cells
Re iew
B inging Mac ophages o he F on line agains Cance : Cu en
Immuno he apies Ta ge ing Mac ophages
Ma iana Reis-Sob ei o , A onso Teixei a da Mo a, Ca olina Ja dim and Ka ine Se e *


Ci a ion: Reis-Sob ei o, M.; Teixei a
da Mo a, A.; Ja dim, C.; Se e, K.
B inging Mac ophages o he
F on line agains Cance : Cu en
Immuno he apies Ta ge ing
Mac ophages. Cells 2021,10, 2364.
h ps://doi.o g/10.3390/
cells10092364
Academic Edi o : Ma ia
Vincenza Ca ie o
Recei ed: 30 June 2021
Accep ed: 29 Augus 2021
Published: 9 Sep embe 2021
Publishe ’s No e: MDPI s ays neu al
wi h ega d o ju isdic ional claims in
published maps and ins i u ional a il-
ia ions.
Copy igh : © 2021 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
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/).
Ins i u o de Medicina Molecula João Lobo An unes, Faculdade de Medicina da Uni e sidade de Lisboa, 1649-028
Lisboa, Po ugal; ma iana.sob ei [email p o ec ed] (M.R.-S.); [email p o ec ed] (A.T.d.M.);
[email p o ec ed] (C.J.)
*Co espondence: ka inese [email p o ec ed]
Abs ac :
Mac ophages a e ound in all issues and display ou s anding unc ional di e si y. F om
emb yo o bi h and h oughou adul li e, hey play c i ical oles in de elopmen , homeos asis,
issue epai , immuni y, and, impo an ly, in he con ol o cance g ow h. In his e iew, we will
b ie ly de ail he mul i- unc ional, p o umo al, and an i umo al oles o mac ophages in he umo
mic oen i onmen . Ou objec i e is o ocus on he e e -g owing he apeu ic oppo uni ies, wi h
p omising p eclinical and clinical esul s de eloped in ecen yea s, o modula e he con ibu ion
o mac ophages in oncologic diseases. While he majo i y o cance immuno he apies a ge T cells,
we belie e ha mac ophages ha e a p omising he apeu ic po en ial as umo icidal e ec o s and in
mobilizing hei su oundings owa ds an i umo immuni y o e icien ly limi cance p og ession.
Keywo ds: mac ophages; myeloid- a ge ed he apies; ep og amming; an i umo unc ions
1. In oduc ion
Rep esen ing indispensable componen s o he inna e immune sys em, mac ophages
possess ema kable s a egic ana omical, and unc ional di e si ies [
1
,
2
], pe o ming a
ple ho a o ac i i ies o p o ec issues when homeos asis is dis up ed [
3
,
4
]. Upon an
insul , mac ophages apidly e oke an immunological esponse o link inna e and adap i e
immune cells, elimina e he dange , and es o e s able condi ions [
5
,
6
]. Tumo s a e seen as a
issue wi h de egula ed ea u es [
7
] composed o issue- esiden cells and a la ge p opo ion
o ec ui ed immune cells. S ikingly, mac ophages a e one o he mos ep esen ed immune
popula ions in cance issues [
8
,
9
], and umo -in il a ing mac ophages (TAMs) hus a ac
a lo o a en ion om immuno-oncologis s o unde s and hei physiological oles in
umo biology.
S udies conduc ed in he 1970s and 1980s demons a ed ha mac ophages ac i a ed
in i o
wi h cy okines (such as IFN-
γ
) and bac e ial-de i ed p oduc s (such as lipopolysac-
cha ide (LPS)) acqui ed umo -cell killing capaci ies [
10
–
13
]. This ini ially sugges ed ha
mac ophages could display umo icidal ea u es in he umo bed and p omo e an i umo
immuni y. In line wi h his, s udies in o a ian, HER2+ b eas cance , and colo ec al cance
pa ien s u he e ealed ha mac ophages associa e wi h be e he apeu ic esponses and
inc eased o e all su i al [
14
–
16
]. Howe e , he as majo i y o expe imen al da a and
clinical epo s indica e ha , in es ablished cance s, mac ophages mainly display p o u-
mo al unc ions, anging om di ec in e ac ions wi h umo cells [
17
] o indi ec shaping
o a umo - acili a ing s oma [
18
,
19
]. Mo eo e , hey can supp ess local endogenous
an i umo immune esponses [
20
–
22
] and limi he e icacy o con en ional and immune-
modula ing he apies [23–25]. Thus, i is no su p ising ha high densi y o mac ophages
in he umo bed is associa ed wi h poo clinical ou come [14].
O e all, h ough a ious manipula ing s a egies (deple ing, inhibi ing ec ui men
o unc ion, o e-educa ing), hese mac ophages ep esen a ac i e he apeu ic a ge s
as pa o combina o ial app oaches in cance ea men . We belie e ha immuno he apy
Cells 2021,10, 2364. h ps://doi.o g/10.3390/cells10092364 h ps://www.mdpi.com/jou nal/cells
Cells 2021,10, 2364 2 o 35
a ge ing mac ophages has he po en ial o induce unc ional e ec o s ha can ac i ely
boos e e y s ep o he cance -immuni y cycle [
26
,
27
] (Figu e 1). Indeed, h ough hei
an i umo unc ions, mac ophages ha e he po en ial o accele a e he cance -immuni y
cycle o unleash a po en an icance immune esponse. In his e iew, we p o ide a de ailed
o e iew o he a ailable he apeu ic s a egies ha a ge mac ophages, especially hei
dele ion, he p e en ion o hei ec ui men in o he umo , he inhibi ion o hei immuno-
supp essi e and umo -p omo ing unc ions, and he eac i a ion o hei an i umo al
ac i i ies o imp o e cu en ea men s.
Figu e 1.
Mac ophages can ac as key e ec o s in he cance -immuni y cycle. (1) TAMs can be ep og ammed o ini ia e he
cycle by killing umo cells h ough p oduc ion o ROS/NO o in a con ac -dependen manne . Cy o oxic TAMs would
induce he elease o umo (neo-)an igens. (2–3) Phagocy osis o dead cance cells by TAMs and ans e o cance -associa ed
an igens o dend i ic cells (DC), in he TME, o a e mig a ion in he d aining lymph nodes (LN). Subcapsula CD169
+
mac ophages in LN ha e also been epo ed o ans e cance -associa ed an igens o DC. This leads o p o icien ac i a ion
o umo -speci ic cy oly ic CD8 T cells. (4) TAMs can sec e e CXCL9, CXCL10, CXCL11, he ligands o CXCR3, and
ac i ely ec ui umo -speci ic CD8 T cells om he ci cula ion. (5) TAMs can p oduce selec i e enzymes ha loosen up
he ex acellula ma ix, limi ib osis, which in u n acili a es T cell in il a ion deep wi hin he umo mass. (6) TAMs
can po en ia e he cy o oxic unc ions o NK and CD8 T cells. Al oge he , his e isi ed model p o ides he a ionale o
a ge ing mac ophages, which, by boos ing di e en s eps o he cance -immuni y cycle, will accele a e he gene a ion o an
an icance esponse. This igu e is adap ed om [26,27].
Cells 2021,10, 2364 3 o 35
2. The Mul i-Func ional Roles o Mac ophages in he Tumo Mic oen i onmen
2.1. M1/M2 Mac ophage Pola iza ions: Classical e sus Al e na i e
M1 (classically ac i a ed) mac ophages di e en ia e in esponse o IFN-
γ
and LPS,
whe eas M2 (al e na i ely ac i a ed) mac ophages a e induced by IL-4 and IL-13. M1
mac ophages exp ess high le els o in lamma o y cy okines as well as inducible ni ic oxide
syn hase (iNOS) [
28
,
29
] and pa icipa e in he elimina ion o pa hogens and malignan
cells. M2 mac ophages exp ess a hemoglobin-hap oglobin sca enge ecep o cys ein-
ich (CD163), he mac ophage-sca enge ecep o -1 (CD204), he C- ype lec in mannose
ecep o (CD206), he mac ophage ecep o wi h collagenous s uc u e (MARCO), a ginase
1 (ARG1), and IL-10 [30], and display issue epai and umo -p omo ing ea u es [31].
Fea u es o he M1/M2 pola iza ion ha e been associa ed wi h di e en cance s ages
and ha e p ognos ic alue. Fo ins ance, in gas ic cance pa ien s, he median alue o
CD68
+
NOS2+ (M1)/CD68
+
CD163
+
(M2) a io was ound o be a posi i e independen
p edic o o su i al [
32
]. High M1/M2 a ios in o a ian umo issues a e co ela ed
wi h ex ended su i al [
33
]. Simila ly, in high-g ade se ous papilla y o a ian cance s, a
p e alence o M1 TAMs and a highe M1/M2 a io was posi i ely associa ed wi h longe
p og ession- ee and o e all su i al [
16
]. In addi ion, HLA-DR+CD68
+
M1-like TAM
le els signi ican ly dec eased du ing cance p og ession, om pa hological s age I o III in
non-small-cell lung ca cinoma (NSCLC) [
34
]. In line wi h his, in o a ian cance , a high
CD206
+
CD68
+
exp ession is associa ed wi h high isk o disease p og ession [
35
] and high
densi y o CD163
+
M2-like mac ophages is associa ed wi h poo p ognosis [
36
]. In NSCLC,
ea ly in umo o ma ion, issue- esiden mac ophages displaying he M2-like ea u es,
CD206 and MARCO, we e shown o p omo e umo cell epi helial–mesenchymal ansi ion
(EMT), in asi eness and o induce a T eg cell esponse ha limi s an icance adap i e
immuni y [37].
2.2. Mac ophages and Clinical Ou come
In mos umo s, mac ophages a e one he mos abundan immune cells which aid
umo de elopmen . Fo ins ance, ele a ed con en o CD68 TAMs in biopsies is linked
o un a o able ou comes in pa ien s wi h b eas , bladde , gas ic, panc ea ic, and head
and neck squamous cell ca cinomas [
38
–
43
]. The de imen al p ognosis in hese pa ien s
can associa e a high numbe o TAMs wi h a ious aspec s o umo p og ession, such
as p ima y umo bu den, umo -in aded nea by lymph nodes, and me as asis. This
is consis en wi h he ac ha mac ophages can exhibi p o umo unc ions, as will be
de ailed in Sec ion 2.4.
Howe e , in some ci cums ances, he p ognos ic impac o TAMs can also be associa ed
wi h pa ien su i al, such as in NSCLC, p os a e, and colo ec al ca cinoma [
14
,
44
–
46
],
and me as asis supp ession in os eosa coma [
47
]. TAM localiza ion wi hin he umo may
be an impo an c i e ion, as in lung cance i was epo ed ha ele a ed TAMs in he
umo isle (as opposed o umo s oma) we e associa ed wi h be e o e all su i al a 3
and 5 yea s [
48
]. In gas ic cance , TAM agg ega ion wi hin he cance cell nes p o ided
a bene icial e ec in e ms o umo cell apop osis and accumula ion o CD8 T cells [
49
].
Thus, e idence exis s ha , in ce ain ypes o cance s, mac ophages can display po en
e ec o unc ions capable o inhibi ing umo g ow h, as will be de ailed in Sec ion 2.5.
Finally, mac ophages can play selec i e oles in esponse o ea men s. In mos cases,
hey a e epo ed o p omo e esis ance o he apy. Fo ins ance, in ea ed mamma y
ca cinomas, mac ophages display immunosupp essi e unc ions ha hinde adap i e
an icance immuni y [
23
,
50
]. Mo eo e , mac ophages can sec e e speci ic lipids and en-
zymes (such as lysophospholipids, ca hepsin p o eases, and cy idine deaminase) capable o
in e e ing wi h chemo he apy [
51
–
53
]. In sha p con as , high TAMs we e independen ly
associa ed wi h be e disease- ee su i al in 5- luo ou acil- ea ed s age III colo ec al
cance pa ien s [
54
]. In human panc ea ic duc al adenoca cinoma PDAC, high densi y
o TAMs a he umo –s oma in e ace, posi i ely dic a ed p ognos ic esponsi eness o
pos su gical adju an chemo he apy, independen ly o T cell densi y [
40
]. In addi ion,
Cells 2021,10, 2364 4 o 35
mac ophages syne gize wi h an i-CTLA-4 immuno he apy [
55
] as well as ac i ely pa -
icipa e in umo cell clea ance wi h umo -speci ic mAb [
56
]. Al oge he , hese esul s
sugges ha e alua ion o , no only he p esence, bu mos impo an ly he pheno ype,
unc ions, and in a- umo dis ibu ion o mac ophages, in un ea ed and ea ed pa ien s,
will p o ide clea e p ognos ic and p edic i e alues, as well as in o ma ion abou how o
manipula e mac ophages in cance .
2.3. Mac ophage Di e si y in Cance , Re ealed by Single Cell RNA Sequencing
I became inc easingly clea ecen ly ha he simple M1-M2 ca ego iza ion [
57
] ails
o po ay he ex en o
in i o
he e ogenei y o TAMs. The ad en o single cell RNA
sequencing (scRNAseq) applied o umo -in il a ing immune cells has e ealed ha he
di e si y o monocy e/mac ophage subse s in he umo mic oen i onmen (TME) does
no compo wi h he pola iza ion model, ei he as disc e e s a es o along a spec um o
pola iza ion ajec o ies in b eas cance [58].
In ac , M1- and M2-associa ed genes we e equen ly co-exp essed in he same cell
and posi i ely co ela ed wi h one ano he along he same ac i a ion ajec o y. The
scRNAseq s udies in a ious cance ypes ha e al eady iden i ied o e 10 di e en mono-
cy e/mac ophage subse s [
37
,
58
–
61
]. Wi hin colo ec al cance , small p opo ions o blood-
de i ed monocy es, including CD14
+
classical, CD14
+
CD16
+
in e media e, and CD16
+
nonclassical subse s we e obse ed [
60
]. Fu he mac ophage subse s we e iden i ied based
on hei high exp ession o CD68, CD163, and MRC1 (encoding CD206), which could
be deno ed as esiden issue mac ophages and seg ega ed in o no mal colon epi helial
issue (NLRP3+ and phospholipid ans e p o ein PLTP+) o umo issue (IL1B+). These
subse s sha ed exp ession o p oin lamma o y genes such as IL1B, NLRP3, HLA-DR, bu
he PLTP+ subse also exp essed LYVE1 and IL10, po en ially holding a c i ical ole in
es aining in lamma ion and ib osis. In addi ion, wo dis inc TAM subse s show in lam-
ma o y/phagocy ic (C1QC
+
, TREM2, MERTK, and CD80) and p o-angiogenic/ umo igenic
(SPP1+, VEGFA, and MARCO) signa u es, espec i ely [
60
]. Deciphe ing he ansc ip-
ional ajec o ies o hese subse s e ealed ha while IL1B+ TAM could gi e ise o C1QC+
TAMs, on he o he hand NLRP3+ may gi e ise o SPP1+ TAMs. An i-CD115 ea men
a ge ing mac ophages was shown o p e e en ially deple e he SPP1
+
TAMs spa ing he
C1QC
+
TAMs and o p omo e umo eg ession [
60
]. A sys ema ic analysis con i med he
exis ence o hese TAM subse s ac oss 15 human cance ypes [
61
]. Su p isingly, he TAM
subse displaying highe p o-angiogenic unc ions (VEGFA, SPP1, MARCO) exhibi ed he
highe di e si ica ion o ma ke s ac oss di e en cance ypes. Mo eo e , he composi ion
o TAMs appea ed o be associa ed wi h ce ain ea u es o cance soma ic mu a ions
and gene exp essions [
61
]. Ano he s udy combined in acellula FACS s aining agains
A g1 wi h scRNAseq and iden i ied a unique T em2+CX3CR1+ TAM subse wi h po en
immunosupp essi e unc ions agains T cells [
62
]. In e es ingly, he au ho s also showed
ha gene ic abla ion o T em2 in mice dec eased immunosupp essi e TAMs and exhaus ed
CD8 T cells, in u nlimi ing umo g ow h.
A no el nomencla u e is s a ing o eme ge ha associa es TAMs wi h selec i e gene
exp ession p o iles and e ec o unc ions. A deep unde s anding o he ex en o he
pheno ypic and unc ional di e si ies o TAMs will be c i ical o de eloping e ec i e
myeloid- a ge ed immuno he apies.
2.4. Roles o Mac ophages in Tumo P og ession
2.4.1. Tumo Angiogenesis and Local Immunosupp ession
Tumo cells de elop nume ous s a egies o g ow, in ade, and me as asize, in pa
h ough he sec e ion o chemokines. Monocy es/mac ophages a e ac i ely ec ui ed by
umo cells ha p oduce chemokines such as CSF-1 [
63
], monocy e chemoa ac an p o ein-
1 (MCP-1/CCL2) [
64
], and CCL5 [
65
–
67
]. In u n, hese in il a ing mac ophages es ablish a
con inuous c oss alk wi h umo cells p oducing umo -su i ing ac o s such as epi helial
g ow h ac o (EGF) [
68
,
69
], hymic s omal lymphopoie in (TSLP) [
70
], and ans o ming
Cells 2021,10, 2364 5 o 35
g ow h ac o be a (TGF-
β
) [
71
] ha leads o cance cell p oli e a ion. In line wi h his,
we p e iously ound ha uncon en ional small pe i oneal mac ophages p oduce soluble
uniden i ied p o umo media o s uniquely and di ec ly capable o p omo ing o a ian
cance cell p oli e a ion [72].
Mac ophages a e also ac i ely in ol ed in emodeling he TME a chi ec u e, display-
ing p o-angiogenic p og ams o p o ide oxygen and nu ien s o he g owing neoplas ic
issue [
73
]. Tumo angiogenesis does no esul only om he in e ac ion o cance cells
wi h endo helial cells, as TAMs also ha e a c i ical ole in igge ing he neo o ma ion
o blood essels. They exp ess angiogenic ac o s such as ascula endo helial g ow h
ac o (VEGF) [
74
,
75
], placen al g ow h ac o (PGF), and pla ele -de i ed g ow h ac o
(PDGF) [
76
], in pa icula in esponse o he hypoxia p esen in a ascula and pe i-nec o ic
a eas [
77
]. TIE2-exp essing TAMs sense endo helial cell-p oduced angiopoie in 2 (ANG2),
align alongside umo ascula u e, and a e c i ical o de no o angiogenesis and cance
cell dissemina ion [78,79].
Mac ophages a e ins umen al in he p og ession o he umo by supp essing he an-
i umo ac ion o o he immune cells. This is mainly achie ed by he p oduc ion o a ious
immunosupp essi e cy okines. IL-10 p oduced by mac ophages can ac in an au oc ine
and local manne o inhibi IL-12 p oduc ion [
80
]. The lack o IL-12 limi s mac ophage
abili y o phagocy ose umo cells and o p oduce p o-in lamma o y ni ic oxide (NO),
skewing mac ophages in o immunosupp essi e pheno ypes [
81
,
82
]. On neighbo cells,
IL-10 also hampe s he ma u a ion o dend i ic cells (DCs) [
50
,
83
], which a e indispensable
o ac i e umo immuni y. TGF-
β
, also p oduced by TAMs, p e en s CD8 T cell-media ed
an i umo esponses [
84
]. Mechanisms o TGF-
β
immunosupp ession include: inhibi ion
o CXCR3 in CD8 T cells, he eby limi ing hei a icking in o umo s [
85
], induc ion o
CCL22 ha con ols he ec ui men o T eg cells o he TME [
86
], and p omo ing su i al
and immunosupp essi e pheno ype o monocy es [87].
Low a ailabili y o nu ien s in he TME is an impo an hu dle o e icien T cell e -
ec o unc ions. Tumo cells and mac ophages can exp ess selec i e enzymes ha deg ade
amino acids, such as indoleamine 2,3-dioxygenase (IDO) [
88
,
89
] and a ginase (A g) [
90
,
91
],
which dep i e he mic oen i onmen om yp ophan and a ginine, espec i ely, and
es ain he equi ed immuni y o igh and elimina e cance .
The immune checkpoin p og ammed cell dea h-1 (PD-1), a co-inhibi o y ecep o
c i ical o main ain sel - ole ance, is mainly exp essed on T cells and nega i ely egula es
pe iphe al T cell esponses. Tumo cells equen ly o e exp ess p og ammed cell dea h
ligand 1 (PD-L1) o escape om he immune sys em. Impo an ly, TAMs a e also a c i ical
sou ce o PD-L1 o supp ess CD8 T cell unc ion agains he umo [
92
]. Ta ge ing he
PD-1/PD-L1 pa hway wi h blocking an ibodies has e olu ionized he ea men o a wide
a ie y o malignancies, leading o du able he apeu ic esponses no ypically achie ed
wi h adi ional cy o oxic an icance agen s.
2.4.2. Mac ophages and Me as asis
A he p ima y cance si e, dissemina ion o umo cells is o en p eceded by he
acquisi ion o mesenchymal ea u es by epi helial cance cells, a p ocess known as epi helial–
mesenchymal ansi ion (EMT). A e in a asa ion in o he bloods eam and ex a asa ion
o he me as a ic niche, cance cells unde go he in e ed p ocess, called mesenchymal- o-
epi helial ansi ion (MET) o es ablish and h i e in he new en i onmen . Rema kably,
TAMs coo dina e he sequence o e en s h oughou his me as a ic dissemina ion p ocess.
In he p ima y si e, TAM-sec e ed TGF-
β
can induce EMT and guide cance cells o lose
adhesion, becoming pheno ypically mo e elonga ed and mo ile in a way ha acili a es
hei en ance in o he bloods eam [
93
,
94
]. Fu he mo e, TAM-de i ed CCL8 induces he
o ma ion o pseudopodia, which a e plasma memb ane p o usions, needed o inc ease
he mo ili y o cance cells. Real- ime imaging e ealed ha local and ansien ascula
pe meabili y and umo cell in a asa ion a e s imula ed by Tie2+ mac ophage-de i ed
VEGFA [95].

Cells 2021,10, 2364 6 o 35
Pionee wo k om Polla d and colleagues showed, using he PyMT ansgenic mouse
model suscep ible o mamma y cance , ha gene ic educ ion o mac ophages d as ically
delayed he p og ession o in asi e and me as a ic ca cinomas wi hou a ec ing he in-
cidence o he g ow h o p ima y umo s [
96
]. These indings led o he de ini ion o
me as asis-associa ed mac ophages (MAMs) sui ed o p omo e b eas cance dissemina-
ion [
97
]. The au ho s u he demons a ed ha he MAMs exp essed CD11b+F4/80+CSF1-
R+CD11cdimCX3CR1highCCR2highVEGFR1high [
97
] as well as CD204+IL4R+ [
98
], and
ha hey o igina ed om in lamma o y Ly6C+ monocy es ec ui ed by CCL2 [
98
–
100
].
Mechanis ically, MAMs a e c i ical o he eng a men and g ow h o b eas cance clones
h ough hei capaci y o p oduce hepa ocy e g ow h ac o [
101
], and o inhibi he an i-
me as a ic unc ions o NK cells ia memb ane-bound TGF-βexp ession [102]. Mo eo e ,
in lamma ion-induced EMT up egula es IL12R
β
2, a subuni o he IL-35 ecep o , in can-
ce cells o help hem espond o IL-35 du ing me as asis [
103
]. Then, a he me as a ic
si e, mac ophages sec e e IL-35 o acili a e me as a ic coloniza ion h ough ac i a ion o
JAK2-STAT6-GATA3 signaling, which induces MET in cance cells.
I is es ima ed ha 90% o cance - ela ed dea hs a e due o me as iza ion [
104
]. Thus,
and conside ing he implica ion o mac ophages in he p ima y umo as well as in
me as a ic dissemina ion, immuno he apies a ge ing hese cells ep esen a c i ical oppo -
uni y o block he dis an seeding o malignan cells and imp o e he su i al o cance
pa ien s.
2.5. Roles o Mac ophages in Tumo Reg ession
Mos o he li e a u e has desc ibed TAMs ha suppo s umo p og ession, o e shad-
owing ha , in ac , mac ophages can limi umo g ow h. Some subse s o mac ophages
wi h a p o-in lamma o y pheno ype we e obse ed in ea ly s age o a ian and colo ec-
al cance s and associa ed wi h good p ognosis [
105
,
106
]. I is likely ha TAMs display
p o-in lamma o y signa u es a an ea ly s age o umo g ow h bu hen acqui e umo -
p omo ing ea u es du ing malignancy p og ession [
107
]. In he nex sec ions, we aim o
desc ibe known an i umo unc ions o TAMs (Figu e 2A).
Figu e 2. Con .
Cells 2021,10, 2364 7 o 35
Figu e 2.
(
A
) Desc ibes he main known an i umo unc ions o mac ophages in pa icula umo
cell killing and phagocy osis as well as ec ui men and ac i a ion o immune cells in he TME.
(
B
) P esen s he cu en he apeu ic s a egies ha a ge TAMs o induce an icance esponses. On
he op le side a e di e en app oaches o kill mac ophages o inhibi hei ec ui men in umo s.
On he bo om le side a e app oaches o inhibi TAM p o umo unc ions. On he igh side a e
s a egies o e-educa e TAMs in o an i umo e ec o s.
2.5.1. P ope ies o Mac ophages o Kill and Phagocy ose Tumo Cells
Mac ophages can kill umo cells h ough he ecogni ion o speci ic cell su ace ma k-
e s and p oduc ion o umo icidal molecules (Figu e 2A Le ). They a e capable o clea ing
apop o ic and iable umo cells. Apop o ic cells unde go a ious changes such as he edis-
ibu ion o phospha idylse ine and cal e iculin o he plasma memb ane. The ecogni ion
o phospha idylse ine o he clea ance o apop o ic cells (a p ocess e med “e e ocy osis”)
igge s immunosupp ession and he con e sion o TAMs in o an i-in lamma o y e ec-
o s [
108
,
109
]. In con as , up ake h ough cal e iculin igge s an immunogenic esponse
agains apop o ic cells [
110
,
111
]. Fu he mo e, apop o ic cells ac i a e he complemen
and a e opsonized wi h iC3b, leading o ecogni ion and up ake by mac ophages [
112
].
Mac ophages can also phagocy ose li e cance cells, bu he la e ha e e ol ed mechanisms
o escape immune phagocy ic ecogni ion and o e exp ess “don’ -ea -me” signals. CD47
is a ma ke o “sel ” exp essed on no mal cells, ha , when binding o signal egula o y
p o ein alpha (SIRP
α
, CD172a) on he su ace o immune cells hinde s phagocy osis [
113
].
The con ibu ion o TAMs, in umo cell elimina ion, was e ealed in mouse models o
umo cells ha became insensi i e o cy o oxic CD8 T cells [
114
]. In line wi h his, he
success o mAbs a ge ing umo su ace an igen, which ep esen s a powe ul s a egy o
he ea men o se e al ypes o cance , elies on he Ab-dependen cellula cy o oxici y
(ADCC) exe ed by mac ophages, which exp ess ac i a ing FcR. An elegan s udy, using in-
a i al imaging in a model o B cell lymphoma, demons a ed he key ole o mac ophages
in umo cell elimina ion in esponse o an i-CD20 i uximab in he bone ma ow [
115
]. Fas
ligand (FasL), a cell su ace molecule belonging o he umo nec osis ac o amily, binds
o i s ecep o Fas, media ing apop osis by caspase ac i a ion. In human colo ec al cance ,
mac ophages we e ound o be he main sou ce o FasL and hey associa ed wi h apop o ic
cance cells along he in asi e ma gin [
116
]. Howe e , he exp ession o FasL by TAMs
has been mainly associa ed wi h immunosupp essi e oles. FasL exp ession by TAMs was
Cells 2021,10, 2364 8 o 35
ound o se e as a ba ie agains he in il a ion o CD8 T cells [
117
]. In pa ien s wi h
li e me as asis, FasL+CD11b+F4/80+ mac ophages could di ec ly elimina e Fas+CD8 T
cells [118].
P o-in lamma o y mac ophages acqui e he capaci y o exp ess iNOS and p oduce
he ee adical NO. Di usion o NO in hei p oximi y esul in umo cell dea h [
119
,
120
].
In addi ion, mac ophage-de i ed NO was ound o induce adhesion molecules on umo
essels a o ing T cell ex a asa ion and umo ejec ion [
121
]. Howe e , NO p oduced
by TAMs has also been linked o esis ance o he apy [
122
] and o supp ession o T cell
unc ions [
123
]. This sugges s a bimodal dose-dependen e ec , wi h NO a mode a ed
concen a ion may display umo cell killing p ope ies, while a high concen a ion in he
TME, NO migh be p opaga ing umo -p omo ing e ec s. Finally, TNF- ela ed apop osis-
inducing ligand (TRAIL) is a cy okine ha can e-educa e TAMs o a M1-like pheno ype
and induce apop osis o umo cells h ough he binding o dea h ecep o s DR4 and
DR5 [124–126].
2.5.2. Mac ophages Ac i a e CD8 T Cells and NK Cells
Mac ophages a e cen al o shaping a umo -limi ing o umo -p omo ing TME due o
hei capaci y o ec ui and/o in e ac wi h a ious immune compa men s (
Figu e 2A
Righ ). Fo ins ance, TAMs p oduce he ligands o CXCR3 (mainly CXCL9 and CXCL10),
which we e ound o associa e wi h high le els o in il a ing T cells in human solid
cance s [
127
,
128
]. Impo an ly, he exp ession o CXCL9 by TAMs p omo es he ec ui men
o CXCR3-exp essing CD8 T cells ha a e c i ical o he esponse o immune checkpoin
blocke (ICB an i-PD-1/an i-CTLA-4 o an i-PDL-1) ea men s [129,130].
Mac ophages also p oduce cy okines ha pa icipa e in CD8 T cell ac i a ion. In e leukin-
12 (IL-12) is a key cy okine ha ac s on mac ophages hemsel es, inducing a p o-in lamma o y
pheno ype cha ac e ized by TNF-
α
, IL-15, and IL-18 p oduc ion in he TME [
131
]. Fu -
he mo e, IL-12-s imula ed mac ophages a e umo icidal in a cell–cell con ac -dependen
manne , leading o a T cell-dependen e adica ion o es ablished umo s [
132
]. En o ced
ac i a ion o No ch signaling also inc eased IL-12-p oducing an i umo mac ophages o
ul ima ely limi umo g ow h [
133
]. IL-12 p oduced by TAM locally enhances he in lam-
ma o y Th1 esponse, which in u n gene a es la ge amoun s o IFN-
γ
and ac i a es NK
and CD8 T cells ha exe an i umo e ec s. Then, and as de ailed abo e, IFN-
γ
p opa-
ga es mac ophage ac i a ion. TNF-
α
is p oduced by ac i a ed an i umo mac ophages.
K a och ill e al. desc ibed ha he loss o he TNF-
α
ecep o in mac ophages esul s in
he exp ession o genes ela ed wi h p o umo al unc ions. In his model, he pola iza ion
s a us o mac ophages was dynamic and dependen on he balanced le els o TNF-
α
and
IL-13 ( ha induces M2-like mac ophages) [
134
]. Ac i a ed mac ophages can also os e
he an i umo po en ial o NK cells. We ound ha pa olling monocy es making IL-15
ac i a e NK cells and IFN-
γ
p oduc ion, ha hen inhibi lung me as ases [
135
]. In addi ion,
inc eased IL-15, IL-18, and ype I IFN sec e ion induced NK cell-media ed cy o oxici y
agains umo cells in an NKG2D-dependen manne [136,137].
Mac ophages ha e been sugges ed o be as e icien as DCs a p esen ing umo an i-
gens o T cells in he TME [
138
], in pa icula a e being ac i a ed wi h TLR agonis s [
139
].
In e es ingly, he in a umo al injec ion o apop o ic umo cells wi h IL-2 led o an 80%
a e o cu e in mice models, con i ming ha he APC in he TME e ain he in insic ca-
paci y o up ake, p esen , and gene a e a umo -speci ic cy o oxic T cell esponse [
140
].
Mac ophages a e also known o in e ac wi h o he immune cells in lymphoid o gans.
Subcapsula CD169
+
mac ophages in egional lymph nodes co ela ed wi h CD8 T cell
in il a ion in melanoma and b eas cance , which associa ed wi h be e p ognosis and
imp o ed su i al a es [
141
,
142
]. These CD169
+
mac ophages we e ound o phagocy ose
dead umo cells anspo ed ia lympha ic low and o c oss-p esen umo an igens o
CD8 T cells [
143
]. Mo eo e , a close collabo a ion be ween CD169
+
mac ophages and DCs
was also p oposed o he ini ia ion o e ec i e CD8 T cell esponses, in which mac ophages
ans e ed Ag o DC in a cell–cell con ac dependen manne [144].
Cells 2021,10, 2364 9 o 35
O e all, mac ophages a e highly poly unc ional in he TME and we a e con inced
ha his e sa ile mul i- asking ea u e is a c i ical p ope y ha poin s ou mac ophages as
key e ec o s wi h he apeu ic po en ials. Mul iple ac ions o mac ophages in he TME, and
in he egional lymph nodes, may posi i ely accele a e each o he consecu i e unc ional
s eps o he cance -immuni y cycle, including: (1) killing o cance cells, (2) cance cell
an igen ans e o DCs, (3) T cell ac i a ion, (4) ec ui men o ci cula ing T cells o he
umo bed, (5) acili a ing T cell in il a ion wi hin he umo and (6) p omo ing he killing
o umo cells by NK and CD8 T cells (Figu e 1).
3. Tumo The apies Ta ge ing Mac ophages
In ense e o s ha e been made in ending o manipula e TAMs, in pa icula s a egies
o deple e hem, o limi hei ec ui men o he umo si e, o o exploi hei plas ici y
o epola ize hem om immune supp essi e owa ds in lamma o y and umo icidal
pheno ypes. In he nex sec ions, we aim o desc ibe he ac ual he apeu ic op ions o
manipula e mac ophages (see Figu e 2B o a summa y and Table S1 o examples o
cu en in e en ional clinical ials a ge ing mac ophages).
3.1. Deple ion o Mac ophages
3.1.1. Bisphosphona es
Bisphosphona es, such as clod ona e and zoled ona e, which a e ex ensi ely used
o ea diseases associa ed wi h bone loss such as os eopo osis, a e also used o deple e
mac ophages [
145
]. The adminis a ion o clod ona e o zoled ona e o mul iple myeloma
o mamma y umo -bea ing mice leads o a educ ion in p o umo al TAMs and umo
ascula iza ion, consequen ly inc easing mice su i al [
146
,
147
]. In F9 e a oca cinoma
and A673 habdomyosa coma mouse models, combina ion o bisphosphona es wi h VEGF-
blocking an ibodies led o TAM deple ion and umo eg ession, al hough only sus ained
du ing he ime o he he apy [148].
3.1.2. Blocking o Su i al Signals
Cells o he monocy e-mac ophage lineage ely on he mac ophage-colony s imula ing
ac o (M-CSF), also known as colony s imula ing ac o -1 (CSF-1), a g ow h ac o essen ial
o hei su i al and hey exclusi ely exp ess he CSF1R (CD115). Gene ic dele ion o
Cs 1 o Cs 1 esul s in loss o monocy es and issue mac ophages, bu expe imen s in hese
mice a e di icul o in e p e because o addi ional se e e pleio opic e ec s, including
in e ili y, os eopo osis, neu onal de ec i e de elopmen , low body weigh , and se e e
skele al abno mali ies [149,150]. The he apeu ic capaci y o a CSF1 signaling blockade o
modula e mac ophage su i al was demons a ed using speci ic kinase inhibi o s ac ing
on CSF1R (GW2580, AMG820, and PLX3397) o blocking an i-CSF1R mAb (RG7155).
No ably, adminis a ion o a blocking an i-mouse CD115 an ibody o MMTV-PyMT mice
delayed umo mani es a ion and p olonged mice su i al, simul aneously po en ia ing
he an icance e ec o Pacli axel [
151
]. Aside om b eas cance , CSF1R blockade appea ed
o be su icien o enhance su i al in a ious cance mouse models [
152
–
155
]. In a mouse
model o colon cance , ea men wi h RG7155 esul ed in s ong educ ion in TAMs
accompanied by an inc ease in T cell in il a ion [
154
]. Mo eo e , GW2580 e e sed he
esis ance o panc ea ic umo cells o con en ional chemo he apy [
156
]. Combina ion
o CSFR1 inhibi o (PLX3397 o an i–CSF1R Ab) wi h an i-PD1 educed TAM numbe s,
enhanced CD8 T cell in il a ion, and consequen ly dec eased umo size [
157
,
158
]. In
human, CSF1R inhibi o s (AMG820 and PLX3397) induced a dec eased in CD14
dim
CD16
+
monocy es [
159
–
161
], bu his e ec was accompanied by an inc ease in plasma CSF1
and his ques ions he impac o ea men discon inua ion on monocy e numbe and
pheno ype.
Cells 2021,10, 2364 16 o 35
oxici y a e sys emic adminis a ion. Va ious s a egies aim a p o iding IFN-
γ
di ec ly o
TAMs o p omo e ac i a ion and an i umo unc ions, such as IFN-
γ
deli e ing nanopa -
icles [
266
,
267
]. Speci ically enginee ed pa icles, e e ed o as a “backpack”, we e also
shown o e ade phagocy osis and elease cy okines o con inuously guide he pola iza ion
o mac ophages owa d an i umo pheno ypes in si u [268].
TNF-
α
is ano he pleio opic cy okine ha induces he ac i a ion o mac ophages
o a umo icidal s a e [
269
]. In e es ingly, his cy okine is also p edominan ly p oduced
by mac ophages, al hough T and NK cells a e also la ge sou ces. Despi e ini ial s ud-
ies o TNF-
α
ea men o cance demons a ed bene i s in a signi ican pe cen age o
pa ien s wi h so issue sa coma [
270
] o un esec able li e me as ases om colo ec al
cance [
271
], he held p omises ailed o conc e ize as clinical he apeu ics. As o IFN-
γ
,
a limi a ion o ecombinan human TNF-
α
is high oxici y a e sys emic adminis a ion.
Va ious app oaches a ge ing he cy okine o he umo , such as colloidal gold-bound
TNF-
α
[
272
], gene ans e [
273
], ecombinan TNF-
α
used o an an ibody an i- ib onec in
o umo endo helium (L19-TNF) [
274
], ailed o each signi ican esul s in clinical ials.
Ne e heless, p og ess may come om oncoly ic adeno i us enginee ed o p oduce TNF-
α
(TILT-123) [275,276] (NCT04217473).
3.4.3. An ibodies: An i-CD40, An i-CSF1R, An i-PD-1, An i-MARCO
CD40 is a co-s imula o y molecule o he TNF- ecep o supe amily exp essed by APCs
ha es ablishes a c oss- alk in which CD40-ac i a ed mac ophages p esen ing an igen o
T cells p o oke up egula ion o CD40L. The CD40-CD40L in e ac ion hen induces in
mac ophages u he up egula ion o MHC molecules, CD80/CD86, and p o-in lamma o y
cy okines, such as IL-12. These signals p ime nai e CD4 T cells in o Th1 T cells and CD8 T
cells in o cy o oxic cells, he immune esponse a o able o umo clea ance.
S imula ion o mac ophages wi h enginee ed CD40L-exp essing mu ine lung cance
cells (3LLSA) enhanced hei cy o oxic e ec [
277
]. Then, agonis an i-CD40 mAb was
shown o s imula e he umo killing ac i i y o mac ophages [
278
] and o induce T cell-
independen an i umo e ec s ha in ol e mac ophages, in neu oblas oma [
279
]. The u-
mo icidal e ec was p oposed o in ol e he p oduc ion o IFN-
γ
, TNF-
α
, o
NO [279,280]
.
In e es ingly, CD40 liga ion leads o a posi i e eedback by inducing he up egula ion
o in acellula TLRs, esul ing in syne gis ic ac i a ion o bo h an i-CD40 and TLR (3, 7,
9) ligands in mac ophages in se e al umo mouse models [
280
,
281
]. CD40 agonis was
also shown o syne gize wi h chemo he apy o induce umo eg ession in a gene ically
enginee ed mouse model o panc ea ic cance [
282
]. Engagemen o CD40 pe mi ed o
o e come esis ance o an i-PD1 he apy h ough epola iza ion o mac ophages owa ds
an in lamma o y pheno ype, leading o s ong CD8 T cell ac i a ion in a in ahepa ic cholan-
gioca cinoma [
283
] gene ic mouse model o bladde [
284
] and panc ea ic cance [
285
]. In
b eas and me as a ic panc ea ic cance mouse models, only combining a T cell-inducing
accine wi h bo h PD-1 an agonis and CD40 agonis Abs was able o e adica e he majo i y
o umo s [
286
]. Logically, hese obse a ions ha e opened he way o he de elopmen o
clinically ele an an i-CD40 Abs (Table S1).
As p esen ed abo e, blocking CSF1-R deple es mac ophages. Su p isingly, mac ophage
disappea ance is no immedia e and CSF-1R inhibi ion (wi h BLZ945 o blocking Abs)
induces a sho - e m ewi ing o TAM unc ionali y ha p omo es hei an i umo unc ions
in he glioma mic oen i onmen [
153
]. Mechanis ically, aside om su i al, CSF1 p omo es
mac ophage pola iza ion owa d a p o umo al s a e [
287
] whe eas, in u n, CSF1 wi h-
d awal unleashes an an i umo po en ial be o e hei dea h. This esul has led o he idea
o combining a p oin lamma o y s imulus, like a CD40 agonis , wi h CSF-1R blockade. This
dual mac ophage- a ge ing combina ion p omo ed an i umo TAMs and ein igo a ed an
e ec i e T cell esponse by inc easing he p oduc ion o IFN-
γ
and TNF-
α
[
288
–
290
]. The
wo humanized mAb di ec ed agains CSF-1R, emac uzumab and AMG820, showed an ac-
cep able sa e y p o ile bu , un o una ely, only eached limi ed e icacy [
160
], ei he alone o

Cells 2021,10, 2364 17 o 35
in associa ion wi h selic elumab (an i-CD40) [
291
], pemb olizumab [
292
] o pacli axel [
293
],
in pa ien s wi h ad anced/me as a ic solid umo s.
Aside om unleashing cy o oxic T cell esponses, ano he consequence o an i-PD-1
he apy is he edi ec ion o mac ophages om p o umo al o an i umo al pheno ype,
inducing he eg ession o lung me as ases [
294
]. Su p ise came, howe e , when PD-1
was ound exp essed by mac ophages and o di ec ly egula e mac ophage phagocy ic
ac i i ies [
22
], as well as T cell-di ec ed immunosupp ession [
295
], al oge he p omo ing
an i umo immuni y [
296
]. E en mo e unexpec edly, myeloid-speci ic PD-1 abla ion was
as e ec i e as plain PD-1 knock-ou (Pdcd1
−
/
−
) and was conside ably mo e e ec i e
han T cell-speci ic PD-1 abla ion [
296
]. Impo an ly, ci cula ing monocy es om pa ien s
wi h hepa ocellula ca cinoma up egula ed PD-1 in a se e i y-dependen manne [
295
].
Mo eo e , upon LPS s imula ion, PD-1 posi i e monocy es p esen ed lowe iNOS and
highe a ginase 1 and IL-10 exp ession han PD-1 nega i e monocy es.
These ema kable esul s indica e ha PD-1 plays a unique ole in mac ophages.
The e o e, an agonis an i-PD-1 an ibody may be ac ing no only on T cells bu also
on mac ophages, bo h in he ci cula ion and in he TME. This knowledge has al eady
been ansla ed in o po en ial he apeu ic app oach h ough he de elopmen o selec-
i e mac ophage- a ge ed PD-1 inhibi ion s a egies. Taking ad an age o he in insic
phagocy ic p ope y o mac ophages, solid lipid nanopa icle-con aining PD-1 siRNA
o Salmonella ca ying PD-1 siRNA we e capable o down egula ing PD-1 exp ession
by TAMs and limi ing melanoma and colon cance p og ession [
297
–
299
]. These p oo -
o -concep expe imen s may open up new a enues o a ge , a will, PD-1 inhibi ion in
lymphocy es o myeloid cells.
MARCO is exclusi ely exp essed by mac ophages. MARCO+ TAMs display im-
munosupp essi e ea u es wi h high exp ession o he ypical an i-in lamma o y genes
a g1, izz1 [
300
]. Consis en ly, MARCO exp ession in human solid cance s co ela es wi h
poo p ognosis [
20
,
301
]. MARCO exp ession is induced by umo -de i ed supe na an ,
IL-10, hypoxic condi ions and IL-37 [
20
]. Howe e , while a ma ke o immunosupp essi e
mac ophages, MARCO engagemen led o he exp ession o he p o-in lamma o y genes
Tn ,Il1b, and Nos2, leading o educed p ima y umo g ow h and me as ases [300]. In e -
es ingly, a ge ing MARCO by mAbs led o NK cell ac i a ion, which in u n inc eased
hei TRAIL-dependen umo cell killing p ope y [137].
3.4.4. I adia ion
Radio he apy ha uses high doses o X- ay adia ion is one o he i s ea men s
di ec ed o physically damage DNA and induce apop osis o cance cells. This cance
ea men induces an immunogenic cell dea h ha elici s an an i umo immune esponse.
On one hand, adia ion induces he elease o umo -associa ed an igens in o he TME as
well as se e al endogenous TLR ligands (DAMPs); on he o he hand, i induces cy osolic
dsDNA accumula ion ha is sensed by he cGAS-STING pa hway in mac ophages [
302
].
Fu he mo e, adio he apy ec ui s monocy es and may impac mac ophage unc ions
in si u. Howe e , he ou come on TAMs may depend on he con ex and on he dose o
adio he apy.
In i o
adio he apy (2 Gy) p imed mac ophages owa ds an iNOS+ M1
pheno ype [
303
]. Human mac ophages a e esis an o ionizing adia ion doses (5*2 Gy)
and, aside om emaining iable and me abolically ac i e, hey adop a p o-in lamma o y-
like p o ile [
304
]. Fu he mo e, low dose o adia ion (0.5 Gy) p og ams he di e en ia ion
o iNOS+ mac ophages ha o ches a e he ec ui men o umo -speci ic T cells, media ing
umo ejec ion in panc ea ic ca cinoma o melanoma xeno ansplan mouse models [
305
].
In line wi h his, we showed ha , in he 4T1 o ho opic b eas umo mouse model, adio-
he apy syne gizes e icien ly wi h immunos imula o y nanopa icles o induce an i umo
immuni y [306].
By con as , human i adia ed (5*2 Gy) mac ophages also sus ain cance cell in asion
and angiogenesis [
304
]. This is also consis en wi h some p eclinical models suppo ing
a de imen al ole o mac ophages du ing adio he apy. Glioblas oma is usually ea ed
Cells 2021,10, 2364 18 o 35
wi h con en ional he apy consis ing o X- ay adio he apy associa ed wi h su ge y. I a-
dia ion (3*4 Gy) o glioma (GL261)-bea ing mice led o deple ion o o al CD68
+
cells bu
o an inc ease in he p opo ion o CD206
+
p o umo mac ophages. The au ho s showed,
using
in i o
bone ma ow-de i ed mac ophages, ha M0 and LPS/IFN-
γ
-s imula ed
mac ophages (an i umo al) a e mo e sensi i e o X- ay adia ion (2 Gy) han IL-4-s imula ed
mac ophages (p o umo al) mac ophages [307]. The p o umo e ec o mac ophages a e
adio he apy was u he demons a ed when, in he B16 melanoma mouse model, using
clod ona e-con aining liposomes o deple e mac ophages be o e adia ion ea men in-
c eased he an i umo e ec s o ionizing adia ion (20 Gy) [
308
]. Fu he wo k is wa an ed
o es ablish he bes mac ophage- a ge ing app oach, ei he s imula ion o deple ion, o
syne gize e icien ly wi h adio he apy in each ype o cance .
3.4.5. Gene ically Enginee ed Mac ophages
To complemen
in i o
p og aming o mac ophages, in ensi e esea ch ocuses on
gene ically enginee ed mac ophages (GEM). This adop i e mac ophage he apy would
hen access he umo si e o could be deli e ed di ec ly wi hin he umo . Al hough his
ea men may no pe sis o a long pe iod o ime, he an icipa ion is ha i should allow
du able local he apeu ic an i umo e icacy while minimizing oxici ies o unwan ed on-
a ge o - umo e ec s. While gene ically enginee ed lymphocy es, wi h TCR-enginee ed
o chime ic an igen ecep o (CAR) T cells, ake he s age in adop i e cell he apy, gene ic
manipula ion o myeloid cells clea ly lags behind. This is due o he di icul ies in ex-
panding and main aining mac ophages in cul u e o gene ic manipula ion. Howe e , he
gene a ion o mac ophages om p oli e a i e p ecu so s de i ed om induced plu ipo en
s em cells [
309
] o monocy es [
310
–
312
] o e a window o ansgene exp ession. Va ious
app oaches ha e been success ully used o modi y mac ophages, including ecombinan
adeno i al o e o i al ec o , len i i us-d i en enginee ing, and CRISPR-Cas9.
Gene ically enginee ed mac ophages may be ideally sui ed o h i e in he TME and
display an i umo unc ions h ough he sec e ion o p o-in lamma o y cy okines o cy o-
oxic bispeci ic T cell engage (BiTE). Mac ophages gene ically enginee ed o exp ess IL-12
e e sed he immunosupp essi e en i onmen de eloped du ing me as a ic p og ession
o glioblas oma by augmen ing T cell esponses and educing me as a ic bu den in p e-
clinical models [
311
]. Mac ophages enginee ed o sec e e a BiTE speci ic o he mu a ed
epide mal g ow h ac o a ian III exp essed by some glioblas oma cells educed ea ly
umo bu den in bo h subcu aneous and in ac anial mouse models [
312
]. Fu he mo e,
CRISPR-Cas9-d i en epigene ic silencing o Hi 1
α
was achie ed by dele ion o he his one
H3 me hylase EZH2, ha is ec ui ed o he Hi 1
α
p omo e egion speci ically. These
Hi 1
α
silenced mac ophages inhibi ed g ow h o he B16-F10 melanoma syngeneic model
a e in a umo al injec ion, h ough ep og amming he immune supp essi e TME o an
ac i e an i umo al mic oen i onmen . This app oach educed he numbe o T eg cells,
ec ui ed cy o oxic T cells, and p olonged he o e all su i al o mice [313].
CAR-exp essing mac ophages may be ac i a ed in an an igen-dependen manne in
he TME and display enhanced phagocy osis o umo cells. CAR-mac ophages di ec ed
o CD19 [
309
–
311
], HER2 [
310
], o meso helin [
309
] o a ge B cell leukemia o o a ian
cance , espec i ely, demons a ed e icacy in mouse models. Gene ic ep og amming o
mac ophages will undoub edly be an impo an a enue o he u u e molecula and cellula
medicine and an i-HER2 CAR-mac ophages a e al eady in clinical ials (NCT04660929).
3.4.6. In acellula Signaling, Epigene ic and Me abolic Manipula ions o Mac ophages
Al hough pha macological inhibi ion o PI3K in mac ophages syne gized wi h ICB
he apy o p omo e umo eg ession [
184
,
185
], o da e, he e is limi ed he apeu ic ap-
plica ion a ge ing in acellula signaling (such as kinases) o ep og am TAMs. In he
nea u u e, he de elopmen o single cell p o eomic p o iling should pe mi he p ecise
dissec ion o he in acellula signaling pa hways ha pa icipa e in he decision swi ch
om p o umo owa ds an i umo pheno ypes. Epigene ic manipula ion also ep esen s an
Cells 2021,10, 2364 19 o 35
a enue o in es iga ion o p og am mac ophages and his one ace yl deace ylase (HDAC)
inhibi o s, which cause changes in he ansc ip ional p o ile o he mac ophages, was
shown o limi umo g ow h [
314
]. Inc easing in e es eme ges o assess he e ec o die
on TAM unc ions. S ikingly, al e na e day as ing o 2 weeks inhibi ed ex acellula
adenosine accumula ion by supp essing he exp ession o CD73 on umo cells, which
in u n limi ed TAM p o umo al pola iza ion [
315
]. Immunome abolism is also becom-
ing a g owing a ea o esea ch gi en ha me abolism s ongly connec s o unc ionali y.
Me abolic ep og amming migh be a p o icien way o p omo e an an i umo pheno ype
in TAMs. Fo ins ance, me o min, a well-known an i-diabe ic, glucose-lowe ing d ug,
induced p og amming owa d a mo e an i umo pheno ype o TAMs in mice, pa ially
h ough AMPK
α
1 ac i a ion [
316
]. This led o he idea o combining an i-PD-1 he apy
wi h me o min-loaded mic opa icles, which e icien ly a ge ed p o umo al TAMs and
pola ized hem owa ds an an i umo pheno ype [
317
]. This elegan s a egy induced TME
emodeling, wi h collagen deg ada ion, and inc eased he ec ui men and in il a ion o
CD8 T cells in o umo in e io s, in a way ha also enhanced pene a ion o an i-PD-1
an ibodies.
4. Conclusions and Pe spec i es
The ema kable plas ici y displayed by mac ophages makes hem a key nexus be ween
he immune sys em and umo cells. As such, we p opose ha a ge ing mac ophages may
eed he cance -immuni y cycle, ini ia ing a snowball e ec ha will eshape o he s omal
compa men s and ul ima ely make he TME a o able o p oduc i e an i umo ac ions o
CD8 T cells (Figu e 1).
ICB, a ge ing CTLA-4 o he PD-1/PD-L1 axis, has been a game change in cance
ea men s; un o una ely, his he apy p olonged he li e o only ew cance pa ien s. In he
u u e, i is expec ed ha ICB will be a backbone he apy o mos pa ien s [
318
] and he e-
o e i is c ucial o imp o e i s e icacy o cu en ly ICB- esis an cance ypes. Al hough
his may come in pa h ough he de elopmen o no el an agonis s o immune checkpoin
such as TIM-3, Lag-3, o TIGIT, we belie e ha his “T-cell cen ic” app oach may p o ide
only limi ed bene i s. Combina ion app oaches may p o ide be e esponses; howe e ,
in b eas cance , e en when associa ed wi h neoadju an chemo he apy, pemb olizumab,
o a ezolizumab imp o ed he o e all esponse a e (ORR) o only abou 10% compa ed
o chemo he apy as single agen [
319
–
321
]. I is likely ha he eac i a ion he CD8 T
cells in ICB- esis an solid cance s is only pa ial due o s ong local immunosupp ession
main ained in he TME by high in il a ion o mac ophages. Fo ins ance, b eas cance
pa ien s wi h low in il a ion o CD163
+
mac ophages achie ed a signi ican ly highe a e
o pa hologic comple e esponse (pCR) o neoadju an chemo he apy [
322
]. The e o e, we
belie e ha he nex b eak h ough in cance ea men may come om iple he apy ha
will combine ICB and chemo he apy wi h s a egies a ge ing mac ophages. Howe e , and
as desc ibed in his e iew, a ious ea men me hods a ge TAMs and he selec ion o
he bes “pa ne ” he apy will equi e u he in es iga ion. Ne e heless, some s a egies
appea mo e p omising han o he s. Fo ins ance, al hough a ac i e and e icien in ce -
ain ci cums ances when associa ed o chemo he apy [
23
,
50
], he s a egy o mac ophage
deple ion has se e al majo d awbacks. The e is no way o exclusi ely a ge he speci ic
mac ophage subse s wi h p o umo ac i i ies, no speci ically hose loca ed in he TME.
Thus, he deple ion o all mac ophages om all issues has sys emic consequences ha
p e en s he p olonged ea men pe iod ha is equi ed o sus ain mac ophage deple ion.
Pe iphe al abla ion o mac ophages is also associa ed wi h inc eased p oduc ion o mono-
cy es and neu ophils om he bone ma ow and ea men cessa ion is usually associa ed
wi h a ebound o mac ophages in he TME [
166
]. Also a ge ing unique immunosupp es-
si e e ec s has p e en ed clinical ansla ion because i ails o limi he cause o hese TAM
subse s o hei o he p o umo al unc ions. In ou opinion, a mo e p omising app oach
will be he con e sion o TAMs in o po en an i umo e ec o s. In his con ex , in ense
in es iga ion is ocusing on TLR/STING as well as CD40 agonis s, and many ha e al eady
Cells 2021,10, 2364 20 o 35
shown sa e y and ole abili y as single agen s, allowing now o es ing hei syne gis ic
e ec s wi h ICB and chemo he apy (Table S1).
In addi ion, TAMs a e key playe s o se e al immuno he apies. Fo ins ance, he e ec
o an i-CTLA-4 is dependen on he p esence o FcR-exp essing TAMs o he elimina ion o
T eg cells in he TME [
55
], and he combina ion PD-1/CTLA-4 elies on he p oduc ion o
CXCL9 by mac ophages [
130
]. Rema kably, i was shown ha mac ophages ac i ely pa ic-
ipa e in umo clea ance in B cell lymphoma ea ed wi h i uximab (an i-CD20 mAb) [
115
]
and HER2+ b eas cance ea ed wi h as uzumab and/o pe uzumab [
56
]. As discussed
abo e, i is plausible ha CD47 blocke s in combina ion wi h an i-HER2 as uzumab (bo h
a ge ing he umo cells) will p omo e ADCC e en o pa ien s whose umo s ha e become
esis an and p og essed a e as uzumab [
323
]. The combina ion ALX148 + as uzumab
+ amuci umab (VEGFR2) + pacli axel is being es ed in a clinical ial (NCT03013218). Thus,
depending on he ea men op ion combined wi h mac ophage-based immuno he apy,
he e-educa ion s a egy would be mo e e ec i e gi en ha mac ophages could ac i ely
pa icipa e in he he apy and induce an e icien an i umo esponse in he TME. This is in
line wi h he ma hema ical modeling o T cell–mac ophage in e ac ions which de e mined
ha mac ophage ep og amming in o he an i umo subse is he mos e ec i e s a egy
(o e deple ion o inhibi ion) [324].
Finally, mac ophages display a unique ap i ude o pene a e deeply in o he co e o
he umo , e en in o hypoxic/nec o ic zones, o whe e an i umo immune cells ha dly
mig a e and cance he apies a e sca cely deli e ed. This led o he idea o exploi ing he
mac ophage p ope y o in a- umo in asion by using hem as "T ojan Ho ses" o deli e
cy o oxic o s imula o y he apies a ge ing malignan cells o immune cells, espec i ely.
Using he phagocy ic capaci y o mac ophage o up ake loaded nanopa icles, as a way
o deli e he apeu ic agen s [
325
] o cy okines [
268
], was al eady shown o be success ul
in mouse models o o e come dense ib oblas ic and s oma- ich s uc u es ha hinde
he apeu ic deli e y o he umo . Fu he mo e, al hough he usage o pa icles o ac i a e
mac ophages an i umo p ope ies has been explo ed o abou 40 yea s [
326
] wi hou
eaching clinical p ac ice ye , we belie e in he po en ial o his s a egy o become a he a-
peu ic eali y o cance pa ien s. The de elopmen o nanopa icles made o bioma e ial
ailo ed o accu a ely a ge and e-educa e TAMs may ha e a lo o po en ial. In pa icula ,
nanopa icles loaded wi h TLR agonis s a ge ing TAMs a e inducing an i umo esponses
in p eclinical models [306].
Wi h all he abo e, we hope ha his e iew makes he case o u ning mo e a en ion
o de eloping no el s a egies owa ds p og amming TAMs as hey ha e clea ly eme ged
as key cance egula o s and po en ial nex -gene a ion immuno he apy a ge s.
Supplemen a y Ma e ials:
The ollowing is a ailable online a h ps://www.mdpi.com/a icle/10
.3390/cells10092364/s1, Table S1: Examples o mac ophage- a ge ing d ugs cu en ly in es iga ed in
cance -associa ed clinical ials.
Au ho Con ibu ions:
All au ho s w o e he manusc ip and designed he igu es. All au ho s ha e
ead and ag eed o he published e sion o he manusc ip .
Funding: The au ho s ecei ed no ex e nal unding o esea ch.
Ins i u ional Re iew Boa d S a emen : No applicable.
In o med Consen S a emen : No applicable.
Acknowledgmen s:
This wo k was suppo ed by an iMM-Laço esea ch g an and he Fundacão
pa a a Ciência e Tecnologia h ough a esea ch g an (PTDC/MED-IMU/30948/2017) and a pe sonal
ellowship (CEECIND/00697/2018) ecei ed by K.S. and a PhD ellowship (SFRH/BD/144792/2019)
o C.J. This wo k was also kindly backed by he COST Ac ion BM1404 Mye-EUNITER (h p://www.
mye-euni e .eu accessed on 26 No embe 2018). COST is suppo ed by he EU F amewo k P og am
Ho izon 2020.
Con lic s o In e es : The au ho s decla e no po en ial con lic o in e es .
Cells 2021,10, 2364 21 o 35
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