Defining the Emergence of New Immunotherapy Approaches in Breast Cancer: Role of Myeloid-Derived Suppressor Cells

Ci a ion: Sánchez-León, M.L.; Jiménez-Co egana, C.; Sil a Romei o, S.; Ga nacho, C.; de la C uz-Me ino, L.; Ga cía-Domínguez, D.J.; Hon ecillas-P ie o, L.; Sánchez-Ma gale , V. De ining he Eme gence o New Immuno he apy App oaches in B eas Cance : Role o Myeloid-De i ed Supp esso Cells. In . J. Mol. Sci. 2023,24, 5208. h ps://doi.o g/10.3390/ijms24065208 Academic Edi o s: Ma co E eni and Dile a Di Mi i Recei ed: 13 Decembe 2022 Re ised: 24 Feb ua y 2023 Accep ed: 6 Ma ch 2023 Published: 8 Ma ch 2023 Copy igh : © 2023 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/). In e na ional Jou nal o Molecula Sciences Re iew De ining he Eme gence o New Immuno he apy App oaches in B eas Cance : Role o Myeloid-De i ed Supp esso Cells Ma ía Luisa Sánchez-León1,2,†, Ca los Jiménez-Co egana 1,2,† , Sil ia Sil a Romei o 2, Ca men Ga nacho 3, Luis de la C uz-Me ino 2,*, Daniel J. Ga cía-Domínguez 1,2,* , Lou des Hon ecillas-P ie o 1,2,‡ and Víc o Sánchez-Ma gale 1,‡ 1Labo a o y Se ice, Depa men o Medical Biochemis y, Molecula Biology and Immunology, School o Medicine, Vi gen Maca ena Uni e si y Hospi al, Uni e si y o Se ille, 41009 Se ille, Spain 2Oncology Se ice, Vi gen Maca ena Uni e si y Hospi al, Depa men o Medicine, School o Medicine, Uni e si y o Se ille, 41009 Se ille, Spain 3Depa men o No mal and Pa hological Cy ology and His ology, School o Medicine, Uni e si y o Se ille, 41009 Se ille, Spain *Co espondence: [email p o ec ed] (L.d.l.C.-M.); dga [email p o ec ed] (D.J.G.-D.) † These au ho s con ibu ed equally o his wo k. ‡ These au ho s also con ibu ed equally o his wo k. Abs ac : B eas cance (BC) con inues o be he mos diagnosed umo in women and a e y he e ogeneous disease bo h in e - and in a umo al, mainly gi en by he a ie y o molecula p o iles wi h di e en biological and clinical cha ac e is ics. Despi e he ad ancemen s in ea ly de ec ion and he apeu ic s a egies, he su i al a e is low in pa ien s who de elop me as a ic disease. The e o e, i is manda o y o explo e new app oaches o achie e be e esponses. In his ega d, immuno he apy a ose as a p omising al e na i e o con en ional ea men s due o i s abili y o modula e he immune sys em, which may play a dual ole in his disease since he ela ionship be ween he immune sys em and BC cells depends on se e al ac o s: he umo his ology and size, as well as he in ol emen o lymph nodes, immune cells, and molecules ha a e pa o he umo mic oen i onmen . Pa icula ly, myeloid-de i ed supp esso cell (MDSC) expansion is one o he majo immunosupp essi e mechanisms used by b eas umo s since i has been associa ed wi h wo se clinical s age, me as a ic bu den, and poo e icacy o immuno he apies. This e iew ocuses on he new immuno he apies in BC in he las i e yea s. Addi ionally, he ole o MDSC as a he apeu ic a ge in b eas cance will be desc ibed. Keywo ds: b eas cance ; umo mic oen i onmen ; myeloid-de i ed supp esso cells; immuno he apy 1. In oduc ion B eas cance (BC) is he leading cause o cance dea h in women wo ldwide and a global public heal h p oblem due o i s high incidence. I is es ima ed ha 2.1 million cases o BC we e diagnosed in he las yea s, which means ha 1 ou o 4 women was diagnosed wi h he disease [ 1 ]. The Wo ld Heal h O ganiza ion es ima ed an inc ease in he incidence o BC o mo e han 2.5 million new cases be ween 2018 and 2040 as well as an inc ease o mo e han 800,000 dea hs in his ime ame [ 2 , 3 ]. B eas umo s a e highly he e ogeneous and can be classi ied acco ding o molecula and ho monal p o iles, exp ession o g ow h ac o s, and o he bioma ke s [ 4 ]. Acco ding o he gene exp ession p o ile using P osigna B eas Cance P ognos ic Gene Signa u e Assay, he e a e i e in insic sub ypes: luminal A, luminal B, human epide mal g ow h ac o ecep o 2 (HER2) en iched, basal-like/ iple- nega i e b eas cance (TNBC), and claudin-low [ 3 – 6 ]. This clus e ing is c i ical o clinical managemen , ea men decisions, and pa ien ca e. Luminal umo s ep esen app oxima ely 70% o all in asi e BCs [ 4 ] and a e cha ac- e ized by es ogen ecep o (ER) and/o p oges e one ecep o (PR) posi i i y and HER2 In . J. Mol. Sci. 2023,24, 5208. h ps://doi.o g/10.3390/ijms24065208 h ps://www.mdpi.com/jou nal/ijms In . J. Mol. Sci. 2023,24, 5208 2 o 28 nega i i y. Speci ically, luminal A is a low-g ade sub ype o BC wi h a low ki67 index, and clinically mani es s slow g owing, which ul ima ely leads o be e p ognosis, whe eas luminal B exp esses highe g ade and Ki67 index and is associa ed wi h wo se p ognosis. A subse o luminal A and luminal B BCs shows HER2 ampli ica ion/o e exp ession [ 3 , 5 – 10 ]. HER2-en iched BCs accoun o 10–15% o diagnosed umo s and do no exp ess ER o PR. Addi ionally, his ype o BC has a high exp ession o genes and p o eins, such as he ERBB2/HER2neu gene and he g ow h ac o ecep o bound p o ein 7, which a e ela ed o p oli e a ion, angiogenesis, and in asion o umo cells, hence HER2-en iched BC pa ien s ha e lowe p og ession- ee su i al (PFS) [ 3 ]. Basal-like/TNBC has a diagnosis a e o 20%, and does no exp ess ER, PR, o HER2 [ 6 – 10 ], bu a high exp ession o genes ela ed o cell p oli e a ion and a high numbe o mu a ions can be obse ed in he genome, such as he b eas cance gene 1 mu a ion, which is ound in 80% o TNBCs. In u n, TNBC can be di ided in o six di e en sub ypes, al hough he impac o his classi ica ion in ea men decision is no comple ely clea and s ill needs u he esea ch: basal-like 1 and 2, mesenchymal, mesenchymal s em-like, immunomodula o y, and luminal wi h and ogen ecep o exp ession [8,11–13]. Simila ly, claudin-low BCs ha e poo p ognosis and a e nega i e o ER, PR, and HER2. They accoun o 7–14% o all in asi e umo s and a e cha ac e ized by a low exp ession o c i ical molecules in he cell–cell adhesion (including claudins 3, 4, and 7, occludin and E-cadhe in) and ha e a high in il a ion o immune and s omal cells [8]. Gi en he as he e ogenei y o BCs, ea men choice is a he complex, so di e - en s a egies ha e been de eloped h oughou hese yea s o cu e he disease and b ing pa ien s he bes quali y o li e [ 14 , 15 ]. In he ea ly se ing, luminal A umo s a e usu- ally ea ed wi h su ge y ollowed by adio he apy (RT) and endoc ine he apy (ET). Chemo he apy (CT) is used o la ge umo s o nodal in ol emen . Luminal B BCs a e ea ed wi h (neo)adju an CT ollowed by su ge y, RT, and ET. HER2+ BCs a e ea ed wi h (neo)adju an CT combined wi h an i-HER2 d ugs, ollowed by su ge y and RT. Ea ly s age TNBC is ea ed wi h (neo)adju an CT ollowed by su ge y and RT [14]. Cu en ly, in he ad anced se ing, inno a i e ea men s a e now a ailable o he di e en sub ypes o BC, including (bu no limi ed o) ET combined wi h cyclin-dependen kinases 4/6 inhibi o s in luminal umo s [ 16 ], new an i-HER2 d ugs o HER2+ umo s, and immune checkpoin inhibi o s combined wi h CT. Al oge he , i may p o ide he maximum bene i o he pa ien s, minimizing ecu ence, ea men esis ance, and oxic e ec s, al hough combina o ial app oaches should be ca e ully chosen o ensu e he bes quali y o li e [ 17 , 18 ]. Howe e , he e a e 15–20% o pa ien s who ecu and die, so e ec i e new he apeu ic s a egies a e needed. In his sense, accumula ing da a based on clinical and p eclinical s udies suppo ha immuno he apy imp o es bo h he esponse and ou comes o BC pa ien s compa ed o s anda d he apies. Un o una ely, only a small p opo ion o BC pa ien s bene i om immuno he apy and he ea men esponse is no uni o m due o he he e ogenei y o his umo . Thus, he cu en e o s y o unde s and he di e en esponses obse ed ac oss BC sub ypes and iden i y bioma ke s ha p edic he pa ien ’s ou come. Howe e , i is a challenge ha s ill needs o be me , because immuno he apy esponses depend on bo h umo immunogenici y and he abili y o he immune sys em o esponse e ec i ely and e adica e he umo [ 17 , 18 ]. Fo all he p e ious easons, he aim o his e iew is o p o ide an o e iew o he new immuno he apies in BC as well as he ole ha he immune sys em plays, mainly ocused on he eme ging myeloid-de i ed supp esso cells (MDSCs), a popula ion o pa hologically ac i a ed and imma u e myeloid cells, as a p omising he apeu ic a ge in his disease. 2. The Role o he Immune Sys em in B eas Cance The immune sys em plays a dual ole in he biology o BC since i no only p omo es umo g ow h h ough di e en mechanisms ha gene a e ch onic in lamma ion [ 19 – 21 ], which is conside ed a new hallma k o cance , bu also media es umo e adica ion. The ela ionship be ween he immune sys em and BC depends on se e al ac o s, such as umo In . J. Mol. Sci. 2023,24, 5208 3 o 28 his ology and size, lymph node in ol emen , as well as he p esence o a wide a ie y o cells, molecules, and chemicals ha shape he umo mic oen i onmen (TME) [ 22 ]. The balance be ween inhibi o y and s imula o y signals is decisi e o explain he inal e ec o ane gy o ac i a ion o he immune sys em agains cance cells [ 23 ]. Ne e heless, mamma y umo s ha e been classically conside ed o ha e low immunogenici y due o he he e ogeneous exp ession o an igens in he p ima y umo o me as ases, modi ica ions in he an igenic p o ile du ing p og ession, low le els o majo his ocompa ibili y com- plex (MHC) exp ession, elease o supp esso cy okines, and exp ession o T-lymphocy e ac i a ion supp esso molecules, such as cy o oxic T lymphocy e-associa ed an igen 4 (CTLA-4) and p og ammed cell dea h p o ein 1 (PD-1) [ 23 ]. All his a o s a supp essi e mic oen i onmen ha helps umo cells o escape om immune su eillance [24]. The e- o e, he TME is a complex ne wo k composed o umo cells; immune cells in ol ed in he inna e esponse, such as na u al kille (NK) cells, monocy es, mac ophages, dend i ic cells (DCs), and MDSCs; cells in ol ed in he adap i e esponse, such as T helpe lym- phocy es (Th) 1, Th2, egula o y T cells (T egs), cy o oxic T cells, B lymphocy es, s omal, endo helial, and epi helial cells; and cy okines [ 25 – 27 ]. All his is communica ed h ough molecula pa hways, gi ing ise o a ch onic in lamma o y esponse a o ing bo h umo ini ia ion and p og ession. This esul s in an immunosupp essi e mic oen i onmen wi h low an igen exp ession, loss o co-s imula o y molecules, and MHC class I sys em, which ul ima ely p omo e an inc ease o immunosupp essi e molecules, such as indoleamine 2,3-dioxygenase (IDO), PD-1, CD39, CD73, o adenosine ecep o s. In e es ingly, IDO exp ession has ecen ly been shown o a o he immunosupp essi e s a us o BC pa ien s wi h posi i e nodes. This cy okine, oge he wi h ans o ming g ow h ac o (TGF)- β o in- e leukin (IL)-10, can be p oduced by supp essi e cell popula ions, such as MDSCs [ 28 – 30 ] o M2-like umo associa ed mac ophages (TAMs) o p omo e disease p og ession in BC pa ien s ia bone me as ases and epi helial–mesenchymal ansi ion [ 31 – 33 ], as shown in Figu e 1. Al oge he , i seems clea ha he TME plays a majo ole in umo ini ia ion, de elop- men , and p og ession o BC cells, which has encou aged he design and de elopmen o new immuno he apeu ic s a egies o ea BC pa ien s in o de o a oid he TME esis ance agains BC ea men s [34]. In . J. Mol. Sci. 2023,24, 5208 4 o 28 In . J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 4 o 28 Figu e 1. Composi ion o he umo mic oen i onmen (TME) in b eas cance . The b eas cance mic oen i onmen is composed by a a ie y o immune cells, including a huge p opo ion o sup- p essi e cells, such as M2 mac ophages, egula o y T cells, and myeloid-de i ed supp esso cells ha ul ima ely induce he sec e ion o p oin lamma o y media o s o p omo e he de elopmen and p og ession o cance cells. Howe e , he TME also includes a low p opo ion o dend i ic cells and umo -in il a ing lymphocy es, such as T, B, and Na u al Kille cells. In his sense, immuno he a- pies ha e shown o boos he ma u a ion and unc ion o hose cells o exe po en immune e- sponses agains he umo . 3. Immuno he apy in B eas Cance BC has adi ionally been conside ed as a cold umo in e ms o immunogenici y. Howe e , much e idence has desc ibed immunogenic ac i i y in he BC sub ypes [35,36]. This inding is allowing a a ie y o s udies o explo e he g ea po en ial o di e en im- muno he apies in BC pa ien s [37] o p o ide p edic i e and p ognos ic in o ma ion, and o op imize s anda d he apies (su ge y, RT, CT, and ET), which has esul ed in imp o ed Figu e 1. Composi ion o he umo mic oen i onmen (TME) in b eas cance . The b eas cance mic oen i onmen is composed by a a ie y o immune cells, including a huge p opo ion o sup- p essi e cells, such as M2 mac ophages, egula o y T cells, and myeloid-de i ed supp esso cells ha ul ima ely induce he sec e ion o p oin lamma o y media o s o p omo e he de elopmen and p og ession o cance cells. Howe e , he TME also includes a low p opo ion o dend i ic cells and umo -in il a ing lymphocy es, such as T, B, and Na u al Kille cells. In his sense, immuno he apies ha e shown o boos he ma u a ion and unc ion o hose cells o exe po en immune esponses agains he umo . 3. Immuno he apy in B eas Cance BC has adi ionally been conside ed as a cold umo in e ms o immunogenici y. Howe e , much e idence has desc ibed immunogenic ac i i y in he BC sub ypes [ 35 , 36 ]. This inding is allowing a a ie y o s udies o explo e he g ea po en ial o di e en immuno he apies in BC pa ien s [ 37 ] o p o ide p edic i e and p ognos ic in o ma ion, and o op imize s anda d he apies (su ge y, RT, CT, and ET), which has esul ed in imp o ed clinical ou comes wi h a signi ican imp o emen in su i al a es [ 38 ]. Howe e , inc easing BC immunogenici y and modula ing he TME a e some necessa y s a egies o imp o ing he apeu ic e icacy. In . J. Mol. Sci. 2023,24, 5208 5 o 28 Immuno he apies can be conside ed as passi e o ac i e. Passi e immuno he apies include an i-HER2 a ge ed monoclonal an ibodies, such as as uzumab, pe uzumab, and ma ge uximab; whe eas ac i e immuno he apies mainly encompass cance accines o boos he an i umo esponse by ac i a ing au ologous immune cells and inducing a he apeu ic e ec [37]. 3.1. Vaccines The immuno he apeu ic e ec o accines is o ac i a e bo h he adap i e immune esponse and immunological memo y, des oying umo cells wi h minimal oxici y [ 37 ]. Vaccines a e o mula ed wi h di e en ypes o an igens, which a e ecognized by he immune sys em o induce bene icial he apeu ic e ec s [ 39 – 41 ]. The main an igens in BC a e HER2 o HER2- ela ed pep ides [ 42 , 43 ], bu o he non-HER2 an igens a e also being s udied, including mucins, elome ase e e se ansc ip ase (hTERT), and p53 [ 39 ]. In addi ion, adju an s play a i al ole since hey enhance he an igen immunogenici y and egula e immune esponses [44–47]. The e a e di e en ypes o accines depending on he ype o an igen. The mos s udied a e hose o mula ed wi h umo -associa ed an igen (TAA) pep ides [ 48 ], bu o he s such as umo p o ein- o ca bohyd a e-associa ed an igens, and an igens based on DNA and DCs a e also being s udied o s imula e bo h inna e and adap i e an i umo immuni ies [39,49–51]. 3.1.1. Pep ide Vaccines E-75 accines a e based on HER2 as he main TAA in BC. Di e en HER2-de i ed im- munogenic pep ides ha e been s udied, wi h di e en speci ic immune esponses [ 37 ]. Neu- ax is he mos s udied BC accine and is o mula ed wi h he E75 pep ide, de i ed om HER2, ecognized by MHC class I, and combined wi h he immune adju an g anulocy e- mac ophage colony-s imula ing ac o (GM-CSF). The g ea ad an age is ha i s imula es CD8+ cy o oxic and memo y T cells, which bind human leukocy e an igen (HLA) molecules on APCs o ecognize, neu alize, and des oy HER2-exp essing p ima y umo cells and me as a ic umo s [ 52 , 53 ]. P eclinical s udies we e ini ially conduc ed o analyze he im- munogenici y o he E-75 pep ide [ 54 – 57 ], ollowed by phase I ials such as NCT00841399, in which pa ien s wi h me as a ic BC and di e en HER2 exp ession we e en olled, and bo h doses and inocula ion ou es we e op imized. This accine p o ed o be sa e and e ec i e in s imula ing E-75 speci ic CTLs [58]. This pep ide is HLA- es ic ed, hence HLA-A2 o HLA-A3 posi i e pa ien s we e acci- na ed, while hose who we e nega i e we e ollowed up as un accina ed con ols. Inc easing doses o he accine we e adminis e ed mon hly, o 4–6 mon hs, wi h good ole ance and minimal oxici y o di e en doses. Phase II ials (such as NCT02297698) demons a ed im- p o ed su i al a es a e accina ion, wi h accep able ole ance and limi ed oxici y. Neu ax inally comple ed a phase III ial, which e alua ed he accine in combina ion wi h GM-CSF s. placebo plus GM-CSF o p e en ecu ence in BC pa ien s wi h node posi i e, HER2 low exp ession, and HLA-A2+/A3+ (NTC01479244). Fo women wi h duc al ca cinoma in si u, a phase II clinical ial is being conduc ed o e alua e he e icacy o Nelipepimu -S plus GM-CSF o Sa g amos im (Leukine®) (NCT02636582) [59,60]. Ano he immunogenic pep ide used in BC accines is GP2, a agmen o he HER2 ansmemb ane domain wi h nine amino acids in leng h. GP2 binds HLA-2 wi h less a ini y han E-75 and also ac i a es CTLs [ 61 ]. A phase I ial (NCT03014276) conduc ed in lymph node-nega i e pa ien s esul ed in he o mula ion o GP2 plus GM-CSF, being well ole a ed and sa e. The phase II ial NCT00524277 was conduc ed in high- isk pa ien s wi h posi i e lymph nodes, HER2 o e exp ession, and ee o disease, demons a ing p omising sa e y and good ole ance [ 43 , 54 ]. Cu en ly, a phase III ial is ongoing o demons a e he e icacy o his ea men . Finally, AE37 is a pep ide ha consis s o 15 amino acids and ac i a es Th lymphocy es [ 62 ]. In he phase II ial NCT00524277, pa ien s wi h HER2 In . J. Mol. Sci. 2023,24, 5208 6 o 28 exp ession a all s ages o disease we e en olled, showing no s a is ical signi icance on disease- ee su i al a e using his ype o accina ion [39,63]. In o he ypes o BC accines, elome ase is in ol ed. This ibonucleop o ein has polyme ase ac i i y o main ain elome e leng h and leads o cellula immo aliza ion, so i s abe an exp ession is associa ed wi h inc eased su i al and p oli e a ion o umo cells [ 38 , 64 ]. I is o e exp essed in mos umo s, including BC [ 38 , 39 , 65 ]. The syn he ic hTERT DNA accine, INO-1400, is composed o a plasmid encoding o he ca aly ic subuni o hTERT wi h wo immunogenic di e en ia ing mu a ions ha elici a b oad CTL-media ed immune esponse agains umo cells. Yan and colleagues demons a ed he po en immuni y exe ed by his accine in p eclinical mu ine and p ima e models [ 37 ]. In mice, hTERT gene a ed a s ong immune esponse cha ac e ized by he inc ease o CD107a-, in e e on (IFN) γ -, and umo nec osis ac o (TNF) α -p oducing cells [ 38 , 66 ]. In monkeys, slowe umo g ow h and longe o e all su i al (OS) we e achie ed, and minimal side e ec s upon adminis a ion we e obse ed [ 66 ]. In he phase I clinical ial NCT02960954, INO-1400 was adminis e ed by in ade mal injec ion alone o wi h an immune sys em ac i a o , IL-12 (INO-9012), in pa ien s wi h b eas , lung, and panc ea ic cance s a high isk o elapse a e su ge y and adju an he apy. In his s udy, dose escala ion was ca ied ou o assess he sa e y and ole abili y o he accine [ 37 ]. O he clinical ials a e unde way, in bo h me as a ic (NCT00573494 and NCT01660529) and adju an (NCT02960594 and NCT00753415) se ings [39]. BC accine cons i u es a new he apeu ic s a egy o enhance an i-cance immuni y. Cu en esul s show ha accines a e sa e, al hough his immuno he apy s ill needs clinical e i ica ion. In addi ion, he e a e se e al limi a ions. One o hem is he need o a sui able adju an o gene a e an e ec i e immune esponse. I is es ic ed o a ew epi opes, esul ing in a limi ed esponse agains umo cells. Fu he mo e, pep ides ha e a sho hal -li e, poo enzyma ic s abili y, and high clea ance a es. Some s udies a e ying o sol e hese limi a ions using mul i alen syn he ic long pep ides, con aining epi opes ha can be ecognized by MHC class I and II in o de o enhance he immune esponse o hese accines by ac i a ing bo h CD8+ and CD4+ T lymphocy es [63]. 3.1.2. P o ein-Based Vaccines These accines in eg a e a p o ein agmen o he umo an igen HER2, whose amino acid sequence is longe han ha o he pep ides. P o ein-based accines u ilize bo h he in a- and ex acellula domains o he HER2 ecep o . In e es ingly, hese accines can up ake, p ocess, and p esen mul iple epi opes o MHC I and II, hus hey a e no HLA- es ic ed accines [ 43 ]. Addi ionally, hey signi ican ly ac i a e T cells, esul ing in an inc eased immune esponse [ 67 , 68 ]. The i s clinical s udy ca ied ou was a phase I ial in which he accine in eg a ed he in acellula domain o HER2 plus GM-CSF [ 69 ] o e alua e he immunogenici y in pa ien s wi h HER2-o e exp essing b eas and o a ian cance s who we e in comple e emission a e being ea ed, esul ing in a p omising ole ance [ 63 ], immuni y main enance [ 43 ], and HER2/neu-speci ic immunoglobulin G an ibody immuni y. Ano he s udy conduc ed by Hamil on e al. aimed o e alua e he immunogenici y, sa e y, and e ec o he an i-HER 2 p o ein [ 27 ] using a accine consis ing o a ecombinan p o ein combined wi h Lapa inib in pa ien s wi h HER2-o e exp essing me as a ic BC and e ac o y o as uzumab, which demons a ed an imp o ed su i al in BC pa ien s (92%) a e 300 days [ 70 ]. Ano he phase I clinical ial (NCT00058526) used a ecombinan HER2 p o ein and AS15 as an adju an and was adminis e ed o 61 p e iously un ea ed pa ien s wi h as uzumab in s age II-III a e su gical esec ion in he adju an se ing [ 71 ]. An associa ion was ound be ween he dose adminis e ed, accina ion schedule, and p e alence o HER2-speci ic humo al esponses. This immuni y was main ained o mo e han 5 yea s in six ou o eigh pa ien s who ecei ed he highes dose. In 40 me as a ic pa ien s, he same accina ion egimen was adminis e ed in he i s o second line o ea men , a e as uzumab main enance, which was well ole a ed and showed p omising clinical ac i i y, wi h wo objec i e esponses and 10 pa ien s wi h s able In . J. Mol. Sci. 2023,24, 5208 7 o 28 disease [ 72 ]. These accines a e s ill in he ea ly s ages o esea ch and u he clinical ials a e needed o inc ease he numbe o pa ien s ec ui ed, which would p obably p o ide mo e da a on he impac on su i al, immuniza ion e icacy, and long- e m ollow-up [ 73 ]. 3.1.3. Tumo Cell Vaccines Whole umo cells o cell lysa es a e also used o s imula e immune esponses. An i- gens a e de i ed om he pa ien ’s own au ologous umo cells. Howe e , his is a e y complex, expensi e me hod and allogeneic cells a e used as an al e na i e [ 72 ]. They can be manipula ed o exp ess cy okines o chemokines o maximize he immune esponse. As an adju an , GM-CSF s imula es he mig a ion o DCs, T cells, eosinophils, and mac ophages o he injec ion si e [ 59 , 74 ], which igge a poly alen immune esponse. Howe e , u- mo cell accines con ain endogenous an igens, may cause au oimmune eac ions, and a s anda dized me hod o he p epa a ion o such accines is s ill lacking [ 43 ]. Di e en s udies ha e explo ed he e icacy o hese accines in BC pa ien s. One o hem in ol ed 121 pa ien s diagnosed wi h b eas and o a ian cance s, who ecei ed a accine o mula ed wi h au ologous umo cells ans ec ed wi h Newcas le disease i us [ 59 , 75 ]. The OS a 4 yea s was 96%, which alida ed he e icacy o he accine [ 75 ]. In ano he s udy, 42 pa ien s ecei ed a accine composed o au ologous and allogeneic cells, h ee TAAs combined wi h GM-CSF and IL-2, which esul ed in a signi ican inc ease o lymphocy e p oli e a ion in mo e han 50% o pa ien s [ 59 , 76 ]. These accines migh be adminis e ed sa ely o pa ien s, imp o ing he immuni y and clinical e icacy. Cu en ly, wo comple ed clinical ials ha e demons a ed he e ec i eness and sa e y o hese accines in BC pa ien s (NCT00317603 and NCT00880464). Howe e , he manu ac u ing p ocess is expensi e and he e may be a high umo an igenic a iabili y [59]. 3.1.4. Dend i ic Cell-Based Vaccines DCs a e highly specialized cells whose main unc ion is o modula e he p ima y immune esponse ia an igen p esen a ion o CD4+ and CD8+ T lymphocy es [ 77 , 78 ], so he e is an in e es in using his cell popula ion o gene a e accines. Blood om cance pa ien s con ains imma u e DCs supplied wi h TAA encoding ecombinan DNA/RNA, and DC- umo hyb ids ha a e s imula ed wi h speci ic cy okines o induce ma u a ion. Those cells a e cu en ly in use o gene a e accines. Kugle e al. demons a ed he e icacy o hese accines in b eas and o a ian cance pa ien s [ 79 ]. A igan e al. used umo cells om au ologous DC pa ien s o gene a e he cell-based accine ha ul ima ely gene a ed s ong immune esponses in b eas and enal cance pa ien [ 80 ]. Zhang e al. gene a ed a BC accine by using DCs wi h TNBC cells, esul ing in a po en an i- umo immune esponse by acili a ing lymphocy e p oli e a ion [ 81 ]. P eclinical s udies o he de elopmen o DC accines o BC ha e also shown p omising esul s: Sakai e al. modi ied DCs by ansducing hem wi h a non-signaling neu oncogene which esul ed in no mamma y umo de elopmen in BALB-neu ansgenic mice [82]. 3.1.5. DNA-Based Vaccines These ypes o accines use DNA sequence encoding umo an igens ha a e usually deli e ed as plasmids o ec o s [ 43 ], mos commonly om bac e ial, cy omegalo i us (CMV), o chime ic p omo e SV40 (simian i us 40) -CMV o igin [ 83 , 84 ]. The mos impo an aspec s a e he selec ion o design o a po en plasmid, an e icien deli e y sys em, and moni o ing he immune esponse a e accina ion. The mos a ge ing an igens used a e oncop o eins HER2/neu and mammaglobin A (Mam-A). In his ega d, he e a e wo ials ha s udied he e ec o such accines, obse ing signi ican humo al esponses. No ell e al. conduc ed a pilo clinical ial wi h eigh pa ien s diagnosed wi h me as a ic BC. The accine was o mula ed wi h HER2/neu, low-dose IL-2, and GM-CSF. They obse ed a s ong humo al esponse, bu T-cell esponses emained unchanged [ 85 ]. Kim e al. se up a phase I clinical ial in which a DNA accine composed o Mam-A cDNA was adminis e ed o 15 Mam-A posi i e cance pa ien s and he immune esponse was moni o ed o 6 In . J. Mol. Sci. 2023,24, 5208 8 o 28 mon hs. Se en ou o 15 pa ien s showed an inc ease in inducible cos imula o (ICOS Hi ) CD4+ T cells and a dec ease in Foxp3C CD4C T cells. Ac i a ed ICOS Hi CD4+ T cells exp essed INF γ ins ead o IL-10, which esul ed in lysis o Mam-A exp essing umo cells [ 86 ]. These s udies showed ha DNA-based accines a e e icien o con ol he disease, al hough he sa e y and immunogenic mechanisms o such accines need u he esea ch [63,72]. 3.1.6. Ca bohyd a e An igen Vaccines One o he disaccha ide ca bohyd a es is Sialy-Tn (STn), which is exp essed in a wide a ie y o umo cells associa ed wi h MUC-1. Due o he good esul s obse ed in animal models ega ding umo eg ession and su i al, a syn he ic conjuga e accine was de eloped, comme cially named The a ope ® (STn-keyhole limpe hemocyanin—KLH). In a double-blind, phase II clinical ial, 1028 pa ien s wi h me as a ic BC om 126 cen e s o 10 coun ies we e andomized o ecei e STn-KLH o KLH alone, co-adminis e ed wi h low-dose cyclophosphamide o inc ease immunogenici y [ 87 ]. The p ima y endpoin was ime o p og ession (TTP) and OS. Despi e he signi ican le els o STn-speci ic an ibodies obse ed, he e was no imp o emen in TTP o OS [ 88 ]. Howe e , he e migh be a lack o eligibili y c i e ia, since only 30–40% o b eas umo s exp essed STn and his was no conside ed when including pa ien s. In a subg oup analysis, he accine a m had be e TTP and OS ou comes when ET was adminis e ed, indica ing ha he combina ion o he STn-KLH accine wi h ET could imp o e clinical ou comes [ 88 , 89 ]. In summa y, accines a e cu en ly a he apeu ic s a egy unde in es iga ion ha ha e shown p omising esul s, al hough a be e unde s anding o he TME, immunosupp essi e, and umo e asion pa hways is s ill needed [ 90 ]. The e is a necessi y o conduc clinical ials wi h la ge coho s o pa ien s, and explo e he combina ion wi h o he ea men s, such as checkpoin inhibi o s o an i-angiogenic d ugs [ 63 ]. In addi ion, accines seem o be mo e use ul in pa ien s wi h minimal disease and, on he con a y, hey appea o be ine ec i e in he me as a ic se ing [91]. 3.2. Monoclonal An ibodies (mAbs) Immuno he apy wi h mAbs has imp o ed he he apeu ic a senal in he igh agains BC, speci ically in HER2 ecep o o e exp essing umo s [ 92 , 93 ]. T as uzumab (He cep in ® ) was he i s humanized mAb app o ed by he FDA in 1998 o he ea men o HER2+ me as a ic BC in combina ion wi h CT. T as uzumab di ec ly a ge s he ex acellula domain o he HER2 ecep o , and se e al mechanisms o ac ion ha e been desc ibed: he i s one in ol es HER2 ecep o deg ada ion by binding i s ex acellula ansmemb ane domain, inducing HER2 in e naliza ion and deg ada ion h ough a E3 ubiqui in ligase (c-CBL) [ 94 , 95 ]; he second mechanism o ac ion is o a ac cy o oxic inna e immune cells o he TME by binding he agmen c ys allizable egion (Fc egion) o IgG1 (T as uzumab) o he Fc γ RIII/CD16 o NK cells, so-called “an ibody-dependen cellula cy o oxici y” (ADCC) [96,97]. NK cells elease p oin lamma o y cy okines, such as INF γ and TNF α , du ing ADCC, a mechanism known as an ibody-dependen cy okine elease (ADCR) [ 97 ]. The c oss alk among NK cells, umo cells, and he p o-in lamma o y en i onmen induced by ADCC may p omo e o he immune cell popula ions, which has been called he “ accina ion e ec ” [ 98 ]. Finally, he ex acellula binding o as uzumab o HER2 leads o he inhibi ion o he RAS/MAPK and PI3K/AKT signaling pa hways [ 94 , 99 ]. Se e al s udies ha e desc ibed i s ole in immune sys em ac i a ion, including an inc ease o TILs, which has been associa ed wi h a dec ease in dis an ecu ence [ 100 ]. O he s epo ed ha TILs had p ognos ic and p edic i e alue as i imp o es he pa hological comple e esponse (pCR) and disease- ee su i al (DFS) [ 101 , 102 ]. Bense e al. showed ha he p esence o di e en ypes o immune cells di e ed acco ding o BC sub ype [ 103 ]. A high T eg ac ion in HER2+ umo s was associa ed wi h a lowe a e o pCR, DFS, and OS [ 104 ]. Inc eased T γδ lymphocy es in all BC pa ien s was associa ed wi h a highe a e o pCR, p olonged DFS, In . J. Mol. Sci. 2023,24, 5208 9 o 28 and OS. High le els o ac i a ed mas cells we e associa ed wi h wo se p ognosis, DFS, and OS in HER2+ pa ien s [ 97 ]. Al hough i is a s anda d d ug in a ge ed he apy agains HER2, i should be no ed ha be ween 27% and 42% o pa ien s de elop de no o and acqui ed esis ance o i in neoadju an and adju an he apy, which hinde s i s clinical bene i . This esis ance mechanism in ol es he TNF α signaling pa hway, which is also induced by he exp ession o mucin 4, a p o ein ha masks he as uzumab binding epi ope on he HER2 ecep o o p omo e he sp ead o umo cells, and which is a bioma ke o poo esponse o adju an as uzumab [ 99 ]. HLA-G exp ession in umo cells has been iden i ied as ano he media o o as uzumab esis ance, which, when coupled o he HLA-G/KIR2DL4 in e ac ion, enhances he ulne abili y o HER2+ b eas umo s o as uzumab ea men in i o [105]. Pe uzumab (Pe je a ® ) is a dual HER2/HER3 mAb ha was app o ed by he FDA in 2012 in combina ion wi h as uzumab and doce axel o i s -line ea men o HER2+ me as a ic BC as i inc eased OS, acco ding o he esul s ob ained in he Cleopa a ial (NCT00567190) [ 95 , 105 ]. I was subsequen ly app o ed in he ea ly disease se ing, based on he esul s om he NeoSphe e (NCT00545688) and Aphini y (NCT01358877) ials. Pe uzumab binds o a di e en epi ope wi hin he ex acellula domain o HER2 han as uzumab, p e en ing ligand-dependen HER2/HER3 he e odime iza ion by inhibi - ing he PI3K and MAPK pa hways [ 95 , 106 , 107 ]. An in i o s udy showed ha bo h as uzumab and pe uzumab alone ac i a ed ADCC wi h equal po ency. Howe e , no inc ease in ADCC ac i i y was obse ed when adminis e ed combined. In i o s udies ha e shown ha he combina o y ea men inc eased NK mig a ion in o he TME, which would delay as uzumab esis ance in BC xenog a models [95]. Ma ge uximab (MGAH22) is a chime ic mAb wi h an i-HER2 ac i i y [ 108 ] whose agmen an igen-binding (Fab) po ion sha es he same HER2 speci ici y as as uzumab, while he Fc po ion is enginee ed by glycosyla ion, which inc eases and imp o es a ini y o he Fc γ RIIIa ecep o , con ibu ing o imp o ed an ibody-dependen cellula cy o oxic- i y [ 96 , 109 ]. The Fc-independen p ope ies o ma ge uximab a e simila o as uzumab, including he same binding a ini y o HER2+ exp essing umo cells, hence ha ing simila an ip oli e a i e ac i i y. I has been shown ha ma ge uximab is mo e e ec i e as an ADCC media o han as uzumab, bo h in i o and ex i o. In i o s udies sugges ha ma ge uximab p omo es g ea e NK cell ac i a ion, expansion, and p oli e a ion han as uzumab and pe uzumab [ 96 ]. In e ms o adap i e immune esponses, inc eases in B- cell-media ed HER2-speci ic an ibody le els we e ound in 42–69% o as uzumab- ea ed pa ien s and 94% o ma ge uximab- ea ed pa ien s. In addi ion, inc eases in T-lymphocy e- media ed esponses we e obse ed in 50–78% o as uzumab- ea ed pa ien s and 98% o ma ge uximab- ea ed pa ien s [ 96 ]. In a phase I ial (NCT01148849), 66 pa ien s wi h HER2-o e exp essing ad anced BC ecei ed in a enous in usion o MGAH22, which was well ole a ed, and pa ial esponses and s able disease we e obse ed in 12% and 50% o pa ien s, espec i ely; also umo educ ion was obse ed in o e hal (18/23, 78%) o esponse-e aluable pa ien s wi h BC [110]. Finally, ma ge uximab was app o ed by he FDA in 2020 in pa ien s wi h HER2+ me as a ic BC who ecei ed wo o mo e egimens o an i-HER2 he apy. This app o al was based on he SOPHIA s udy (NCT02492711), a phase III ial ha compa ed ma ge uximab plus CT e sus as uzumab plus CT in 536 pa ien s wi h HER2+ me as a ic BC who had ecei ed a leas wo p io an i-HER2+ he apies [ 108 , 111 ]. E icacy analysis using Fc γ RIIIa- 158 allele exp ession in 506 pa ien s showed bene icial esul s wi h ma ge uximab on PFS e sus as uzumab in Fc γ RIIIa-158F ca ie s. In con as , no bene i wi h ma ge uximab was obse ed o e as uzumab in FcγRIIIa-158V homozygo es [96,111]. Zanida amab (ZW25) is a biospeci ic an ibody ha binds wo di e en epi opes on HER2, HER2 ex acellula domains (EDC2 and ECD4) [ 108 ]. As a esul o hese modi i- ca ions, ZW25 shows a mo e speci ic binding o umo cells, hus inhibi ing bo h ligand- and ligand-independen umo g ow h and enhancing ecep o in e naliza ion and deg a- da ion compa ed o as uzumab [ 108 ]. In i o assays demons a ed ha ZW25 leads o In . J. Mol. Sci. 2023,24, 5208 16 o 28 PF-04518600, wi h ni olumab. Simila ly, he e icacy and sa e y o i omilumab is being de e mined in he phase I ial NCT03364348 in HER2+ ad anced BC in combina ion wi h as uzumab [97,145]. 4. Myeloid-De i ed Supp esso Cells as a The apeu ic Ta ge in B eas Cance MDSCs a e a g oup o cells widely s udied in cance , including BC. This is a he - e ogeneous popula ion o imma u e, pa hologically ac i a ed myeloid cells de ined by hei mo phology, su ace pheno ype, and unc ions. MDSCs can be mainly di ided in o monocy ic MDSCs (M-MDSCs), wi h a ypical monocy e mo phology, and g anulocy ic o polymo phonuclea MDSCs (G-MDSCs o PMN-MDSCs, espec i ely), wi h a mo phology ha esembles g anulocy es [ 163 – 165 ]. In humans, M-MDSCs a e CD11b+CD14+HLA-DR- /lowCD15- cells and G-MDSCs a e cha ac e ized by he CD11b+CD14+CD15+ o CD11b+ CD14- CD66b+ pheno ype. Cells cha ac e ized as Lin- (including CD3, CD14, CD15, CD19, CD56) HLA-DR-CD33+ a e mo e imma u e g oups o MDSCs, he so-called “ea ly s age-MDSC” (e-MDSC), a e m p e iously p oposed [ 30 , 166 ]. In addi ion, he exp ession o bo h CD45 and CD33 has been conside ed o de ine MDSC subpopula ions in some diseases, including cance [ 167 , 168 ]. In BC se ings, he e a e some cy okines associa ed wi h he de elopmen , di e en ia ion, and expansion o MDSCs, including g anulocy e colony-s imula ing (G-CSF), mac ophage colony-s imula ing ac o (M-CSF), GM-CSF, IL-6, IL-1 β , mac ophage mig a ion inhibi o y ac o (MIF), and TGF β [ 169 – 171 ]. Simila ly, a se o chemokines pa icipa es o p omo e he ec ui men o MDSCs in o he TME, such as C-X-C mo i chemokine ligand (CXCL)5, C-C mo i chemokine ligand (CCL)1, CCL2, CCL5, o he monocy e chemo ac ic p o ein-1, among many o he s. Impo an ly, o he ac o s ha a o he ec ui men o MDSCs o he main me as a ic niches o BC include he S100 calcium-binding p o ein A8 and A9 (S100A8 and S100A9, espec i ely) [169–171]. In b eas umo s, MDSCs exe hei po en immunosupp essi e unc ions h ough se e al pa hways: (a) STAT3-NF-Kb-IDO, which is ac i a ed by umo -de i ed IL-6 o ac i a e STAT3 in MDSCs [ 171 ]. STAT3 modula es he exp ession o genes in ol ed in in lamma o y p ocesses and p omo es IDO exp ession, a o ing he inhibi ion o immune su eillance, and immune ole ance by supp essing T-cell ac i a ion h ough he TCR and inducing he ampli ica ion o T egs [ 171 ]. (b) STAT3/in e e on egula o y ac o 8 (IRF8) pa hway, in which G-CSF and GM-CSF p omo e he low exp ession o IRF-8, ia STAT3 and STAT5 pa hways. Speci ically, IRF-8 is a nega i e egula o o human MDSCs, whe eby low le els o IRF-8 a e accompanied by an inc eased numbe o MDSCs. (c) PTEN/AKT pa hway, since a low PTEN exp ession p omo es he ac i a ion o he AKT pa hway (including mTOR and NF- κ B pa hways), and inc eased exp ession o me allop o einases (MMPs), including MMP2, MMP13, and MMP14, o p omo e in asion and me as asis [ 169 , 170 ]. MDSCs ac as p ogeni o s o os eoclas s, p omo ing bone me as asis h ough he ni ic oxide (NO) p oduc ion and c oss- communica ion wi h umo cells, which ep esen s a majo p oblem causing high mo bidi y and mo ali y in BC pa ien s, a leas in pa due o he c oss alk be ween MDSCs and umo cells [171]. In ecen yea s, a mul i ude o s udies ha e been ca ied ou , which ha e epo ed he ela ionship be ween MDSCs and BC cells. Tumo cells ec ui elemen s, such as MDSCs, T eg, and M2 mac ophages, o shape a p o- umo igenic mic oen i onmen ha de egula es he an i umo immune esponses. F om a clinical pe spec i e, he le els o ci cula ing MDSCs ha e been co ela ed wi h umo s age and me as a ic disease bu den in pa ien s [ 170 ]. New esea ch in his ield es ablishes he ela ionship be ween ele a ed le els o MDSC in pe iphe al blood o BC pa ien s and he p ognosis o he disease, as i is associa ed wi h ad anced s ages, highe umo bu den, and lowe PFS and OS, as well as lowe esponse o CT, RT, immuno he apy, and a ge ed he apies [ 172 ]. The g oup o J. Ma kowi z demons a es ha MDSC le els in pe iphe al blood a e associa ed wi h umo bu den o me as a ic BC pa ien s and he dec eased o ci cula ing MDSCs imp o ed he apeu ic esul s [ 173 ]. A s udy ca ied ou by Be gen elz e al. epo ed ele a ed ci cula ing monocy es and M-MDSCs in pa ien s wi h p ima y BC, loco- egional In . J. Mol. Sci. 2023,24, 5208 17 o 28 ecu ence, and me as asis, who we e compa ed wi h a heal hy coho o indi iduals [ 174 ]. Addi ionally, monocy e le els we e al e ed in pa ien s wi h ea ly-s age BC, sugges ing ha small, localized umo s showed a sys emic esponse a ec ing ci cula ing myeloid cells du ing umo de elopmen . In addi ion, T-cell p oli e a ion was supp essed in pa ien s wi h ea ly BC and hose wi h a mo e ad anced s age o he disease, which was posi i ely co ela ed wi h M-MDSC le els. In he same line, he monocy e/T-lymphocy e a io was highe in ad anced BC pa ien s han in heal hy con ols, sugges ing ha b eas umo s also ec ui p oin lamma o y cells in o he TME and he abili y o T cells o pe o m s ong immune esponses is low. In his s udy, hey also highligh ed ha he high equency o M-MDSCs was associa ed wi h wo se p ognos ic disease in pa ien s, wi h a high numbe o me as ases, including lymph nodes and isce al o gan [ 174 ]. In he same line, p e ious s udies had epo ed ha monocy es inc eased he in asi e and me as a ic po en ial o BC cells. Speci ically, he wo k o ou esea ch g oup in he ield o MDSCs in a a ie y o diseases, including bo h ad anced and ea ly-s age BCs, demons a es ha high le els o hese cells in pe iphe al blood o pa ien s a o s an immunosupp essi e mic oen i onmen ha p omo es he de elopmen o me as asis and umo p og ession [ 175 ]. Thus, no only M-MDSCs bu also G-MDSCs in pe iphe al blood may be a p omising, in e es ing bioma ke o assess disease p og ession in BC pa ien s, as well as a possible he apeu ic a ge . Gi en he eme ging impo ance o MDSCs in BC, his makes hem an a ac i e a ge o u he esea ch in o hei unc ionali y as a he apeu ic a ge in his disease. MDSC-Ta ge ed Immuno he apies in B eas Cance Cu en ly, he e a e a a ie y o immuno he apeu ic s a egies in BC o a ge MD- SCs, ei he educing hei numbe o inhibi ing hei immunosupp essi e unc ions [ 176 ]. Blocking he p oduc ion o cy okines and chemokines ha p omo e he de elopmen and mig a ion o MDSCs o he TME is one o he s a egies ha is being s udied in BC o educe MDSC niche [169]. Cu cumin is an IL-6 inhibi o ha has been p e iously used in a human gas ic cance xenog a model and mouse colon cance model, demons a ing he inhibi ion o umo g ow h, MDSCs in bo h blood and umo issue, and IL-6 le els. Fu he mo e, cu cumin ea men pola ized MDSCs o an M1 mac ophage pheno ype wi h inc eased exp ession o CCR7 and dec in 1 in i o and in i o [ 177 ]. Cu cumin is eme ging as a p omising an icance agen as i modula es mamma y ca cinogenesis h ough i s e ec on cell cycle and p oli e a ion, apop osis, senescence, and me as asis de elopmen [ 178 ]. The an i umo pa hways o cu cumin include he PI3K/Ak /mTOR, JAK/STAT, MAPK, NF-kb, p53, and Wn / β -ca enin [ 178 , 179 ]. In addi ion, cu cumin modula es he TME, immuni y, BC s em cells, and BC- ela ed miRNAs [178]. Bone Mo phogene ic P o eins (BMP4), a membe o he TGF β g ow h ac o am- ily, educes he exp ession o G-CSF in human and mouse BCs [ 169 ]. BMP4 is a po en supp esso o me as asis de elopmen in BC. In a mu ine model, i has been obse ed ha he de elopmen o b eas umo s wi h high me as a ic po en ial is associa ed wi h a high accumula ion o MDSCs, which could be speci ically induced by ea men wi h G-CSF/Cs 3 o by G-CSF sec e ion om umo si es. I is known ha MDSCs a e associa ed wi h poo p ognosis in BC pa ien s and he apies based on BMP4 ac i a ion and may o e a new ea men s a egy in clinical se ings [180]. 84 is an an i-VEGF inhibi o ha educes he p oduc ion o IL-1 β , IL-6, and CXCL1 [ 169 ]. Roland’s g oup conduc ed a s udy using h ee di e en BC mu ine models (MDA-MB-231 xenog a , 4T1 syngeneic, and a ansgenic model wi h MMTV-PyMT mice) o s udy he e ec s o di e en an i-VEGF he apies, including 84, on umo ascula u e, immune cell in il a ion, and cy okine le els. Speci ically, 84 selec i ely inhibi ed he binding o VEGF o i s ecep o VEGFR2, esul ing in he dec ease o he le els o cy okines, such as IL-1b, IL-6, and CXCL1, he inhibi ion o immune supp esso cell in il a ion, he inc ease o he DC ac ion, and he down egula ion o angiogenesis, which we e co ela ed wi h he esponse o ea men s. In . J. Mol. Sci. 2023,24, 5208 18 o 28 In addi ion, ano he an i-VEGF ea men , called be azizumab, is in use o HER2-nega i e me as a ic BC pa ien s [181]. Sul o aphane is a MIF inhibi o [ 169 ] ha blocks he pleio opic, in lamma o y, and p o umo e ec s o ha cy okine [ 182 ]. In p eclinical models o BC, MIF no only p omo es umo g ow h and lung me as asis, bu also a o s he di e en ia ion o MDSCs in TME. In i o , pha macological inhibi ion o MIF educed he accumula ion o MDSCs in he TME and blocked hei di e en ia ion. Thus, MIF inhibi o s may be conside ed as a he apeu ic a ge ei he alone o in combina ion wi h o he ea men s, including immuno he apy [ 183 ]. Sibylline down egula es he exp ession o CCR2 in MDSCs o dec ease le els o his cell popula ion. NG-monome hyl-L a ginine ace a e, an inducible NO syn hase inhibi o , blocks he di e en ia ion o MDSCs in o os eoclas s, and may be e ec i e in he cha ac e is ic os eolysis o MDSCs. Rega ding he elimina ion o MDSCs, bo h p eclinical and clinical s udies a e unde way, mos o hem p oposing he ma u a ion o MDSCs [ 169 ]. S udies ha e been conduc ed o e alua e he an i umo e ec o Sybilline on MDSCs. Fo his pu pose, mice bea ing mamma y umo s ha e been used and a lowe accumula ion o MDSCs was obse ed bo h in blood and in he umo issue [184]. Ac i a ed T cells (ATCs) combined wi h an i-CD3 x an i-He 2/neu bispeci ic an ibod- ies (aATC) ha e been able o elimina e MDSCs mainly ia INF γ and IL-2 [ 79 ]. Thaku e al. in es iga ed whe he ATC a med wi h bispeci ic an ibodies (aATCs) could inhibi umo g ow h and in u n dec ease MDSC le els in a mic oen i onmen en iched wi h IL-2 and INF gamma cy okines. The da a ob ained con i med ha aATC e ec i ely inhibi ed umo g ow h, also, IL-2 and INF gamma supp essed he ac ions and unc ions o MDSCs and T eg di e en ia ion. Thus, aATCs can be a ge ed o ea men in BC [185]. Adop i e cellula he apy o ep og ammed umo -sensi ized immune cells includes CD25+NKT, NK, and memo y T cells, and a enua es he immunosupp essi e unc ion o MDSCs due o hei ma u a ion in o DCs ia NKT cells h ough NKG2D-dependen signaling. F om a clinical pe spec i e, i has been shown ha he combined injec ion o IL-7 and IL-15 in o BC lesions a e adio equency he mal abla ion can educe he numbe o MDSCs and hus inhibi umo g ow h and me as asis [186]. O he s a egies es ed in p eclinical models in ol e a accine composed o Lis e ia monocy ogenes exp essing he TAA Mage-b and c-di-GMP as a s imula o y ligand. Wi h his, an enhanced immune esponse was obse ed due o he esponse o TAA-speci ic T lymphocy es [ 187 ]. P eclinical s udies wi h Lis e ia ha e epo ed dec eased le els o MDSCs on bo h blood and umo s, as well as enhancing T- and NK-cell immune esponses, sugges ing he e ec i eness o Lis e ia immuno he apy in me as a ic BC [188]. Clinical ials combining immuno he apy wi h o he ypes o he apies in BC o a ge MDSC a e cu en ly unde way, as shown in Table 3. The e o e, conside ing ha MDSCs play a key ole in he BC mic oen i onmen o a o bo h umo g ow h and me as asis, MDSC- a ge ing he apies may be po en ial ea men s in clinical se ings. Table 3. Myeloid-de i ed supp esso cell- a ge ing he apies in b eas cance clinical ials. The apy Combina ion The apy MDSC Ta ge Addi ional Tumo s Tes ed Clinical T ial Phase S a us Clinical T ial Re e ence En inos a Ni olumab Class I HDCA - I Ac i e, no ec ui ing NCT02453620 IPI-549 Ni olumab PI3K NSCLC, SCCHN, AdC, MEL, MES I/Ib Ac i e, no ec ui ing NCT02637531 IPI-549 Tecen iq + Ab axane PI3K - II Ac i e, no ec ui ing NCT03961698 Repa ixin Pacli axel CXCR2 - II Comple ed NCT02370238 AB928 IPI-549, PLD, NP AzaR and AzbR O a ian I/Ib Comple ed NCT03719326 In . J. Mol. Sci. 2023,24, 5208 19 o 28 Table 3. Con . The apy Combina ion The apy MDSC Ta ge Addi ional Tumo s Tes ed Clinical T ial Phase S a us Clinical T ial Re e ence PD-0360324 A elumab CSF-1 A a ie y o ad anced umo s (including BC) Ib/II Ac i e, no ec ui ing NCT02554812 Ciclophosphamide/ Deci abine/ ca bopla n/ pacli axel/ Doxo ubicin Pemb olizumab PD-1 - II Ac i e, no ec ui ing NCT02957968 Le onlimab Ca bopla in CCR5 - Ib/II Ac i e, no ec ui ing NCT03838367 Imiquimod Pacli axel TLR7 - II Comple ed NCT00821964 AdC: Ad enoco ical ca cinoma; BC: B eas cance ; CCR5: C-C mo i chemokine ecep o 5; CSF-1: colony- s imula ing ac o 1; CXCR2: C-X-C mo i chemokine ecep o 2; HDCA: His one deac ylase; IPI-549: Inhibi o o PI3K- γ -549 (Eganelisib); MDSC: myeloid-de i ed supp esso cell; MEL: melanoma; MES: Meso helioma; NP: Nanopa icle albumin-bound pacli axel; NSCLC: non-small cell lung cance ; PD-1: P og ammed cell dea h 1; PDL: pegyla ed liposomal doxo ubicin; PI3K: phospha idylinosi ol 3-kinase; SCCHN: Squamous cell cance o head and neck; TLR7: Toll-like ecep o 7. 5. Conclusions and Fu u e Pe spec i es Immuno he apy has eme ged as a e y p omising he apeu ic app oach in cance because i has shown o boos su i al a es no only in p eclinical se ings, bu also in oncological pa ien s wi h di e en ypes o umo s. Un il now, immuno he apy has no been p io i ized o BC ea men because BC has been conside ed a poo ly immunogenic umo o many yea s. Howe e , inc easing e idence in ecen yea s has sugges ed some immunogenic ac i i y in di e en BC sub ypes [ 189 , 190 ], exhibi ing he iple-nega i e pheno ype, he s onges immunogenici y [ 191 ], which sugges s ha immuno he apy may be inc easingly impo an in he ea men o BC. In line wi h his no ion, highly e ec i e ea men s a e cu en ly being used ou inely in he clinical p ac ice (Figu e 3), including bu no limi ed o mAbs o ADCs. Howe e , some o hose immuno he apies s ill need o be u he in es iga ed in e ms o de elopmen and design, such as an i-cance accines, which ha e demons a ed p omising esul s in ea ly clinical ials and may change he cou se o BC. In addi ion o de eloping new immuno he apeu ic d ugs, hei combina ion wi h con en ional BC he apies, such as ho mone he apy o CT, a e eme ging and a e conside ed as p omising s a egies o imp o e clinical esul s in e ms o umo g ow h, esponse o ea men , and su i al a es. Al hough immuno he apy may be an e ec i e app oach, u u e s udies should e al- ua e he e ec s o hose ea men s conside ing ha BC p esen s a a ie y o di e en immunological p o iles. In addi ion, he numbe o cance pa ien s and heal hy indi- iduals ec ui ed should be inc eased o alida e ele an immunological bioma ke s clinically [ 186 ]. This is impo an because he well-es ablished bioma ke s could p o ide a s a i ica ion o he BC pa ien s, which a e c i ical o he e ec i eness o immuno he apy bo h in mono he apy and in combina ion. Howe e , o de e mine he bes s a egy o hese pa ien s will be a long- e m challenge. We hink ha he immunogenici y o BC will con ibu e o mo e e ec i e and pe sonalized he apeu ic s a egies o a ge he mos immunogenic sub ypes [170], wi h mo e a o able oxici y p o iles. Finally, i should be kep in mind ha he e ec i eness o immuno he apies depends on no only cance cells, bu also he immune composi ion o he TME, which usually is shaped by TILs, immunosupp esso cells, cy okines, CAFs, o o he molecules ha modula e immune esponses. A be e unde s anding o he in e play be ween b eas umo cells and he mic oen i onmen will p o ide new di ec ions o he apeu ic s a egies. Speci ically, i has been desc ibed ha MDSCs a enua e an i- umo immuni y o p omo e umo g ow h and me as asis in a a ie y o diseases, including BC, and he eby educing he e ec o immuno he apies. Fo ha eason, he e is g ea in e es in disco e ing he complexi y In . J. Mol. Sci. 2023,24, 5208 20 o 28 and he e ogenei y o MDSCs o de elop and design new he apeu ic s a egies. Indeed, se e al he apeu ic app oaches ha e been add essed in o de o modi y he beha io o MDSCs, such as hei down egula ion o inhibi ion o hei unc ions. Fu he mo e, in he coming yea s, i will be possible o assess whe he he combina ion o MDSCs wi h ICIs can o e come he exis ing limi a ions o immuno he apy in cance ea men [ 171 ]. Conside ing ha MDSCs play an impo an ole in he de elopmen o BC and, in u n, MDSCs can be supp essed by using di e en s a egies, we belie e ha immuno he apy a ge ing MDSCs has a b oad u u e pe spec i e. In . J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 20 o 28 conside ed as p omising s a egies o imp o e clinical esul s in e ms o umo g ow h, esponse o ea men , and su i al a es. Al hough immuno he apy may be an e ec i e app oach, u u e s udies should e al- ua e he e ec s o hose ea men s conside ing ha BC p esen s a a ie y o di e en im- munological p o iles. In addi ion, he numbe o cance pa ien s and heal hy indi iduals ec ui ed should be inc eased o alida e ele an immunological bioma ke s clinically [186]. This is impo an because he well-es ablished bioma ke s could p o ide a s a i i- ca ion o he BC pa ien s, which a e c i ical o he e ec i eness o immuno he apy bo h in mono he apy and in combina ion. Howe e , o de e mine he bes s a egy o hese pa- ien s will be a long- e m challenge. We hink ha he immunogenici y o BC will con ib- u e o mo e e ec i e and pe sonalized he apeu ic s a egies o a ge he mos immuno- genic sub ypes [170], wi h mo e a o able oxici y p o iles. Figu e 3. The apeu ic app oaches in b eas cance se ings. Finally, i should be kep in mind ha he e ec i eness o immuno he apies depends on no only cance cells, bu also he immune composi ion o he TME, which usually is shaped by TILs, immunosupp esso cells, cy okines, CAFs, o o he molecules ha mod- ula e immune esponses. A be e unde s anding o he in e play be ween b eas umo cells and he mic oen i onmen will p o ide new di ec ions o he apeu ic s a egies. Speci ically, i has been desc ibed ha MDSCs a enua e an i- umo immuni y o p omo e umo g ow h and me as asis in a a ie y o diseases, including BC, and he eby educing he e ec o immuno he apies. Fo ha eason, he e is g ea in e es in disco e ing he complexi y and he e ogenei y o MDSCs o de elop and design new he apeu ic s a e- gies. Indeed, se e al he apeu ic app oaches ha e been add essed in o de o modi y he beha io o MDSCs, such as hei down egula ion o inhibi ion o hei unc ions. Fu - he mo e, in he coming yea s, i will be possible o assess whe he he combina ion o Figu e 3. The apeu ic app oaches in b eas cance se ings. Au ho Con ibu ions: Concep ualiza ion, M.L.S.-L., L.H.-P., D.J.G.-D., L.d.l.C.-M. and V.S.-M.; W i ing he o iginal d a , M.L.S.-L., L.H.-P., C.J.-C., S.S.R. and D.J.G.-D.; Re ision, M.L.S.-L.; L.H.-P.; D.J.G.-D.; L.d.l.C.-M.; V.S.-M.; S.S.R.; C.G. All au ho s ha e ead and ag eed o he published e sion o he manusc ip . Funding: The wo k o he g oup is suppo ed by PAIDI, Jun a de Andalucia (CTS-151). L. Hon ecillas- P ie o is suppo ed by he Conseje ía de Salud y Familias, Jun a de Andalucía (RH-0047-2021). CJC is suppo ed by a Ma ga i a Salas ellowship, g an ed by he Uni e si y o Se ille (Se ille, Spain). Con lic s o In e es : The au ho s decla e no con lic o in e es . The au ho s ha e designed and concei ed bo h igu es. These igu es ha e no copy igh pe mission o any hi ds. Re e ences 1. 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