Cance Le e s 591 (2024) 216879
A ailable online 16 Ap il 2024
0304-3835/© 2024 The Au ho s. Published by Else ie B.V. This is an open access a icle unde he CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-
nc-nd/4.0/).
O iginal A icles
Galec in-3 deple ion ames p o- umou al mic oglia and es ains cance
cells g ow h
Albe o Ri e a-Ramos
a
,
b
,
1
, Luis C uz-He n´
andez
a
,
b
,
1
, Rocío Tala e ´
on
a
,
b
,
1
,
Ma ía Te esa S´
anchez-Mon e o
a
,
b
, Juan Ga cía-Re illa
a
,
b
, Ma a Mule o-Ace edo
a
,
b
,
Tomas Deie bo g
c
, Jos´
e Luis Vene o
a
,
b
, Manuel Sa mien o So o
a
,
b
,
*
a
Ins i u o de Biomedicina de Se illa, IBiS/Hospi al Uni e si a io Vi gen Del Rocío/CSIC/Uni e sidad de Se illa, Se illa, Spain
b
Depa amen o de Bioquímica y Biología Molecula , Facul ad de Fa macia, Uni e sidad de Se illa, Spain
c
Expe imen al Neu oin lamma ion Labo a o y, Depa men o Expe imen al Medical Science, Lund Uni e si y, Lund, Sweden
ARTICLE INFO
Keywo ds:
Galec in-3
Mic oglia
B ain me as asis
Glioblas oma
Tumou -associa ed mic oglia and mac ophages
(TAMs)
ABSTRACT
Galec in-3 (Gal-3) is a mul i unc ional p o ein ha plays a pi o al ole in he ini ia ion and p og ession o a ious
cen al ne ous sys em diseases, including cance . Al hough he in ol emen o Gal-3 in umou p og ession,
esis ance o ea men and immunosupp ession has long been s udied in di e en cance ypes, mainly ou side
he cen al ne ous sys em, i s ele a ed exp ession in myeloid and glial cells unde sco es i s p o ound impac on
he b ain’s immune esponse. In his con ex , mic oglia and in il a ing mac ophages, he p edominan non-
cance ous cells wi hin he umou mic oen i onmen , play c i ical oles in es ablishing an immunosupp essi e
milieu in di e se b ain umou s. Th ough he u ilisa ion o p ima y cell cul u es and immo alised mic oglial cell
lines, we ha e elucida ed he cen al ole o Gal-3 in p omo ing cance cell mig a ion, in asion, and an immu-
nosupp essi e mic oglial pheno ypic ac i a ion. Fu he mo e, employing wo dis inc in i o models encom-
passing p ima y (glioblas oma) and seconda y b ain umou s (b eas cance b ain me as asis), ou his ological
and ansc ip omic analysis show ha Gal-3 deple ion igge s a obus p o-in lamma o y esponse wi hin he
umou mic oen i onmen , no ably based on in e e on- ela ed pa hways. In e es ingly, his esponse is p om-
inen ly obse ed in umou -associa ed mic oglia and mac ophages (TAMs), esul ing in he supp ession o cance
cells g ow h.
1. In oduc ion
The umou mic oen i onmen (TME) o e s a ich spa io empo al
dynamic o cellula componen s. Apa om malignan cance cells,
he e a e a complex my iad o issue esiden and in il a ed pe iphe al
immune sys em cells. This e ol ing mosaic o mul i unc ional cells
makes he TME a a he complica ed opic o s udy in he ield, wi h
g ea impo ance o de elop no el he apies ha may e ec i ely be used
in he mos challenging cance s o ea .
B eas cance has been ecen ly placed as he mos diagnosed cance
wo ldwide, wi h g ea a ini y o colonise dis an o gans, specially he
b ain [1]. Thus, depending on umou sub ype, b eas cance b ain
me as ases (BCBM) a e e y common and ep esen a majo heal h issue
wi h meag e su i al a es amongs pa ien s (ca. 12 mon hs) [1]. In u n,
one o he mos common o ms o p ima y b ain umou s is glioblas oma
(GB), he deadlies ypes o b ain cance , whe e pa ien s who unde go
ea men ha e an a e age su i al measu ed jus in mon hs (ca. 15
mon hs) [2]. Despi e p ima y b ain umou s and he me as a ic coloni-
sa ion o he b ain a e comple ely di e en diseases, hey g ow unde
he in luence o he unique immune mic oen i onmen ound in he
cen al ne ous sys em, which clea ly d i es he disease p og ession [1,
3].
BCBM and GB a e conside ed immunologically “cold”, owing o hei
lack o umou an igens, ine icien T-cell in il a ion and an abundan
p esence o myeloid-de i ed immune cells [4]. Amongs he b oad
ple ho a o cells ha cons i u e he TME, he mos abundan
non-cance ous cell ype a e mic oglia and in il a ed mac ophages.
Tumou -associa ed mic oglia and mac ophages (TAMs) ha e been
* Co esponding au ho . Dep . Biochemis y and Molecula Biology. P o eso Ga cía Gonz´
alez s , Uni e si y o Se ille, Spain.
E-mail add ess: [email p o ec ed] (M. Sa mien o So o).
1
These au ho s con ibu ed equally.
Con en s lis s a ailable a ScienceDi ec
Cance Le e s
jou nal homepage: www.else ie .com/loca e/canle
h ps://doi.o g/10.1016/j.canle .2024.216879
Recei ed 26 Oc obe 2023; Recei ed in e ised o m 8 Ap il 2024; Accep ed 9 Ap il 2024
Cance Le e s 591 (2024) 216879
2
desc ibed o de elop an an i-in lamma o y pheno ype and ul ima ely
suppo BCBM and GB g ow h [5,6].
In ha sense, one key componen o he immunosupp essi e
beha iou o TAMs is Galec in-3 (Gal-3) [7,8]. This β-galac oside-bind-
ing p o ein is long known o be a poo p ognos ic ma ke in b eas
cance [9] and glioblas oma [10]. Gi en he immunomodula o y ole o
Gal-3, a po en ial ole o his lec in in d i ing a p o- umou al TME in
b ain umou s is wo h being conside ed. P e ious wo ks ha e shown
how ac i a ed mic oglia a e he majo sou ce o Gal-3 wi hin he b ain
[7,11], oge he wi h as ocy es [12]. The e o e, his s udy has ocused
on he ole o TAMs-associa ed Gal-3, in d i ing immunosupp ession in
BCBM and GB.
Ou esul s desc ibe a s ong p esence o Gal-3 closely associa ed o
TAMs in BCBM and GB human esec ions, and in i o models. Blocking
Gal-3 in i o, we managed o shape mic oglia ac i a ion, supp essing
hei an i-in lamma o y pheno ype and inhibi ing cance cells mig a ion
and in asion. Ou in i o models p o ided compelling e idence sup-
po ing a c i ical ole o Gal-3 in d i ing TAMs-associa ed immunosup-
p ession wi hin BCBM and GB mic oen i onmen . T ansc ip omic
analysis showed a s ong ac i a ion o key inna e and adap i e immune
esponse pa hways, hus, empe ing p o- umou al TAMs pheno ype o-
wa ds a mo e p o-in lamma o y s a e, con ibu ing o cy o oxic T-cell
in il a ion and umou size educ ion. Las ly, GB-bea ing Gal-3 KO mice
who unde wen he s anda d chemo he apeu ic agen , Temozolomide
(TMZ), showed a syne gis ic e ec in a es ing umou g ow h [13].
2. Ma e ial and me hods
2.1. Cell cul u e and ans ec ion
GL261 (mouse glioblas oma), BV2 (mouse mic oglia) and EO771
(mouse mamma y ca cinoma) cell lines we e pu chased om he
Ame ican Type Cul u e Collec ion (ATCC, USA). Fo de ailed cul u e
condi ions see Supp. da a. BV-2 cells Gal-3 knockou we e kindly p o-
ided by P o Tomas Deie bo g. Fo p ima y mic oglia cul u e, mig a-
ion and in asion, please ead Suppplemen a y da a.
2.2. RNA ex ac ion and quan i a i e PCR
RNA was ex ac ed om he cell lines using he RNeasy Ki (Qiagen,
Ne he lands). Fo p o ocol and p ime s in o ma ion see Supp. da a.
2.3. Wes e n blo ing assay
Memb anes we e incuba ed wi h an i-Gal 3 an ibody (1:1000).
GAPDH an ibody (Sigma-Ald ich; 1:2000; USA) was used as a loading
con ol. Fo p o ocol in o ma ion see Supp. da a.
2.4. Animals and su ge y
Expe imen s we e pe o med in 12-week-old mice C57BL/6 (wild-
ype, w , n =8) and Galec in-3 null mu an mice (Gal-3 KO, n =8) wi h
he same backg ound, bo h lines ob ained om Cha les Ri e s. Female
mice we e used o he BCBM model [14] and male mice o he GB
model [15]. Animal expe imen a ion was ca ied ou in acco dance wi h
he Eu opean Communi y Council Di ec i es (86/609/EU) and Spanish
law (R.D. 53/2013 BOE 34/11370-420, 2013) o he use and ca e o
labo a o y animals. Fo in i o p o ocol de ails, please see Supplemen-
a y da a.
2.5. Immunohis ochemis y and mic oscopy
S aining p o ocol was pe o med as p e iously desc ibed [15]. Fo
ull in o ma ion, please see Supp. da a.
2.6. Quan i a ion o umou bu den
All da a we e analysed blinded o expe imen al condi ions. Tumou
g ow h was his ologically assessed as p e iously desc ibed [16,17],
(Supp. Figu e 2). Fo ull in o ma ion see Supp. da a.
2.7. TAMs analysis
Colocalisa ion s udies we e analysed using he ImageJ so wa e
package (NIH) as p e iously desc ibed [18]. Full in o ma ion on he
plugins and so wa e can be ound in he Supp. Da a.
2.8. Mic oa ay analysis
To s udy he di e ences in genes exp ession be ween w (n =3) and
Gal-3 KO mice (n =3), T ansc ip ome Analysis Console (TAC) So wa e
was used. Gene Se En ichmen analysis (GSEA) and he Da abase o
Anno a ion, Visualiza ion and In eg a ed Disco e y (DAVID) we e also
used o u he analyse gene clus e s compa isons and key biological
pa hways. Fo ull in o ma ion see Supp. da a.
2.9. Human da a and samples om umou Bank collec ion
Samples and da a we e p o ided by Biobank HUB-ICO-IDIBELL, in-
eg a ed in he Spanish Biobank Ne wo k and unded by Ins i u o de
Salud Ca los III (PT20/00171) and by Xa xa de Bancs de Tumo s de
Ca alunya (XBTC, Pla Di ec o d’Oncologia de Ca alunya). We down-
loaded he Cance Genome A las (TCGA) da a o he b eas cance
(TCGA-BRCA) and he lowe -g ade glioma and glioblas oma (GBMLGG)
da abase om UCSC Xena (h ps://xena.ucsc.edu/).
2.10. S a is ical analysis
All da a a e p esen ed as mean ±SD. Unless o he wise s a ed, da a
we e analysed using G aphPad P ism ( 8.4). Fo 2 g oups, S uden ’s -
es (2- ailed) was used. Fo mo e han 2 g oups, 1-way ANOVA wi h
Tukey’s mul iple compa ison es o wo-way Ano a wi h Bon e oni
es we e used and adjus ed P alues a e epo ed. S a is ical signi icance
was de ined as p <0.05.
2.11. Da a a ailabili y s a emen
T ansc ip omic da a we e gene a ed a he Genome and Sequencing
acili ies in he Ins i u e o Biomedicine o Se ille (IBiS, Spain). Raw da a
suppo ing he indings o his s udy a e comple ely a ailable om he
co esponding au ho (MSS) on eques .
3. Resul s
3.1. TAMs a e a majo sou ce o Gal-3 in he TME o BCBM and GB
Fi s ly, we in es iga ed whe he he TME o BCBM and GB showed
signi ican le els o Gal-3 and, was closely ela ed o TAMs. The in i o
EO771 (Fig. 1A–C, BCBM) and GL261 (Fig. 1D–F, GB) models showed a
s ong p esence o Gal-3 in he TME. Using Iba-1, a pan-ma ke o TAMs,
we obse ed ha mos Gal-3 exp ession colocalised wi h hese cells.
Fu he mo e, o alida e he Gal-3 KO (Fig. 1C and F) model, no p es-
ence o Gal-3 was ound, excep o a negligible amoun likely de i ed
om he umou cells (Fig. 1A and D).
We also aimed a deciphe ing whe he Gal-3 (LGALS3) exp ession
had a p ognos ic alue in b eas cance pa ien s. Thus, a e using The
Cance Genome A las (TCGA) da abase, we ound ha high exp ession
o LGALS3 was signi ican ly linked wi h poo e su i al (Fig. 1G).
Fu he mo e, human b ain esec ions om pa ien s wi h BCBM (Fig. 1H)
had a s ong exp ession o Gal-3 in he umou oci, wi h clea inc ease
in he co e compa ed wi h he ma gins o he umou . Tha Gal-3
A. Ri e a-Ramos e al.
Cance Le e s 591 (2024) 216879
3
Fig. 1. A.-) Quan i a ion o TAMs exp essing Gal-3 in he TME in w and Gal-3 KO mice (n =8) injec ed wi h EO771 cells. B.-) Imuno uo escence (IF) image (me ge
–le - inse – igh -) showing he me as a ic a ea in a w mouse. (DAPI, blue), Gal-3 (G een) and TAMs (Iba1, ed). C.-) Me as a ic a ea o a Gal-3 KO mouse. D.-)
Quan i a ion o TAMs exp essing Gal-3 in he TME in w and Gal-3 KO mice (n =8) injec ed wi h GL261. E.-) IF image (me ge –le - inse – igh -) showing he GB a ea
in a w mouse. F.-) Me as a ic a ea o a Gal-3 KO mouse. G.-) GDC TCGA b eas cance da abase analysing he dele e ious impac o high le els o LGALS3. H.-)
Quan i a ion (n =6) o Gal-3-exp essing TAMs. BCBM esec ion om a 59-yea s-old woman, showing a s ong p esence o TAMs (Iba1, ed) colocalising wi h Gal-3
(g een). I.-) TCGA da abase showing he s iking nega i e in luence o high le els o LGALS3 in low and high-g ade glioma pa ien s. J.-) Quan i a ion (n =6) o Gal-3-
exp essing TAMs. GB esec ion om a 67-yea s-old man, highligh ing (inse ) he a ea wi h s ong p esence o TAMs (Iba1, ed) and Gal-3 (g een). Scale ba s: 100
μ
m.
*, p <0,0001 (Welch’s - es .
A. Ri e a-Ramos e al.
Cance Le e s 591 (2024) 216879
4
exp ession was closely ela ed wi h TAMs and umou cells, amongs
o he cell ypes. Rega ding TCGA s udies in GB samples, high LGALS3
was also closely linked wi h meag e su i al in low g ade and glio-
blas oma pa ien s, sugges ing he dele e ious impac ha his p o ein in
hose pa ien s (Fig. 1I). Again, Gal-3 p esence was ound in GB e-
sec ions, wi h he same pa e n ound in BCBM samples (Fig. 1J). In
gene al, he he e ogeneous p esence o Gal-3 in he TME, likely o ming
a eas o pseudopalisades, unde sco es i s in ol emen in he adap i e
s a egies o umou cells and TAMs wi hin he s essed TME (e.g.
nu ien dep i a ion o hypoxia). Al oge he , wi h hese esul s, we
demons a ed a s ong p esence o Gal-3-exp essing TAMs in wo
di e en o ms o b ain umou s.
3.2. Gal-3 igge s an an i-in lamma o y pheno ype in mic oglia cell line
P e ious wo ks om ou esea ch eam desc ibed he key ole o Gal-
3 in TREM2-associa ed mic oglial ac i a ion [7,11,19]. Tha pheno ypic
change in mic oglial esponse upon Gal-3 p esence s ongly sugges ed
he need o elucida e he po en ial ole o his lec in in he ac i a ing
pheno ype in TAMs.
3.2.1. EO771-condi ioned medium
We aimed a deciphe ing how b eas cance -de i ed Gal-3 may
egula e mic oglia pola isa ion. To his end, we moni o ed iNOS, A g1,
CD206 and PD-L1.
iNOS, he p o o ypic ma ke o p o-in lamma o y mic oglia [20]
(Fig. 2A), su e ed a signi ican d op when soluble Gal-3 was added o
he medium. In e sely, A g1, CD206 and PD-L1 (Fig. 2B–D), p o o ypic
ma ke s o an i-in lamma o y (p o- umou al) mic oglia [6,21,22], once
TCM was added, hei exp ession was signi ican ly inc eased. Howe e ,
when Gal-3 was silenced in umou cells and hei condi ioned medium
was hen added (Gal3KD), le els o e e y ma ke signi ican ly d opped.
Fig. 2. Top ow is de o ed o BV-2 cells ea ed wi h EO771-condi ioned medium. Ba g aphs show he quan i a i e analysis o p o-in lamma o y ma ke iNOS (A),
and immunosupp essi e A g1 (B), CD206 (C) and PD-L1 (D). Cells we e ea ed (24h) wi h umou -condi ioned medium om pa en al EO771 (EO) o EO771 cells
wi h Gal-3 knockdown (siRNA, Gal3KD). Las expe imen al condi ion was used o s udy he e ec o he exogenous addi ion o Gal-3 o EO-Gal3KD medium. Con ocal
Images showing he exp ession o iNOS (E, ed), A g1 (G een, F), CD206 (G een, G) and PD-L1 (H, ed). Le images e e o con ol (DMEM) and igh images o cells
ea ed wi h exogenous Gal-3. As pe EO771 g oup, Bo om ow is de o ed o BV-2 cells ea ed wi h GL261-condi ioned-medium. Con ocal images show exp ession
o iNOS ( ed, I), A g1 (G een, J), CD206 (K, G een) and PD-L1 (L, ed). Ba g aphs show quan i a i e analysis o he exp ession o iNOS (M), A g1 (N), CD206 (O) and
PD-L1 (P). Scale ba s: 50
μ
m.
A. Ri e a-Ramos e al.
Cance Le e s 591 (2024) 216879
5
In con as o iNOS, when exogenous Gal-3 was added back o he me-
dium, he le els o e e y immunosupp essi e ma ke we e up egula ed.
Rep esen a i e images o he abo e-men ioned ma ke s a e depic ed in
Fig. 2E–H.
A a gene le el, iNOS showed a simila beha iou as p e iously
desc ibed, since i s gene exp ession was signi ican ly up egula ed (>3-
old) a e EO-TCM and Gal-3 exposi ion. Simila ly, A g-1, CD206 and
PD-L1 we e up egula ed once BV2 we e exposed o TCM and Gal-3
(Supp. Figu e 3).
Rega ding he s udy using BV-2 Gal-3 KO cells, hese cells we e less
esponsi e o all expe imen al challenges (Supp. Figu e 4). Howe e ,
iNOS was down egula ed when exogenous Gal-3 was added. In e sely,
A g1, CD206 and PD-L1 we e up egula ed upon Gal-3 addi ion,
mimicking he p e ious esul s ound wi h pa en al BV-2 cells.
All hese da a suppo he ole o Gal-3 as a c i ical p omo e o an
an i-in lamma o y pheno ype in mic oglia, by up egula ing classical
immune-supp essi e ma ke s.
3.2.2. GL261-condi ioned medium
When TCM om Gal-3 KD umou cells was added (Gal3KD), iNOS
was signi ican ly inc eased in BV-2 (Fig. 2M). In e es ingly, when Gal-3
was added, iNOS d opped o con ol le els. Con e sely, he absence o
Fig. 3. A.-) Sc a ch-wound assay pe o med wi h EO771 cells in DMEM ( ed do ed lines) condi ions. Time-cou se s udy wi h pic u es a 0, 6 and 24h. EO771 cells
a e 24h exposi ion o GB1107 (black do ed line). B.-) G aph measu ing gap closu e abili y o cells a di e en expe imen al condi ions: con ol (DMEM, ed line),
mic oglia-condi ioned medium (BCV2, G een line), selec i e Gal-3 inhibi o (GB1107, black line) and a e addi ion o exogenous Gal-3 (blue line). C.-) Pic u es o
he sc a ch assay pe o med o GL261 cells in con ol (DMEM, ed do ed line) and GB1107 condi ion (black do ed line). D.-) Quan i a ion o gap closu e unde he
same condi ions as desc ibed in B. E.-) In asion assay pe o med in Boyden Chambe s o EO771 cells, s ained wi h c ys al iole . Cells we e coa ed wi h pa en al BV2
cells (DMEM) a he bo om well, o BV2 wi h Gal3 knockdown (siRNA –Gal3KD-). Nex expe imen al condi ion was pe o med using exogenous Gal-3 wi h Gal3KD
BV2 cells seeded a he bo om (+Gal3), and inally, using pa en al BV2 cells wi h GB1107 (0,1
μ
M). F.-) Quan i a ion o numbe o in ading cells. G.- Same as E, wi h
GL261 cells. H.-) Same as F wi h GL261 cells. *p <0,05; **p <0,01; ***p <0,005. One-way ano a wi h Tukey pos hoc analysis. Scale ba 750
μ
m.
A. Ri e a-Ramos e al.

Cance Le e s 591 (2024) 216879
6
Gal-3 in he TCM (Gal3KD) d opped A g1 le els (Fig. 2N), whils Gal-3
addi ion had an opposing e ec , inc easing A g1 exp ession. Rega ding
CD206, i s le els emained unal e ed excep when cells we e coa ed
wi h Gal3KD-medium (ca. 2- old inc ease, Fig. 2O). This esul s may be
expec ed since p e ious wo ks showed ha glioma cells up egula e
CD206. Finally, PD-L1 was up egula ed a all expe imen al condi ion
(Fig. 2P), wi h special ele ance upon Gal-3 addi ion (ca. 6- old
inc ease).
qPCR analysis showed how Gal-3 inc eased he gene exp ession o
PD-L1 and A g-1 e en highe han he p esence o TCM. In he case o
iNOS, bo h Gal-3 and TCM inhibi ed i s exp ession, hus, sugges ing an
an i-in lamma o y beha iou in BV-2 cells upon Gal-3 p esence (Supp.
Figu e 3)
BV-2 Gal-3 KO cells showed almos no pheno ypic changes a e he
expe imen al challenges, sugges ing he impo an ole o endogenous
Gal-3 o igge he mic oglial esponse in he p esence o TCM. In e -
es ingly, PD-L1 was signi ican ly up egula ed when Gal-3 was added
(Supp. Figu e 4).
These da a a e in ag eemen wi h ou p e ious indings (EO771-
condi ioned medium), highligh ing he key ole o Gal-3 up egula ing
classical immunosupp essi e ma ke s (e.g. A g1 and PD-L1) in BV-2
[23].
In e es ingly, we epea ed he same expe imen al challenges in p i-
ma y mic oglia cells, and he same end owa d he ac i a ion p o-
in lamma o y and he supp ession o immunosupp essi e ma ke s was
ound [24,25] in cells om Gal-3 KO pups (Supp. Fig. 5 and Supp. Da a).
3.3. Pha macological blocking o Galec in-3 hinde s umou cells
mig a ion
We pos ula ed ha pha macological blocking o Gal-3 (GB1107)
[26], could es ain umou cells mig a ion. We used EO771 and GL261
o s udy hei mig a ing p ope ies in esponse o he cell pe meable
GB1107.
In e es ingly, bo h cell lines (Fig. 3A–D) showed simila sensi i i y o
Gal-3 modula ion. Bo h umou cells exposed o mic oglial-condi ioned
medium (BV-2, Fig. 3B and D, g een line) had simila gap closu e speed
compa ed wi h con ol (DMEM) condi ions (Fig. 3A–D, ed line). A
sligh inc ease in he mig a ion pa e n was obse ed when 0.1
μ
M
soluble Gal-3 (Gal-3, Fig. 3B–D, blue line) was added. Impo an ly, when
bo h umou cell lines we e exposed o GB1107, hei mig a ion was
g ea ly educed (Fig. 3A–D, black line). O e all, ou esul s sus ain he
ele an ole o Gal-3 in umou cell mig a ion.
3.4. Mic oglial Galec in-3 suppo s umou cell in asion
We aimed a s udying he ole o mic oglial Gal-3 in he in ading
p ope ies o EO771 and GL261 cells. Pe o ming an in asion assay wi h
an a i icial basemen memb ane [27], umou cells we e co-cul u ed
wi h pa en al BV2 cells, and BV2 cells wi h Gal-3 exp ession
knocked-down (Gal3 KD) (Supp. Figu e 1B). EO771 cells ha eached
he luminal side o he memb ane was signi ican ly lowe wi h BV2
Gal-3 KD cells (Fig. 3E–F and Supp. Figu e 1C, Gal3KD). Adding back
exogenous Gal-3 had a signi ican inc ease in he numbe o in ading
cells (+Gal3). The use o GB1107 (0.1
μ
M), mimicked he esul s om
Gal-3 KD condi ion, and success ully educed he numbe o in aded
umou cells (Fig. 3E–F, GB1107).
Simila ly, GL261 cells mi o ed esul s ound in EO771 cells
(Fig. 3G). When coa ed wi h Gal-3KD BV-2 cells (Gal3KD), numbe o
in ading cells d opped ((Fig. 3G–H). Again, when Gal-3 was added back
o he medium (+Gal3), he numbe o cells eached con ol alues
(DMEM). Impo an ly, when GB1107 was added, he numbe o in asi e
cells signi ican ly d opped, hus, con i ming he impo an e ec o Gal-
3 ega ding in ading capaci y o bo h umou cell lines (Fig. 3H,
GB1107). These esul s unde sco e po en ial ole o Gal-3 egula ing he
TME, exe ing a obus in luence on umou cells mig a ion and
in asion.
3.5. Glioblas oma and b eas cance b ain me as asis bu den is educed in
Galec in-3 knockou mice
EO771 and GL261 cells we e implan ed in wild- ype C57Bl/6 and
Gal-3 KO mice (n =8, pe expe imen al condi ion), and he umou
bu den was quan i ied a di e en ime poin s (Fig. 4, 10 and 21 days).
3.5.1. B eas cance b ain me as asis
Since 99 % o b eas cance cases accoun o women [28], we
ocused ou BCBM in i o s udy in emale mice. E alua ion o he me -
as a ic bu den wi hin he b ain o w and Gal-3 KO mice emained
simila a day 10 (Fig. 4A–B). Howe e , a day 21, Gal-3 KO mice
showed a signi ican d op o he me as a ic bu den (Fig. 4C–E, ca.
8- old).
3.5.2. Glioblas oma
Simila o he BCBM model, GB size showed no di e ence a day 10
(Fig. 4F–G). Again, a day 21, he umou size was signi ican ly educed
in Gal-3 KO mice (Fig. 4H–J, ca. 5- old).
3.6. Galec in-3 a ec s ascula densi y and angiogenesis in he TME
The ole o Gal-3 in p omo ing angiogenesis in cance s ou side he
b ain has been ex ensi ely s udied [24]. Ou indings show ha Gal-3
KO mice exhibi ed educed angiogenic essels compa ed o w (Supp.
Figu e 6). Fu he mo e, ascula densi y was signi ican ly dec eased in
he BCBM model, while he numbe o blood essels emained simila in
he GB model. These indings highligh he po en ial impac o Gal-3 on
angiogenesis and ascula isa ion in di e en b ain cance models.
3.7. Galec in-3 deple ion shi ed TAMs ac i a ion owa ds a p o-
in lamma o y pheno ype
We quan i ied immuno luo escen colocalisa ion o key p o- and
an i-in lamma o y ma ke s in TAMs. In bo h models, we clea ly
obse ed a pheno ype shi owa ds a p o-in lamma o y pheno ype o e
immunosupp ession (Fig. 5).
No di e ences we e ound a day 10 a e umou implan a ion in
ei he model (da a no shown). Howe e , a day 21, in he BCBM model,
he exp ession o iNOS wi hin he TAMs popula ion was inc eased om
15 % in w animals, o 40 % in he Gal-3 KO model (Fig. 5A–C).
Impo an ly, A g-1-exp essing TAMs we e educed 50 % in Gal-3 KO
mice (Fig. 5D–F). Again, hese indings sugges he impo an ole o Gal-
3 in d i ing he p o- umou al TAMs pheno ype.
Wi h ega d o he GB model, pe cen age o iNOS-exp essing TAMs
inc eased om 23 % (w ) up o 58 % (Gal-3 KO) (Fig. 5G–I). On he
con a y, he s ong immunosupp essi e pheno ype in w mice (high
exp ession o A g-1), was signi ican ly d opped in Gal-3 KO mice (4- old
dec ease, Fig. 5J-L).
Unde s anding he complexi y o cha ac e ising he TME, owing in
g ea pa o he b oad numbe o po en ial p o- s an i-in lamma o y
ma ke s o be used, he iNOS/A g-1 balance (Supp. Figu e 7A) may
sugges ha a e Gal-3 deple ion, TAMs showed a shi owa ds an an i-
umou al ac i a ion by inc easing p o-in lamma o y iNOS and educing
he exp ession o immunosupp essi e A g-1 [15]. In e es ingly, he
iNOS/A g-1 balance in o al numbe o cells (beyond TAMs) was il ed
owa ds he p o-in lamma o y side (Supp. Fig. 7). Fu he suppo ing
he an i- umou al scena io gene a ed by TAMs in Gal-3 KO mice,
CD68-posi i e cells we e inc eased in he BCBM Gal-3 KO model, sug-
ges ing ha Gal-3 deple ion migh enhance phagocy ic ea u es in
TAMs. (Supp Fig. 8).
Las ly, in alignmen wi h ou p e ious indings, we employed
GB1107 (28), o in es iga e i s po en ial impac on TAMs pheno ype.
Gi en ha b ain me as ases g ow in close p oximi y o ce eb al blood
A. Ri e a-Ramos e al.
Cance Le e s 591 (2024) 216879
7
essels, we aimed o de e mine whe he GB1107 could shi TAMs
beha iou owa ds a p o-in lamma o y s a e. Rema kably, despi e no
signi ican educ ion in b ain me as asis size (da a no shown), he TAMs
pheno ype in GB1107-injec ed mice mi o ed ou esul s om Gal-3 KO
mice. We obse ed a signi ican inc ease in iNOS and a down egula ion
o A g-1 ac oss he TAMs popula ion (Supp. Fig. 9). These indings
sugges ha GB1107 has he po en ial o modula e TAMs pheno ype in
he TME.
3.8. Gal-3 KO mice showed a obus an i- umou T cell in il a ion
Tumou -in il a ing lymphocy es (TILs) a e also impo an
componen s o he TME. High le el o CD4
+
TILs combined wi h low
CD8
+
TILs is associa ed wi h poo p ognosis in b ain umou s [28]. Since
A g-1 exp essing TAMs impai T cell esponses inducing immunosup-
p ession [15], we pu sui he po en ial e ec o Gal-3-dependen TAMs
pola isa ion in TILs popula ion.
In BCBM, he in a umou al CD4
+
T cells in il a ion was signi i-
can ly inc eased in w mice (Fig. 6A–B). Impo an ly, CD8
+
T cells
in il a ion had an opposing pa e n and we e mo e abundan in Gal3 KO
mice (Fig. 6C–D). This CD8
+
/CD4
+
T Cell a io has long been desc ibed
as a good p ognosis ma ke in b ain umou s [28].
In he GB model, TILs mi o ed he esul s ound in he BCBM mice.
CD4
+
T cells we e less in il a ed in he Gal3 KO mice (Fig. 6F–G), whils
Fig. 4. Con ocal images o he me as a ic a ea o w (n =8) (A) and ansgenic Gal3KO (n =8) (B) emale C57Bl/6 a day 10 and day 21 (C and D). E.-) Quan i a ion
o he me as a ic a ea in w ( ed) and Gal-3 KO mice (ligh ed ba s). Con ocal images o glioblas oma a ea in w (F) and Gal3KO (G) mice injec ed wi h GL261 cells a
day 10. H and I a e images aken a day 21 pos injec ion. J.-) quan i a ion o glioblas oma a ea compa ing w (da k G een) and Gal3KO mice (ligh G een) a 10 and
21 days. S a is ical analysis o umou olume p og ession was made independen ly ac oss bo h imes. One-way ano a and Tukey pos hoc es s was pe o med. ***,
p <0,005. ****, p <0,001.
A. Ri e a-Ramos e al.
Cance Le e s 591 (2024) 216879
8
CD8
+
T we e signi ican ly inc eased (Fig. 6H–I).
We obse ed a ypical clus e ing pa e n in TILs, p esumably a si es
o an igen p esen a ion [29]. TAMs, as an igen-p esen ing cells, had a
deep impac in TILs, since he p o-in lamma o y pheno ype ound in
Gal-3 KO mice, suppo ed a umou icidal CD8
+
/CD4
+
TILs a io (Fig. 6E
and J).
3.9. Tumou size was u he dec eased a e emozolomide ea men
Following he signi ican educ ion in umou size obse ed in Gal-3
KO mice, we sough o de e mine whe he TMZ adminis a ion could
po en ially enhance he al eady desc ibed an i- umou al mic oen i-
onmen . Ou indings e ealed ha animals ea ed wi h wo ounds o
TMZ exhibi ed a signi ican educ ion in GB compa ed o hose ea ed
wi h TMZ alone (Supp. Figu e 10). Mo eo e , ou da a indica e ha TMZ
shi ed TAMs owa ds a p o-in lamma o y s a e. Speci ically, TMZ
e oked an inc ease in iNOS exp ession in w and Gal-3 KO mice, while
A g1 was signi ican ly educed in TMZ- ea ed Gal-3 KO mice (Supp.
Figu e 10). These indings sugges ha Gal-3 de iciency may enhance
he an i- umou al e ec s o TMZ, which could ha e impo an
implica ions o GB ea men .
3.10. Gal-3 deple ion enhanced a mul i a ious p o-in lamma o y gene
exp ession signa u e in he TME
In an ini ial e o o explo e he p e-clinical ele ance o ou ind-
ings, we pe o med nex gene a ion ansc ip ome-wide gene-le el
exp ession p o iling s udies in he TME. Tumou s om BCBM and GB
animals we e ha es ed a day 21 pos -implan a ion, and hen analysed.
3.10.1. B eas cance b ain me as asis
Compa ing he TME o w and Gal3 KO mice, we obse ed 180
dys egula ed genes (100 genes up egula ed and 80 genes down-
egula ed). The mos o up egula ed genes in he Gal-3 KO model we e
immune sys em- ela ed, closely linked o p o-in lamma o y ma ke s (e.
g. Tn s 10, Tn s 1b), commonly exp essed in mic oglia and mac o-
phages (e.g. CD68, P2 y14), T cells (e.g. CD2, CD2, CD8b, G anzyme A-
B) and Kille cells (Kl b1, KL 1a). On he con a y, w animals showed
p o-me as a ic genes exp ession (e.g. Se pinb9 , Se pinb3b) [30] and
a ginase-1 [31] (Fig. 7A). Gene se en ichmen analysis (GSEA)
Fig. 5. A.-) Con ocal images o he me as a ic a ea o w mouse a day 21 a e being injec ed wi h EO771 cells. Pic u es shows he TME (le image) and he inse
( igh ) wi h nuceli s ained in blue (DAPI), TAMs in ed (Iba-1) and iNOS (g een). B.-) Quan i a ion o TAMs exp essing iNOS wi hin he TME (n =5). C.-) As pe A,
images showing he TME o Gal3KO mice. D.-Same s udy as abo e-men ioned bu analysing TAMs exp essing A g1 (E) in w (D) and Gal3KO (F). G.-) Glioblas oma
a ea o w mouse injec ed wi h GL261 cells a day 21. Pic u es shows he TME (le image) and he inse ( igh ) wi h nuceli s ained in blue (DAPI), mic oglia in g een
(Iba-1) and iNOS in ed. H.-) Quan i a ion o TAMs exp essing iNOS (n =5). I.-) As pe G, images showing he TME o Gal3KO mice. J.-) Same s udy as abo e-
men ioned bu analysing TAMs exp essing A g1 (K) in w (J) and Gal3KO mice (L). Scale ba s 250
μ
m (50
μ
m inse s).
A. Ri e a-Ramos e al.
Cance Le e s 591 (2024) 216879
9
compa ing o e 22000 genes ac oss bo h expe imen al models, showed
se e al p o-in lamma o y gene clus e s in he Gal-3 KO model. Specially
mic oglial esponse o in e e on gamma was one o he mos dys egu-
la ed clus e s ac oss g oups (Fig. 7B–C), which is well-known o play a
pi o al ole in d i ing he p o-in lamma o y pheno ype in TAMs [32].
Mo eo e , using wikipa hways analysis om TAC so wa e, Gal-3 KO
e oked an inc ease in di e en in lamma o y gene signa u es, wi h
ADAR1 edi ing de iciency immune esponse and Type II in e e on sig-
nalling as he mos up egula ed pa hways (Fig. 7D). These pa hways a e
mu ually ela ed since ADAR1 is a c i ical IFN-inducible o m, display-
ing a pi o al ole in d i ing p o-in lamma o y esponses o he immune
sys em [33,34].
3.10.2. Glioblas oma
We obse ed 194 dys egula ed genes, 111 we e down egula ed and
83 up egula ed. Again, mos dys egula ed genes a e ypically exp essed
in ac i a ed immune sys em cells including mic oglia and mac ophages
(Clec7a, CD68, CD180) (Fig. 8A). GSEA showed he in e e on gamma
mic oglial pa hway as one o he mos up egula ed clus e s in he
immunologic signa u e gene se s (Fig. 8B)
Using he Da abase o Anno a ion, Visualiza ion and In eg a ed
Disco e y (DAVID) we obse ed ha GO TERM biological p ocesses
showed chemokine-media ed pa hways, oge he wi h in e e on
gamma and in lamma o y esponses (Fig. 8C). Finally, gene clus e
analysis ound up egula ion in Vi al p o ein in e ac ions, which is
closely ela ed wi h ADAR-1 [34], and cellula esponse o in e e on
gamma (Fig. 8D).
Al oge he , ou ansc ip omic analysis o he TME clea ly show an
immune- ela ed p o-in lamma o y mic oen i onmen , which signi i-
can ly a ec ed BCBM and GB g ow h.
4. Discussion
Galec in-3 is a membe o he e olu iona y conse ed animal lec ins
amily. I is exp essed in se e al cells ypes playing key oles in di e en
biological p ocesses such as apop osis, au ophagy, phagocy osis,
in lamma ion, and cance [8,35–38]. Howe e , in he las yea s, ou
g oup has unmasked he pi o al ole o Gal-3 in immune sys em cells
ac i a ion, speci ically mic oglia [7,19]. Thus, his wo k desc ibes a
no el ole o his pleio opic p o ein, ac i a ing an immunosupp essi e
pheno ype in TAMs in a b ain umou con ex .
Gal-3 induces an i-in lamma o y mic oglial ac i a ion in p ima y
and immo alised mic oglia cell lines. We ound a complex beha iou ,
unde lining he ac i a ing e ec ha exogenous addi ion o Gal-3
exe ed on classical immunosupp essi e ma ke s (e.g. A g-1 and PD-
L1), simila o he e ec s obse ed a e he exposi ion o umou -
condi ioned medium. In e es ingly, such Gal-3-dependen an i-in lam-
ma o y esponse was ab oga ed once Gal-3 was inhibi ed. These esul s
a e in ag eemen wi h o he wo ks whe e blocking Gal-3 diminished
le els o classical immunosupp essi e ma ke s [25,39]. Howe e , ou in
i o esul s also sugges ha umou al Gal-3 plays an impo an ole
d i ing such immunosupp essi e mic oglial esponse. In pa icula , he
Fig. 6. A.-) Quan i a ion o CD4
+
T cells wi hin he TME (g een) o BCBM. Nuclei in blue (DAPI) B.-) Con ocal images o a BCBM showing CD4
+
T cells (g een) in he
TME in w and Gal3KO mice. C.-) Con ocal images showing CD8
+
T cells (g een) in he TME o EO771 mice. D.-) Quan i a ion o CD8
+
T cells wi hin he TME o
BCBM mice. E.-) Ra io o CD8 s CD4 T cells wi hin he TME o BCBM animals. F.-) Con ocal images o CD4
+
T cells in he TME o GL261-injec ed mice. G.-)
Quan i a ion o CD4
+
T cells (g een) in he TME o GB model. H.-) con ocal images o CD8
+
T cells (g een). I.-) Quan i a ion o in a umou al CD8
+
T cells in ou GB
model J.-) CD8/CD4 T cells a io wi hin he TME o ou GB model. Scale ba 250
μ
m (100
μ
m inse ).
A. Ri e a-Ramos e al.