Academic Edi o : Alexande
E. Kalyuzhny
Recei ed: 2 Augus 2025
Re ised: 25 Augus 2025
Accep ed: 29 Augus 2025
Published: 30 Augus 2025
Ci a ion: Vule i´c, A.; Mi jaˇci´c
Ma ino i´c, K.; Ju iši´c, V. The Role o
Tumo Mic oen i onmen in
T iple-Nega i e B eas Cance and I s
The apeu ic Ta ge ing. Cells 2025,14,
1353. h ps://doi.o g/10.3390/
cells14171353
Copy igh : © 2025 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license
(h ps://c ea i ecommons.o g/
licenses/by/4.0/).
Re iew
The Role o Tumo Mic oen i onmen in T iple-Nega i e B eas
Cance and I s The apeu ic Ta ge ing
Ana Vule i´c 1, Ka a ina Mi jaˇci´c Ma ino i´c 1and Vladimi Ju iši´c 2,*
1Depa men o Expe imen al Oncology, Ins i u e o Oncology and Radiology o Se bia, Pas e o a 14,
11000 Belg ade, Se bia
2Facul y o Medical Sciences, Uni e si y o K aguje ac, P.O. Box 124, 34000 K aguje ac, Se bia
*Co espondence: [email p o ec ed]
Abs ac
T iple-nega i e b eas cance (TNBC) is an agg essi e sub ype o b eas cance cha ac e ized
by apid p oli e a ion and in il a ion o immune cells in o umo mic oen i onmen
(TME). The ea men o TNBC s ill emains challenging due o he lack o exp ession o
e ec i e molecula a ge s pe aining o he umo cell i sel . In TNBC s anda d o ca e,
he apies such as chemo he apy, oge he wi h ecen ly in oduced immuno he apy wi h
checkpoin inhibi o s, o en do no esul in du able clinical esponse. The e o e, be e
unde s anding o complex in e ac ions be ween umo cells, immune cells, and s omal cells
media ed by mul iple cy okines, chemokines, enzymes, and me aboli es in TME is c ucial
o unde s anding he mechanisms ha unde lie umo cell immune e asion s a egies.
The aim o his e iew is o gi e comp ehensi e o e iew o immune cell ne wo k and hei
in e ac ions wi h cells in TME and possibili ies o he apeu ic a ge ing o TME in TNBC.
We discuss cance -associa ed ib oblas s (CAFs) as an impo an ecen ly cha ac e ized
playe in TNBC wi h espec o hei ole in in e ac ions wi h immune cells and hei impac
on umo in asion. Based on he ecen ly accumula ed knowledge, he apies a ge ing
immune supp essi e mechanisms and CAF- ela ed umo -p omo ing mechanisms in TME
hold g ea po en ial o clinical e alua ion in TNBC.
Keywo ds: iple nega i e b eas cance ; umo mic oen i onmen ; cance associa ed
ib oblas s
1. In oduc ion
Tumo mic oen i onmen (TME) o umo s oma is a complex biological ne wo k in
which malignan cells coexis wi h a a ie y o nonmalignan cells, including immune cells,
umo -associa ed ib oblas s (CAFs), mesenchymal s em cells (MSCs), umo -associa ed
adipocy es, endo helial cells, and pe icy es in glycop o ein- ich ex acellula ma ix (ECM).
The c oss alk be ween malignan cells and TME con ibu es o epi helial- o-mesenchymal
ansi ion (EMT), acquisi ion o in asi e pheno ype, inc eased mo ili y o umo cells, and
subsequen o ma ion o seconda y umo deposi s [1].
B eas cance is one o he mos p e ailing malignancies [
2
]. T iple-nega i e b eas
cance (TNBC) is cha ac e ized by he lack o exp ession o es ogen ecep o
α
(ER-
α
),
p oges e one ecep o (PR), and he absence o exp ession/ampli ica ion o human epi-
de mal g ow h ac o ecep o 2 (HER2). TNBC exhibi s a highe p oli e a ion a e and
highe incidence o me as ases compa ed o o he b eas cance ypes [
3
]. The he e oge-
neous molecula cha ac e is ics o TNBC educe he op ions o a ge ed he apies, making
Cells 2025,14, 1353 h ps://doi.o g/10.3390/cells14171353
Cells 2025,14, 1353 2 o 31
his umo pa icula ly agg essi e and esul ing in poo p ognosis [
4
]. Acco dingly, sys-
emic chemo he apy based on an h acyclines and axanes emains he p ima y ea men
op ion [5]
. In he pas decade, based on s udies in ol ing genomic analyses, TNBC was
classi ied in o dis inc molecula sub ypes ha we e u he e ined acco ding o he im-
munomodula o y gene exp ession p o ile a ibu ed o he umo in il a ing lymphocy es
(TILs) and s omal cells [
6
,
7
]. The classi ica ion p oposed sub ypes displaying dis inc
clinical and pa hological cha ac e is ics wi h disease p ognosis di e ing signi ican ly be-
ween he sub ypes, and each sub ype showing a speci ic pa e n o gene exp ession [
8
,
9
].
Howe e , he classi ica ion o TNBC due o he complexi y and he e ogenei y o he umo ,
emained wi hou he ue consensus. Ne e heless, he ou sub ypes iden i ied based on
gene exp ession p o iles named basal-like immune ac i a ed (BLIA), basal-like immune
supp essed (BLIS), luminal and ogen ecep o (LAR), and mesenchymal (MES) p oposed by
Bu s ein e al. a e he mos ho oughly alida ed [
8
,
10
]. In one s udy TNBC was classi ied
acco ding o genomic immune p o ile in TME [11].
Recen ly, immuno he apy based on he inhibi ion o immune checkpoin (IC)
molecules, mos no ably o he p og ammed cell dea h ecep o (PD)-1 and cy o oxic
T lymphocy e an igen (CTLA)-4, has shown po en ial o enhance immune esponse agains
umo cells and he eby modula e TME [
12
,
13
]. In his sense, PD-/PD-L1 blockade wi h
Food and D ug Adminis a ion (FDA)-app o ed monoclonal an ibodies such as an i-PD-1
(pemb olizumab, ni olimab, cemiplimab, cam elizumab), an i-PD-L1 (a ezolizumab, du -
alumab, a elumab), and CTLA-4 blockade wi h ipilimumab, we e in oduced in clinical
ials o b eas cance in TNBC and ha e shown he apeu ic po en ial [
14
]. Howe e ,
mono he apy wi h IC inhibi o s esul ed in limi ed clinical bene i as umo cells a e p one
o immune escape and d ug esis ance due o highly dynamic in e play be ween he cells in
TME. In he con ex o a ailable he apeu ic op ions including chemo he apy, adio he apy
and immuno he apy, he e is a cons an need o be e unde s anding o cellula and
molecula componen s o TME and hei in e ac ions ha could be a ge ed in o de o
o e come he apeu ic limi a ions and imp o e ea men ou come.
2. Immune Cells in TME
Immune cells in TME include immuno eac i e cells such as CD8 cy o oxic T lym-
phocy es (CTL)s, CD4 helpe T cell, B lymphocy es, na u al kille (NK) cells, dend i ic
cells (DC)s, M1 mac ophages, and supp essi e immune cells such as myeloid-de i ed sup-
p esso cells (MDSC)s, umo -associa ed mac ophages (TAM)s, egula o y T cells (T eg)s,
e c. [
15
] (Figu e 1). TILs in TME encompass s omal TILs, which a e posi ioned in he
umo ma gins wi hou di ec con ac wi h cance cells and in a umo al TILs, de ined
as lymphocy es in di ec cell- o-cell con ac wi h umo cells wi h no in e ening s oma.
In a umo al TILs make he smalle p opo ion o TILs as endo helial cells and s omal
issue agg a a e hei in il a ion in o he umo [16].
The abundance and he composi ion o TILs ega ding he p esence o dis inc lym-
phocy e popula ions has been e alua ed in mul iple s udies in he con ex o b eas cance
disease p ognosis and esponse o chemo he apy [
16
–
19
]. TNBC is cha ac e ized by a
g ea e abundance o TILs compa ed o o he molecula sub ypes o b eas cance [
20
].
Mo eo e , in TNBC and HER2+ b eas umo s, highe p opo ions o TILs ha e been associ-
a ed wi h be e esponse o chemo he apy. The same s udy indica ed he p esence o TILs
as a a o able p ognos ic ma ke in ea ly-s age TNBC [
21
]. Fu he mo e, he de iciency o
s omal TILs and low numbe o CD8+ T cells we e ound o independen ly p edic mo al-
i y in TNBC while he p ognos ic alue o TILs and hei CD8+ subse a ied wi h espec
o he cance compa men [
22
]. In pa ien s wi h TNBC dis an me as ases we e shown o
exhibi sca ce p esence o TILs compa ed o he p ima y umo . The same s udy epo ed
Cells 2025,14, 1353 3 o 31
he longe median o e all su i al (OS) in he coho o pa ien s wi h me as a ic lesions
con aining TILs>10% compa ed o pa ien s wi h TILs<10% [
23
]. In dis an me as ases o
TNBC, he p esence o TILs iden i ied pa ien s wi h enhanced esponse o immuno he apy,
sugges ing he impo ance o immune ac i a ion o imp o ing su i al ou comes [24].
Figu e 1. Immunosupp essi e and immuno eac i e cells in umo mic oen i onmen (TME). Cells
in TME: M2 mac ophages, cance -associa ed ib oblas s (CAF), N2 subse o neu ophils, egula o y
T cells (T eg), myeloid-de i ed supp esso cells (MDSC), M1 mac ophages, na u al kille (NK) cells,
cy o oxic T lymphocy es (CTL), CD4 helpe T cells, and B lymphocy es.
CD8+ T cells play an ini ial ole in an i umo immune de ense. The naï e T cells
unde go p iming by umo -de i ed an igens p esen ed by an igen-p esen ing cells (APCs)
in egional lymph nodes, and mig a e o TME ia blood s eam o lympha ics, whe e hey
di e en ia e in o cy o oxic T cells (CTLs). CTLs di ec ly kill umo cells by p oducing
cy o oxic enzymes pe o in and g anzymes. CD4+ T cells play an immuno egula o y
ole. Based on he cy okines hey gene a e, CD4+ T cells a e di ided in o Th1, Th2, Th17,
and egula o y T (T eg) cell subse s, each o which has a unique unc ion in immune
esponses [
25
]. Th1 T cells a e he p ima y sou ce o he in lamma o y cy okines in e e on
(IFN)-
ꝩ
, in e leukin (IL)-2, and umo nec osis ac o (TNF), which induce ma u a ion o
APCs, mo e e ec i e p esen a ion o umo an igens, and ma u a ion o CD8+ cells o
ully unc ional CTLs. Con e sely, Th2 cells sec e e IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13,
which a e esponsible o ac i a ion o humo al immuni y, eosinophil ac i a ion, inhibi ion
o mac ophage unc ions and supp ession o an i umo immune esponses [
26
]. Th17
cy okines IL-17, IL-21, and IL-22 a e epo ed o acili a e cance p og ession by p omo ing
in lamma ion and angiogenesis. The ele a ed le el o hese cy okines in pe iphe al blood
has been ela ed o p og ession o b eas cance [27].
Cells 2025,14, 1353 4 o 31
B cells in TME o b eas cance ha e been less s udied in p ima y umo s compa ed
o axilla y lymph nodes. In an i umo immune esponse, B cells ha e a p ima y ole in
p esen ing an igens o CD8 T cells and sec e ing an ibodies ha induce umo cell des uc-
ion ia an ibody-dependen cellula cy o oxici y (ADCC), as well as p oducing cy okines
ha u he suppo an igen p esen a ion by p o essional APCs. In in asi e b eas can-
ce , B cells in TIL popula ion a e associa ed wi h highe g ade umo s and lymph node
posi i i y, indica ing he ole o B cells in umo agg essi eness [
28
]. The e a e opposing
esul s ega ding he associa ion o B cells in TME wi h disease p ognosis in TNBC. While
se e al s udies epo he associa ion o in il a ing B cell wi h la ge umo size, high
his ological g ade, lympho ascula in asion, and lymph node me as ases in ea ly b eas
cance [
29
], he e a e also da a showing associa ion o high densi ies o in il a ing B cells
in p ima y umo s wi h a be e esponse o neoadju an chemo he apy [
30
]. Acco ding o
some esea ch da a, in e ac ions be ween TNBC cells and B cells inc ease gene exp ession
o a numbe o in lamma o y cy okines, mos no ably IL-1
β
, which esul s in ch onic
in lamma ion [31]
. IL-1
β
by ac i a ing nuclea ac o -
κ
B (NF
κ
B), p omo es angiogenesis
and EMT. Fu he mo e, NF
κ
B signaling was associa ed wi h la ge umo size, high his-
ological g ade, and nega i i y o ER and PR exp ession in in asi e b eas cance [
32
].
Newly de ined sub ype o B cells, he egula o y B cells (B egs), ha e a ole in immune
ole ance, simila o egula o y T cells. In his sense, CD25+ and IL10+ B cells we e ecen ly
ela ed o he induc ion o T egs in TIL agg ega es and he de elopmen o me as asis in
b eas cance [33].
NK cells a e he subpopula ion o inna e lymphoid cells (ILCs) ha ha e a unique
abili y o di ec ly ecognize malignan ly ans o med cells ia cogna e NK cell ecep o s
and kill umo cells by eleasing cy o oxic enzymes pe o in and g anzymes. Aside om cy-
o oxic ac i i y, which is pe o med wi hou majo his ocompa ibili y complex (MHC) class
I es ic ion, NK cells p oduce mul iple cy okines—IFN-
ꝩ
, TNF, IL-10, IL-13—and g anulo-
cy e mac ophage colony-s imula ing ac o (GM-CSF) ha egula e immune esponses [
34
].
NK cell an i umo ac i i y is igh ly egula ed by he balance o signals ansmi ed by
ac i a ing and inhibi o y ecep o s bound o hei ligands on umo cells. NK cells a e able
o dis inguish no mal om ans o med cells by kille cell immunoglobulin-like ecep o s
(KIRs) ha inhibi NK cell cy o oxic ac i i y by binding o MHC class I molecules [
35
,
36
].
In umo issues, NK cells a e mos ly p esen du ing ea ly umo igenesis, bu hey become
sca ce in ad anced s ages o umo de elopmen due o inc easing immunosupp ession in
TME [37].
In his sense, se e al umo e asion mechanisms o e coming NK cell-media ed umo
con ol ha e been iden i ied, including he impai ed NK cell ec ui men o he umo bed,
shedding o ligands o NK cell ac i a ing ecep o s, and he induc ion o he inhibi o y
ligands on umo cells [
38
,
39
]. The supp essi e e ec o TME on NK cy o oxic ac i i y
o NK cells can be media ed by cy okines such as TGF-
β
and IL-10, L-kynu enine, a
p oduc o yp ophan deg ada ion, and p os glandin E2 (PGE2), sec e ed by umo cells,
supp essi e immune cells, and CAFs [
40
]. These soluble ac o s in TME down egula e he
exp ession o NK cell-ac i a ing ecep o s, including NK g oup 2D (NKG2D), NKp30, and
DNAM1, as well as hei signaling pa hways [
41
–
43
]. Mo eo e , pe sis en s imula ion
o NKG2D ecep o by i s umo cell ligands, as well as soluble ligands gene a ed by
p o eoly ic shedding media ed by ma ix me allop o einases (MMPs) p oduced by umo
cells o CAFs, may also lead o unc ional exhaus ion o NK cells [42,44]. Howe e , IFN-γ
eleased du ing acu e ea ly in lamma ion in he con ex o an i umo immune esponse
induces he esis ance o umo cells o NK cell-media ed lysis by up egula ing classical
and non-classical MHC class I molecules [45].
Cells 2025,14, 1353 5 o 31
Cell- o-cell con ac s in TME signi ican ly a ec NK cell unc ions. In his sense, ligand–
ecep o in e ac ion be ween CAFs and NK cells was epo ed o inhibi NK cell cy o oxici y
owa d cance cells, down egula e he exp ession o ac i a ing NK cell ecep o s, and
he eby p omo e cance cell escape om NK cell su eillance, acco ding o a s udy ha used
a mouse model o b eas cance . Mo eo e , he umo samples ob ained om pa ien s wi h
TNBC showed en ichmen o NK cells in CAF- ich egions, accompanied wi h up egula ed
binding o NK cell ecep o s o ligands on CAFs ha al oge he co ela ed wi h poo disease
ou come [46].
Tumo in il a ing NK cells appea o ha e lowe an i umo ac i i y compa ed o
ci cula ing NK cells [
47
,
48
]. Mo eo e , in ea ly s ages o umo de elopmen , NK cells
exhibi an i umo ac i i y, while in he la e cance s ages hey show impai ed cy o oxic
capaci ies and become senescen cells, as shown in he mu ine model o TNBC [
49
]. In one
s udy in mice, a unique subclus e o Socs3highCD11b-CD27- o imma u e NK cells ound
speci ically in TNBC samples showed educed exp ession o cy o oxic g anzyme signa u e
and he abili y o ac i a e cance s em cells h ough Wn signaling. Fu he mo e, he same
s udy epo ed inc eased numbe s in unc ionally less ma u e CD56b igh NK cell subse
in umo samples, which co ela ed wi h poo OS in pa ien s wi h TNBC [
50
]. A ecen
s udy iden i ied he dis inc i e IL-10+sec e ing immunosupp essi e NK cell popula ion
in TILs wi h a s ong ela ion o poo su i al p ognosis in TNBC pa ien s ea ed wi h
neoadju an IC blockade immuno he apy [51].
2.1. Immune Checkpoin Recep o s
IC ecep o s a e up egula ed as a consequence o immune esponses, wi h hei p i-
ma y physiological ole being he p e en ion o excessi e immune eac ions. IC ecep o
PD-1 and i s ligand PD-L a e impo an nega i e egula o s o immune ac i i y. Du ing
umo igenesis, oncogenic pa hways, gene ic, and epigene ic ac o s in insic o umo cells
up egula e he exp ession o PD-L1 (B7H1) and PD-L2 (PD-L2) on umo cells. In his
sense, mic oRNA-200/ZEB1 axis, abe an ac i a ion o Wn signaling, loss o phospha ase
and ensin homolog (PTEN), ac i a ion o phosphoinosi ide 3-kinase (PI3K)/p o ein ki-
nase B (AKT)/mechanis ic a ge o apamycin (mTOR) signaling pa hway, and MUC1-
C/Myc/NF-K
β
axis we e shown o up egula e he exp ession o PD-L1 in TNBC [
52
–
55
].
In lamma o y cy okines in TME, such as IFN-
ꝩ
, IL-1
β
, IL-6, and TNF, u he a o ize
cance immune escape by augmen ing PD-L1/PD-L2 exp ession on umo cells including
TNBC [
55
,
56
]. Aside om umo cells, mul iple cells in TME, including immune cells (DCs,
mac ophages, T egs) and CAFs, exp ess PD-L1 which u he con ibu es o supp essing an-
i umo immuni y [
57
–
59
]. In his sense, in TME and umo -d aining lymph nodes, PD-L1
is o en up egula ed by IFN-
ꝩ
on APCs, leading o he inhibi ion o T cell ac i a ion [60].
The p ocess o EMT is associa ed wi h augmen a ion o PD-L1 exp ession in mul iple
umo s. EMT-inducing ansc ip ion ac o s such as ZEB1 ha e been shown o bind o
gene p omo e egion o PD-L1 gene [
61
]. By blocking he glycogen syn hase kinase
(GSK)3
β
-media ed phospho yla ion, ubiqui ina ion, and deg ada ion o he EMT-inducing
ansc ip ion ac o Snail, PD-L1 has been shown o enhance EMT in TNBC cells, hence
inc easing he likelihood o me as asis [62].
Aside om PD-1 and CTLA-4, ch onic in lamma ion in TME can esul in up egula ed
exp ession o se e al o he IC ecep o s con ibu ing o immunosupp ession and esis ance
o umo cells o he an i umo ac i i y o lymphocy es in il a ing he TME. In his sense,
T cell immunoglobulin and mucin domain-con aining p o ein 3 (TIM3), lymphocy e ac i-
a ion gene-3 (LAG-3), T cell immuno ecep o wi h immunoglobulin and ITIM domain
(TIGIT), and V-domain Ig-con aining supp esso o T cell ac i a ion (VISTA) a e up eg-
ula ed on T and NK cells [
13
]. The e o e, he apeu ic a ge ing o mul iple IC molecules
Cells 2025,14, 1353 6 o 31
on immune cells ep esen s a challenge and a po en ial op ion o ea men o TNBC
[ e iewed in 10].
2.2. Supp essi e Immune Cells in TME
2.2.1. Regula o y T Cells
Regula o y T cells ep esen he unc ionally dis inc i e subpopula ion o CD4+ T cells
ha exp ess o khead box P3 (FoxP3) ansc ip ion ac o and high-a ini y IL-2 ecep o
α
chain (CD25), deple ing IL-2 om he TME and he eby dis u bing T cell p oli e a ion
and ac i a ion. T egs play he key ole in immunologic ole ance p ima ily by inhibi ing
he p oli e a ion, cy o oxic ac i i y, and cy okine p oduc ion o T cells. T egs supp ess
immune eac ions by mul iple mechanisms such as he p oduc ion o immunosupp essi e
cy okines and o he soluble ac o s, including TGF-
β
, IL-10, IL-35, and PGE2. Addi ionally,
T egs exp ess IC molecules ha al oge he c ea e an immunosupp essi e en i onmen by
impai ing an i umo ac i i y o T and NK cells [
63
,
64
]. B eas cance and s omal cells in
TME cells sec e e chemokines CCL2, CCL5 (RANTES), CCL20, CCL22, and CXCL12 which,
by binding o he co esponding ecep o s, induce T eg homing o umo issue. I has been
epo ed ha by eleasing CCR5-associa ed chemokines, mainly CCL3 (MIP-1
α
), CCL4
(MIP-1
β
), and CCL5, which a e s ong a ac an s o lymphocy es and mac ophages, T egs
acili a e me as a ic in asion o TNBC o lymph nodes and bone ma ow [64–66].
T egs cons i u i ely and highly exp ess IC ecep o CTLA-4 and a e able o down-
egula e he exp ession o cos imula o y molecules CD80/CD86 on APCs he eby hinde ing
an i umo ac i i y o T cells [
67
]. In lamma o y condi ions in TME such as ype I IFN
signa u e, PD-L1, and exp ession o indoleamine 2,3-dioxygenase (IDO), which cha ac e ize
immunomodula o y sub ype o TNBC, con ibu e o acquisi ion o a s ong immune-
supp essi e unc ional T eg pheno ype [
8
,
9
]. Fu he mo e, i was epo ed ha PD-1 in
con ac wi h PD-L1 could con e nai e CD4+ T cells o T egs h ough he down egula ion
o Ak , mTOR, and ERK2 and simul aneous up egula ion o PTEN signaling pa hways [
68
].
Al hough he p esence o lymphocy e in il a es seems o e lec a o able hos an i-
umo immune esponses, sugges ing ha immune ac i a ion is impo an o imp o ing
su i al ou comes, he composi ion o lymphocy e in il a e a ies ac oss molecula sub-
ypes o b eas cance [
69
]. In his sense, he inc eased p esence o Foxp3+ T eg subse s
was epo ed in TNBC and HER-2-posi i e umo s compa ed o luminal sub ypes [
70
].
Mo eo e , he same s udy indica ed he associa ion o g ea e T eg o CD8+ T cell a-
ios wi h highe g ade umo s and highe Ki-67 exp ession. Changes in T eg in il a ion
we e epo ed du ing he cou se o me as a ic p og ession, indica ing he ele ance o
T eg in il a ion o un a o able disease p ognosis in b eas cance [
71
]. Fu he mo e, he
p esence o FOXP3+ lymphocy es in umo issues, along wi h TNM s age, has been iden-
i ied as an independen p ognos ic ac o o OS in TNBC [
72
]. Aside om hei ole in
p omo ing me as a ic in asion, high in il a ion a e o T egs in pa ien s wi h TNBC has
been associa ed wi h inhibi ed immune ac i a ion pa hways and esis ance o an i-PD1
immuno he apy [66].
2.2.2. Tumo -Associa ed Mac ophages
Tumo -associa ed mac ophages (TAMs) may include mac ophages o M1 an i umo
pheno ype ha a e p esen mos ly in ea ly phases o immune esponse o umo and
immunosupp essi e M2 subse which is mo e abundan in TME o he g owing umo .
M2 mac ophages enhance immune ole ance, angiogenesis, umo g ow h, and in asion.
This subse o mac ophages p oduces nume ous an i-in lamma o y ac o s (TGF-
β
, IL-4,
IL-13, IL-10, IL-1RA), lowe le els o in lamma o y cy okines (IL-6, IL-12, IL-23, TNF-
α
),
and conside able amoun s o g ow h ac o s (EGF, ascula endo helial g ow h ac o
Cells 2025,14, 1353 7 o 31
(VEGF)) [
73
]. Fu he mo e, by lowe ing he le els o a ginine h ough he con e sion o
L-a ginine o o ni hine h ough a ginase (A g) ac i i y, TAMs supp ess T cell an i umo
unc ion [74]. The o ni hine is u he ansla ed in o polyamines (pu escine, spe midine,
and spe mine), which a e linked o mo e immunosupp essi e TME as hey p omo e he
g ow h and unc ion o MDSCs, mac ophages and T egs, as well as collagen p oduc ion in
he p ocess o issue epai and su i al o umo cells [
75
,
76
]. Mo eo e , he exp ession
le el o A g-1, which is one o he molecula ma ke s o M2 mac ophages, was ele a ed in
pe iphe al blood, lymph nodes and umo issue o b eas cance pa ien s [
77
]. Fu he mo e,
TAMs p oduce mul iple enzymes ha pa icipa e in me as a ic umo in asion, such as
MMPs, se ine p o eases, and ca hepsins, and decompose collagen and o he componen s o
he ECM, he eby helping he mig a ion o umo and s omal cells [
73
]. The e a e also da a
indica ing he ole o M2 mac ophages in EMT, as CCL2 p oduced by cance and myeloid
cells was shown o a ac M2 mac ophages and induce Wn -1 up egula ion. Wn -1 in u n
down egula es E-cadhe in junc ions in HER2+ ea ly b eas cance [
78
]. Acco dingly, in
TNBC, he p esence o M2 TAMs in TME was co ela ed wi h s omal ib oblas in il a ion,
EMT, and poo pa ien su i al [79].
2.2.3. MDSC
MDSCs a e imma u e myeloid cells ha a ise a e p olonged exposu e o monocy es
o neu ophils o in lamma o y condi ions in TME. Va ious umo -de i ed ac o s induce
di e en ia ion o MDSCs
in i o
, including PGE2, IL-6, IL-10, IL-1
β
, TGF-
β
, s em cell
ac o (SCF), and p oangiogenic VEGF [
80
]. By exp essing IC molecules and sec e ing
immunosupp essi e cy okines, mos no ably TGF-
β
, IL-1
β
, and IL-10, MDSCs supp ess he
ac i i y o T and NK cells [
81
,
82
]. MDSCs also block T cell ac i a ion by seques e ing cys ine
and limi ing he a ailabili y o cys eine in TME [
83
]. Addi ionally, MDSCs con ibu e o
umo -induced immune supp ession by blocking he p oduc ion o IL-12 by TAMs and
DCs ha induces pola iza ion o mac ophages owa d a umo -p omo ing M2 pheno ype.
Fu he mo e, M2 mac ophages play a ole in he de elopmen and p oli e a ion o FoxP3+
T eg cells [84].
Inc eased in il a ion o MDSCs was epo ed in TNBC compa ed o o he molecula
sub ypes o b eas cance [
85
]. The same s udy epo ed ha he numbe o MDSCs
popula ion in pa ien wi h basal-like TNBC co ela ed wi h he le el o ansc ip ion ac o
∆
Np63, which p omo es umo g ow h, p og ession, and me as asis in humans and in he
mouse model o TNBC. The mechanism in ol ed in MDSC ec ui men was he
∆
Np63-
dependen ac i a ion o chemokines CXCL2 and CCL22. Fu he mo e, i has been e ealed
ha MDSCs ec ui ed in o he TME o TNBC elease he enzymes chi inase 3-like and
MMP9, which a e implica ed in me as a ic in asion and he p omo ion o cance s em cell
unc ion [85,86].
2.2.4. Tumo -Associa ed Neu ophils
Tumo media ed signals such as TGF-
β
p omo e he di e en ia ion o a p o-
umo igenic N2 sub ype o neu ophils. Tumo -associa ed neu ophils (TANs) p oduce
high le els o TNF, inducible ni ic oxide syn hase (iNOS), NO, and H
2
O
2
, damaging
DNA and inducing gene ic ins abili y. The e o e, N2 neu ophils ep esen ac i e playe s in
in lamma ion- ela ed umo igenesis and umo de elopmen . Fu he mo e, his popula ion
o neu ophils suppo s umo in asion, angiogenesis, and me as asis by sec e ing VEGFA
and MMP9. Besides he sec e ion o soluble ac o s, TANs ha e been shown o impai he
an i umo immune esponse by exp essing immunosupp essi e molecules, such as PD-L1.
TANs a e p esen in mos TNBC umo s, indica ing hei po en ial o impac b eas cance
p ognosis [87].
Cells 2025,14, 1353 8 o 31
3. Cance -Associa ed Fib oblas s
Fib oblas s a e mesenchymal cells wi h di e se physiological unc ions such as ECM
syn hesis and emodeling, issue epai , mesenchymal lineage main enance, sec e ion o
signaling molecules, and immune egula ion. Cance -associa ed ib oblas s (CAFs) play a
complex ole in he TME because o hei abili y o in luence many aspec s o umo igenesis,
including he p oli e a ion o malignan cells, mo ili y, angiogenesis, me as a ic in asion,
in lamma ion, and me abolic ep og amming [
88
]. In b eas cance , up o 80% o no mal
ib oblas s in b eas issue acqui e CAF pheno ype du ing umo p og ession [89,90].
Aside om “no mal” ib oblas s, di e se cells o mesenchymal o igin, such as
adipocy es and mesenchymal s em cells, can gi e ise o CAF di e en ia ion when ex-
posed o he soluble ac o s in TME due o in insic in e cellula plas ici y. In his sense,
bone ma ow mesenchymal s em cells can be ans o med in o CAFs when exposed o
condi ioned medium in which umo cells we e cul i a ed [
91
]. Simila ly, adipocy es and
pe icy es, as cells o mesenchymal o igin, we e epo ed o di e en ia e in o ib oblas s
when exposed o condi ioned media om umo cells [
92
,
93
]. Fu he mo e, in he p oximi y
o he de eloping umo and h ough he p ocess o EMT igge ed by soluble ac o in
TME, epi helial and endo helial cells can down egula e epi helial and endo helial ma ke s,
espec i ely, and acqui e ib oblas -like cha ac e is ics [88].
Mo phologically, CAFs a e spindle-shaped mesenchymal cells cha ac e ized by o e -
exp ession o
α
-smoo h muscle ac in (
α
-SMA) ha egula es cy oskele on ea angemen s
con ibu ing o cellula mobili y. In b eas cance , he exp ession o
α
-SMA was epo ed
o co ela e wi h lymph node me as ases and poo p ognosis [
89
]. CAFs also exp ess
cy oskele al p o ein imen in, which is also a ma ke o EMT [94].
In umo su oundings, g ow h ac o s, cy okines, a ious signaling molecules,
me aboli es eleased by cance and immune cells, mechanical s ess, al e a ions in his-
one ace yla ion, and DNA damage, all igge di e en ia ion o CAFs [
95
]. TGF-
β
plays
a leading ole in he gene a ion o CAFs by ac i a ing canonical (SMAD-dependen ) and
noncanonical (non-SMAD) pa hways [
96
,
97
]. In addi ion, TGF
β
-1 sec e ed by ac i a ed
ib oblas s (TGF-
β
RII) c ea es a posi i e eedback loop by binding o he ype 2 TGF-
β
ecep o , u he inc easing ib oblas ac i a ion. Aside om TGF-
β
, os eopon in (OPN),
IL-1
β
and IL-6, sec e ed by cance o immune cells, induce he con e sion o s omal
ib oblas s o CAFs by ac i a ing downs eam TGF-
β
/Smads, NF-kB signaling, and signal
ansduce and ac i a o o ansc ip ion (STAT)3 ha a e pi o al o modula ing exp ession
o genes linked o he CAF lineage [
98
]. Fu he mo e, me abolic ep og aming owa ds
ae obic glycolysis induced by lysophospha idic acid (LPA), TGF-
β
1 o pla ele -de i ed
g ow h ac o (PDGF) p oduced by umo cells, ac i a e hypoxia-inducible ac o (HIF)-1
α
pa hway [
96
] con ibu ing o he di e en ia ion o ib oblas s in o CAFs. The cen al ole in
he es ablishmen and main enance o CAFs is associa ed wi h unc ion o Yes-associa ed
p o ein (YAP)1 and i s pa alog, ansc ip ional coac i a o wi h a PDZ-binding mo i (TAZ).
In CAFs and umo cells, he lack o YAP1 phospho yla ion esul s in i s ansloca ion o
he nucleus, i s subsequen binding o TEAD ansc ip ion ac o s, and he ac i a ion o
mi ogenic pa hways [
99
]. YAP egula es he exp ession o se e al cy oskele al p o eins.
The ac i a ion o YAP occu s as a consequence o he ECM emodeling and s i ening
induced by ac o s such as LPA and TGF-
β
. S c- amily kinase unc ion is equi ed o
YAP ac i i y and exp ession o myosin ligh chain (MYL) 9, ma ix s i ening, and many
p o- umo igenic p ope ies o ib oblas s [
100
]. Mo eo e , cance -de i ed exosomes induce
ib oblas con e sion o CAFs by shu ling no only mic oRNAs (miRNAs) and long non-
coding RNA Gm26809, bu also g ow h ac o s such as TGF-
β
1 [
101
]. Fu he mo e, PDGF
d i es pe icy e di e en ia ion in o ib oblas s [90,92].
Cells 2025,14, 1353 9 o 31
Aside om p e iously men ioned cy oskele al ma ke s, CAFs we e also ound o
exp ess ib oblas ac i a ion p o ein (FAP), CD29 (in eg in
β
1), PDGF ecep o s
α
and
β
(PDGFR
α
and PDGFR
β
), CD90 (THY-1), podoplanin (PDPN), ib oblas -speci ic p o ein 1
(FSP-1, S100A4), and ca eolin 1. Howe e , hese ma ke s a e nei he uniquely exp essed
no necessa ily coexp essed in CAFs [102].
The FAP se ine p o ease is exclusi ely p esen in he TME and absen in heal hy issues.
I s unc ion is o enzyma ically emodel he ECM, he eby enabling cellula mig a ion. In
HER2+ and TNBC, his umo -p omo ing p o ein is en iched in CAFs exp essing PD-L1/2
which, by binding o PD-1, ha e a po en ial o inhibi T cell ac i i y [
102
]. Based on a
ecen s udy comp ising in eg a ed analysis o single-cell RNA sequencing da a, clini-
cal specimens,
in i o
, and
in i o
expe imen s, FAP+ CAFs ha e been iden i ied as he
p edominan s omal popula ion associa ed wi h poo clinical ou comes and immuno-
supp essi e ea u es in b eas cance pa ien s. FAP+ subse o CAFs by sec e ing high
le els o ib onec in 1 which binds o in eg in
α
5
β
1 on mac ophages was ound o igge s
mac ophage pola iza ion owa d immunosupp essi e M2-like pheno ype by ac i a ing
ocal adhesion kinase (FAK)-AKT-STAT3 signaling [103].
The CD90 glycop o ein plays a majo ole in cell mig a ion, adhesion, and ib osis
by egula ing in e ac ions be ween cells o be ween cells and he ECM. High exp ession
o CD90 in b eas cance CAFs was associa ed wi h inc eased cell ans o ma ion and
un a o able disease p ognosis, pa icula ly in he basal-like TNBC sub ypes [104].
The high exp ession o in eg in
α
11/PDGFR
β
on s omal cells was associa ed wi h
high umo g ades and un a o able clinical ou come in b eas cance pa ien s. P o-
in asi e unc ion o in eg in
α
11 elies on i s abili y o in e ac wi h PDGFR
β
ha p o-
mo es downs eam JNK ac i a ion, leading o he p oduc ion o p o-in asi e ECM p o ein
enascin C [105].
FSP-1, he small calcium-binding in eg al memb ane p o ein, is a se ine p o ease
o en exp essed in CAFs in p ima y b eas cance and in ma ching lymph node me as asis
as well as in mac ophages, o he immune cells, and cance cells. FSP-1+ CAFs p oduce
he ECM componen enascin C, cy okines, MMPs, and VEGF-A, he eby acili a ing cell
mo ili y, angiogenesis and me as a ic in asion [
106
]. Mo eo e , he exp ession o FSP-1 was
associa ed wi h un a o able p ognosis and may be conside ed a p ognos ic indica o o
me as a ic disease in ea ly b eas cance [107,108].
P o- umo igenic CAFs in b eas cance o en exp ess CD10, a Zn-dependen ma ix
me allop o einase which was p edominan ly ela ed o ER-nega i e in asi e b eas cance ,
whe eas CD10- CAFs we e associa ed wi h luminal ype in asi e b eas cance [
109
].
GPR77, he non-G p o ein-coupled complemen ecep o , when exp essed in combina ion
wi h CD10, de ines dis inc CAF subpopula ion ha was ela ed o chemo he apy esis ance
and poo p ognosis in pa ien s wi h lung and b eas cance . CD10+GPR77+ CAFs supply
he TME wi h IL-8 and IL-6, which h ough he ac i a ion o NF-
κ
B signaling ia p65
phospho yla ion and ace yla ion, p o ec cance s em cells om chemo he apy-induced
cell dea h [110–112].
Howe e , he low exp ession o some ma ke s, such as ca eolin 1 sca old p o ein,
in CAFs is ela ed o a mo e ad anced umo s age, ea ly cance ecu ence, lymph node
me as asis, and poo disease p ognosis in b eas cance [113].
3.1. Subpopula ions o Fib oblas s in TNBC
CAFs a e a he e ogeneous popula ion wi h espec o hei emb yonic and spa ial
o igin and unc ional cha ac e is ics. Se e al a emp s ha e been made o classi y CAFs in
b eas cance acco ding o he exp ession o molecula ma ke s and unc ional p ope ies.
In his sense, single-cell RNA sequencing and his ological cha ac e iza ion pe o med on
Cells 2025,14, 1353 16 o 31
Mos no ably, an i-TGF-
β
he apy has been used o imp o e he e ec o IC inhibi o s.
In his sense, clinical phase Ib s udy (NCT02423343) in es iga ing he combina ion o
an i-PD-1 he apy (ni olumab) wi h no el TGF-
β
RI kinase inhibi o galunise ib mono-
hyd a e (LY2157299) in ad anced solid umo s has been comple ed and showed good
ole abili y [167].
In he pas decade, bi unc ional an ibodies a ge ing IC and TGF-
β
signaling ha e
been de eloped. In his sense, bi unc ional an ibody SHR-1701 di ec ed agains ex acel-
lula domain o TGF-
β
RII and PD-L1 is cu en ly being in es iga ed in a phase I s udy
(NCT03710265) in subjec s wi h me as a ic o locally ad anced solid umo s o assess he
sa e y and ole abili y o di e en dose le els [168].
Fu he mo e, bin a usp al a, ano he bi unc ional an ibody a ge ing TGF-
β
RII and
PD-L1 in phase Ib/II clinical (NCT03579472) s udy in pa ien s wi h me as a ic TNBC, was
in es iga ed in combina ion wi h e ibulin mesyla e. The s udy showed ha he agen was
gene ally well ole a ed and showed p omising an i umo ac i i y [169].
HCW9218, a bi unc ional an ibody combining simul aneous TGF-
β
-neu alizing and
immune-s imula ing p ope ies, was designed o a ge ex acellula domains o human
TGF-
β
RII and he IL-15 ecep o
α
. HCW9218 is p esen ly being in es iga ed in a phase
I i s in-human clinical ial (NCT05322408) o de e mine he maximum ole a ed dose
in ad anced o me as a ic solid umo s. Fu he mo e, he s udy in mice ea ed wi h
HCW9218 in combina ion wi h an i-PD-1 he apy demons a ed modula ion o immune
landscape in umo -d aining lymph nodes and enhanced T cell an i umo ac i i y [170].
5.1.1. Ta ge ing IL-6
Ta ge ing IL-6/JAK/STAT-3 pa hway is ano he he apeu ic app oach di ec ed agains
he TME, as IL-6 egula es many umo -p omo ing unc ions. Acco ding o p e ious s udies,
TNBC cells sec e e au oc ine IL-6 and a e less esponsi e o pa ac ine IL-6 signaling. The
exposu e o IL-6 leads o he ch onic induc ion o STAT3 phospho yla ion, which p omo es
u he g ow h and in asion o hese umo cells [
171
]. Monoclonal an ibody agains IL-6
ecep o , ocilizumab (TCZ), inhibi ed
in i o
g ow h o MDA-MB-231 umo cells and
educed he o ma ion o me as asis in mice. Fu he mo e, phase I and II clinical ials
demons a ed he e icacy o monoclonal an ibodies agains IL-6 and i s ecep o , ei he as
single agen s o in combina ion wi h o he chemo he apeu ic agen s, adia ion, and a ge ed
he apies in a ious ypes o cance [
172
]. In ecen yea s he clinical use o TCZ was mainly
es ic ed o mi iga ion o immune- ela ed ad e se e en s in pa ien s ea ed wi h IC
inhibi o s and showed p omising esul s in phase II clinical s udy (NCT04375228) [173].
The inhibi ion o he STAT3 signaling ep esen s ano he expe imen al he apeu ic
s a egy. In his sense, OPB-51602, he small-molecule inhibi o o STAT3 phospho yla ion
was e alua ed in phase I (NCT01423903) mul iple dose escala ion clinical ial o de e mine
sa e y and ole abili y in subjec s wi h ad anced cance s o whom he e is no s anda d
ea men a ailable. Fu he mo e, STAT3 inhibi o VVD-130850 is cu en ly being in es i-
ga ed in a phase I clinical s udy (NCT06188208), bo h as a single agen and in combina ion
wi h IC inhibi ion o e alua e i s sa e y, ole abili y, and p elimina y an i umo ac i i y in
pa icipan s wi h ad anced solid and hema ological umo s [174].
Addi ionally, he polykinase inhibi o IMX-110 a ge ing STAT3 in combina ion wi h
an i-PD1 agen islelizumab is cu en ly being in es iga ed a phase I/IIa in dose escala-
ion/dose expansion clinical s udy (NCT05840835) designed o assess i s sa e y, ole abili y,
pha macokine ics and an i umo ac i i y in pa ien s wi h ad anced solid umo s [125].
The nex -gene a ion an isense oligonucleo ide inhibi o s o STAT3, AZD9150, and
AZD5069, a e cu en ly being e alua ed in combina ion wi h MEDI4736 (du alumab),
in a phase I clinical s udy (NCT02499328) in ad anced solid malignancies o compa e he
Cells 2025,14, 1353 17 o 31
e ec s o STAT3 inhibi ion mono he apy and i s combina ion wi h an i-PD-L1 he apy. A
simila phase I clinical s udy (NCT03394144) conduc ed on Japanese adul pa ien s wi h
ad anced solid malignancies showed good ole abili y o mono he apy and combina ion
o his agen wi h du alumab [
175
]. Howe e , compa ing he numbe o STAT3 inhibi o s
de eloped, only a small ac ion is cu en ly in clinical ials, pe haps due o he se e e
oxici ies o he mos o hem [176].
5.1.2. Ta ge ing IL-8
Due o i s ole as a chemokine ha p omo es EMT o umo cells, he ec ui men
o MDSCs o umo si e, subsequen immune escape, and i s equen o e exp ession in
malignancies, including TNBC, IL-8 has been e alua ed as a he apeu ic a ge . P eclinical
da a showed he apeu ic po en ial o clinical-s age monoclonal an ibody ha neu alizes
IL-8 (HuMax-IL8) as i was shown o e e EMT in claudin-low TNBC models. The
same s udy showed a signi ican dec ease in he ec ui men o MDSCs o umo si es, an
e ec subs an ia ed in ea men combina ion wi h doce axel in TNBC [
171
,
177
]. An i-IL-8
monoclonal an ibody HuMax-IL8 (BMS-986253) was e alua ed in phase Ib clinical s udy
(NCT02536469) and showed good sa e y and ole abili y, while he ongoing s udies a e
p esen ly e alua ing he combina ion o IL-8 blockade and IC inhibi o s in melanoma [
178
].
5.1.3. CXCR4 An agonis
A po en selec i e CXCR4 an agonis , he bilixa o ide (B)- POL6326 is a syn he ic
cyclic pep ide. POL6326 showed encou aging sa e y/e icacy da a in phase I clinical ial
(NCT01837095) in combina ion wi h e ibulin, he syn he ic analog o a na u al p oduc
ha inhibi s mic o ubule dynamics and induces apop osis o cance cells, in second line
ea men op ions o pa ien s wi h elapsed TNBC and ho mone e ac o y ER-posi i e
me as a ic b eas cance [
179
]. The p elimina y ac i i y o he combina ion showed p omis-
ing esul s in pa ien s wi h HER-nega i e me as a ic b eas cance ha led o phase III
clinical ial (NCT03786094) in locally ecu en o me as a ic b eas cance .
5.1.4. IDO1 Inhibi ion
IDO1 allows umo escape h ough kynu enine p oduc ion which s imula es T eg
ac i i y while supp essing he p oli e a ion o e ec o T cells. P eclinical indings ha e
e ealed ha IC inhibi o s, while emo ing molecula b akes on cy o oxic immune cells,
also s imula e he p oduc ion o IDO1 in TME, he eby ac i a ing a nega i e eedback loop
in immune esponses. Based on his, pha macological IDO1 inhibi o s a e cu en ly being
es ed in clinical s udies (Table 2), mos ly in combina ion wi h IC inhibi o s [
180
]. In his
sense, phase I clinical s udy (NCT02658890) e alua ing BMS-986205 (Lin odos a ) IDO-1
inhibi o , in combina ion wi h ni olumab alone, and bo h ni olumab and ipilimumab
(an i-CTLA-4) (NCT02658890), showed a o able sa e y and e icacy in hea ily p e ea ed
pa ien s wi h ad anced solid umo s [181]. Fu he mo e, wo clinical s udies in es iga ed
he possibili y o using BMS-986205 IDO1 inhibi o in combina ion wi h LAG-3 inhibi o
ela limab, CTLA-4 inhibi o ipilimumab, and PD-1 inhibi o s in ad anced and me as a ic
solid umo s (NCT03335540; NCT03459222), concluding ha his he apeu ic app oach
should be u he in es iga ed [182].
Epacados a ep esen s ano he IDO1 inhibi o ha compe es wi h T p o binding
o he ca aly ic si e o he enzyme. Recen ly, epacados a was e alua ed in addi ion o
chemo he apy and pemb olizumab in phase I/II clinical s udy (NCT03085914) and showed
accep able sa e y p o ile and an i umo ac i i y ac oss mul iple ypes o ad anced o
me as a ic solid umo s [
183
]. Howe e , a phase Ia/1b clinical s udy (NCT03343613),
in es iga ing an an i-IDO1 agen LY3381916 adminis e ed alone o in combina ion wi h
Cells 2025,14, 1353 18 o 31
PD-L1 inhibi o LY3300054 in ad anced solid umo s including TNBC, was e mina ed
wi hou a conclusion [184].
Table 2. Summa y o clinical ials a ge ing IDO1 in TNBC.
Agen The apy Design G oup o Pa ien s Clinical T ial Numbe
and Phase S a us
BMS-986205 (Lin odos a )
BMS-986205+ ni olumab
BMS-986205 + ni olumab +
ipilimumab
BMS-986205 + ipilimumab
Ad anced malignan
umo s NCT02658890
Phase I/IIa Comple ed
BMS-986205 BMS-986205+
ni olumab/ ela limab/ipilimumab
Ad anced malignan
umo s NCT03335540 (ADVISE)
Phase I Comple ed
BMS-986205 BMS-986205 + ela limab
(an i-LAG-3) + ni olumab Ad anced malignan
umo s NCT03459222
Phase I/II Comple ed
Epacados a Epacados a + pemb olizumab +
chemo he apy Ad anced o me as a ic
solid umo s
NCT03085914 (ECHO-
207/KEYNOTE-723)
Phase I/II Comple ed
LY3381916 LY3381916 + an iPD-L1
(LY3300054) Solid umo s NCT03343613
Phase I Te mina ed
E7046 Mono he apy Selec ed ad anced
malignancies NCT02540291
Phase I Te mina ed
HTL0039732 Mono he apy
HTL0039732 + a ezolizumab Ad anced solid
umo s NCT05944237
Phase Ib/IIa Rec ui ing
Bupa lisib (AN0025) Bupa lisib (AN2025) + AN0025
AN2025 + AN0025 +
a ezolizumab Ad anced solid umo s NCT04975958
Phase Ia Comple ed
eganelisib (IPI-549) Mono he apy
PI-549 + ni olumab Ad anced solid
umo s NCT02637531
Phase I/Ib Unknown s a us
IPI-549
IPI-549+ ecen iq (a ezolizumab)
+ nab-pacli axel F on -line TNBC NCT03961698
MARIO-3
Phase II
Ac i e, no
ec ui ing
T iple-nega i e b eas cance (TNBC).
5.1.5. Ta ge ing PGE2
PGE2, ano he po en ial he apeu ic a ge in TME, media es i s ac i i y mainly
h ough i s G p o ein-coupled ecep o s EP2 and EP4 [
185
]. PGE2 supp esses he im-
mune esponse by inhibi ing he unc ion o T cells, NK cells, and M1 mac ophages, hus
p omo ing issue egene a ion and inducing he di e en ia ion o T egs.
In i o
s udies
ha e shown ha he adminis a ion o EP4 an agonis s es o ed he cy o oxic ac i i y o NK
cells in he con ex o p og essi e umo g ow h [
186
]. P eclinical s udies in b eas cance
ha e shown NK cell-dependen an ime as a ic ac i i y o wo PGE2 inhibi o s: ondoside-
A, he an agonis o EP4/EP2 ecep o s de i ed om he sea cucumbe , and RQ-15986,
he newly syn he ized an agonis o he EP4 ecep o [
186
,
187
]. Fu he s udies led o he
de elopmen o se e al an agonis s o he EP4 ecep o o clinical use. In his sense, he
i s in-human phase I s udy o E7046, a highly selec i e small-molecule an agonis , showed
manageable ole abili y in pa ien s wi h ad anced malignancies, immunomodula o y e -
ec s, he bes clinical esponse o s able disease, and ecommended he dose o phase II
clinical in es iga ion (Table 3) [
188
]. Phase I/IIa clinical ial (NCT05944237) is cu en ly
in es iga ing he bes dose o HTL0039732, a po en small-molecule an agonis o EP4, o
i s sole applica ion in me as a ic solid umo s and i s join use wi h a ezolizumab [
189
].
Fu he mo e, a no el EP4 an agonis AN0025 was in es iga ed in combina ion wi h pan-
class I PI3K inhibi o bupa lisib (AN2025) and a ezolizumab in a phase Ia clinical s udy
(NCT04975958) in pa ien s wi h ad anced solid umo s [190].
Cells 2025,14, 1353 19 o 31
Table 3. Summa y o clinical ials a ge ing PGE2 in TNBC.
Agen The apy Design G oup o Pa ien s Clinical T ial
Numbe and Phase S a us
E7046 Mono he apy Ad anced
malignancies NCT02540291
Phase I Te mina ed
HTL0039732 Mono he apy
HTL003973+ a ezolizumab Ad anced solid
umo s NCT05944237
Phase I/IIa Rec ui ing
AN0025 AN0025 + AN2025 (pan-class I
PI3K inhibi o )
AN0025 +a ezolizumab
Ad anced malignan
umo s NCT04975958
Phase Ia Comple ed
T iple-nega i e b eas cance (TNBC).
5.1.6. PI3KγInhibi ion
In p eclinical s udies, a selec i e inhibi o o PI3K-
γ
, eganelisib (IPI-549), showed
po en abili y o eshape he TME by educing myeloid cell ec ui men o umo s and
ep og amming TAMs om immune-supp essi e o immune-ac i a ing pheno ype. This
e ec enhanced he ac i i y o an i-PD-1/PD-L1 he apy [
191
]. Two complemen a y clinical
s udies ha e in es iga ed eganelisib in combina ion wi h IC inhibi o s. In his sense,
eganelisib alone, and in i s combina ion wi h ni olumab, was e alua ed o sa e y and
ole abili y in a phase I/Ib clinical s udy (NCT02637531) in pa ien s wi h ad anced solid
umo s. Simul aneously, he phase II MARIO-3 clinical s udy (NCT03961698) in es iga ed
he inhibi ion o PI3K-
ꝩ
, in combina ion wi h ecen iq (a ezolizumab) and ab axane (nab-
pacli axel) in on -line TNBC (Table 4). The MARIO-3 s udy e ealed ele a ed gene
signa u es o TAM ep og amming and immune ac i a ion in pa ien s wi h TNBC wi h
longe p og ession- ee su i al, ega dless o he baseline PD-L1 s a us [192].
Table 4. Summa y o clinical ials a ge ing PI3K-γin TNBC.
Agen The apy Design G oup o Pa ien s Clinical T ial Numbe
and Phase S a us
Eganelisib
(IPI-549) Mono he apy Ad anced solid umo s NCT02637531
Phase I/Ib Te mina ed
IPI-549 IPI-549 + nabpacli axel +
a zolizumab F on -line iple TNBC NCT03961698
Phase II Unknown s a us
T iple-nega i e b eas cance (TNBC).
5.2. CAF-Di ec ed The apies
Based on ecen ly de ined oles o CAFs in immune supp ession and esis ance o
he apy, di ec a ge ing o CAFs and hei su ace ma ke s was es ablished as anno el and
a ac i e a ge o an icance he apies in ad anced b eas cance (Table 5) [102,110].
RO7300490 ep esen s a bi unc ional an ibody (Table 3) wi h po en ial immunos imula-
o y and an ineoplas ic ac i i y. RO7300490 is a CD40 agonis ha also a ge s he inducible
umo s omal an igen FAP-
α
p o ease, which is b oadly exp essed in CAFs in a ious
solid umo s. RO7300490 displays i s immunos imula o y e ec by ac i a ing CD40 cell
su ace s imula o y TNF amily ecep o exp essed on immune cells. CD40 engagemen
induces he p oli e a ion and ac i a ion o B lymphocy es, di e en ia ion o immunos-
imula o y mac ophages, and ac i a ion o DCs o sec e e in lamma o y cy okines. This
p omo es he p oli e a ion and ac i a ion o CTLs o kill umo cells. A phase I clinical s udy
(NCT04857138) e alua ing sa e y, pha macokine ics, and an i umo ac i i y o RO7300490,
as a single agen o in combina ion wi h a ezolizumab, in pa icipan s wi h ad anced
and/o me as a ic solid umo s, showed a a o able sa e y p o ile, and suppo ed u he
clinical in es iga ions o his agen in combina ion wi h o he an icance he apies [193].
Cells 2025,14, 1353 20 o 31
Table 5. Summa y o clinical ials a ge ing cance -associa ed ib oblas s in TNBC.
Ac i i y/Mechanism Agen The apy Design G oup o Pa ien s Clinical T ial S a us
Ta ge ing FAPα
FAPα- a ge ed CD40
agonis an ibody RO7300490 Mono he apy
RO7300490 +
a ezolizumab
Ad anced and/o
me as a ic solid
umo s NCT04857138 Phase I Comple ed
Ta ge ing FGFR signaling
Pan FGFR 1-4 inhibi o E da i inib Mono he apy Ad anced solid umo s
NCT04083976 RAGNAR
Phase II Comple ed
E da i inib +
Ful es an + Palbociclib ER+/HER2-/FGFR-
ampli ied BC NCT03238196
Phase I
Ac i e, no ec ui ing
Ta ge ing Hedgehog signaling
Smoo hened ecep o
(SMO) an agonis Sonidegib (LDE225) Sonidegib + doci axel Ad anced TNBC
NCT02027376 EDALINE
Phase Ib Comple ed
Inhibi o o SMO Vismodegib (GDC-0449) Vismodegib + s anda d
neoadju an
chemo he apy TNBC NCT02694224 Phase II Unknown s a us
Re e ing CAFs in o quiescen s a e
Vi amin D
ecep o agonis 19-no -1,25-
dihyd oxy i amin D2
19-no -1,25-
dihyd oxy i amin D2 +
s anda d Neoadju an
Chemo he apy
Me as a ic B eas Cance NCT00637897 Phase I Comple ed
RARβ
agonis All ans e inoic acid
(ATRA)
ATRA +
non-s e oidal a oma ase
inhibi o anas ozole
Ho monal
ecep o +/HER2- ea ly
BC NCT04113863 Phase II Rec ui ing
Ta ge ing ECM
Inhibi ion o
hyalu onidase ac i i y
PEGPH20 PEGyla ed
Recombinan Human
hyla onidaze
PEGPH20 +
e ibulin mesyla e
HER2-, high-hyalu onan
me as a ic BC NCT02753595
Phase Ib/II Te mina ed
Inhibi ion o colagen and
hyalu on p oduc ion
Losa an
(angio ensin ecep o
blocke )
Losa an +
cam elizumab
+doxo ubicin
TNBC ea ed wi h no
mo e han 1 P io line o
chemo he apy
NCT05097248
Phase II Unknown s a us
(TNBC), es ogen ecep o (ER), es ogen ecep o (ER), b eas cance (BC), human epide mal g ow h ac o
ecep o 2 (HER2), ib oblas g ow h ac o ecep o (FGFR), e inoic acid ecep o (RAR).
P eclinical s udies ha e consis en ly implica ed FGFR signaling in b eas cance p o-
g ession, while clinical e idence ailed o suppo hese indings. This may indica e ha
he clinical signi icance o FGFR should be analyzed in he con ex o he s oma, as he
ac i a ion o esiden ib oblas s can be a c ucial ac o o CAF gene a ion and umo
p og ession [
194
]. E da i inib, a y osine kinase inhibi o o FGFR, was in es iga ed in a
phase Ib clinical s udy (NCT03238196) in combina ion wi h ul es an and palbociclib in
ER+/HER2-/FGFR-ampli ied me as a ic b eas cance . The s udy sugges ed some an i u-
mo e icacy o his combina ion. Fu he in es iga ions o e da i inib in phase II RAGNAR
(NCT04083976) clinical ial showed clinical bene i in umo -agnos ic se ing in pa ien s
wi h ad anced solid malignancies wi h suscep ible FGFR al e a ions who ha e exhaus ed
o he ea men op ions. The esul s o his ial suppo he con inued de elopmen o
pha macological inhibi o s o FGFR o pa ien s wi h ad anced solid umo s [195].
The hedgehog signaling pa hway is ac i a ed in he s oma o TNBC in esponse
o hedgehog ligand sec e ed by cance epi helial cells. Acco dingly, a he apeu ic ap-
p oach ha combines a ge ing cance epi helial cells wi h chemo he apy and CAFs wi h
hedgehog pa hway inhibi o eme ged as a po en ial app oach o imp o e TNBC ea men
ou come [
196
]. Recen ly, i was shown ha he hedgehog signaling inhibi o sonidegib o
down egula es he exp ession o CSC ma ke s and inc eases he sensi i i y o TNBC o
pacli axel in phase I clinical s udy (NCT02027376), hus imp o ing pa ien su i al and
educing me as asis in ad anced TNBC [
197
]. Ano he inhibi o o hedgehog signaling,
ismodegib (GDC-0449), was added o s anda d neoadju an chemo he apy in phase II
clinical s udy (NCT02694224) due o i s abili y o inhibi b eas CSC sel - enewal and
mammosphe e o ma ion [198]. The s a us o his clinical ial emains unknown.
Cells 2025,14, 1353 21 o 31
5.2.1. Re e ing CAFs in o Quiescen S a e
The apies e e ing CAFs o a quiescen s a e, such as a ge ing i amin D ecep o ha
ep esen s a ansc ip ional supp esso o ac i a ed CAFs, ha e been shown o con ibu e
o enhanced deli e y o an icance d ug in animal models. In his sense, a phase I clinical
ial (NCT00637897) in es iga ed he e ec o pa icalci ol (19-no -1,25-dihyd oxy i amin
D2) in assis ing chemo he apeu ic d ugs o mo e e icien ly kill umo cells. The s udy
showed sa e y and easibili y in women wi h me as a ic b eas cance ecei ing axanes o
ixabepilone [199].
All ans e inoic acid (ATRA) ac i a es e inoic acid ecep o (RAR)
β
and in oduces
CAFs in o quiescen s a e by down egula ing ac in myosin con ac ili y, he eby educing
CAF ac i i ies ela ed o ECM emodeling and cell mig a ion [
200
]. An I alian single-cen e
andomized phase II clinical s udy (NCT04113863) is p esen ly in es iga ing p eope a i e
ac i i y o ATRA in HR+/HER2- ea ly b eas cance in combina ion wi h non-s e oidal
a oma ase inhibi o anas ozole.
5.2.2. Ta ge ing ECM
As desmoplasia and inc eased in e s i ial p essu e exe comp ession on umo ascu-
la u e impeding he e ec i e dis ibu ion o chemo he apeu ic agen s in o umo issue,
he inhibi ion o ECM p oduc ion by CAFs ep esen s ano he CAF- a ge ed he apeu ic
s a egy. Fo example, enzyma ic ac i i y o hyalu onidase can be used o modi y umo
a chi ec u e o a mo e e ec i e deli e y o chemo he apeu ics. In his sense, hyalu onidase
PEGPH20 in combina ion wi h e ibulin e sus e ibulin alone showed in phase II clinical
ial (NCT02753595) imp o ed an i umo e ec s in subjec s wi h HER2-nega i e b eas
cance [201]
. Fu he mo e, angio ensin ecep o blocke losa an, also known o inhibi -
ing he p oduc ion o collagen and hyalu onan and supp essing TGF-
β
-media ed ib o ic
signaling, ep esen s ano he agen a ge ing he ECM ha showed some e icacy in he
p eclinical model o TNBC [
202
]. Losa an was included in he ea men p o ocol in a
phase II clinical ial (NCT05097248) e alua ing PD-1 inhibi o cam elizumab and liposomal
doxo ubicin in pa ien s wi h ad anced o locally ad anced TNBC, who we e p e iously
ea ed wi h no mo e han one line o chemo he apy.
6. Conclusions
The accumula ing e idence shows he impo ance o complex c oss alk be ween cance
cells, immune cells, ib oblas s, endo helial cells, molecula s omal componen s, and ex a-
cellula ma ix componen s o umo p og ession and esponse o he apy. The impo ance
o TME o esponse o he apy is e y p onounced in TNBC due o he lack o adequa e
molecula he apeu ic a ge s con ined o umo cells and he absence o e ec i e ea men
op ions excep o chemo he apy. Al hough mul iple clinical ials ha e ecen ly implied
he bene i s o in oducing immuno he apy in o he ea men o TNBC, he esponse a es
emained low due o ac i a ion o addi ional supp essi e mechanisms in TME du ing he
cou se o he apy ha unde mine he he apeu ic bene i . In his sense he apeu ic a ge ing
o he TME and i s ac o s, including cy okines, enzymes, me aboli es, chemokines, and
signaling pa hways in TME, ep esen s po en ial complemen a y he apeu ic s a egy in
TNBC. Fu he mo e, CAFs eme ge as ele an a ge s due o hei mul iple oles in umo
ini ia ion and p og ession. Mo eo e , CAFs con e cance cell esis ance o he apy, as hey
o ches a e immunosupp essi e TME by in e ac ing wi h immune cells and play a c ucial
ole in he o ma ion o ECM ha se es as a physical ba ie o he apeu ic d ugs and
immune cells om eaching he umo .
Howe e , based on he ecen da a ob ained om clinical in es iga ions, he mos o
cu en ly a ailable agen s a ge ing cy okines, cy okine ecep o s, p os aglandin ecep o s,
Cells 2025,14, 1353 22 o 31
IDO-1 ac i i y and CAF- ela ed a ge ing agen s ha e eached phase I/II clinical ials.
The leading numbe o de eloped pha macological agen s and clinical ials in es iga ing
hem was ela ed o blockade o TGF-
β
signaling and IDO inhibi ion. Fo e icien clinical
applica ion, u he de elopmen o he apeu ics wi h high speci ici y o hei a ge in
TME is needed, as well as expe imen al e alua ions o s a egies ha simul aneously a ge
se e al componen s in he TME. Fu he mo e, be e unde s anding o he in e ac ions
be ween cells and molecula componen s in he TME is c ucial o disco e ing no el
d uggable a ge s ela ed o molecula and immunological aspec s o TNBC.
Au ho Con ibu ions: Concep ualiza ion, A.V. and V.J.; w i ing—o iginal d a p epa a ion, A.V.,
K.M.M. and V.J.; w i ing— e iew and edi ing, A.V., K.M.M. and V.J.; supe ision, V.J.; unding
acquisi ion, V.J. All au ho s ha e ead and ag eed o he published e sion o he manusc ip .
Funding: Minis y o Science, Technological De elopmen and Inno a ion o he Republic o Se bia
(Ag eemen s No. 451-03-136/2025-03/200111 and 451-03-136/2025-03/200043).
Ins i u ional Re iew Boa d S a emen : No applicable.
In o med Consen S a emen : No applicable.
Da a A ailabili y S a emen : No new da a we e c ea ed o analyzed in his s udy. Da a sha ing is
no applicable o his a icle.
Con lic s o In e es : The au ho s decla e no con lic s o in e es .
Re e ences
1.
Fu ukawa, N.; S ea ns, V.; San a-Ma ia, C.A.; Popel, A.S. The umo mic oen i onmen and iple-nega i e b eas cance
agg essi eness: Shedding ligh on mechanisms and a ge ing. Expe Opin. The . Ta ge s 2022,26, 1041–1056. [C ossRe ]
2.
B ay, F.; La e sanne, M.; Sung, H.; Fe lay, J.; Siegel, R.L.; Soe joma a am, I.; Jemal, A. Global Cance S a is ics 2022: GLOBOCAN
Es ima es o Incidence and Mo ali y Wo ldwide o 36 Cance s in 185 Coun ies. CA Cance J. Clin. 2024,74, 229–263. [C ossRe ]
3.
Ensenya -Mendez, M.; Llinàs-A ias, P.; O ozco, J.I.J.; Íñiguez-Muñoz, S.; Salomon, M.P.; Sesé, B.; DiNome, M.L.; Ma zese, D.M.
Cu en T iple-Nega i e B eas Cance Sub ypes: Dissec ing he Mos Agg essi e Fo m o B eas Cance . F on . Oncol. 2021,
11, 681476. [C ossRe ] [PubMed]
4.
Kennecke, H.; Ye ushalmi, R.; Woods, R.; Cheang, M.C.U.; Voduc, D.; Spee s, C.H.; Nielsen, T.O.; Gelmon, K. Me as a ic beha io
o b eas cance sub ypes. J. Clin. Oncol. 2010,28, 3271–3277. [C ossRe ] [PubMed]
5.
Obidi o, O.; Ba og okh, G.; Akala, E.O. T iple Nega i e B eas Cance T ea men Op ions and Limi a ions: Fu u e Ou look.
Pha maceu ics 2023,15, 1796. [C ossRe ]
6.
Lehmann, B.D.; Shy , Y.; Pie enpol, J.A.; Lehmann, B.D.; Baue , J.A.; Chen, X.; Sande s, M.E.; Chak a a hy, A.B.; Shy , Y.;
Pie enpol, J.A. Iden i ica ion o Human T iple-Nega i e B eas Cance Sub ypes and P eclinical Models o Selec ion o Ta ge ed
The apies. J. Clin. In es ig. 2011,121, 2750–2767. [C ossRe ]
7.
Lehmann, B.D.; Jo ano i’c, B.; Chen, X.; Es ada, M.V.; Johnson, K.N.; Shy , Y.; Moses, H.L.; Sande s, M.E.; Pie enpol, J.A.
Re inemen o T iple-Nega i e B eas Cance Molecula Sub ypes: Implica ions o Neoadju an Chemo he apy Selec ion. PLoS
ONE 2016,11, e0157368. [C ossRe ]
8.
Bu s ein, M.D.; Tsimelzon, A.; Poage, G.M.; Co ing on, K.R.; Con e as, A.; Fuqua, S.A.W.; Sa age, M.I.; Osbo ne, C.K.; Hilsen-
beck, S.G.; Chang, J.C.; e al. Comp ehensi e Genomic Analysis Iden i ies No el Sub ypes and Ta ge s o T iple-Nega i eB eas
Cance . Clin. Cance Res. 2015,21, 1688–1698. [C ossRe ] [PubMed]
9.
Jiang, Y.-Z.; Ma, D.; Suo, C.; Shi, J.; Xue, M.; Hu, X.; Xiao, Y.; Yu, K.-D.; Liu, Y.-R.; Yu, Y.; e al. Genomic and T ansc ip omic
Landscape o T iple-Nega i e B eas Cance s: Sub ypes and T ea men S a egies. Cance Cell 2019,35, 428–440.e5. [C ossRe ]
10.
Nedeljko i´c, M.; Vule i´c, A.; Mi jaˇci´c Ma ino i´c, K. Di ide and Conque -Ta ge ed The apy o T iple-Nega i e B eas Cance . In .
J. Mol. Sci. 2025,26, 1396. [C ossRe ]
11.
He, Y.; Jiang, Z.; Chen, C.; Wang, X. Classi ica ion o T iple-Nega i e B eas Cance s Based on Immunogenomic P o iling. J. Exp.
Clin. Cance Res. 2018,37, 327. [C ossRe ]
12.
Beldi-Fe chiou, A.; Cailla -Zucman, S. Con ol o NK Cell Ac i a ion by Immune Checkpoin Molecules. In . J. Mol. Sci. 2017,
18, 2129. [C ossRe ]
13. Kwa, M.J.; Adams, S. Checkpoin Inhibi o s in T iple-nega i e B eas Cance (TNBC): Whe e o Go om He e. Cance 2018,124,
2086–2103. [C ossRe ] [PubMed]
Cells 2025,14, 1353 23 o 31
14.
Li, L.; Zhang, F.; Liu, Z.; Fan, Z. Immuno he apy o T iple-Nega i e B eas Cance : Combina ion S a egies o Imp o e Ou come.
Cance s 2023,15, 321. [C ossRe ]
15.
Chung, W.; Eum, H.H.; Lee, H.-O.; Lee, K.-M.; Lee, H.-B.; Kim, K.-T.; Ryu, H.S.; Kim, S.; Lee, J.E.; Pa k, Y.H.; e al. Single-Cell
RNA-Seq Enables Comp ehensi e Tumou and Immune Cell P o iling in P ima y B eas Cance . Na . Commun. 2017,8, 15081.
[C ossRe ]
16.
Coleman, C.; Sel akuma , T.; Thu lapa i, A.; G a , K.; Pa ulu i, S.; Meh o a, S.; Sahin, O.; Si api agasam, A. Ha nessing
Tumo -In il a ing Lymphocy es in T iple-Nega i e B eas Cance : Oppo uni ies and Ba ie s o Clinical In eg a ion. In . J. Mol.
Sci. 2025,26, 4292. [C ossRe ]
17.
Loi, S.; Si aine, N.; Pie e, F.; Salgado, R.; Viale, G.; Van Eenoo, F.; Rouas, G.; F ancis, P.; C own, J.P.A.; Hi e, E.; e al. P ognos ic
and p edic i e alue o umo -in il a ing lymphocy es in a phase III andomized adju an b eas cance ial in node-posi i e
b eas cance compa ing he addi ion o doce axel o doxo ubicin wi h doxo ubicin-based chemo he apy: BIG 02-98. J. Clin.
Oncol. 2013,31, 860–867. [C ossRe ] [PubMed]
18.
Adams, S.; G ay, R.J.; Dema ia, S.; Golds ein, L.; Pe ez, E.A.; Shulman, L.N.; Ma ino, S.; Wang, M.; Jones, V.E.; Saphne , T.J.;
e al. P ognos ic Value o Tumo -In il a ing Lymphocy es in T iple-Nega i e B eas Cance s F om Two Phase III Randomized
Adju an B eas Cance T ials: ECOG 2197 and ECOG 1199. J. Clin. Oncol. 2014,32, 2959–2966. [C ossRe ] [PubMed]
19.
Dieci, M.V.; Ma hieu, M.C.; Gua ne i, V.; Con e, P.; Delaloge, S.; And e, F.; Gouba , A. P ognos ic and p edic i e alue o
umo -in il a ing lymphocy es in wo phase III andomized adju an b eas cance ials. Ann. Oncol. 2015,26, 1698–1704.
[C ossRe ]
20.
Solinas, C.; Ca bognin, L.; De Sil a, P.; C isci iello, C.; Lambe ini, M. Tumo -in il a ing lymphocy es in b eas cance acco ding
o umo sub ype: Cu en s a e o he a . B eas 2017,35, 142–150. [C ossRe ]
21.
Gao, Z.H.; Li, C.X.; Liu, M.; Jiang, J.Y. P edic i e and p ognos ic ole o umou -in il a ing lymphocy es in b eas cance pa ien s
wi h di e en molecula sub ypes: A me a-analysis. BMC Cance 2020,20, 1150. [C ossRe ]
22.
Vihe uo i, H.; Au e e, T.A.; Repo, H.; Ku ki, S.; Kallio, L.; Lin unen, M.M.; Tal inen, K.; K onq is , P. Tumo -in il a ing
lymphocy es and CD8+ T cells p edic su i al o iple-nega i e b eas cance . J. Cance Res. Clin. Oncol. 2019,145, 3105–3114.
[C ossRe ] [PubMed]
23.
Dieci, M.V.; Ts e ko a, V.; O ie o, E.; Piacen ini, F.; Fica a, G.; G iguolo, G.; Miglie a, F.; Gia a ano, T.; Oma ini, C.;
Bonagu o, S.
; e al. Immune cha ac e iza ion o b eas cance me as ases: P ognos ic implica ions. B eas Cance Res. 2018,20, 62.
[C ossRe ]
24.
Cia ka, A.; Pi ˛a ek, M.; P˛eksa, R.; Kunc, M.; Senkus, E. Tumo -In il a ing Lymphocy es (TILs) in B eas Cance : P ognos ic and
P edic i e Signi icance Ac oss Molecula Sub ypes. Biomedicines 2024,12, 763. [C ossRe ] [PubMed]
25.
Xie, Q.; Ding, J.; Chen, Y. Role o CD8+ T lymphocy e cells: In e play wi h s omal cells in umo mic oen i onmen . Ac a Pha m.
Sin. B 2021,11, 1365–1378. [C ossRe ]
26.
Schmi z, J.; Assenmache , M.; Radb uch, A. Regula ion o T helpe cell cy okine exp ession: Func ional dicho omy o an igen-
p esen ing cells. Eu . J. Immunol. 1993,23, 191–199. [C ossRe ]
27.
Ka pisheh, V.; Ahmadi, M.; Abbaszadeh-Gouda zi, K.; Mohammadpou Sa ay, M.; Ba shidi, A.; Mohammadi, H.;
Youse i, M.
;
Jadidi-Nia agh, F. The ole o Th17 cells in he pa hogenesis and ea men o b eas cance . Cance Cell In . 2022,22, 108.
[C ossRe ] [PubMed]
28.
Guan, H.; Lan, Y.; Wan, Y.; Wang, Q.; Wang, C.; Xu, L.; Chen, Y.; Liu, W.; Zhang, X.; Li, Y.; e al. PD-L1 media ed he di e en ia ion
o umo -in il a ing CD19+ B lymphocy es and T cells in In asi e b eas cance . Oncoimmunology 2015,5, e1075112. [C ossRe ]
29.
Miligy, I.; Mohan, P.; Gabe , A.; Aleskanda any, M.A.; Nolan, C.C.; Diez-Rod iguez, M.; Mukhe jee, A.; Chapman, C.; Ellis, I.O.;
G een, A.R.; e al. P ognos ic signi icance o umou in il a ing B lymphocy es in b eas duc al ca cinoma in si u. His opa hology
2017,71, 258–268. [C ossRe ]
30.
Qin, Y.; Peng, F.; Ai, L.; Mu, S.; Li, Y.; Yang, C.; Hu, Y. Tumo -in il a ing B cells as a a o able p ognos ic bioma ke in b eas
cance : A sys ema ic e iew and me a-analysis. Cance Cell In . 2021,21, 310. [C ossRe ]
31.
Toney, N.J.; Opdenake , L.M.; F e ichs, L.; Moda ai, S.R.; Ma, A.; A chinal, H.; Ajayi, G.O.; Sims-Mou ada, J. B cells enhance IL-1
be a d i en in asi eness in iple nega i e b eas cance . Sci. Rep. 2025,15, 2211. [C ossRe ] [PubMed]
32.
Toney, N.J.; Opdenake , L.M.; Cicek, K.; F e ichs, L.; Kenning on, C.R.; Obe ly, S.; A chinal, H.; Somasunda am, R.; Sims-
Mou ada, J. Tumo -B-cell in e ac ions p omo e iso ype swi ching o an immunosupp essi e IgG4 an ibody esponse h ough
up egula ion o IL-10 in iple nega i e b eas cance s. J. T ansl. Med. 2022,20, 112. [C ossRe ] [PubMed]
33.
Ishigami, E.; Sakakiba a, M.; Sakakiba a, J.; Masuda, T.; Fujimo o, H.; Hayama, S.; Nagashima, T.; Sangai, T.; Nakagawa, A.;
Naka ani, Y.; e al. Coexis ence o egula o y B cells and egula o y T cells in umo -in il a ing lymphocy e agg ega es is a
p ognos ic ac o in pa ien s wi h b eas cance . B eas Cance 2019,26, 180–189. [C ossRe ]
34.
Coope , M.A.; Colonna, M.; Yokoyama, W.M. Hidden alen s o na u al kille s: NK cells in inna e and adap i e immuni y. EMBO
Rep. 2009,10, 1103–1110. [C ossRe ] [PubMed]
Cells 2025,14, 1353 24 o 31
35.
Campbell, K.S.; Pu dy, A.K. S uc u e/ unc ion o human kille cell immunoglobulin-like ecep o s: Lessons om polymo phisms,
e olu ion, c ys al s uc u es and mu a ions. Immunology 2011,132, 315–325. [C ossRe ]
36.
Konje ic, G.; Ju isic, V.; Jo ic, V.; Vule ic, A.; Mi jacic Ma ino ic, K.; Radenko ic, S.; Spuzic, I. In es iga ion o NK cell unc ion
and hei modula ion in di e en malignancies. Immunol. Res. 2012,52, 139–156. [C ossRe ]
37.
Ran, G.H.; Lin, Y.Q.; Tian, L.; Zhang, T.; Yan, D.M.; Yu, J.H.; Deng, Y.C. Na u al kille cell homing and a icking in issues and
umo s: F om biology o applica ion. Signal T ansduc . Ta ge . The . 2022,7, 205. [C ossRe ]
38.
Raule , D.H.; Gasse , S.; Gowen, B.G.; Deng, W.; Jung, H. Regula ion o ligands o he NKG2D ac i a ing ecep o . Annu. Re .
Immunol. 2013,31, 413–441. [C ossRe ]
39.
Mamessie , E.; Syl ain, A.; Thibul , M.L.; Hou enaeghel, G.; Jacquemie , J.; Cas ellano, R.; Gonçal es, A.; And é, P.; Romagné, F.;
Thibaul , G.; e al. Human b eas cance cells enhance sel ole ance by p omo ing e asion om NK cell an i umo immuni y. J.
Clin. In es ig. 2011,121, 3609–3622. [C ossRe ]
40.
Ba lle, E.; Massagu’e, J. T ans o ming g ow h ac o -
β
signaling in immuni y and cance . Immuni y 2019,50, 924–940. [C ossRe ]
41.
Cas iconi, R.; Can oni, C.; Della Chiesa, M.; Vi ale, M.; Ma cena o, E.; Con e, R.; Biassoni, R.; Bo ino, C.; Mo e a, L.; Mo e a, A.
T ans o ming g ow h ac o be a 1 inhibi s exp ession o NKp30 and NKG2D ecep o s: Consequences o he NK-media ed
killing o dend i ic cells. P oc. Na l. Acad. Sci. USA 2003,100, 4120–4125. [C ossRe ] [PubMed]
42.
Konje i´c, G.; Vule i´c, A.; Mi jaˇci´c Ma ino i´c, K. Na u al kille cell ecep o s: Al e a ions and he apeu ic a ge ing in malignancies.
Immunol. Res. 2016,64, 25–35. [C ossRe ]
43.
Konje i´c, G.M.; Vule i´c, A.M.; Mi jaˇci´c Ma ino i´c, K.M.; La sen, A.K.; Ju iši´c, V.B. The Role o Cy okines in he Regula ion o NK
Cells in he Tumo En i onmen . Cy okine 2019,117, 30–40. [C ossRe ]
44.
Chi adze, G.; Bha , J.; Le au, M.; Janssen, O.; Kabeli z, D. Gene a ion o soluble NKG2D ligands: P o eoly ic clea age, exosome
sec e ion and unc ional implica ions. Scand. J. Immunol. 2013,78, 120–129. [C ossRe ]
45.
Ho man, T.; Ng, S.W.; Ga cés-Láza o, I.; Heigwe , F.; Bou os, M.; Ce wenka, A. IFN
γ
media es he esis ance o umo cells o
dis inc NK cell subse s. J. Immuno he . Cance 2024,12, e009410. [C ossRe ]
46.
Ben-Shmuel, A.; G upe , Y.; Halpe in, C.; Le i-Galibo , O.; Rosenbe g-Fogle , H.; Ba ki, D.; Ca ado i, G.; S ein, Y.; Yagel, G.;
Naumo a, M.; e al. Cance -associa ed ib oblas s se e as decoys o supp ess NK cell an i-cance cy o oxici y in b eas cance .
Cance Disco . 2025,15, 1247–1269. [C ossRe ]
47.
S ojano ic, A.; Co eia, M.P.; Ce wenka, A. Shaping o NK cell esponses by he umo mic oen i onmen . Cance Mic oen i on.
2013,6, 135–146. [C ossRe ] [PubMed]
48.
Balzasch, B.M.; Ce wenka, A. Mic oen i onmen al signals shaping NK-cell eac i i y in cance . Eu . J. Immunol. 2023,53, e2250103.
[C ossRe ]
49.
Liu, X.; Li, L.; Si, F.; Huang, L.; Zhao, Y.; Zhang, C.; Ho , D.F.; Peng, G. NK and NKT cells ha e dis inc p ope ies and unc ions
in cance . Oncogene 2021,40, 4521–4537. [C ossRe ] [PubMed]
50.
Thacke , G.; Hen y, S.; Nandi, A.; Debna h, R.; Singh, S.; Nayak, A.; Susnik, B.; Boone, M.M.; Zhang, Q.; Kesmodel, S.B.; e al.
Imma u e na u al kille cells p omo e p og ession o iple-nega i e b eas cance . Sci. T ansl. Med. 2023,15, eabl4414. [C ossRe ]
51.
Wang, Y.; Jiang, Y.; Ding, F.; Lu, J.; Huang, T.; Zhong, G.; Zhu, P.; Ma, Y.; Li, J.; Wang, X.; e al. Pheno ypes and cy okines o NK
cells in iple-nega i e b eas cance esis an o checkpoin blockade immuno he apy. B eas Cance Res. 2025,27, 51. [C ossRe ]
[PubMed]
52.
Mi endo , E.A.; Philips, A.V.; Me ic-Be ns am, F.; Qiao, N.; Wu, Y.; Ha ing on, S.; Su, X.; Wang, Y.; Gonzalez-Angulo, A.M.;
Akcakana , A.; e al. PD-L1 exp ession in iple-nega i e b eas cance . Cance Immunol. Res. 2014,2, 361–370. [C ossRe ]
53.
Chen, L.; Gibbons, D.L.; Goswami, S.; Co ez, M.A.; Ahn, Y.H.; Bye s, L.A.; Zhang, X.; Yi, X.; Dwye , D.; Lin, W.; e al. Me as asis
is egula ed ia mic oRNA-200/ZEB1 axis con ol o umou cell PD-L1 exp ession and in a umo al immunosupp ession. Na .
Commun. 2014,5, 5241. [C ossRe ]
54.
Cas agnoli, L.; Cancila, V.; Co doba-Rome o, S.L.; Fa aci, S.; Tala ico, G.; Belmon e, B.; Io io, M.V.; Milani, M.; Volpa i, T.;
Chiodoni, C.; e al. WNT signaling modula es PD-L1 exp ession in he s em cell compa men o iple-nega i e b eas cance .
Oncogene 2019,38, 4047–4060. [C ossRe ]
55.
Maeda, T.; Ba am, T.; O en, N.; E lichman, N.; Meshel, T.; Ben-Ba uch, A. In lamma ion-D i en Regula ion o PD-L1 and PD-L2,
and Thei C oss-In e ac ions wi h P o ec i e Soluble TNF
α
Recep o s in Human T iple-Nega i e B eas Cance . Cance s 2022,
14, 3513. [C ossRe ]
56.
Ju, X.; Zhang, H.; Zhou, Z.; Chen, M.; Wang, Q. Tumo -associa ed mac ophages induce PD-L1 exp ession in gas ic cance cells
h ough IL-6 and TNF-a signaling. Exp. Cell Res. 2020,396, 112315. [C ossRe ] [PubMed]
57.
Cu iel, T.J.; Wei, S.; Dong, H.; Al a ez, X.; Cheng, P.; Mo am, P.; K zysiek, R.; Knu son, K.L.; Daniel, B.; Zimme mann, M.C.;
e al. Blockade o B7-H1 imp o es myeloid dend i ic cell-media ed an i umo immuni y. Na . Med. 2003,9, 562–567. [C ossRe ]
[PubMed]
Cells 2025,14, 1353 25 o 31
58.
Naza e h, M.R.; B ode ick, L.; Simpson-Abelson, M.R.; Kellehe , R.J., J .; Yoko a, S.J.; Banke , R.B. Cha ac e iza ion o human
lung umo -associa ed ib oblas s and hei abili y o modula e he ac i a ion o umo -associa ed T cells. J. Immunol. 2007,178,
5552–5562. [C ossRe ]
59.
Zou, W.; Wolchok, J.D.; Chen, L. PD-L1 (B7-H1) and PD-1 pa hway blockade o cance he apy: Mechanisms, esponse
bioma ke s, and combina ions. Sci. T ansl. Med. 2016,8, 328 4. [C ossRe ]
60.
Wang, Y.; Wang, H.; Yao, H.; Li, C.; Fang, J.Y.; Xu, J. Regula ion o PD-L1: Eme ging ou es o a ge ing umo immune e asion.
F on . Pha macol. 2018,9, 536. [C ossRe ]
61.
Tsu sumi, S.; Saeki, H.; Nakashima, Y.; I o, S.; Oki, E.; Mo i a, M.; Oda, Y.; Okano, S.; Maeha a, Y. P og ammed dea h-ligand
1 exp ession a umo in asi e on is associa ed wi h epi helial-mesenchymal ansi ion and poo p ognosis in esophageal
squamous cell ca cinoma. Cance Sci. 2017,108, 1119–1127. [C ossRe ]
62.
Chen, C.; Li, S.; Xue, J.; Qi, M.; Liu, X.; Huang, Y.; Hu, J.; Dong, H.; Ling, K. PD-L1 umo -in insic signaling and i s he apeu ic
implica ion in iple-nega i e b eas cance . JCI Insigh 2021,6, e131458. [C ossRe ] [PubMed]
63.
Ohue, Y.; Nishikawa, H. Regula o y T (T eg) cells in cance : Can T eg cells be a new he apeu ic a ge ? Cance Sci. 2019,110,
2080–2089. [C ossRe ] [PubMed]
64.
Malla, R.R.; Vasude a aju, P.; Vempa i, R.K.; Rakshmi ha, M.; Me chan , N.; Naga aju, G.P. Regula o y T cells: Thei ole in
iple-nega i e b eas cance p og ession and me as asis. Cance 2022,128, 1171–1183. [C ossRe ]
65.
Huang, P.; Zhou, X.; Zheng, M.; Yu, Y.; Jin, G.; Zhang, S. Regula o y T cells a e associa ed wi h he umo immune mic oen-
i onmen and immuno he apy esponse in iple-nega i e b eas cance . F on . Immunol. 2023,14, 1263537. [C ossRe ]
[PubMed]
66.
Nunez, N.G.; Tosello Boa i, J.; Ramos, R.N.; Riche , W.; Cagna d, N.; Ande uh en, C.D.; Nibo ski, L.L.; Bigo , J.; Meseu e, D.; De
La Roche e, P.; e al. Tumo in asion in d aining lymph nodes is associa ed wi h T eg accumula ion in b eas cance pa ien s. Na .
Commun. 2020,11, 3272. [C ossRe ]
67.
Tekguc, M.; Wing, J.B.; Osaki, M.; Long, J.; Sakaguchi, S. T eg-exp essed CTLA-4 deple es CD80/CD86 by ogocy osis, eleasing
ee PD-L1 on an igen-p esen ing cells. P oc. Na l. Acad. Sci. USA 2021,118, e2023739118. [C ossRe ]
68.
Cai, J.; Wang, D.; Zhang, G.; Guo, X. The Role O PD-1/PD-L1 Axis In T eg De elopmen And Func ion: Implica ions Fo Cance
Immuno he apy. OncoTa ge s The . 2019,12, 8437–8445. [C ossRe ]
69.
Sa as, P.; Salgado, R.; Denke , C.; So i iou, C.; Da cy, P.K.; Smy h, M.J.; Loi, S. Clinical ele ance o hos immuni y in b eas
cance : F om TILs o he clinic. Na . Re . Clin. Oncol. 2016,13, 228–241. [C ossRe ]
70.
Papaioannou, E.; Sakellakis, M.; Melach inou, M.; Tzo acole he akis, E.; Kalo onos, H.; Kou ea, E. A S anda dized E alua ion
Me hod o FOXP3+ T egs and CD8+ T-cells in B eas Ca cinoma: Associa ion wi h B eas Ca cinoma Sub ypes, S age and
P ognosis. An icance Res. 2019,39, 1217–1232. [C ossRe ]
71.
S ens öm, J.; Heden alk, I.; Hage ling, C. Regula o y T lymphocy e in il a ion in me as a ic b eas cance -an independen
p ognos ic ac o ha changes wi h umo p og ession. B eas Cance Res. 2021,23, 27. [C ossRe ] [PubMed]
72.
Tian, W.; Wang, L.; Yuan, L.; Duan, W.; Zhao, W.; Wang, S.; Zhang, Q. A p ognos ic isk model o pa ien s wi h iple nega i e
b eas cance based on s omal na u al kille cells, umo -associa ed mac ophages and g ow h-a es speci ic p o ein 6. Cance Sci.
2016,107, 882–889. [C ossRe ]
73.
Wang, S.; Wang, J.; Chen, Z.; Luo, J.; Guo, W.; Sun, L.; Lin, L. Ta ge ing M2-like umo -associa ed mac ophages is a po en ial
he apeu ic app oach o o e come an i umo d ug esis ance. npj P ecis. Oncol. 2024,8, 31. [C ossRe ]
74.
Huang, X.; Cao, J.; Zu, X. Tumo -associa ed mac ophages: An impo an playe in b eas cance p og ession. Tho ac Cance 2022,
13, 269–276, E a um in Tho ac Cance 2022,13, 3437. [C ossRe ]
75.
Ma, H.; Gao, L.; Chang, R.; Zhai, L.; Zhao, Y. C oss alk be ween mac ophages and immunome abolism and hei po en ial oles in
issue epai and egene a ion. Heliyon 2024,10, e38018. [C ossRe ]
76.
Holbe , C.E.; Case o, R.A., J .; S ewa , T.M. Polyamines: The pi o al amines in in luencing he umo mic oen i onmen . Disco .
Oncol. 2024,15, 173. [C ossRe ]
77.
de Boni ace, J.; Mao, Y.; Schmid -Mende, J.; Kiessling, R.; Poschke, I. Exp ession pa e ns o he immunomodula o y enzyme
a ginase 1 in blood, lymph nodes and umo issue o ea ly-s age b eas cance pa ien s. Oncoimmunology 2012,1, 1305–1312.
[C ossRe ]
78.
Linde, N.; Casano a-Acebes, M.; Sosa, M.S.; Mo ha, A.; Rahman, A.; Fa ias, E.; Ha pe , K.; Ta dio, E.; Reyes To es, I.; Jones, J.;
e al. Mac ophages o ches a e b eas cance ea ly dissemina ion and me as asis. Na . Commun. 2018,9, 21. [C ossRe ]
79.
Zhang, W.J.; Wang, X.H.; Gao, S.T.; Chen, C.; Xu, X.Y.; Sun, Q.; Zhou, Z.H.; Wu, G.Z.; Yu, Q.; Xu, G.; e al. Tumo -associa ed
mac ophages co ela e wi h phenomenon o epi helial-mesenchymal ansi ion and con ibu e o poo p ognosis in iple-nega i e
b eas cance pa ien s. J. Su g. Res. 2018,222, 93–101. [C ossRe ] [PubMed]
80.
G o h, C.; Hu, X.; Wee , R.; Fleming, V.; Al e og , P.; U ikal, J.; Umansky, V. Immunosupp ession media ed by myeloid-de i ed
supp esso cells (MDSCs) du ing umou p og ession. B . J. Cance 2019,120, 16–25. [C ossRe ] [PubMed]
