his com-
bina ion o ma hema ical and s a is ical me hods will answe he
ques ion on he ela ionships be ween ECM bio enseg i y and he
changes media ed by he cell in il a e.
EFFECT OF TREATMENT ON TUMOR MICROENVIRONMENT
AND CONSEQUENCES
The e is much e idence ha he lack o o al speci ici y o can-
ce he apeu ic agen s (chemo he apy and adio he apy) causes
colla e al damage o he mechanical p ope ies o he umo
ECM and benign s omal cells (which we e p e iously igh ing
he umo ), c ea ing esis ance o he apy and a o ing elapse
and me as asis. This ac becomes e iden while analyzing pos -
ea men biopsies, which con ain a high deg ee o ib osis and
calci ica ion. Some s udies ha e shown ha cance he apy can
some imes damage umo DNA and s omal cells, which esul s in
he sec e ion o a spec um o p o eins, including he Wn am-
ily membe s. Fo example, in p os a e cance , he exp ession o
his p o eins in he umo mic oen i onmen , egula ed by lymph
B cells, a enua es he e ec s o cy o oxic chemo he apy in i o,
p omo ing umo cell su i al and disease p og ession (63,81).
I has also been epo ed ha in ollicula lymphoma and di -
use la ge cell lymphoma, ea men wi h lenalidomide a ec s
he immune synapses o in a- umo al T lymphocy es (82). In
b eas cance , ea men wi h doxo ubicin esul s in an inc ease in
ibuline-1, an ECM p o ein, and i s binding p o eins, ib onec in
and laminin-1, which cons i u e a sou ce o chemo esis ance (83)
and igge s o e exp ession o maspin p o ein, which induces he
accumula ion o collagen ibe s, hus causing disease p og ession
(84). A no el Toll ecep o -9-dependen mechanism ha ini ia es
umo eg ow h a e local adio he apy has also been epo ed
(85). Monoclonal an ibodies agains ibulin-1 a e able o e e se
such chemo esis ance, and he inhibi ion o MMP seems o ha e
a he apeu ic e ec (86).
An example o he e ec o ea men in NB is shown in
Figu e 4. When compa ing a p ima y NB wi h i s non-p ima y
sample, we can app ecia e ha Re F, GAGs, and Col I F accu-
mula e in he ECM o he pos - ea men sample. The amoun
o blood mac o ascula u e is sligh ly dec eased. All hese indings
desc ibe a s i e ECM a e mul imodal ea men .
POTENTIALITY OF MECHANOTHERAPY
The changes exe ed on ECM by he apeu ic agen s and he pe -
spec i e o he epi helial–mesenchymal ansi ion in epi helial
umo s ha e opened he doo o a new line o ea men , which
conside s he gene ic and epigene ic mechanisms associa ed wi h
esis ance o chemo he apy (59,87). The e is a need o pe sonalize
he apeu ics aking in o accoun no only he ea u es known o
ha e p ognosis impac , bu also new ma ke s, such as he mechan-
ical s ess o he umo ECM elemen s. Indeed, because o i s
impo ance o he umo , he ECM ep esen s an “Achilles heel”
ha can be exploi ed in designing cance he apy. The bione -
wo k be ween he ECM and umo cells is dynamic, and o e e y
ac ion, such as exposu e o geno oxic s ess, he e a e eac ions
and consequences h oughou he mic o and mac osys em (65).
Remo alo ECM ba ie s willei he ha eadi ec nega i e e ec on
umo cells o acili a e an i- umo immune esponses and d ug
ea men , h ough be e in a- umo al pene a ion and acces-
sibili y o a ge cells. In his ega d, a numbe o expe imen al
app oaches a e aimed owa d he ansien deg ada ion o down
egula ion o ECM p o eins using injec ion o ECM-deg ading
enzymes in o he umo o hei in a- umo al exp ession a e
i al- o s em cell-based gene ans e (65). O he app oaches
a emp o indi ec ly dec ease umo -associa ed ECM by killing
umo s omal cells ha p oduce ECM p o eins (64) o aim
a enhancing he hos immune esponse (88). The po en iali y
o he apeu ic agen s o modi y ECM can be u ned a ound in
such a way ha new chemicals can be applied o modi y a gi en
ECM s i ness o composi ion in o one shown o igge a be e
p ognosis.
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Tadeo e al. Bio enseg i y o he ex acellula ma ix
FIGURE 4 | Changes in he ECM a e ea men in one neu oblas ic
umo . Se e al s ainings (HC and IHC) o (A–E) a p ima y neu oblas oma and
(A’–E’) i s pos - ea men biopsy a e shown.The p esen ed ma k-up images
o (B,B’) Re F, (C,C’) Col I F, and (D,D’) GAGs show ha hese ECM elemen s
a e inc eased in NPNB. Rega ding blood essels (E,E’), he amoun o does
no seem o change a e ea men bu he ype o essels has changed in
such a way ha hey a e smalle in NPNB.The emodeling cha ac e is ics o
he ECM componen s, wi h di e en mechanical p ope ies, a e di e en ially
ela ed o p ognos ically signi ican clinical and biological ea u es in NB. The
pe cen ages o s ained a ea a e indica ed. PNB, p ima y neu oblas oma;
NPNB, non-p ima y neu oblas oma (a e ea men ); HE, hema oxylin and
eosin.
CONCLUDING REMARKS
Bio enseg i y is he s uc u al p inciple o mechano he apy. Cells
a e linked bo h o each o he and o he ECM o ming a mechan-
ical bio enseg al sys em in homeos asis. Cell–cell junc ions a e
ancho ed o neighbo ing cells and ocal adhesions o ECM, allow-
ing o ces o c oss ia in a-cellula cy oskele al and nuclea ne -
wo ks. These s uc u es luc ua e, and he mul iple esponses
appea o s ongly a ec issue emodeling and cell ans o -
ma ion. As desc ibed, no mal o gans issue, p ima y NB, and
pos - ea men NB ha e a di e en amoun and opog aphy o
bio enseg al ECM elemen s. Con en ional app oaches ha e a-
di ionally ocused on he neoplas ic cell. Mo eo e , an a senal o
mechano he apeu ic app oaches o enhance he e icacy o mo e
classical cance he apeu ics and o e come ea men esis ance
has al eady been disco e ed. To achie e mo e e ec i e pe son-
alized s a egies, u he s udies should conside o imp o e he
de ini ion o he in e ac ions be ween umo and s omal cells and
he ECM elemen s and ascula cons i uen s o he umo , as well
as hei in luence on ea men . We p opose ha in eg a ing he
umo opo- unc ional ne wo ks o he ECM elemen s wi h he
clinical, his opa hology, and gene ic in o ma ion could p o ide
new in o ma ion abou he impac o bio enseg i y on pa ien
ca e. Unde s anding he mechanical p ope ies o umo ECM
componen s, ela ed o a ia ions in quan i y, deg ee o in e e -
ence, and ypes o o ganiza ion, is key o de ining new po en ial
mechano he apeu ic a ge s and agen s.
ACKNOWLEDGMENTS
This s udy was suppo ed by g an s om he FIS (con ac
PI10/15) and RTICC (Red Tema ica de In es igacion Coope -
a i a en Cance , con ac s RD06/0020/0102; RD12/0036/0020),
Ins i u o Ca los III Mad id, and ERDF (Eu opean Regional De el-
opmen Fund). Luis Ma ía Escude o was suppo ed by he Miguel
Se e p og am (Ins i u o Ca los III).We would like o hank Ma -
cial Ga cia-Rojo (Pa hology Uni , Gene al Hospi al, Ciudad Real,
Spain) o excellen echnical assis ance; SEHOP (Spanish Soci-
e y o Hema ology and Pedia ic Oncology) and Desi ee Ramal,
Vic o ia Cas el, and Adela Cañe e (Pedia ic Oncology Uni , Uni-
e si y Hospi al La Fe, Valencia, Spain) o pa ien clinical da a
managemen ; and Da id Ha ison o English language assis ance.
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