Bringing macrophages to the frontline against cancer: current Immunotherapies targeting macrophages

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. 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