Cellular and Molecular Mechanisms Underlying Glioblastoma and Zebrafish Models for the Discovery of New Treatments

cance s
Re iew
Cellula and Molecula Mechanisms Unde lying Glioblas oma
and Zeb a ish Models o he Disco e y o New T ea men s
Ped o Reimunde 1,2,*,† , Alba Pensado-López 3,4,† , Ma ín Ca ei a C ende 3, Vanesa Lombao Iglesias 3,
Lau a Sánchez 3, Ma a To ecilla-Pa a 5, C is ina M. Ramí ez 5, Clémen An ay 6
and Fe nando To es Andón4,6,*


Ci a ion: Reimunde, P.;
Pensado-López, A.; Ca ei a C ende,
M.; Lombao Iglesias, V.; Sánchez, L.;
To ecilla-Pa a, M.; Ramí ez, C.M.;
An ay, C.; To es Andón, F. Cellula
and Molecula Mechanisms
Unde lying Glioblas oma and
Zeb a ish Models o he Disco e y o
New T ea men s. Cance s 2021,13,
1087. h ps://doi.o g/10.3390/
cance s13051087
Academic Edi o s: Da id Wong and
Pie e A. Robe
Recei ed: 27 Janua y 2021
Accep ed: 1 Ma ch 2021
Published: 3 Ma ch 2021
Publishe ’s No e: MDPI s ays neu al
wi h ega d o ju isdic ional claims in
published maps and ins i u ional a il-
ia ions.
Copy igh : © 2021 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
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1Depa men o Medicine, Campus de Oza, Uni e sidade da Co uña, 15006 A Co uña, Spain
2Depa men o Neu osu ge y, Hospi al Uni e si a io Lucus Augus i, 27003 Lugo, Spain
3
Depa men o Zoology, Gene ics and Physical An h opology, Campus de Lugo, Uni e sidade de San iago de
Compos ela, 27002 Lugo, Spain; [email p o ec ed] (A.P.-L.); [email p o ec ed] (M.C.C.);
[email p o ec ed] (V.L.I.); [email p o ec ed] (L.S.)
4Cen e o Resea ch in Molecula Medicine and Ch onic Diseases (CiMUS), Uni e sidade de San iago de
Compos ela, 15706 San iago de Compos ela, Spain
5IMDEA Resea ch Ins i u e o Food and Heal h Sciences, 28049 Mad id, Spain;
[email p o ec ed]g (M.T.-P.); [email p o ec ed]g (C.M.R.)
6IRCCS Is i u o Clinico Humani as, Via A. Manzoni 56, 20089 Rozzano, Milan, I aly;
clemen .an ay@humani as esea ch.i
*Co espondence: ped [email p o ec ed] (P.R.); e nando. o [email p o ec ed] (F.T.A.)
† These au ho s ha e con ibu ed equally o his wo k.
Simple Summa y:
Glioblas oma (GBM) is one o he g ea es challenges acing neu o-oncology oday.
Cu en ea men s a e a om sa is ac o y and, gi en he poo p ognosis o he disease, he apeu ic
e o s a e ocused on pallia i e managemen a he han cu a i e in e en ion. He e, we e iew he
cellula he e ogenei y o GBM, including umo cells and mic oglia/mac ophages among o he s, as
well as he gene ic, epigene ic and me abolic al e a ions con olling i s ini ia ion and p og ession.
Then, we desc ibe he gene ic and xeno ansplan a ion zeb a ish models es ablished in he las ew
yea s o he s udy o GBM physiopa hology and o es ing new d ugs o imp o e he ea men o
he disease. Taking his in o ma ion in o accoun , o hcoming s udies using zeb a ish models o GBM
a e expec ed o shed ligh on be e diagnosis and ea men s, hus p o iding hope o GBM pa ien s.
Abs ac :
Glioblas oma (GBM) is he mos common o all b ain malignan umo s; i displays a
median su i al o 14.6 mon hs wi h cu en comple e s anda d ea men . High he e ogenei y, ag-
g essi e and in asi e beha io , he impossibili y o comple ing umo esec ion, limi a ions o d ug
adminis a ion and he apeu ic esis ance o cu en ea men s a e he main p oblems p esen ed by
his pa hology. In ecen yea s, ou knowledge o GBM physiopa hology has ad anced signi ican ly,
gene a ing ele an in o ma ion on he cellula he e ogenei y o GBM umo s, including cance and
immune cells such as mac ophages/mic oglia, gene ic, epigene ic and me abolic al e a ions, comp is-
ing changes in miRNA exp ession. In his scena io, he zeb a ish has a isen as a p omising animal
model o p og ess u he due o i s unique cha ac e is ics, such as anspa ency, ease o gene ic
manipula ion, e hical and economic ad an ages and also conse a ion o he majo b ain egions
and blood–b ain–ba ie (BBB) which a e simila o a human s uc u e. A ew pape s desc ibed in
his e iew, using gene ic and xeno ansplan a ion zeb a ish models ha e been used o s udy GBM
as well as o es he an i- umo al e icacy o new d ugs, hei abili y o in e ac wi h a ge cells,
modula e he umo mic oen i onmen , c oss he BBB and/o hei oxici y. P ospec i e s udies
ollowing hese lines o esea ch may lead o a be e diagnosis, p ognosis and ea men o pa ien s
wi h GBM.
Keywo ds:
glioblas oma; cance ; umo mic oen i onmen ; glioma-associa ed mic oglia/mac ophages;
gene ics; me abolism; miRNA; zeb a ish; d ug disco e y
Cance s 2021,13, 1087. h ps://doi.o g/10.3390/cance s13051087 h ps://www.mdpi.com/jou nal/cance s
Cance s 2021,13, 1087 2 o 37
1. In oduc ion
Glioblas oma (GBM) is one o he g ea es challenges acing neu o-oncology oday.
This disease ep esen s he mos common o all b ain malignan umo s, comp ising mo e
han 50% o exis ing high-g ade gliomas. GBM has an annual global incidence o 3–5 cases
pe 100,000 inhabi an s and a sligh p edominance in males. This incu able malignan
umo , wi h almos non-exis en long- e m su i o s, can occu a any age bu p esen s a
clea peak wi h he highes incidence in he six h decade o li e [1,2].
Mos GBM a ise de no o, while seconda y GBM umo s commonly de elop om
lowe g ade gliomas [
1
]. The complexi y o managing GBM pa ien s depends on many
ac o s, including umo size and loca ion, age, he Ka no sky Pe o mance Scale Index,
umo his ology and he s a us o molecula ma ke s. The diagnosis o GBM is based on
his ological echniques, wi h a high-g ade glioma being de ined by a ypia, cellula pleo-
mo phism, mi osis, ascula p oli e a ion and nec osis, acco ding o he c i e ia es ablished
by he Wo ld Heal h O ganiza ion (WHO) classi ica ion. By de ini ion, GBM co esponds o
a g ade IV and has he wo s p ognosis among in il a ing gliomas [
3
]. In clinical p ac ice,
isocy a e dehyd ogenase (IDH) mu a ions and me hyla ion o he p omo e O(6)-me hyl
guanine me hyl ans e ase (MGMT) gene a e ou inely e alua ed o imp o e diagnosis
and he classi ica ion o umo s and o es ima e he sensi i i y o he umo o alkyla ing
agen s such as emozolomide (TMZ) [
4
,
5
]. Despi e his, he agg essi e and in asi e de el-
opmen and g ow h o b ain umo s, he impossibili y o comple ing umo esec ion, he
limi a ions o d ug adminis a ion and he apeu ic esis ance o ea men a e he main
p oblems p esen ed by his pa hology. While o e all su i al a e su gical esec ion o
he umo is 3 o 6 mon hs, he inclusion o adio he apy in he ea men plan inc eases
his pa ame e o 12.1 mon hs (2-yea su i al o 10.4%) and a sligh inc ease in su i al o
14.6 mon hs (2-yea su i al 26.5%) can be achie ed by he addi ion o concomi an and
adju an chemo he apy wi h TMZ. In addi ion, cu en ea men s a e a om sa is ac o y
and, gi en he poo p ognosis o he disease, he apeu ic e o s a e mainly ocused on
pallia i e managemen a he han cu a i e in e en ion.
In his scena io, di e en p omising lines o esea ch ha e been ini ia ed o he de-
elopmen o new he apeu ic s a egies o ea GBM [
2
]. F om a cellula and molecula
poin o iew, GBM umo s p esen high he e ogenei y [
6
–
9
], which con ibu es o hei
ecu ence and he apeu ic esis ance [
10
,
11
]. In addi ion, unde s anding he umo mi-
c oen i onmen (TME), he cellula o igin and he molecula al e a ions o he umo cells
and immune ela ed cells will po en ially help o imp o e he diagnosis, p ognosis and
ea men o he disease.
The zeb a ish (Danio e io) has become a well-es ablished model o s udying he
physiopa hological ea u es and sc eening o new ea men s o se e al human diseases,
including cance . Rapid emb yo de elopmen , i s small size and i s anspa ency, gene ic
and physiological conse a ion and e hical and economic ad an ages ha e made zeb a ish
s and ou om all o he
in i o
models [
12
]. Conce ning GBM, he simplici y o pe o m
gene ic manipula ions and he ansplan a ion o human umo cells, oge he wi h he
conse a ion o he majo b ain subdi isions in his species, enables esea che s o eca-
pi ula e he cha ac e is ics o human umo s and hei ela ed TMEs, in u n, allowing he
mechanism o ac ion o new he apeu ic s a egies o be e alua ed [13].
In his manusc ip , we e iew he physiopa hology o GBM om a cellula and
molecula pe spec i e, wi h a pa icula ocus on mac ophages/mic oglia in he TME,
gene ic, epigene ic and me abolic al e a ions and possibili ies o hei in e en ion by
mic oRNA (miRNA) manipula ion. Then, we p o ide ou iew on he oppo uni ies and
challenges o zeb a ish models o imp o ing ou unde s anding o he disease and he
e alua ion o new ea men s.
Cance s 2021,13, 1087 3 o 37
2. Cellula Pa hology and Tumo Mic oen i onmen in Glioblas oma:
Mac ophages/Mic oglia
I is desi able o unde s and he cellula o igin o he disease and he composi ion
o he TME o es ablish an ea ly diagnosis, iden i y he apeu ic a ge s and imp o e i s
ou come. The genomic, epigenomic and ansc ip omic cha ac e iza ion o GBMs has
p o ided in o ma ion which allowed o es ablish he di e en umo sub ypes: p oneu al,
mesenchymal and classical [
14
–
19
]. Abe a ions in he exp ession o pla ele -de i ed
g ow h ac o ecep o alpha (PDGFRA), neu o ib oma osis ype I (NF1) and epide mal
g ow h ac o ecep o (EGFR) we e associa ed wi h he p oneu al, mesenchymal and
classical sub ypes, espec i ely [
19
]. Howe e , hese mu a ions can co-exis wi hin a single
umo , bo h a he egional and single-cell le els, hus, in his case he designa ed sub ypes
e lec he dominan ansc ip ional p og am o a speci ic umo wi hin a pa icula ime
and space o sample isola ion [
8
,
18
–
20
]. A e oncogenic al e a ions, GBM cells can be
di e en ia ed and de-di e en ia ed and can acqui e s em cell p ope ies [
21
,
22
]. All o
his sugges s ha gene ic, epigene ic and me abolic al e a ions ( e iewed in Sec ion 3)
can be a ge ed, a he han simply killing a pa icula popula ion o umo cells, so a e
aluable s a egies o ea he disease. In addi ion, o umo cells, he he apeu ic a ge ing
o immune cells has gained high ele ance in he las decade wi h he ou s anding esul s
achie ed by an i- umo al immuno he apy in o he ypes o cance (i.e., melanoma and
lung cance ) [23].
The glioma issue is in il a ed by many cells o di e en on ogenies, mos ly esi-
den mic oglia and umo -in il a ing monocy es, which a e deno ed glioma-associa ed
mac ophages/mic oglia (GAMs) and which can ep esen om 30 o 50% o he o al cells
in he umo [
24
–
26
]. His o ically, mac ophages and mic oglia had been hough o be
ansposable in he TME as hey sha e immunologic unc ions such as phagocy osis o
an igen p esen a ion. Howe e , in ecen yea s, single-cell RNA sequencing (RNA-seq) pe -
o med on GAMs om human glioma issues ha e iden i ied wo pheno ypically dis inc
subse s associa ed wi h gene signa u es o mic oglia-en iched and bone ma ow-de i ed
mac ophages [
27
]. These indings ha e also e ealed key di e ences in he way he glioma
mic oen i onmen shapes he ansc ip ional exp ession o hese cells, in pa icula a he
le el o genes in ol ed in he sec e ion o in lamma o y cy okine and an igen p esen a-
ion [
28
–
30
]. Mo eo e , a e mapping analysis has e ealed ha mic oglia cells de i e
om p imi i e myeloid p ogeni o s which en e he b ain du ing emb yogenesis [
31
].
Con e sely, ci cula ing blood-monocy es ypically en e he b ain in pa hological si ua ions
and hey di e en ia e in o mac ophages [
32
]. The exac con ibu ion o mic oglia and ci cu-
la ing blood-monocy es o he o al pool o GAMs is s ill an open ques ion. In some models,
mic oglia p edominance has been epo ed [
33
], whe eas o he s ha e demons a ed ha
in il a ing monocy es a e accoun able o he majo i y o he GAM popula ion [
29
,
34
].
Ne e heless, he key d i e s o GAM ec ui men a e con ex ual, as hese chemokines a e
he e ogeneously exp essed among glioma, p obably e lec ing he di e si y in glioma ge-
ne ics. T ansc ip ional classi ica ions ha e e ealed ha his di e si y in luences he ex en
o GAM in il a ion. Fo ins ance, phospha ase and ensin homolog (PTEN) de iciency in
glioma d i es mac ophage in il a ion ia up- egula ion o lysyl oxidase (LOX) [
35
] and in
mesenchymal GBMs neu o ib omin 1 (NF1) de iciency esul s in inc eased GAM in il a ion
compa ed wi h p oneu al o classical sub ypes [
18
,
36
,
37
]. No ably, i is now ecognized
ha mic oglia and mac ophages colonize di e en egions o gliomas. Monocy e-de i ed
mac ophages a e en iched in he umo co e whe e hey occupy pe i ascula egions, while
mic oglia-de i ed umo associa ed mac ophages a e ypically ound a he umo pe iph-
e y [
29
,
38
]. In a i al 2-pho on mic oscopy has e ealed ha hese wo subse s a e also
mo phologically dis inc , monocy e-de i ed cells, being small and highly mig a o y, while
mic oglia a e la ge, b anched cells wi h highly ac i e p ocesses con inuously ex ending
and e ac ing wi hin umo s [
39
]. Fu u e s udies will be necessa y o in es iga e whe he
his di e gence is ela ed o a di e en ac i i y and i hese wo popula ions a ec umo
g ow h di e en ly.
Cance s 2021,13, 1087 4 o 37
Al hough he mechanisms by which GAMs mig a e o he umo si e a e no en i ely
clea , some s udies ha e shown ha hei ec ui men is media ed by a ious glioma-
de i ed ac o s such as CCL2, CX3CL1, SDF-1, CSF-1, GM-CSF and EGF, which ac as
chemoa ac an s o GAMs and media e he c oss alk be ween umo cells and he in-
na e immune sys em [
40
] (Figu e 1). In an NF1 mu an op ic glioma mu ine model, Guo
e al. showed ha CX3CL1 is a key chemokine esponsible o he a ac ion o mic oglia
o he umo [
36
]. Using CX3CR1- and CCR2-enginee ed mu ine models, Chen e al.
demons a ed ha in glioma, mic oglia only exp ess CX3CR1, whe eas mos in lamma-
o y monocy es/mac ophages exp ess bo h CX3CR1 and CCR2. This sugges s ha CCL2
is a majo a ac an o monocy es. Acco dingly, in his model, CCL2 deple ion led o
p o-longed su i al [
29
]. CCL2 p oduced by bo h glioma cells and GAMs, has also been
shown o be essen ial o he ec ui men o T eg cells and myeloid-de i ed supp esso
cells [
41
], which ac as d i e s o he immune-supp essi e pheno ype ypical o hese
umo s. Ano he ecen s udy has sugges ed ha os eopon in migh also ac as an im-
po an chemokine o mac ophage ec ui men o GBM umo s igge ing i s binding o
in eg in
α
β
5 [
42
]. T-cell dys unc ion is also a hallma k o high-g ade glioma, e lec ing
he ac i e immunosupp essi e mic oen i onmen which con ibu es o umo immune
escape in pa ien s. Takenaka e al. showed ha exp ession o CCR2 is p omo ed in GAMs,
by he ac i a ion o he a yl hyd oca bon ecep o (AHR), causing cy o oxic T-cell dys-
unc ion h ough he CD39/CD73/adenosine pa hway [
43
]. An i- umo al unc ions o
umo -in il a ing lymphocy es (TILs) a e impai ed by mul iple immunosupp essi e ac o s
p oduced by GAMs and glioma cells and hese ac o s a e also mo e likely o up- egula e
mul iple immune checkpoin molecules such as PD-1, TIM-3 and LAG-3 [44].
Pheno ypically, i is now well documen ed ha GAMs do no i wi h he classical
M1/M2 mac ophage dicho omy [
45
]. O iginally sugges ed o be simpli ied as mac ophages,
he pheno ypic dicho omy assigned o T-helpe cells (Th1/Th2), he M1/M2 denomina ions
o mac ophages, we e la e desc ibed by Man o ani e al. as he wo ex eme poles o a
con inuum o pheno ypes and unc ions which could be acqui ed by e y plas ic immune
cells [
46
]. In his ou line, classically ac i a ed M1-like mac ophages p esen a p o o yp-
ical p o-in lamma o y esponse and an i- umo unc ions, while al e na i ely, ac i a ed
M2-like mac ophages, esembling umo associa ed mac ophages (TAMs), p esen an i-
in lamma o y p ope ies, a he same ime as suppo ing umo p og ession, angiogenesis
and me as asis and las o all, p e en adap i e immune esponses [47].
In he con ex o glioma, using genome-wide mic oa ay analysis, Szulzewsky e al.
e ealed ha a signi ican amoun o genes up- egula ed in GAMs do no i wi h he
M1/M2 classi ica ion [
48
] and he single cell RNA-seq demons a ed ha GAMs exp ess
a he a mix o M1/M2 ma ke s [
27
,
49
]. Despi e such con o e sy, some ypical M2 ma ke s
o mac ophage pola iza ion which a o umo p og ession ha e also been ound in glioma,
some examples o which a e p o ided below. Glioma he e ogenei y may explain why
cu en ly, he co ela ion be ween TAM in il a ion in glioma and pa ien ou come is s ill
no clea . Some s udies ha e epo ed on a posi i e co ela ion be ween pa ien ou comes
and he p esence o TAMs in he i al umo co e in IDH1R132H-non-mu an GBMs [
49
]. In
ano he s udy, M2-like TAM numbe s, iden i ied wi h CD204, inc eased wi h malignancy
g ade and we e associa ed wi h a poo p ognosis, while high IBA-1 in ensi y, a ma ke o
ac i a ed mac ophages, co ela ed wi h a longe su i al [
50
]. Simila ly, compa ed wi h
IDH-wild ype GBM, a educed numbe o mac ophages which we e mo e o ien ed owa ds
a p o-in lamma o y M1-like ac i a ion s a e we e ound in IDH-mu an GBM pa ien s,
possibly con ibu ing o hei p olonged su i al [
51
]. As a whole, o unde s and GAM
biology in gliomas, i is c i ical o e alua e an in eg a ed ision, including hei on ogeny,
genomic and pheno ypic di e si y, di e en ial loca ion in he TME and unc ional ac i i y.
P eclinical s udies using animal models ha e shown ha GAMs play a majo ole in
gliomagenesis and sus ain umo g ow h in bo h low-g ade and high-g ade gliomas. In
mu ine models, deple ion o mac ophages/mic oglia has led o a educ ion in umo
g ow h [
32
] and in line wi h his, some ac o s eleased by GAMs such as IL-6, IL-1
β
,
Cance s 2021,13, 1087 5 o 37
EGF, STI-1 and TGF-
β
can p omo e umo g ow h [
32
]. Indeed, he ole o mic oglia in
glioma in asi eness has also been ela ed o he sec e ion o SIP1, EGF and TGF-
β
[
52
].
Mo eo e , he ac i a ion o TLR2 exp essed on mic oglia, has igge ed he elease o ma ix
me allop o einases (MMP2 and MMP9) [
53
,
54
], deg ading he ex acellula ma ix and
acili a ing he in asion o glioma cells. Toll-like ecep o 4 (TLR4) signaling in mic oglia
has been shown o induce he elease o IL-6 [
55
], a ligand o he signal ansduce and
ac i a o o he ansc ip ion 3 (STAT3) pa hway in gliomas, which is also in ol ed in
in asi eness [
56
]. Up- egula ion o TGF-
β
and M-CSFR in he con ex o hypoxia, he
main ea u e o high-g ade gliomas, has been shown o induce he M2-like p o umo al
pola iza ion o GAMs in mu ine glioma models [
57
,
58
]. The adminis a ion o ac i la ine,
a hypoxia-inducible ac o (HIF) inhibi o , hampe ed GAM en ichmen and pola iza ion
and signi ican ly inhibi ed umo p og ession [38].
Cance s 2021, 13, x 5 o 40
Figu e 1. Rep esen a ion o in e ac ions be ween glioma cells, mac ophages/mic oglia and o he componen s o he u-
mo mic oen i onmen . Mac ophages and mic oglia (GAMs) a e a ac ed o he umo by se e al glioma cells-de i ed
ac o s such as CCL2, CX3CL1, SDF-1, CSF-1, GM-CSF, EGF o OSTP, exp essed in esponse o en i onmen al s ess
(such as hypoxia) o GAMs-de i ed ac o s hemsel es. In he umo mic oen i onmen , GAMs exe a umo -suppo ing
ac i i y h ough sec e ion o ac o s such as IL-6 (pa icula ly in esponse o TLR4 ac i a ion in mic oglia), IL-1β, EGF,
STI-1, TGF-β o VEGF ha ac i a e di e en signaling pa hways (such as JNK, MAPK o STAT3 in glioma cells) p o-
mo ing p oli e a ion, angiogenesis o in asion o umo cells. In mic oglia, TLR2/6 ac i a ion has been shown o induce
MMP2 and MMP9 exp ession, con ibu ing o ex acellula ma ix deg ada ion and umo in asi eness. GAMs a e also
immunosupp essi e e ec o s, especially ia he exp ession o molecules which lead o T-cell dys unc ion (CD80/86,
PD-L1 o CD39). The phagocy ic ac i i y o GAMs is also impai ed by he exp ession o CD47 (binding o SIRPα) by
glioma cells.
In he con ex o glioma, using genome-wide mic oa ay analysis, Szulzewsky e al.
e ealed ha a signi ican amoun o genes up- egula ed in GAMs do no i wi h he
M1/M2 classi ica ion [48] and he single cell RNA-seq demons a ed ha GAMs exp ess
a he a mix o M1/M2 ma ke s [27,49]. Despi e such con o e sy, some ypical M2
ma ke s o mac ophage pola iza ion which a o umo p og ession ha e also been
ound in glioma, some examples o which a e p o ided below. Glioma he e ogenei y
may explain why cu en ly, he co ela ion be ween TAM in il a ion in glioma and pa-
ien ou come is s ill no clea . Some s udies ha e epo ed on a posi i e co ela ion be-
ween pa ien ou comes and he p esence o TAMs in he i al umo co e in
IDH1R132H-non-mu an GBMs [49]. In ano he s udy, M2-like TAM numbe s, iden i ied
wi h CD204, inc eased wi h malignancy g ade and we e associa ed wi h a poo p ogno-
sis, while high IBA-1 in ensi y, a ma ke o ac i a ed mac ophages, co ela ed wi h a
longe su i al [50]. Simila ly, compa ed wi h IDH-wild ype GBM, a educed numbe o
mac ophages which we e mo e o ien ed owa ds a p o-in lamma o y M1-like ac i a ion
s a e we e ound in IDH-mu an GBM pa ien s, possibly con ibu ing o hei p olonged
su i al [51]. As a whole, o unde s and GAM biology in gliomas, i is c i ical o e alu-
a e an in eg a ed ision, including hei on ogeny, genomic and pheno ypic di e si y,
di e en ial loca ion in he TME and unc ional ac i i y. P eclinical s udies using animal
models ha e shown ha GAMs play a majo ole in gliomagenesis and sus ain umo
Figu e 1.
Rep esen a ion o in e ac ions be ween glioma cells, mac ophages/mic oglia and o he componen s o he umo
mic oen i onmen . Mac ophages and mic oglia (GAMs) a e a ac ed o he umo by se e al glioma cells-de i ed ac o s
such as CCL2, CX3CL1, SDF-1, CSF-1, GM-CSF, EGF o OSTP, exp essed in esponse o en i onmen al s ess (such as
hypoxia) o GAMs-de i ed ac o s hemsel es. In he umo mic oen i onmen , GAMs exe a umo -suppo ing ac i i y
h ough sec e ion o ac o s such as IL-6 (pa icula ly in esponse o TLR4 ac i a ion in mic oglia), IL-1
β
, EGF, STI-1, TGF-
β
o VEGF ha ac i a e di e en signaling pa hways (such as JNK, MAPK o STAT3 in glioma cells) p omo ing p oli e a ion,
angiogenesis o in asion o umo cells. In mic oglia, TLR2/6 ac i a ion has been shown o induce MMP2 and MMP9
exp ession, con ibu ing o ex acellula ma ix deg ada ion and umo in asi eness. GAMs a e also immunosupp essi e
e ec o s, especially ia he exp ession o molecules which lead o T-cell dys unc ion (CD80/86, PD-L1 o CD39). The
phagocy ic ac i i y o GAMs is also impai ed by he exp ession o CD47 (binding o SIRPα) by glioma cells.
To da e, he as majo i y o s udies ha e demons a ed ha mac ophages/mic oglia
suppo GBM p og ession, al hough, in some cases, he abili y o hese cells o igh agains
he umo has been epo ed. This wo k has also e ealed some molecula a ge s o e ing
possibili ies o he apeu ic in e en ion. Al hough, o ou knowledge, s udies es ing
mac ophage-di ec ed he apies o ea GBM ha e s ill no been pe o med in zeb a ish,
se e al in es iga ions ha e al eady e alua ed he ole o mac ophages and mic oglia in
zeb a ish egene a ion, umo o igin and p og ession [
59
,
60
] and in he con ex o o he

Cance s 2021,13, 1087 6 o 37
diseases [
61
,
62
]. Rele an zeb a ish models o he s udy o GBM and he e alua ion o
new ea men s a e e iewed in Sec ions 4and 5, espec i ely.
3. Molecula Pa hology in Glioblas oma
3.1. Gene ic Mu a ions in Glioblas oma
Th ee impo an gene ic e en s d i e he de elopmen o GBM: he dys egula ion o
g ow h ac o signaling (EGF, PDGF, VEGF, e c.), he ac i a ion o he phospha idylinosi ol-
3-OH-kinase (PI(3)K) pa hway and he inac i a ion o he p53 and e inoblas oma (Rb)
umo supp esso pa hways [
15
]. IDH mu a ion occu s ea ly in his p ocess and s ands
ou as he main bioma ke used in he 2016 CNS WHO [
3
] o iden i y and classi y GBM
in o he ollowing ypes: (1) Glioblas oma IDH-wild ype (abou 90% o cases): p ima y
o de no o GBM ha p edomina es in pa ien s o o e 55 yea s o age. (2) Glioblas oma
IDH-mu an (abou 10% o cases): seconda y GBM ha a ises in younge pa ien s. (3)
Glioblas oma no o he wise speci ied (NOS), when he e is no in o ma ion on his gene. The
mu a ion o a ginine o his idine on codon 132 (p.R132H) is mos equen . In GBM, IDH1/2
mu a ions co ela e wi h be e p ognosis, while no IDH3 mu a ions ha e been associa ed
ye . IDH mu a ion is s ongly associa ed wi h 1p/19q codele ion and MGMT p omo e
me hyla ion, ano he common diagnos ic bioma ke , bu a e mu ually exclusi e wi h EGFR
ampli ica ion (gain o ch omosome 7) and PTEN dele ion (loss o ch omosome 10) [
63
].
While he me hyla ion o he MGMT gene p omo e helps o es ima e he sensi i i y o
he umo o alkyla ing agen s such as TMZ, he ela ionship o IDH1/2 mu a ions wi h
esponse o chemo he apy emains con o e sial [64].
As an example o he dys egula ion o g ow h signaling commonly occu ing in GBM,
57% o GBM shows e idence o gains in unc ion mu a ion and/o ocal ampli ica ion o
EGFR, associa ed wi h an inc ease in he agg essi eness o hese gliomas [
14
]. The mos
equen EGFR mu a ion in GBM ha occu s, EGFR
III
, con ains an in- ame dele ion wi hin
he ex acellula domain ha p o ides cons i u i e ac i a ion in a ligand-independen
ashion and p omo es cell p oli e a ion ia he Ras-MAPK and PI3K pa hways. Dele ion o
PTEN displays a simila e ec [65].
Cycle checkpoin p o eins a e o en al e ed in cance . p53 is a classic umo supp esso
ha egula es many genes in ol ed in he cell cycle and apop osis cascades. Inac i a ion
o p53 in GBM happens wi h a a ie y o mechanisms, including ampli ica ion o p53
inhibi o s such as mu ine double minu e (MDM) 2 and MDM4 ( he la e appea s mos
commonly in umo s wi h no TP53 o elome ase e e se ansc ip ase (TERT) mu a ions),
dele ion o p53 s abilize s such as p14/ARF and mu a ion in he TP53 gene which occu s
in 85% o GBMs [
14
]. Cyclin-dependen kinase inhibi o 2A and B (CDKN2A/B) copy
numbe losses (homozygous dele ion o CDKN2A-p16
INK4α
in ch omosome 9p) a e linked
o he ac i a ion o he Rb pa hway and o he p oli e a i e niches ha a e obse ed in
gliomas [
66
]. O he al e a ions a e esponsible o he p og ession and di icul ea men o
GBM. Mu a ions in he p omo e o he elome ase e e se ansc ip ase (TERTp) o ATRX
ch oma in emodele (ATRX) (mu ually exclusi e since hey ha e simila unc ions), lead
o an inc eased leng hening o elome es, allowing cells o o e come cellula senescence
and p omo ing immo aliza ion [67,68].
3.2. Epigene ic Al e a ions in Glioblas oma
Gene ic mu a ions a e no he sole pa icipan s in he de elopmen and p og ession o
GBM. Epigene ics, as shown in many o he ypes o cance , plays an impo an ole oo.
GBM commonly p esen s an ex ended genomic epigene ic hypome hyla ion ha allows
he ansc ip ion o mul iple genes and is associa ed wi h apid p og ession. Some o he
egions a ec ed include D4Z4 (a polymo phic epea s uc u e), oncogenic genomic loci
such as SAT2 and he oncogene MAGE-A1 [
69
]. Mu a ions in he IDH1/2 genes induce
he p oduc ion o an al e na i e me aboli e, 2-hyd oxyglu a a e (2-HG) a he expense
o alpha ke oglu a a e (
α
-KG) [
70
]. Since nume ous enzymes in ol ed in epigene ics
a e dependen on
α
-KG (such as his one deme hylases o en-ele en ansloca ion (TET)
Cance s 2021,13, 1087 7 o 37
enzymes), he absence o his compound inhibi s hem, inc easing he me hyla ion o CpG
islands and his ones. This in ense me hylome emodeling induces wha is known as he
glioma CpG island me hyla o (G-CIMP) pheno ype [
71
] and con as s wi h he gene al
s a e o hypome hyla ion obse ed in GBM. The a e occu ence o IDH-mu a ion, a gene ic
ma ke o seconda y GBM ha is mos ly absen in p ima y GBM and i s co ela ion wi h
be e p ognosis could indica e ha his mu a ion is a hind ance in he p og ession om
low g ade glioma (LGG) o GBM since i changes he epigene ic s a e o mul iple genes
in ol ed in impo an cellula p ocesses. A glioma wi h IDH-mu a ion could equi e
u he al e a ions o de elop a mo e agg essi e pheno ype han gliomas wi hou i .
A key example ha highligh s he impo ance o epigene ics in GBM comes om
MGMT al e a ions, one o he gold s anda d ma ke s used o he cha ac e iza ion o he
disease. The me hyla ion o he MGMT p omo e is a bioma ke wi h g ea diagnos ic
alue ha co ela es wi h good p ognosis. Fu he mo e, as wi h he DNA alkyla ion
epai enzyme, he inhibi ion o MGMT exp ession enhances he an i- umo al e icacy o
TMZ [72].
The epigene ic inac i a ion o many umo supp esso genes has also been obse ed
in GBM. PTEN, p53, RB, p14 and p16 appea hype me hyla ed, along wi h some o he
genes in ol ed in key p ocesses such as in e cellula con ac (PCDH-
γ
A11), he MAPK
pa hway (SOCS1) and apop osis (caspase-8) [
69
]. His one deace ylases (HDACs) ha e
been implica ed in many GBM umo s owing o he ac ha hei dys egula ion in luences
umo p og ession, in asion and esis ance o he apy. This is o special ele ance in GBM
s em cells (GSC), whe e dys egula ed HDAC exp ession has been associa ed wi h al e ed
signaling mechanisms like he sonic hedgehog (SHH) pa hway (essen ial o s emness, ia-
bili y and adio- esis ance) and he main enance o mi ochond ial unc ions and me abolic
adap ions [73].
3.3. Me abolic Changes in Glioblas oma
Me abolic swi ching is a well-known hallma k o cance , including GBM and unde -
s anding and ep og amming me abolic pa hways in umo s may yield new ea men
op ions [
74
]. Many cance s p e e ae obic glycolysis as hei me abolic p og am o choice
o ul ill hei bioene ge ic and anabolic equi emen s o apid g ow h and enhance hei
su i al in esponse o mic oen i onmen al s ess [
75
]. Abe an exp ession o oncogenes
and umo supp esso genes in GBM has co ela ed wi h changes in he exp ession and
ac i i y o glycoly ic anspo e s. Ae obic glycolysis, desc ibed in he 1920s by O o Wa -
bu g and his colleagues, known as he Wa bu g E ec , is de ined as an inc ease in he a e
o glucose up ake and p e e en ial p oduc ion o lac a e in he p esence o oxygen in cance
cells in compensa ion o insu icien OXPHOS [
76
,
77
]. The ad an age ha he Wa bu g
E ec con e s o cance cells is no comple ely clea . Howe e , since ATP le els do no seem
o ep esen a limi ing ac o o umo cells, i is gene ally accep ed ha ae obic glycolysis
in cance p omo es he use o NADH as a by-p oduc o lac a e o gene a e biomass and
lac a e o acidi y he mic oen i onmen , acili a ing umo in asion [
78
]. Ae obic glycolysis,
which a o s he deadly p og ession o GBM [
79
] (Figu e 2), is con olled by glucose ans-
po e s, GLUT1-4 and key glycoly ic enzymes such as hese: HK1-3, PFK1, GAPDH, PKM2
and LDHA; hey a e also in luenced by se e al o he cellula pa hways and egula o y
p o eins, including he ollowing: PI3K/AKT, LKB1/AMPK, HIF-1/2, p53, EGFR, PDGFR
and c-MYC, among o he s. Da a ob ained om The Cance Genome A las (TCGA) ha e
iden i ied h ee co e pa hways which a e equen ly al e ed in mo e han 75% o GBMs: (i)
ecep o y osine kinase/RAS/phospha idylinosi ol 3 kinase (RTK/RAS/PI3K) signaling
(ii) p53 (iii) Rb signaling ne wo ks [
15
]. The analysis o 96 GBM human samples showed
abe an o e ac i a ion in a numbe o RTKs and gain o unc ion mu a ions such as in
EGFR (obse ed in 45% o GBM cases) which igge s RAS and AKT/PI3K cascades. As a
consequence, hese downs eam p oli e a i e and me abolic signaling cascades ha e been
ound o be up- egula ed in GBM [
15
]. The ac i a ion o he mTOR signaling cascade by
AKT in GBM has led o he up- egula ion o ansc ip ion ac o s such c-Myc [
37
], which
Cance s 2021,13, 1087 8 o 37
up- egula es he exp ession o glycoly ic genes [
80
]. AKT can also up- egula e glycol-
ysis by ac i a ing ansc ip ion and ansloca ion o glucose anspo e s (GLUTs), also
up- egula ed in GBM and o he key enzymes such as Hexokinase II, in ol ed in he i s
s ep o glycolysis [
81
]. The e o e, al e a ions in oncogenes and umo supp esso genes
con olling me abolism and me abolic enzymes unde lying umo p og ession in GBM a e
a ge s o in e es o he ea men o he disease.
Cance s 2021, 13, x 10 o 40
Figu e 2. Rep esen a ion molecula mechanisms unde lying he umo al p ope ies o glioma cells. A. Canonical pa hway
o miRNA biogenesis. miRNA a e ansc ibed by RNA Pol II o p oduce he p i-miRNA ha is la e p ocessed by he
endonuclease D osha/DGCR8 complex esul ing in he o ma ion o he hai pin p ecu so (p e-miRNA) ha goes unde a
second clea age by he endonuclease Dice and gene a es a miRNA duplex. One o he s ains, he ma u e ~22 n (guide
s ain) is la e loaded in o he RISC (RNA Induced Silencing Complex) in associa ion wi h Ago p o eins. Via impe ec
base-pai ing o he 3′ UTR o i s a ge mRNAs, miRNAs egula e he exp ession o i s a ge genes by ep essing mRNA
ansla ion and/o p omo ing mRNA deg ada ion. B. miRNAs and a ge genes in ol ed in GBM. Examples o miRNAs
ound o be down egula ed o up egula ed (indica ed wi h he a ow) in GBM and hei a ge genes in ol ed in GBM
pa hology. C. Me abolic pa hways al e ed in GBM. Me abolic swi ching owa ds ae obic glycolisis is a hallma k o cance
which con ols he p ope ies and unc ions o umo cells, such as hei inc eased p oli e a ion o esis ance o ea -
men s. The he apeu ic in e en ion o me abolic pa hways, desc ibed in his igu e, may o e new oppo uni ies o he
ea men o GBM.
The ole o di e en miRNAs in egula ing classic pa hways and genes c i ical o
he GBM genesis a he pos - ansc ip ional le el has been desc ibed. These include p53,
EGFR, PDGFR, PTEN, PI3K and AKT and MGMT, among o he s [94] (Figu e 2). Mi-
c oRNA signa u es wi h de icien pa e ns o exp ession ha e been ound o se e al
miRNAs such as he miR-7, miR-34a, miR-128, miR-124, miR-137 and miR-181 amilies
Figu e 2.
Rep esen a ion molecula mechanisms unde lying he umo al p ope ies o glioma cells. A. Canonical pa hway
o miRNA biogenesis. miRNA a e ansc ibed by RNA Pol II o p oduce he p i-miRNA ha is la e p ocessed by he
endonuclease D osha/DGCR8 complex esul ing in he o ma ion o he hai pin p ecu so (p e-miRNA) ha goes unde a
second clea age by he endonuclease Dice and gene a es a miRNA duplex. One o he s ains, he ma u e ~22 n (guide
s ain) is la e loaded in o he RISC (RNA Induced Silencing Complex) in associa ion wi h Ago p o eins. Via impe ec
base-pai ing o he 3
0
UTR o i s a ge mRNAs, miRNAs egula e he exp ession o i s a ge genes by ep essing mRNA
ansla ion and/o p omo ing mRNA deg ada ion. B. miRNAs and a ge genes in ol ed in GBM. Examples o miRNAs
ound o be down egula ed o up egula ed (indica ed wi h he a ow) in GBM and hei a ge genes in ol ed in GBM
pa hology. C. Me abolic pa hways al e ed in GBM. Me abolic swi ching owa ds ae obic glycolisis is a hallma k o cance
which con ols he p ope ies and unc ions o umo cells, such as hei inc eased p oli e a ion o esis ance o ea men s.
The he apeu ic in e en ion o me abolic pa hways, desc ibed in his igu e, may o e new oppo uni ies o he ea men
o GBM.
Cance s 2021,13, 1087 9 o 37
I is o no e, ha mi ochond ia a e a key cellula o ganelle egula ing ene gy me abolism,
gene a ion o ee adicals and apop osis. Addi ional me abolic signs o malignancy in
GBM ha e been linked o mi ochond ial dys unc ion and mo phological abno mali ies,
which ha e been co ela ed wi h comp omised ene gy/ATP p oduc ion ia OXPHOS
and educed apop osis. Impai ed mi ochond ial me abolic capaci y in glioma cells has
been e iden om he iden i ica ion o mu a ions in gene coding o IDH, desc ibed in
Sec ion 3.1 and a p incipal componen o he K ebs cycle. Mu a ions in he NADPH-
linked mi ochond ial iso o ms o IDH1 and IDH2 ha e led o impai ed ene gy p oduc ion
in he mi ochond ia and hus, p o ide clea e idence o mi ochond ial dys unc ion in
gliomas [
82
]. Indeed, IDH1 mu a ion no only causes 2-HB build-up, bu also b oad changes
in cellula me abolism. Due o changes in he exp ession o se e al key enzymes, GBMs
con aining IDH1 mu a ions ha e been shown o exhibi a educed glycoly ic pheno ype
compa ed o GBM wi hou he mu a ion. The e o e, IDH s a us can also indica e dis inc
glycoly ic pheno ypes o GBM, which may con ibu e o di e en clinical beha io o
umo s wi h and wi hou he IDH1 mu a ions [
80
]. Ca diolipin, an impo an phospholipid
concen a ed a he con ac si es o ou e and inne mi ochond ial memb anes and a he
elec on anspo chain (ETC) associa es wi h espi asome complexes ha a e c ucial
o mi ochond ial unc ion. E idence om expe imen al and clinical s udies has shown
de ec s in mi ochond ial ETC associa ed wi h ca diolipin biosyn hesis in gliomas. Thus,
he deg ee o aul y ca diolipin syn hesis and i s unc ion indi ec ly a ec s he mi ochon-
d ial me abolic capaci y and in insic apop osis execu ion which ha e been ound o be
impai ed/mal unc ioned in ce ain g ades o glioma cells [82].
In addi ion, o gene ic and epigene ic in e en ion, a b oad a ie y o biological
molecules ha e been e alua ed o he he apeu ic manipula ion o me abolic pa hways,
including suga s, lipids and p o eins [
83
]. Fu he mo e, wi h he same pu pose, no el
pos - ansc ip ional egula o s, such as miRNAs ( e iewed below), a e o u mos in e es .
3.4. miRNAs in Glioblas oma
miRNAs a e small (18–25 nucleo ides), e olu iona ily conse ed, non-coding RNAs
wi h impo an unc ions in gene egula ion, ac ing p edominan ly a he pos ansc ip-
ional le el. miRNAs a e ansc ibed in he nucleus by RNA polyme ase II in o p ima y
ansc ip s (p i-miRNAs) ha a e hen p ocessed sequen ially in he nucleus and cy oplasm
by a complex o RNase III-endonucleases, namely D osha and Dice [
73
,
84
,
85
], (Figu e 2).
D osha, speci ically, p ocesses he p i-miRNA ansc ip o a 70–100 nucleo ide s em-loop
p ecu so (p e-miRNA), which is hen deli e ed o he cy oplasm by Expo in 5, whe e i is
subsequen ly clea ed by Dice o p oduce a miRNA duplex [
73
,
84
]. The esul ing duplex is
hen inco po a ed in o he RNA-induced silencing complex (RISC) in associa ion wi h an
Ago amily membe . One o he s ands ( he passenge s and) is deg aded, while he o he
s and ( he ma u e miRNA) emains associa ed wi h he Ago p o ein and binds o pa ially
complemen a y si es in mRNAs. By way o binding o he 3
0
UTR o a ge messenge
RNAs (mRNAs), miRNAs ep ess ansla ion o induce mRNA deg ada ion [86,87].
O e he pas wo decades, miRNAs ha e been shown o egula e many physiological
p ocesses in he cells and hey ha e been implica ed in a wide ange o pa hological condi-
ions, including cance . A ound 50% o he human-miRNA-encoded genes a e loca ed in
gene ic loci associa ed wi h cance [
88
] and nea ly all umo s show dys egula ed miRNA
exp ession signa u es [
89
]. These al e a ions equen ly co ela e wi h up- egula ion o
oncogenes and/o down egula ion o umo supp esso -genes, he e o e, p omo ing he
de elopmen o he umo . The pa e n o miRNA exp ession is now being used, oge he
wi h gene-exp ession p o iling o s a i y GBM pa ien s in o di e en g oups [
90
]. In addi-
ion, miRNAs ha e mul iple a ge s, allowing hem o modula e many pa hological aspec s
c i ical o cance p og ession, including p oli e a ion, cell dea h, me as asis, angiogene-
sis and d ug esis ance. A numbe o miRNAs ha e been iden i ied ha in e ac wi h
known genes and pa hways ha unde lie GBM [
91
] and some o hem ha e been placed as
bioma ke s [92] and p edic o s o su i al and ou comes [93].
Cance s 2021,13, 1087 16 o 37
he in ape i oneal ca i y in he case o adul xenog a s [
113
,
139
,
142
–
149
]
(Figu e 3).
In he
pa icula con ex o b ain umo s, he likelihood o de eloping o ho opic umo s p omp s
mos esea che s o injec cells di ec ly in o he en icles o he hindb ain-midb ain bound-
a y. Al hough his is no op imal, i allows o s udy he g ow h o cance cells in an
en i onmen which esembles he ne ous sys em.
Cance s 2021, 13, x 17 o 40
in e oga ed, mos implan a ions a e pe o med in he yolk sac, he duc o Cu ie , he
pe i i elline space o he in ape i oneal ca i y in he case o adul xenog a s
[113,139,142–149] (Figu e 3). In he pa icula con ex o b ain umo s, he likelihood o
de eloping o ho opic umo s p omp s mos esea che s o injec cells di ec ly in o he
en icles o he hindb ain-midb ain bounda y. Al hough his is no op imal, i allows o
s udy he g ow h o cance cells in an en i onmen which esembles he ne ous sys em.
Figu e 3. Injec ion si es commonly used in zeb a ish xeno ansplan a ion assays: (a) Blas ula (3.5
hp ); (b) Duc o Cu ie , pe i i elline space and yolk sac in zeb a ish emb yo (48 hp ); (c) Emb yo
b ain (48 hp ); (d) In ape i oneal ca i y in adul zeb a ish. Images (a–c) we e acqui ed in ou lab
wi h a s e eomic oscope (AZ-100 Mul izoom, Nikon). Image (d) was acqui ed in ou lab wi h a
Samsung Galaxy A70.
Xenog a models ha e p o ided in o ma ion abou GBM ini ia ion and p og es-
sion (Table 2) and ha e allowed in es iga ions o no el he apeu ic app oaches ( e-
iewed in Sec ion 5). To in es iga e b ain cance biology, human a - ed-labeled GBM
cells we e injec ed in zeb a ish la ae o ollow umo p og ession, size, shape and
b igh ness [150]. In his s udy, la ge numbe s o umo cells and cellula di isions o e
ime we e quan i ied by a combina ion o s e eomic oscopy, ligh shee luo escence mi-
c oscopy and low cy ome y. A obus , as and au oma able ansplan a ion app oach
has ecen ly been epo ed by Pudelko e al. o es ablish o ho opic GBM umo s in
zeb a ish. By injec ing GBM cell lines o pa ien -de i ed samples in o zeb a ish blas ulas,
hey obse ed hei obus mig a ion in o he de eloping ne ous sys em, al eady es ab-
lishing an o ho opic in ac anial umo a 24 h pos -injec ion (hpi). This app oach
a oids he echnically challenging in ac anial ansplan a ion o single emb yos, hus
enabling he ansplan a ion o hund eds o emb yos pe hou and p o iding an o ho-
opic e eb a e GBM model use ul o d ug disco e y sc eens [151]. O he s ha e used
s em cells cul u es de i ed om pedia ic high-g ade glioma umo s o gene a e o ho-
opic b ain umo s, conse ing s emness p ope ies and esembling human gliomas
[152]. The eng a men o pa ien -de i ed GBM cells in adul zeb a ish has been assessed
in an op ically clea , immunocomp omised homozygous compound mu an (p kdc−/−,
il2 ga−/−). Tumo cells obus ly eng a ed and p oli e a ed showing simila g ow h kine -
ics and his opa hology o hose obse ed in a mouse model. Ne e heless, in his case,
he equi emen o p e- ea animals wi h clod ona e liposomes in o de o inhibi ea ly
mac ophage inges ion migh be oo oxic, es ic ing he use ulness o his model [153].
The injec ion o glioma cells in ac anially o in he yolk sac o 2 dp emb yos was used
o demons a e ha chond oi in 4-sul a e (C4S) and chond oi in 6-sul a e (C6S) p omo e
Figu e 3.
Injec ion si es commonly used in zeb a ish xeno ansplan a ion assays: (
a
) Blas ula (3.5
hp ); (
b
) Duc o Cu ie , pe i i elline space and yolk sac in zeb a ish emb yo (48 hp ); (
c
) Emb yo
b ain (48 hp ); (
d
) In ape i oneal ca i y in adul zeb a ish. Images (
a
–
c
) we e acqui ed in ou lab
wi h a s e eomic oscope (AZ-100 Mul izoom, Nikon). Image (
d
) was acqui ed in ou lab wi h a
Samsung Galaxy A70.
Xenog a models ha e p o ided in o ma ion abou GBM ini ia ion and p og ession
(Table 2) and ha e allowed in es iga ions o no el he apeu ic app oaches ( e iewed in
Sec ion 5). To in es iga e b ain cance biology, human a - ed-labeled GBM cells we e
injec ed in zeb a ish la ae o ollow umo p og ession, size, shape and b igh ness [
150
]. In
his s udy, la ge numbe s o umo cells and cellula di isions o e ime we e quan i ied by
a combina ion o s e eomic oscopy, ligh shee luo escence mic oscopy and low cy ome y.
A obus , as and au oma able ansplan a ion app oach has ecen ly been epo ed by
Pudelko e al. o es ablish o ho opic GBM umo s in zeb a ish. By injec ing GBM cell lines
o pa ien -de i ed samples in o zeb a ish blas ulas, hey obse ed hei obus mig a ion
in o he de eloping ne ous sys em, al eady es ablishing an o ho opic in ac anial umo
a 24 h pos -injec ion (hpi). This app oach a oids he echnically challenging in ac a-
nial ansplan a ion o single emb yos, hus enabling he ansplan a ion o hund eds o
emb yos pe hou and p o iding an o ho opic e eb a e GBM model use ul o d ug
disco e y sc eens [
151
]. O he s ha e used s em cells cul u es de i ed om pedia ic high-
g ade glioma umo s o gene a e o ho opic b ain umo s, conse ing s emness p ope ies
and esembling human gliomas [
152
]. The eng a men o pa ien -de i ed GBM cells in
adul zeb a ish has been assessed in an op ically clea , immunocomp omised homozygous
compound mu an (p kdc
−/−
,il2 ga
−/−
). Tumo cells obus ly eng a ed and p oli e a ed
showing simila g ow h kine ics and his opa hology o hose obse ed in a mouse model.
Ne e heless, in his case, he equi emen o p e- ea animals wi h clod ona e liposomes
in o de o inhibi ea ly mac ophage inges ion migh be oo oxic, es ic ing he use ul-
ness o his model [
153
]. The injec ion o glioma cells in ac anially o in he yolk sac o
2 dp emb yos was used o demons a e ha chond oi in 4-sul a e (C4S) and chond oi in
6-sul a e (C6S) p omo e GBM cell mig a ion and in asion [
154
]. The impac o ni ic oxide
(NO) in umo p og ession was con i med in 2 dp Tg( li1:EGFP) emb yos injec ed in he

Cance s 2021,13, 1087 17 o 37
yolk sac wi h a GBM-labeled cells (GV1A1) [
155
]. Cells we e able o g ow and p oduce
NO, as con i med wi h diamino- luo esceins and diamino- hodamines, and he inc ease in
he NO syn hases (nos1 and nos2a). Fu he mo e, neo ascula iza ion and inc ease in eg a
and cyclin D1 exp ession we e obse ed in 85% o he emb yos wi h NO p oduc ion, and
he addi ion o a NO sca enge (CPTIO) educed eg a and cyclin D1 exp ession as well as
he numbe o endo helial cells, sugges ing ha he educ ion in NO le els by ni ic oxide
sca enging could be an e icien app oach o he ea men o glioma.
Table 2. Xenog a app oaches using zeb a ish o modeling GBM.
Injec ion
Si e Cell Line S age Zeb a ish S ain No able Resul s Re e ence
Yolk sac
Fa - ed CCF-STTG1 48 hp WT
Tumo p og ession size, shape, b igh ness and
quan i ica ion o umo cells, by combina ion o
LSFM and low cy ome y
[150]
GV1A1-CM-DiI 48 hp Tg( li1:EGFP) Impac o NO p oduc ion ia eg a and cyclin
D1 exp ession [155]
U87-RFP
(TGF-β1- ea ed) 48 hp Tg( li:GFP) Inc ease in newly- o med blood essels and
mac ophage accumula ion in he b ain egion [156]
U87-RPF
(CSCs-en iched cells) 48 hp Tg( li:GFP) Up- egula ion o CD133 and MMP9 leads o
glioma in asi eness [157]
U373-GFP
(GBM s em-like cells) 48 hp Tg(kd :mChe y) RAC p o eins p omo e agg essi eness and poo
p ognosis o GBM [158]
Yolk sac/
B ain U251- CM-DiI 48 hp WT C4S and C6S p omo e GBM cell mig a ion
and in asion [154]
-
Pa ien -de i ed/
U343-MGA-GFP/
GBM p ima y cul u es
Blas ula
(3.5 hp )
Tg( li1a:EGFP)/Tg(ela l3:GFP)/
Tg(mpeg1:mChe y)
No el ansplan a ion p ocedu e wi h
de elopmen o o ho opic in ac anial umo s
and mac ophage/mic oglia in e ac ions
[151]
Ven icles
BPC-A7-RFP (s em cell
cul u es de i ed om
pedia ic b ain umo s)
48 hp WT Ini ia ion o glioma-like umo s om s em cell
cul u es, conse ing s emness p ope ies [152]
Op ic
ec um
U87-mChe y/U251-
mChe y 72 hp Tg(mpeg1:EGFP)/i 8−/−)Mic oglia p o umo al ac i i y and di e en ial
esponse o pa icula cell ypes [136]
B ain
RECQ1- silenced U87-DsRed 52 hp WT
RECQ1 plays an impo an ole in
umo p og ession.
P omising app oach o GBM ea men
[159]
DiI/DiO- Bone ma ow
-de i ed MSCs/U373-
eGFP/U87-dsRED
52 hp WT MSC-GBM cell c oss alk a ec s in asion in a
cell ype-speci ic manne [160]
D54-MG/D2159MG 72 hp Tg( li1a:eGFP); caspe
Cell a achmen and mig a ion h ough he
b ain ascula u e. Impo ance o
mic oen i onmen
[161]
KMT2A- knockdown
U87MG 48 hp Tg( li1:EGFP) Inc ease in umo g ow h and angiogenesis.
KMT2A ac s nega i ely on umo g ow h [162]
Hindb ain
en icle
U251MG in lama5
knockdown emb yos 48 hp Tg( li:EGGP) lama5 supp esses in asion bu inc eases umo
o ma ion ia VEGF [163]
In ape i oneal Pa ien -de i ed Adul Caspe , p kdc−/−,il2 ga−/−Success ul umo eng a men a physiological
empe a u e (37 ◦C) [146]
The mechanisms o glioma- essel in e ac ions, angiogenesis and umo in asion ha e
also been s udied in zeb a ish models. Umans e al. in ac anially injec ed an adul glioma
cell line and a pedia ic pa ien de i ed xenoline in o Tg( li1a:eGFP) caspe emb yos a
3 dp and obse ed he a achmen o cells o he b ain ascula u e wi hin 24 hpi, as well
as hei expansion and mig a ion along he ascula ne wo k by 7 dp [
161
]. The same cells
injec ed in o he unk ailed o in e ac wi h he ascula u e and mo ed os ally owa ds
he b ain, highligh ing he impo ance o he mic oen i onmen . Simila ly, zeb a ish
xenog a models we e used o elucida e he ole o mesenchymal s ems cells (MSCs) in
GBM p og ession [
160
,
164
]. By co-injec ing bone ma ow-de i ed MSCs wi h wo di e en
GBM cell lines (U373 o U87) in he b ain o zeb a ish emb yos, in a umo he e ogenei y
was obse ed as a consequence o he di e en ial c oss alk be ween MSC-GBM cells,
a ec ing in asion in a cell ype-speci ic manne [
160
]. Such di e en umo p og ession
depending on he cell ype and i s in e ac ion wi h mic oglia was demons a ed using a
Cance s 2021,13, 1087 18 o 37
mac ophage/mic oglia-labeled ansgenic zeb a ish line o he in e e on egula o y ac o
8 mu an (i 8
−/−
), which does no con ain mic oglia [
136
]. The injec ion o U87 o U251
cells in o he op ic ec um esul ed in umo p og ession and he a ac ion o mic oglia
in bo h cell ypes al hough in e ac ions and in il a ion di e ed in numbe and na u e.
The injec ion o cells in i 8
−/−
dec eased p oli e a ion and su i al, demons a ing he
p o umo al ac i i y o mic oglia and how impo an i is o conside hei di e en ial
esponse o dis inc cell ypes when de eloping immuno he apies o ea gliomas. TGF-
β
was also shown o enhance umo -induced angiogenesis ia he JNK pa hway and
mac ophage in il a ion in a zeb a ish glioma xenog a model [
156
]. P e ea men o
U87 GBM cells wi h TGF-
β
1 and subsequen injec ion in o he yolk sac o Tg( li:GFP)
emb yos a 2dp led o a signi ican inc ease in newly- o med blood essels, as well as in
mac ophage accumula ion in he b ain egion, ail and yolk sac. Addi ionally, he ea men
o xenog a ed emb yos wi h a JNK inhibi o esul ed in a dec ease in angiogenesis, which
may well be a powe ul model o an i-angiogenesis d ug sc eenings. The same g oup
es ablished a umo in asion model in 2dp Tg( li:EGFP) emb yos by ansplan ing he U87
cell line and i s de i ed cance s em cells (CSCs) [
157
]. CSCs en iched om U87 cells sp ead
apidly h ough he essels, acqui ing a p o usi e appea ance o es ablish me as asis and
hei in asi eness co ela ed wi h he up- egula ion o he s em cell ma ke CD133 and
he MMP9. Fu he mo e, CSC in asion was ma kedly inhibi ed by an MMP9 inhibi o ,
alida ing he use o zeb a ish models o s udy he mechanisms unde lying he in asion
and me as a ic beha io o glioma cells.
To examine he e ec s o Rac p o eins on GBM p og ession
in i o
, Lai e al. injec ed
GFP-labeled U373- umo sphe e cells (GBM s em-like cells) in he yolk sac o 2 dp
Tg(kd :mChe y) emb yos wi h labeled endo helial cells, ha bo ing con ol sc amble sho
haipin (shRNA), Rac shRNAs (shRacs) and Rac 1–3 cDNAs, o silence o o e exp ess Rac
p o eins espec i ely [
158
]. They obse ed highe su i al a es and lowe incidence o
angiogenesis in emb yos bea ing he umo sphe es de i ed om U373-MG cells wi h
shRacs han con ols, co ela ing he exp ession o Rac p o eins wi h agg essi eness and
poo p ognosis in GBM. RECQ1 helicase plays an impo an ole in umo p og ession,
as i s exp ession is highly ele a ed in GBM [
159
]. shRNA-silencing o RECQ1 in U87
cells ansplan ed in o 2 dp zeb a ish b ains esul ed in dec eased umo g ow h. MOs
we e used o knock down lysine (K)-speci ic me hyl ans e ase 2A (KMT2A), in ol ed in
glioma p og ession [
162
], esul ing in down egula ed p oli e a ion o neu al p ogeni o s,
p ema u e di e en ia ion o neu ons and impai ed gliogenesis [
165
]. KMT2A-knockdown
in U87MG cells ansplan ed in o 2 dp Tg( li1:EGFP) zeb a ish b ains inc eased bo h umo
g ow h and angiogenesis, e en when emb yos we e ea ed wi h an immune supp essan
(dexame hasone), e ealing ha KMT2A ac s nega i ely on umo g ow h in con as wi h
he p e ious concep ion o KMT2A as an oncogene in umo igenesis [
162
]. Gamble e al.
disco e ed ha adhesion o laminin subuni alpha 5 (lama5), an impo an componen o
blood essels, dec eases GBM cell in asion and p omo es he o ma ion o blood essel
dependen mic o umo s. Wi h 4D indi idual cell acking echnology and U251MG cells,
xeno ansplan a ion in he hindb ain en icle o Tg( li1:EGFP) 2 dp emb yos, p oli e a ion,
dispe sion, mic o umo o ma ion and cell/blood essel associa ion we e con i med [
163
].
Ne e heless, lama5 knockdown by MOs esul ed in signi ican ly highe cell dispe sion
and mobili y whe eas mic o umo o ma ion was lowe . Thus, lama5 inc eases GBM cell
a achmen o blood essels by ele a ing VEGF ac i i y, which in u n supp esses in asion
bu inc eases umo o ma ion.
O e all, a b oad a ie y o xenog a zeb a ish models o GBM ha e been es ablished
wi h di e en pu poses. Al hough o ho opic models a e p e e able o be e mimic he
human pa hology, he e o opic models allowing o he easie implan a ion o cells can be
used o a p elimina y sc eening o d ugs and/o o s udy pa icula mechanisms.
Cance s 2021,13, 1087 19 o 37
5. E alua ion o New T ea men s o GBM Using Zeb a ish Models
GBM is a de as a ing disease, no only because o i s pa icula agg essi e na u e, bu
also due o he e y limi ed e icacy o he he apeu ic op ions cu en ly a ailable [
166
,
167
].
The s anda d o ca e is su ge y, ollowed by adio he apy and TMZ, a DNA alkyla ing
agen ha can be adminis e ed o ally [
166
,
168
]. Recu ence o GBM is e y high and
su i al a e ea men is commonly 12–15 mon hs due o he ollowing issues [
166
,
167
]:
(i) di icul ies o emo e all umo s due o hei g ea in asi e and p oli e a i e capac-
i y [
167
,
168
], (ii) hei high mu a ional capaci y, which apidly gene a es esis ance o
chemo he apies, such as TMZ [
166
,
169
]. In addi ion o he apies a ge ing cance cells,
no el an i- umo al immuno he apies ha e been e alua ed and nume ous clinical ials
using immune checkpoin inhibi o s (ICIs, i.e., an i-PD1 and an i-CTLA4) a e ongoing.
Fu he mo e, he apeu ic s a egies o ep og am he umo mic oen i onmen o o no -
malize angiogenesis, including a ge ing and e-educa ing TAMs owa ds M1 an i- umo al
mac ophages a e o pa icula in e es . Fo example, in a umo al deli e y o IL-12 using
a gene ically-modi ied i us, alone o in combina ion wi h ICIs, showed good esul s in
p eclinical mu ine models [
84
]. Fo he gene ic, epigene ic and me abolic ep og amming o
GBM umo s, se e al app oaches a e being in es iga ed. Fo ins ance, his one deace yla e
inhibi o s and shRNAs owa ds HDAC1 and 2 [
170
] o SIRT1, alone, o in combina ion
wi h adio he apy, showed an i umo al e icacy [
171
,
172
]. Ei he o kill cance cells o o
ep og am he TME, miRNA manipula ion ep esen s a e y a ac i e a ge ( e iewed in
Sec ion 3.4). O he majo issues o he e ec i e ea men o b ain umo s a e he abili y o
he d ugs o c oss he blood–b ain–ba ie (BBB) and/o he absence o sys emic oxici y.
In his scena io, zeb a ish models ha e a isen as e y use ul p e-clinical models o e alua e
in i o
he an i- umo al e icacy o new d ugs and hei abili y o each and in e ac wi h
a ge cells (i.e., cance cells and mac ophages), as well as o modula e he TME (i.e., an-
giogenesis) and hei biodis ibu ion abili y o c oss he blood–b ain–ba ie (BBB) and/o
oxici y. A b ie desc ip ion o ele an s udies using mainly zeb a ish xenog a models
o es ing GBM he apies is p o ided below and in Table 3.
Cance s 2021,13, 1087 20 o 37
Table 3. T ea men s o GBM e alua ed using xenog a zeb a ish models.
T ea men Zeb a ish S ain Cell Line S age Injec ion Si e Rema kable Resul s Re e ence
TMZ Caspe
shNRP-1 o
shVEGF-GBM1A and
GBM22
36 hp B ain en icle
TMZ enhances su i al and dec eases
umo g ow h. NRP-1 ab oga ion imp o es
he e ec o TMZ
[173]
TNB Tg( lk:eGFP)- Caspe U87-RFP o U251-RFP 72 hp B ain TNB is able o c oss he BBB and inhibi s
umo p og ession [174]
MAM WT U251- DiI 48 hp Yolk sac Inhibi ion o umo g ow h, possibly in an
apop osis-independen manne [175]
TMZ/Bo ezomib Caspe
GBM9-GFP
neu osphe es and
X12- 2
36 hp MHB
Di e se di e en ia ion pa e ns in cells, bu
bo h posi i e o Sox2 and esponsi e
o he apeu ics
[168]
TMZ Caspe GBM9-GFP
neu osphe es 36 hp MHB Pu a i e GBM s em cells a e mo e esis an
and migh con ibu e o umo eg ow h [169]
TMZ/Onalespib WT U251HF-GFP 36 hp MHB Combina ion o Onalespib wi h TMZ
educes umo bu den and ex ends su i al
[176]
HK WT U87MG-CM-DiI 48 hp Yolk sac Inhibi ion o umo g ow h and me as asis [177]
Dox-HK-MPEG-PCL
micelles Tg ( lk: eGFP)/WT U87- GFP 14 and 48 hp Pe i i elline space An i-angiogenic and an i- umo p ope ies [178]
Clo oc ol WT U87MG- SLCs-GFP 48 hp Yolk sac Tumo inhibi ion wi hou oxici y [179]
PRMT5 inhibi o s
(CMP12, CMP5,
HLCL65, HLCL66)
Caspe
Pa ien -de i ed
neu osphe es
(GBMNS-30–GFP)
36 hp MHB An i- umo e icacy o CMP5,
wi hou oxici y [180]
HDAC class
III/SIRT1/2 WT
Hs683 and U373-CM-DiI
36 hp Yolk sac Abili y o ab oga e umo de elopmen [181]
ERR-βagonis s
(TG-003/DY131)
Tg(kd l:GRCFP)zn1;
mi ab692/b692;
edn b1b140/b140
42MGBA-TMZ es-DiI 36 hp In ac anial
Shi o ERRb2 iso o m and supp ession o
g ow h and mig a ion in
TMZ- esis an cells
[182]
Cance s 2021,13, 1087 21 o 37
Table 3. Con .
T ea men Zeb a ish S ain Cell Line S age Injec ion Si e Rema kable Resul s Re e ence
5-FU/E lo inib Tg( li1:eGFP) Condi ioned
GBMERBB2-RFP 30 dp Ce eb um (in anasally)
Mouse b ain umo s can g ow
o ho opically in ish and a e esponsi e
o ea men
[183]
MTH1 inhibi o
(TH1579) WT
CD33+en iched ac ion
o pa ien -de i ed CMV-
LUC/U343-MGA:GFP
In ac anial
Real- ime dea h o glioma s em cells (GSCs)
and umo olume dec ease [184]
ZnO NP/LY294002 Tg( li1:eGFP) U87MG-CM- DiI 48 o 72 hp Hindb ain ZnO NPs enhance cance cell p oli e a ion [167]
C60 ulle ene
de i a i es Tg( li:eGFP) C6-PKH26 (mu ine
neu al s em cell) 24 hp B ain Reduc ion in GBM o ma ion [185]
Axi inib, Sun inib,
Va alani/No dy Tg ( li1:EGFP) GSCs U87- de i ed 48 hp Yolk sac
Inhibi ion o umo -induced essel
o ma ion. Model o an i-GSC
d ug e alua ion
[186]

Cance s 2021,13, 1087 22 o 37
5.1. T ea men s Tes ed in Zeb a ish by Xeno ansplan a ion o GBM Cell Lines
Zeb a ish xeno ansplan s ha e allowed GBM g ow h and esponse o ea men o be
s udied in eal ime, which, in u n, enables an e alua ion o he p oli e a i e, mig a o y,
in asi e and angiogenic s a us o he umo . In hese s udies, TMZ was commonly used
as he e e ence ea men and o posi i e con ol. Fo example, 2-me hoxy-6-ace yl-
7me hyljuglone (MAM), a na u al p oduc ha induced nec op osis in colon and lung
cance cells, injec ed in o U251-xenog a zeb a ish models showed simila an i- umo al
e icacy o TMZ a e y low doses [
175
]. A compa ison be ween TMZ and bo ezomib,
a p o easome inhibi o , in GBM9-xenog a zeb a ish, showed simila umo educ ion
o bo h d ugs bu lowe su i al o bo ezomib [
168
]. Using he same model, umo
eg ow h a e TMZ ea men was demons a ed o cance s em cells exp essing Sox2
and GFAP, e ealing molecula mechanisms unde lying esis ance o ea men [
169
].
In ano he s udy, he VEGF and i s co ecep o and p oangiogenic ac o neu opilin-1
(NRP-1) we e knocked down in wo human pa ien –de i ed GBM cell lines, GBM1A and
GBM22, p e iously epo ed o be esis an o TMZ, be o e implan a ion in o he b ain
en icle egion o 36 hp emb yos [
173
]. Al hough dele ion o bo h VEGF and NRP-1
we e able o inhibi umo g ow h, TMZ ea men showed be e esul s in combina ion
wi h NRP-1 ab oga ion. Onalaspib, an inhibi o o hea shock p o ein 90 (HSP90), alone
o in combina ion wi h TMZ, was es ed in GBM zeb a ish models ha we e injec ed in
he mid-hindb ain wi h U251HF-GFP cells, showing be e ac i i y o he combina ion
he apy [
176
]. Honokiol, a na u al compound wi h an i-in lamma o y, an i-mic obial, an i-
oxida i e and an i-dep essan p ope ies, was es ed o i s an i- umo al ac i i y in zeb a ish
models using U87MG cells injec ed in o he yolk sac. Honokiol p e en ed he mig a ion
o he cance cells o he b ain and ail, educed mig a o y, in asi e and p oli e a i e
umo ac i i y wi h an inc ease in caspase-2 and inhibi ed he EGFR, CD133, Nes in, STAT3
phospho yla ion and AKT/ERK signaling pa hways [
177
]. In he U87MG-zeb a ish model,
micella -nanopa icles (NPs) loaded wi h honokiol and doxo ubicin p e en ed umo
p og ession; in an emb yonic angiogenesis-zeb a ish model Tg( lk1: EGFP), he same NPs
inhibi ed he g ow h o in e segmen al essels (ISVs) hanks o a con olled elease o
bo h d ugs, in a mo e e ec i e way han NPs loaded wi h jus one o he ea men s [
178
].
Zeb a ish models also o e ad an ages in e ms o e hical and economic issues o he
sc eening o new ea men s. A e an ini ial
in i o
selec ion o d ugs om adi ional
Chinese medicine o ea GBM s em cells, he 13 bes candida es we e e alua ed o oxici y
and an i- umo al e icacy in U87MG-zeb a ish models, esul ing in he selec ion o clo oc ol
as he compound wi h he bes ac i i y asc ibed o o e exp ession o a p o-apop o ic ac o
(KLF13) [179].
Gene ic and epigene ic he apies ha e been also es ed using zeb a ish GBM models.
P o ein a ginine me hyl ans e ase 5 (PRMT5) inhibi o s, which egula e gene exp ession by
me hyla ion o his onic and non-his onic p o eins, imp o ed su i al a e mic oinjec ion
o GBM human de i ed cells exp essing GFP in he midb ain-hindb ain o 36hp caspe
zeb a ish [
180
]. Schnekenbu ge e al. syn he ized and es ed inhibi o s o HDAC and
si uin (SIRT) in GBM-xenog a s, showing hei e icacy in p e en ing umo g ow h [
181
].
An in e es ing pha macological app oach has been based on he ollowing obse a ion by
Pudelko e al.: human mu T homologue 1 (MTH1), an enzyme esponsible o deg ading
oxidized nucleo ides—which ha e high oxida i e p essu e and p o ec umo cells—is
up- egula ed in GBM and ela ed o a poo p ognosis [
184
]. To ea his condi ion, MTH1
inhibi o s, which o ce he cance cell o inco po a e oxidized nucleo ides in o he DNA,
ha e been es ed in o ho opic GBM-zeb a ish models, showing sa is ac o y an i- umo al
e icacy. Tiek e al. s udied
in i o
he unc ions o he h ee iso o ms o he es ogen- ela ed
ecep o
β
(ERR-
β
) in he con ex o GBM. By using a TMZ- esis an GBM cell line labeled
wi h DiL and mic oinjec ed in o he b ain o zeb a ish, signi ican an i- umo al ac i i y was
e ealed when an ERR-
β
agonis (DY131, which displaces he ERR-
β
owa ds he ERR-
β
2
iso o m) was combined wi h a CLK inhibi o (TG-003) [
182
]. In ano he s udy, 5- luo ou acil
and e lo inib ( y osin kinase inhibi o ) educed umo size in zeb a ish xeno ansplan s
Cance s 2021,13, 1087 23 o 37
wi h mu ine-GBM-cells o e exp essing ERR-
β
2 [
183
]. Thus, hese ea men s could be a
good al e na i e o TMZ- esis an umo s.
Zeb a ish models ha e been applied o e alua e he ou comes o me abolic manip-
ula ion in cance p og ession and GBM [
187
]. Sh aizen e al. showed how mannose
phospha e isome ase, as a me abolic enzyme, can main ain Wa bu g me abolism in ze-
b a ish emb yos wi h GBM [
188
]. Using he same zeb a ish umo model, Wehmas e al.
obse ed signi ican umo inhibi ion a e ea men wi h LY294002, a selec i e PI3K in-
hibi o [
167
]. These esul s alida e he same indings obse ed in o gano ypic mouse b ain
issues [189].
5.2. Use o Zeb a ish o Tes An i-Angiogenic Ac i i y
Inhibi ion o angiogenesis is applied o he ea men o cance o educe he supply
o oxygen and nu ien s o he umo , commonly in combina ion wi h o he he apies [
190
].
Se e al zeb a ish models ha e been used as ools o e alua ing angiogenesis as well as
b ain pa hologies in eal ime [191,192]. An in e es ing p o ocol o s udy umo angiogen-
esis using zeb a ish emb yos was es ablished in 2007 [
139
] and ad an ages/limi a ions
o his model e sus o he s ha e been e iewed by Nowak-Sliwinska e al. [
193
]. In he
con ex o GBM, Wang e al. es ed na u al inhibi o s o cyclooxygenase-2 (COX-2), mic o-
somal p os aglandin E syn hase-1 (mPGES-1) and cy och ome P450 (CYP4A11) as enzymes
in ol ed in angiogenic de elopmen [
194
]. Among di e en la onoids, isoliqui i igenin
(ISL) showed he mos po en an i-angiogenic ac i i y in zeb a ish and abbi co neal mod-
els. This an i-angiogenic e ec led o an imp o ed an i- umo al e icacy and no malized
glioma ascula u e in combina ion wi h TMZ in mu ine models o glioma. In ano he
s udy, he chemo he apeu ic agen dianhyd ogalac i ol (DAG) was e alua ed in zeb a ish
models o phase II clinical GBM ials whe e inhibi ion o mig a ion and in asion o
glioma cells was seen as well as dose-dependen educ ion in he exp ession o VEGF,
VEGFR2, FGF2 and FGFR2, all o which co ela ed wi h educed umo angiogenesis [
195
].
Bousseau e al. demons a ed he abili y o a phosphi e (PST3.1a) o block angiogenesis
by in e ac ion wi h VEGFR2 and galec in-1 [
196
], using zeb a ish. No dy, an inhibi o
o a achinoda e 5-lipoxygenase, was compa ed wi h well-known VEGF ecep o y osin
kinase inhibi o s (i.e., Ve alanib, Sun inib and Axi inib) in zeb a ish emb yos whose yolk
sacs had been injec ed wi h GSCs; he esul s showed how hey we e able o block umo -
induced essel o ma ion and inhibi he in asion and p oli e a ion o GSCs by p omo ing
hei di e en ia ion [186].
5.3. Use o Zeb a ish o Tes he Abili y o D ugs o C oss he Blood–B ain–Ba ie
A majo limi a ion in he applica ion o new d ugs in he igh agains GBM is hei
abili y o c oss he Blood–b ain–ba ie (BBB). This physiological ba ie limi s he passage
o molecules om he ci cula o y sys em o he cen al ne ous sys em (CNS) and is he
cause o ailu e o 98% o he d ugs ha a ge he CNSs es ed in clinical ials [197].
P e ious s udies on he de elopmen and ma u a ion o he BBB in zeb a ish ha e
shown a sophis ica ed BBB, which is unc ionally and s uc u ally simila o ha o highe
e eb a es. Al hough i is no exac ly he same, hese s udies sugges he use o zeb a ish
as an expe imen al model o ganism o BBB-pene a ing d ug sc eenings [
174
,
198
]. Con-
sequen ly, se e al d ug deli e y sys ems ha e been es ed wi h he aim o imp o ing he
abili y o pha macological molecules o c oss i . Acco ding o s udies ca ied ou on bo h
mammals and zeb a ish, hei simila BBBs ha e been a ibu ed no only o he neu o as-
cula cellula composi ion, namely endo helial cells, pe icy es, glia, neu ons and mic oglia,
bu also o hei associa ions, igh junc ion p o eins and ac i e anspo sys ems [
199
].
Likewise, high conco dance has been epo ed in he de elopmen and unc ion o CNS
capilla ies and unde lying molecula e en s d i ing hese p ocesses [
192
]. Addi ionally,
he exp ession o Claudin-5 and ZO-1, which is concomi an wi h he ma u a ion o he
BBB, was de ec ed in ce eb al mic o essels om 3 days dp [
174
,
198
]. Ne e heless, se e al
issues ha e been encoun e ed in ce ain s udies. Fo ins ance, zeb a ish p esen a popula-
Cance s 2021,13, 1087 24 o 37
ion o adial glia ins ead o he classic s ella e as ocy es in mammals and al hough his
glia exp esses ele an as ocy ic signals and also plays a ole o ion homeos asis in he
b ain, some di e ences which emain o be ully unde s ood migh be encoun e ed [
200
].
Some di e ences be ween mammalian and zeb a ish pe icy es ha e been ound, such as
lack o exp ession o canonical ma ke s in he la e (i.e., Rgs5a o Desmin a/b) [
201
]. The
o igin o c anial pe icy es is exclusi e o he neu al c es in mammals, while in zeb a ish
pe icy es may also be o mesenchymal o igin [
202
]. Despi e hese limi a ions, he lexibili y,
p edic abili y and ansla ional alue o igo ous expe imen s o zeb a ish o humans
should be conside ed when using zeb a ish BBB models o p eclinical sc eenings o new
he apies. Below we p o ide some examples.
Doxo ubicin (DOX), a chemo he apeu ic commonly used in cance and unable o
c oss he BBB, is encapsula ed in an apo e i in nanocage o a ge he ans e in ecep o 1
(T R1) o e exp essed in b ain endo helial cells and GBM cells. These NPs, labeled wi h he
luo escen dye Cy5.5, upon injec ion in he hea s o zeb a ish a e able o c oss he BBB,
so a e an in e es ing s a egy agains GBM [
197
]. The same T R1 ligand is used in ca bon
ni ide do s, conjuga ed wi h gemci abine and ans e in (CN-GM-T ) NPs, showing
s ong an i- umo al e icacy in GBM zeb a ish models [
203
]. Ano he app oach consis s o
inc is ine sul a e in powde o m added o low-densi y lipop o ein (LDL, exp essed in
he endo helial cells o he BBB and glioma cells) NPs modi ied wi h he T7 pep ide T R
ligand. This dual in e ac ion is also e ec i e in c ossing he BBB and ac ing on GBM, bu
using LDL nanoca ie s could be p oblema ic in pa ien s wi h high choles e ol le els [
204
].
Zou e al. used monosialo e ahexosylganglioside (GM1) as a a ge ing ligand o imp o e
he anspo o DOX-loaded micelles h ough he BBB [
205
]. LysoGM1, a p oduc o GM1
hyd olysis wi h a hyd ophilic g oup o imp o e he anspo ac oss he BBB, is used
o unc ionalize poly(lac ic-co-glycolic acid) (PLGA) NPs loaded wi h DOX. DOX/GM1
and PLGA-lysoGM1/DOX a e able o inc ease he an i- umo al e icacy o DOX in GBM
zeb a ish models and imp o e ne e unc ions [205,206].
5.4. Use o Zeb a ish o Tes he Toxici y o D ugs
In addi ion o he s udies de ailed abo e, zeb a ish ha e been widely used o oxici y,
biocompa ibili y and/o biodis ibu ion sc eening o d ugs, bene i ing om i s ease o use
and e hical and economic ad an ages e sus o he animal models. Fo example, cobal (III)
bound o nimesulide (Co-NMS), a COX-2 inhibi o , has been p o en o inc ease he e ec s
o adio he apy in GBM and i s neu o oxici y has been e alua ed in zeb a ish models
om he changes in neu obeha iou capaci y (swimming ac i i y and mo emen ) and
mo phological abno mali ies in he CNS om hema oxylin/eosin s aining o his ological
sec ions o he b ain. Reduc ion in b ain unc ion and de elopmen has been obse ed a
Co-NMS concen a ions highe han 10
µ
M, while 5
µ
M concen a ion has been consid-
e ed o be sa e; he e has also been a mo e in ense adio he apy e ec on GBM in e ms
o a ise in he gene a ion o ROSs and mi ochond ia damage in umo cells [
207
]. Se -
e al alkaloids (i.e., moschamine, N-p-couma oyl se o onin) ha e been es ed in zeb a ish,
which showed no oxici y, al hough cy os a ic and cy o oxic e ec s owa ds GBM cells
we e induced [
208
,
209
]. Cu cumin, a neu op o ec i e phy o he apeu ic, encapsula ed
in me hoxy-poly(e hylene-glycol)-poly(
ε
-cap olac one) (MPEG-PCL) NPs showed be e
abso p ion and biodis ibu ion e sus he ee d ug, in pa icula in lipophilic a eas, such
as he CNS and he yolk sac [210].
As a whole, se e al s udies using zeb a ish ha e been pe o med o la ge d ug
sc eenings and also o unde s and he mechanism o ac ion o pa icula ea men s in
zeb a ish GBM. Fu he mo e, op imized zeb a ish models, desc ibed in Sec ion 4, o e
aluable ools o u he s udies, expec ed in he nea u u e, on he e ec o new d ugs in
pa icula cases (i.e., gene ic modi ica ions) and he TME (i.e., mac ophages).
Cance s 2021,13, 1087 25 o 37
6. Conclusions
In ecen yea s, seminal s udies on glioblas oma (GBM) ha e un a eled he in e ac-
ions be ween umo cells and immune cells om he ini ial s eps and along he p og ession
o he pa hology. In his ega d, mac ophages/mic oglia, ep esen ing up o 30-50% o
cells in some umo s, p esen p o umo al p ope ies and unc ions which can, in heo y, be
ep og ammed wi h app op ia e ea men s, no ye a ailable in he clinic, as sugges ed by
some p e-clinical esul s desc ibed in Sec ion 2. In ac , 11 open phase III clinical ials o
GBM pa ien s a e ongoing, mos ly ela ed wi h immuno he apy [
211
]. Despi e signi ican
scien i ic e o s, he s anda d o ca e ea men o GBM is s ill su ge y, ollowed by a-
dio he apy and TMZ, wi h only e alua ion o IDH mu a ions and me hyla ion o MGMT
as molecula ma ke s o ou ine o classi y and ea he disease. Nume ous s udies ha e
e ealed gene ic, epigene ic and me abolic al e a ions encoun e ed in GBM. Among hem,
we highligh he iden i ica ion o miRNAs and hei changes in GBM as e y a ac i e
a ge s o diagnosis and/o he apeu ic pu poses, ollowing a simila end o o he ypes
o cance [59–61,212].
In his con ex , gene ic and xeno ansplan zeb a ish models ha e been success ully
used o gene a e new knowledge abou GBM pa hology and some s udies ha e been
ini ia ed o sc eening o new ea men s. While he e is s ill some oom o imp o emen
ela ed o he modeling o he human immune sys em and op imiza ion o o ho opic
models o GBM in zeb a ish, sa is ac o y esul s ha e al eady been es ablished o de ailed
unde s anding o gene and me abolic al e a ions o he disease.
O e all, we expec ha zeb a ish models will be u he exploi ed by aking in o
accoun hei biological, e hical and economic ad an ages o he sc eening and comp e-
hensi e e alua ion o new d ugs o ul ima ely imp o e he ea men o pa ien s wi h
glioblas oma.
Au ho Con ibu ions:
M.C.C., V.L.I., M.T.-P. and C.A. ha e con ibu ed o he w i ing and he
elabo a ion o Figu es 1and 2, A.P.-L. o he w i ing and he elabo a ion o Figu e 3, he ables, he
e iew and he edi ing, and P.R., L.S., C.M.R. and F.T.A., o he w i ing, he e iew and he edi ing.
All au ho s ha e ead and ag eed o he published e sion o he manusc ip .
Funding:
F.T.A. has been suppo ed by he AECC (“Asociación Española Con a el Cánce ”, Spain).
We would also like o hank he ollowing: he Talen o P og am om Mad id Go e nmen , Spain
(2017-T1/BMD-5333); Con oca o ia 2018 de p oyec os de I+D+i «RETOS INVESTIGACIÓN» (RTI2018-
095061-B-I00) ( o C.M.R.); “Con oca o ia de ayudas pa a la con a ación de ayudan es de in es i-
gación” (PEJ-2018-AI/BMD-9724) ( o M.T.-P.); he Xun a de Galicia P e-doc o al Fellowship (ED481A-
2018/095) ( o A.P.L.).
Con lic s o In e es :
The au ho s decla e ha he e is no con lic o in e es . The au ho s ce i y
ha hey ha e no a ilia ions wi h o in ol emen in any o ganiza ion o en i y wi h any inancial
in e es o non- inancial in e es in he subjec ma e o ma e ials discussed in his manusc ip . The
unde s had no ole in he s udy design, collec ion, analyses, in e p e a ion o he da a, w i ing o he
manusc ip , o in he decision o publish he esul s.
Abb e ia ions
2-HG 2-hyd oxyglu a a e
AHR A yl hyd oca bon ecep o
AKT1 AKT se ine/ h eonine kinase 1
ATP Adenosine iphospha e
ATP5A1 ATP syn hase, H+ anspo ing, mi ochond ial F1 complex, alpha subuni 1
ATP5B ATP syn hase, H+ anspo ing, mi ochond ial F1 complex, be a polypep ide
ATRX ATRX ch oma in emodele
BBB Blood-b ain ba ie
C4S Chond oi in 4-sul a e
Cance s 2021,13, 1087 32 o 37
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