Iglesias‑Escudero et al. Molecular Cancer (2023) 22:26 Molecular Cancer https://doi.org/10.1186/s12943-023-01714-0 REVIEW Open Access Regulatory cells and the effect of cancer immunotherapy María Iglesias‑Escudero1,2*, Noelia Arias‑González1 and Eva Martínez‑Cáceres1,2* Abstract Several mechanisms and cell types are involved in the regulation of the immune response. These include mostly regulatory T cells (Tregs), regulatory macrophages (Mregs), myeloid suppressor cells (MDSCs) and other regulatory cell types such as tolerogenic dendritic cells (tolDCs), regulatory B cells (Bregs), and mesenchymal stem cells (MSCs). These regulatory cells, known for their ability to suppress immune responses, can also suppress the anti-tumor immune response. The infiltration of many regulatory cells into tumor tissues is therefore associated with a poor prognosis. There is growing evidence that elimination of Tregs enhances anti-tumor immune responses. However, the systemic depletion of Treg cells can simultaneously cause deleterious autoimmunity. Furthermore, since regulatory cells are characterized by their high level of expression of immune checkpoints, it is also expected that immune checkpoint inhibitors perform part of their function by blocking these molecules and enhancing the immune response. This indicates that immunotherapy does not only act by activating specific effector T cells but can also directly or indirectly attenuate the suppressive activity of regulatory cells in tumor tissues. This review aims to draw together our current knowledge about the effect of immunotherapy on the various types of regulatory cells, and how these effects may be beneficial in the response to immunotherapy. Keywords Tumor, Immunotherapy, Regulatory cells, Checkpoint inhibitors, MDSCs, Tregs, TAMs Introduction [1–3]. Histological analysis of tumors has highlighted Tumors can modify the microenvironment by releas- the importance of immunological infiltrates, including ing extracellular molecules, inducing tumor angiogen- macrophages, dendritic cells, polymorphonuclear cells, esis and promoting peripheral immune tolerance, while natural killer (NK) cells, B cells and T cells, and revealed the immune cells in the microenvironment can affect a wide diversity of these among patients [4]. Although the the growth and evolution of cancerous cells. Increas- extent of the immune infiltrate can be a good prognos- ing amounts of T CD3 + , cytotoxic CD8 + and memory tic indicator in some cancers, the anti-tumor response CD45RO + T cells are associated with greater disease- is clearly insufficient to prevent disease progression. free survival and overall survival (OS) in most studies In inflamed tumors, negative immune regulatory fac- tors tend to be dominant due to the chronic nature of the immune infiltrate. For instance, in a study aimed at *Correspondence: identifying biomarkers associated with clinical outcome María Iglesias‑Escudero in melanoma patients, the decrease in FOXP3 + /regu- miglesiase.germanstrias@gencat.cat Eva Martínez‑Cáceres latory T cells (Tregs) was associated with better clinical Emmartinez.germanstrias@gencat.cat responses in the group treated with ipilimumab [5]. 1 Immunology Division, LCMN, Germans Trias i Pujol University Hospital Therefore, patients with tumors containing infil- and Research Institute, Campus Can Ruti, Badalona, Spain 2 Department of Cellular Biology, Physiology and Immunology, Universitat trates could be induced to respond to immunotherapy if Autònoma de Barcelona, Cerdanyola del Vallès, Spain immune cells within the microenvironment are reacti- vated. On the other hand, the presence of regulatory cells © The Author(s) 2023. 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The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Iglesias‑Escudero et al. Molecular Cancer (2023) 22:26 Page 2 of 15 such as Tregs, MDSCs (myeloid-derived suppressor cells) immunotherapy does not only act by activating specific and TAMs (tumor-associated macrophages) has gener- effector T cells but also can directly or indirectly attenu- ally been associated with a poor clinical prognosis. The ate the suppressive activity of regulatory cells in tumor tis- actions on the vast majority of these components that sues. The aim of this review is to draw together the current have been reviewed here are not as advanced in head knowledge about the effect of immunotherapy on the vari- and neck cancer (HNC) as in other neoplasms such as ous types of regulatory cells, and how these effects may be melanoma. beneficial in the response to immunotherapy (Fig. 1). Although therapy based on immune checkpoint block- ade has achieved outstanding results in terms of OS [6], Tregs a high percentage of patients still show intrinsic resist- The activities of Tregs are the most widely studied of ance [7]. Some features are required to achieve a success- the mechanisms regulating the immune response. They ful response to programmed cell death protein 1 (PD-1) were discovered several decades ago [15] and are known blockade, such as a high frequency of tumor neoantigens, to have a strategic role in the maintenance of immune the amount of infiltrating effector T cells, a high level of homeostasis [16]. Their function has been closely linked expression of PD-L1, or an IFN-related gene signature to the development of diverse pathologies, including [8]. Due to their potent suppressive activities against autoimmunity [17] and cancer [18]. As Tregs are known effector lymphocytes and their abundance in the tumor to express many immune checkpoint inhibitor (ICI) tar- microenvironment, immunosuppressive cells act as a gets, the effect of these immunotherapies could alter major barrier to cancer immunotherapy. A variety of Treg numbers and function. For this reason, a compre- therapeutic approaches directed towards immunosup- hensive understanding of their action in cancer set- pressive cells are actively being tested in preclinical and tings could help increase the efficacy and reduce the clinical studies [9–14]. incidence of immune-related adverse effects after ICI Since regulatory cells are characterized by their high treatment (Fig. 2). level of expression of immune checkpoints, it is also In the clinical setting, the use of daclizumab (a human- expected that immune checkpoint inhibitors perform ized neutralizing monoclonal antibody against the part of their function by blocking these molecules and α-chain of the interleukin-2 receptor) in patients with enhancing the immune responses. This suggests that metastatic melanoma resulted in depletion of Tregs and Fig. 1 Strategies to reduce regulatory cells in cancer patients. Several strategies alone or combining the targeting of different biochemical pathways have been reported to modulate regulatory cells in cancer patients. Some approaches aim to reduce the frequency, function and extent of blocking mobilization, while others focus on enabling phagocytosis, polarization, or potentiating differentiation. Blue crosses indicate potentiation/increase; forbidden sign indicates blockade/reduction Iglesias‑Escudero et al. Molecular Cancer (2023) 22:26 Page 3 of 15 Fig. 2 Effect of immunotherapy on Tregs. Several ICIs and other treatments can directly affect the number and function of regulatory T cells consequently, improving anti-tumoral function and preventing tumor growth. Some treatments act by reducing the number of Tregs (by antibody dependent cellular citotoxicity, ADCC) while others affect their suppressive activity or positively modulate the functions of CD8 T, NK cells and dendritic cells. The modulation towards pro-inflammatory cytokines and the increase in the CD4 + effector/Treg ratio are other mechanisms by which immunotherapy can potentiate the immune response within the tumor microenvironment. Created with Biorender.com Iglesias‑Escudero et al. Molecular Cancer (2023) 22:26 Page 4 of 15 effector T cells, so that augmented T cell responses in of Treg and effector T cells, in other words, whether the the tumor are not induced [19]. In contrast, in a study longer the treatment, the more likely effector T cells are of breast cancer patients, administration of daclizumab to be depleted, thereby hindering the generation of effec- followed by vaccination with multiple tumor-associated tive tumor immunity [18, 35]. The anti-CCR4 antibody, peptides reduced the frequency of Tregs and gave rise to mogamulizumab, has demonstrated an effective reduc- a durable, stable disease with little progression [20–22]. tion in the frequency of effector Tregs that selectively aug- Denileukin diftitox (a ligand–toxin fusion protein that ment the induction of tumor antigen-specific CD4 + and consists of full-length IL-2, and which has been approved CD8 + T cells in vivo [36, 37]. for the treatment of CD25 + cutaneous T-cell leukemia Moreover, anti CCR8 monoclonal antibodies were and lymphoma) was used for lung, ovarian and breast shown to cure tumors in mice by selectively depleting cancer, demonstrating that a single dose reduced the tumor Tregs and increasing CD8 + effector T cells [38]. prevalence and absolute numbers of peripheral Tregs and Van Damme et al. also demonstrated that anti-CCR8 increased effector T-cell activation in all patients [23, 24]. antibodies had antitumor effects and were seen to dis- Considering all the previous results, it was inferred that play synergistic antitumor effects combined with anti- therapeutic success may depend on the phase and state of PD-1 mAbs [39]. More recent studies describe the use of the anti-tumor immune responses [25]. microRNAs -15a/16–1 to regulate immunosuppression Several reports have suggested that the effect of ipili- in hepatocellular carcinoma by reducing CCL22 binding mumab (anti CTLA-4, human antibody IgG1 isotype) to C–C chemokine receptor type 4 on Tregs [40]. on Tregs is mediated only through antibody-dependent New therapeutic strategies are being developed to tar- cell-mediated cytotoxicity, by which their suppressive get the major co-inhibitory and co-stimulatory molecules functions are maintained [26–28]. On the other hand, of Tregs. Some TIGIT monoclonal antibodies (BMS- tremelimumab (anti CTLA-4 antibody IgG2 isotype) has 986207) are under clinical trial in combination with been shown to be able to replenish effector and memory nivolumab for the treatment of advanced solid tumors CD4 + and CD8 + T-cell numbers by suppressing Treg (NCT02913313) and as monotherapy or in combination activity without influencing the proportion of Tregs [29]. A with atezolizumab (anti PD-1 antibody) (MTIG7192A) recent study showed that ipilimumab and tremelimumab [41–43]. Studies of patients with melanoma show that both increase infiltration of intratumoral CD4 + and LAG-3 + Tregs selectively expand in PBMCs and TILs, CD8 + cells without depleting FOXP3 + cells in human bestowing potent suppressive activity on them in a cell- tumors, suggesting that their efficacy could be enhanced to-cell, contact-dependent manner [42]. by modifying the Fc portions of the monoclonal antibodies Other approaches, such as blockade of TIM-3 [44], (mAbs) to enhance Fc-mediated depletion of intratumoral V-domain Ig suppressor of T cell activation (VISTA) [45, regulatory T cells [30]. Also, it has been described that a 46], treatment with glucocorticoid-induced TNFR-related combined treatment with anti B7x may help to overcome (GITR) protein agonistic antibody [47, 48], anti-OX40 anti- the B7x-mediated resistance to anti-CTLA-4 [31]. bodies [49–51], IDO-1 inhibitors [52, 53], TGF-β inhibi- However, the mechanism underlying the action of tors [54]VEGF-targeting therapy receptor 2 (VEGFR2) [55, these antibodies is unknown. The discrepancies could be 56], PI3K inhibitor and HSP inhibitor (phosphoinositide explained by the fact that the number of peripheral Tregs 3-kinase (PI3K) pathway, or heat shock protein (HSP) [57], depends on the genetic background of the patients and the have also yielded promising results. Some drugs in combi- time since immunotherapy that the analysis is performed. nation with immune checkpoint inhibitors are currently the PD1 blockade can induce CD8 + T-cell proliferation and subject of ongoing clinical trials for cancer therapy [58]. cytokine production, thereby negatively regulating Treg Others approaches such as vaccines, nanodrugs [59], numbers by increasing the CD8 + Teff-to-Treg ratio [32]. the generation of chimeric antigen receptor (CAR-)T The suppressive capacity of effector Tregs was enhanced cells, [60] or directly targeting FOXP3 in Tregs with an in some advanced gastric cancer patients treated with antisense oligonucleotide are also under investigation PD1 blockade and was associated with rapid cancer pro- with the aim of reprogramming Tregs [61]. gression [33]. This pattern has also been described by other authors [34]. Considering these findings together, MDSCs the reports suggest that a combination of anti-CTLA4- MDSCs are recognized as one of the major cell compo- depleting Tregs with PD1 blockade could be used to nents in the tumor microenvironment, where they pro- enhance CD8 activation and Treg depletion. It would mote tumor growth by exerting their immunosuppressive also be interesting to study the effects of the timing and functions. MDSCs have emerged as major regulators of duration of such a Treg-targeting antibody treatment, to immune responses in cancer and key targets for treating determine whether it is critical for the differential control cancer [62] (Fig. 3). Iglesias‑Escudero et al. Molecular Cancer (2023) 22:26 Page 5 of 15 Fig. 3 Effect of immunotherapy on MDSCs. The figure depicts different treatments that can modulate MDSC expansion and function. The drugs affect the frequency of MDSCs, reduce MDSC suppressive activity, improve CD8 T cell function, and modulate the release of cytokines from the tumor. A combination of these therapies may have a synergistic effect that promotes better outcomes. Created with Biorender.com Suk Lee et al. reported that anti-thymocyte globulin (ATG)- regulation of pathways involved in the expansion of MDSCs treated mice showed a reduction in MDSCs frequency and [64]. The beneficial effects mediated by AMPK activation function, suggesting that ATG could be used to suppress could therefore be induced by the inhibition of MDSCs func- MDSCs [63]. On the other hand, the role of mitogen-activated tions, and the authors proposed that AMPK activators could protein kinase (MAPK) activation is known to be crucial in the be promising drug candidates for cancer therapy [64, 65]. Iglesias‑Escudero et al. Molecular Cancer (2023) 22:26 Page 6 of 15 In a murine model of neuroblastoma, the adoptive of increasing IL-1ra and suppressing MDSCs-promoting recipient leukocyte infusion (RLI) triggered a potent cytokines. The authors combined PDL1 blockade and the cytotoxic T cell response against the tumor. Conversely, kinase inhibitors cabozantinib and BEZ235, which induce the RLI also provoked a systemic expansion of MDSCs a reduction in MDSCs function. This combined therapy that weakened the CD8 anti-tumoral effect. When these proved to be more effective than single therapy [72]. MDSCs were depleted with 5-FU the inhibition of tumor These findings highlight the necessity of including growth and OS were improved significantly, suggesting MDSCs neutralization in novel strategies of combined that MDSCs targeting is a novel approach for increasing cancer treatment. RLI effectiveness [66]. In another study, Eriksson et al. The combination of immunoregulatory treatments reported reduced levels of Tregs and PMN-MDSCs in with immune checkpoint blockade might represent a pancreatic cancer patients treated with gemcitabine [67]. novel beneficial approach. The regulatory effects of dia- Weiss et al. demonstrated, in two murine models, the rylheptanoid curcumin on STAT3 and JAK2 signaling importance of targeting MDSCs and Tregs in anti-tumor involved the decrease of IL-6 production by MDSCs but immunotherapy. They described how this MDSCs and with no adverse effects detected [73]. Conversely, when Treg reduction was Fas-dependent and, consistent with STAT3 inhibitors were applied in clinical settings for tar- the conclusions of other authors, noted the importance geting tumor-associated myeloid cells, the studies were of targeting these regulatory cells in order to ensure that disrupted by the side effects [74]. Additional approaches the therapies produced successful anti-tumoral activ- aimed at blocking STAT3 exploit the administration ity [68]. Dang et al. investigated the expression of TLR8 of siRNA and decoy oligonucleotides. For instance, on MDSCs and the effect of motolimod, a TLR8 ago- AZD9150 is a STAT3 oligonucleotide inhibitor under nist, on MDSCs survival and function. They found that investigation in combination with immune-checkpoint motolimod treatment reduced MDSCs levels in healthy inhibitors in phase I/II clinical trials [10]. donors and cancer patients, and concluded that TLR8 Various approaches that aim to target MDSCs have agonists could be deployed in conjunction with can- explored the blockade of MDSCs mobilization from the cer immunotherapeutic approaches in order to enhance bone marrow. In this regard, bisphosphonates, drugs the anti-tumor effects of the adaptive immune response administered in cancer patients with bone metasta- [69]. Davis et al. demonstrated functional inhibition of ses, have an MMP-9 inhibitory effect that is related MDSCs with IPI-145, an inhibitor of PI3Kδ and PI3Kγ to reduced MDSCs expansion in peripheral blood isoforms that enhances responses to PD-L1 blockade. and bone marrow [75]. This reduction overcame the These results offer a proof of concept for the low-dose immune suppression and potentiated the anti-tumor use of isoform-specific PI3Kδ/γ inhibitors to suppress response induced by immunization against the p185/ MDSCs and thereby enhance responses to the immune HER-2. In spite of these results, most clinical trials of checkpoint blockade [70]. MMP inhibitors in recent decades have failed because Other approaches focus on promoting MDSCs dif- of side effects: MMP regulates multiple signaling targets ferentiation. A few years ago, Gabrilovich´s group dem- and the inhibition of some MMPs could have pro-tum- onstrated how all-trans-retinoic acid (ATRA) induces origenic effects that impair the benefits of target inhibi- MDSCs differentiation into macrophages and dendritic tion. These effects might be responsible for the failure cells (DCs) [71]. In 2018, Tobin et al. conducted a ran- of MMP inhibitors in clinical trials [76]. More recently, domized phase II clinical trial in which they treated more specific MMP inhibitors with improved toxicity advanced melanoma patients with ipilimumab mono- have been developed [77]. To date, several studies have therapy, or ipilimumab plus ATRA. They showed that indicated that modulating MMP activity can improve treatment with ATRA reduced MDSCs function in mixed immunotherapy. As a consequence, several MMP inhib- lymphocyte reactions. ATRA also reduced the expres- itors are the subject of clinic trials [62, 77]. sion of immunosuppressive genes by MDSCs. Finally, More strategies aimed at decreasing the recruitment of ATRA significantly decreased the frequency of circulat- MDSCs such as targeting tumor glutamine metabolism ing MDSCs relative to ipilimumab treatment alone in [78] and targeting chemokines [79] have been described. advanced-stage melanoma patients [9]. Huber et al. described microRNAs associated with Results published by Xin Lu et al. shed light on the MDSCs features and shorter progression-free survival field of metastatic castration-resistant prostate cancer (PFS) and OS in melanoma patients treated with immune (mCRPC) in relation to the use of immune checkpoint checkpoint inhibitors. These miRs were responsible for blockade for the purpose of blocking MDSCs. They devel- the conversion of monocytes into MDSCs mediated by oped a chimeric mouse model of mCRPC with which melanoma extracellular vesicles (EVs) [80]. The authors they demonstrated that combined therapies were capable argued that the role of the identified MDSC-miRs may Iglesias‑Escudero et al. Molecular Cancer (2023) 22:26 Page 7 of 15 reflect functional features that MDSCs display upon into several populations based on their specific func- immune activation triggered by ICIs, because resistance tions: M2a induced by Type2 cytokines, are responsible to ICIs can be reverted by myeloid cell depletion. This for mediating tissue repair; M2b induced by immune highlights the need for drugs that are capable of blocking complexes, TLRs and IL-1R are known for their role in myeloid cells dysfunction. Hence, MDSC-related miRs immunoregulation; M2c macrophages induced by anti- are potential biomarkers for assessing systemic immu- inflammatory cytokines affect phagocytosis and M2d nosuppression and immunotherapy outcomes in cancer induced by IL-6 like cytokines participate in angiogenesis patients [80]. [91]. Limagne et al. reported the outcomes of a prospective It is accepted that TAMs influence the tumorigenic immunomonitoring study of 25 metastatic colorectal can- process because they promote immunosuppression in the cer (mCRC) patients treated with a first-line combination tumor microenvironment. In this regard, several stud- regimen of 5-fluorouracil 5-FU, oxaliplatin and bevaci- ies have demonstrated that regulation of TAM responses zumab (FOLFOX–bevacizumab), compared with healthy may enhance immunotherapy [92]. Therefore, a wide volunteers. FOLFOX–bevacizumab treatment produced range of strategies to deplete TAMs have been investi- a drop in gMDSCs in most patients and was associated gated in experimental settings and are now considered a with better survival. Moreover, those gMDSCs expressing promising therapeutic approach in the clinic [92] (Fig. 4). higher levels of PD-L1, CD39 and CD73 showed potent Bisphosphonates have been successful in reducing immunosuppressive activity that could be reversed by TAM infiltration in vivo as they promote transformation blocking the CD39/CD73 and PD-1/PD-L1 axes [81]. of M2-like macrophages into M1-like macrophages. A The inhibition of prostaglandin E2 receptor 4 (EP4) in large number of bisphosphonate derivatives are used to MDSCs may offer a strategy for enhancing the efficacy inhibit TAMs and treat tumors [93]. Likewise, an RNA of immunotherapy in colorectal cancer [82]. Li T et al. aptamer that blocks the murine or human IL-4receptor-α showed that pharmaceutical inhibition of c-Rel in mice (IL4Rα or CD124) can promote TAM elimination, an markedly inhibited cancer growth and suggest c-Rel as effect that is associated with an increased number of a myeloid checkpoint that may be targeted for treating tumor-infiltrating T cells and a reduction in tumor cancer [83]. Similarly, S100A9-derived peptides conju- growth [94]. Allavena et al. have shown that trabectedin gated to antibody Fc [84] and targeting the TNF-related activates caspase-8 and induces apoptosis in the presence apoptosis-induced ligand (TRAIL) receptor could be a of death receptor 5 (DR5), which is present in TAMs with potent and selective method for MDSCs depletion [85]. death receptors [95]. Several drugs against CSF1R have shown promising anti- In addition, specific antibodies can be used to reduce tumor efficacies by inhibiting the survival of M-MDSCs TAMs. Zhang et al. [96] synthesized IRD-αCD206, a and tumor associated macrophages (TAMs) and are TAM probe, that conjugated anti-CD206 antibodies with being tested in in cancer patients [86]. near-infrared phthalocyanine dye and successfully inhib- In summary, several candidate drugs have been pro- ited growth in a sorafenib-resistant tumor model. CD11b posed for reducing MDSCs frequency and function; is an important molecule expressed in myeloid cells that other approaches aim to modulate MDSCs differentia- works as an oncogene that can be targeted in colorectal tion or mobilization. A combination of immunoregula- cancer (CRC) [97]. Also, a recombinant immunotoxin tory drugs with ICBs might be more effective in targeting consisting of FR-β monoclonal antibodies (mAbs), which MDSCs as anti-tumor immunotherapy. are expressed in TAMs, suppressed tumor growth in a glioma model by depleting TAMs [98]. TAMs Other mechanisms targeting TAMs have been devel- Macrophages play an important role in regulating the oped to inhibit monocyte recruitment. In this regard, the innate immune system. On the one hand, they promote inhibition of CSF-1/CSF-1 receptor (CSF-1R) signaling inflammation and eliminate pathogens. On the other, has been effective because this axis is essential for mac- depending on their specific responses and cytokines, rophage survival. Also, targeting CSF-1/CSF-1R alters macrophages can induce immune stimulation and immu- macrophage polarization and blocks glioma progres- nosuppression as well as promote or inhibit inflam- sion [99]. RG7155, a monoclonal antibody that inhibits mation [87, 88]. Macrophages display a high degree of CSF-1R, has been shown to inhibit CSF-1R and thereby plasticity in response to different microenvironments decrease F4/80 + TAMs in vitro and in vivo [100]. Pre- [89]. Classic macrophages (M1) are known for their clinical studies have demonstrated that another CSF-1R role in promoting immune responses. Conversely, M2 inhibitor, BLZ945, inhibits tumor growth in different macrophages are associated with a reduction of tissue mouse models [101] and its potential use in treating solid inflammation [90]. M2 macrophages can be classified tumors is being studied in clinical trials [12]. Iglesias‑Escudero et al. Molecular Cancer (2023) 22:26 Page 8 of 15 Fig. 4 Effect of immunotherapy on TAMs. The figure displays treatments that aim to prevent the expansion of TAMs, consequently enhancing the anti-tumoral effect. The drugs work by promoting apoptosis (which reduces TAM frequencies)and TAM differentiation to M1 macrophages, and by blocking TAM mobilization, infiltration and metastasis. On the other hand, there is an increase in pro-inflammatory cytokine release and a reduction in the phagocytic activity of macrophages. Also, the use of these treatments combined with chemotherapy or other ICIs may be synergistic, bursting the immune response against the tumor. Created with Biorender.com The anti-tumor agent dequalinium-14 can reduce mac- locally irradiated mice. Interferon-γ (IFN-γ) may induce rophage motility, inhibit macrophage infiltration of irra- recruitment of monocytes/macrophages into the tumor diated tumors, and reduce the extent of metastasis in microenvironment but inhibits their differentiation into Iglesias‑Escudero et al. Molecular Cancer (2023) 22:26 Page 9 of 15 TAMs in vivo; this effect may reduce the concentration TLR agonists induce pro-inflammatory cytokines and of VEGF and angiogenesis in a tumor [102]. Anti-CCL2 reprogram macrophages [111, 112]. In addition to TLR antibodies have been shown to inhibit tumor growth agonists inducing cytokine release, stimulator of IFN in several animal models, such as glioma, colon cancer, gene (STING) agonists have also been shown to repro- prostate cancer, and melanoma. Tumor growth inhibition gram macrophages [113–115]. is observed with the use of Anti-CCL2 antibodies alone In tumor models of colon and pancreatic cancers, or in combination with chemotherapeutic drugs [13, 14]. TAMs produce IL-6 by activating the STAT3 signaling In humans, a phase 1b clinical study showed that the pathway to promote the proliferation of colon tumor cells combination of chemotherapy with the anti-CCL2 anti- [116, 117]. Therefore, STAT3 inhibitors affect polariza- body, carlumab, for treatment of solid tumors was well tion of TAMs in tumor therapy. Several studies have tolerated but could sequester CCL2 for only a short time shown that application of STAT3 inhibitors to several [13]. Inhibition of CCR2 can also block the CCL2/CCR2 tumor models has therapeutic effects [118–120]. Some axis to reduce the TAM population. PF-136309, a CCR2 studies report the importance of the cellular metabolism inhibitor, proved to be safe and tolerable when used in in TAMs as an approach to abrogating the immunosup- combination with chemotherapy, yielding an objective pressive effects of TAMs [121, 122]. tumor response in clinical trials [14]. The phagocytic activity of macrophages can be inhib- A different approach involves regulating TAM polari- ited by blocking the interaction signals with cell sur- zation. For example, dimethyl sulfoxide can revert TAM face proteins. Weissman et al. described three different polarization from M2 to M1 in a mouse 4T1 breast pathways that inhibit phagocytosis: the signal regula- cancer model [103]. Overmeire et al. demonstrated tory protein alpha (SIRPα)/CD47 pathway, the major that M-CSFR signaling shapes the M2-like phenotype histocompatibility complex class I/leukocyte immuno- in TAMs, and its blockade favored the shift towards globulin (Ig)-like receptor subfamily B member 1 (MHC- M1-like TAMs [104]. IFN-γ and celecoxib increased the 1/LILRB1) pathway, and the CD24/sialic acid-binding percentage of M1 macrophages and decreased that of M2 Ig-like lectin 10 (Siglec-10) pathway [123–125]. macrophages in lung tumors, suggesting that IFN-γ and Currently, since blocking those pathways enables celecoxib have the potential to be further optimized as a phagocytosis, “don’t eat me” signals are considered to be new anti-cancer therapy [105]. phagocytic checkpoints in macrophages, which serve as As mentioned above, the release of pro-inflamma- specific immune checkpoints for innate immunity. Some tory cytokines such as IFN-γ, TNF-α, and IL-12 induce drugs targeting the SIRPα/CD47 signaling pathway have transformation of TAMs into M1-like macrophages. been widely studied in anti-tumor treatment, and most Therefore, promoting the release of inflammatory fac- have been subjected to clinical trials [126, 127]. However, tors, specifically through antibodies and small molecu- drugs targeting the MHC-1/LILRB1 and Siglec-10 path- lar agents, can promote the tumor immune response. A ways are still being investigated. large number of studies have shown that the use of CD40 In a report published in 2021, the authors identified mAb can upregulate the level of proinflammatory factors Fc domain-enhanced anti-TREM2 monoclonal antibody and enhance the body’s response to tumor cells by regu- therapy promotes anti-tumor responses by modulation lating the adaptive and inherent immune systems [106, of TAM populations [128]. In non-small cell lung can- 107]. In addition, the combination of anti-CD40 and anti- cer (NSCLC) patients ILT4 inhibition prevented immu- CSF-1R not only promotes the maturation and differen- nosuppression and tumor promotion by bloquing the tiation of inflammatory macrophages and DCs but also recruitment of M2-like TAMs [129]. Monoamine oxidase drives the effective initiation of effector T cells during A (MAO-A) inhibition treatment induces TAM repro- cancer immunotherapy [108]. In addition to CD40 anti- gramming and suppresses tumor growth in preclinical bodies, other small molecules can enhance the secretion mouse syngeneic and human xenograft tumor models of pro-inflammatory cytokines. Thymosin-α activates [130]. Another recent report published by Hezaveh K and converts TAMs into pro-inflammatory cell subsets et al. [131] demonstrated that pharmacologic inhibi- that produce IL-1, TNF-α, reactive oxygen species (ROS), tion of aryl hydrocarbon receptor (AhR) in myeloid cells and nitric oxide (NO). Furthermore, several clinical trials reduced pancreatic ductal adenocarcinoma growth and have confirmed that thymosin-α can prolong the survival improved efficacy of immune checkpoint blockade, and time of patients with metastatic melanoma and advanced increased intra-tumoral frequencies of IFNγ+CD8+ T non-small-cell lung cancer (NSCLC) [109]. cells. The application of nanoparticles to target the tumor TLRs stimulation can lead to the expression and secre- microenvironment is also being explored in combination tion of a variety of pro-inflammatory cytokines such as with other therapies [132]. TNF-α, IL-12, and IL-1 [110]. Several studies proved that Iglesias‑Escudero et al. Molecular Cancer (2023) 22:26 Page 10 of 15 In conclusion, several strategies for depleting TAMs death-derived, whole-tumor antigen can synergize with in the tumor microenvironment are being explored and anti-PD1 treatment [138]. are producing promising results. The use of these strat- Tumor antigen-loaded cDC1s were transferred into egies combined with chemotherapy or immunotherapy three cancer models in combination with anti-PD1 treat- could potentiate the anti-tumoral effect by promoting the ment, which had a strong synergistic effect [139]. Moreo- immune response to the tumor. ver, tumors grafted onto BATF3-deficient mice, which lack cDC1s, did not respond to anti-PD1, anti-PDL1, or Other regulatory cells anti-CD137 treatments, and SEC22B-mediated cross- The effect of immunotherapy in other cells with immu- presentation of TAAs by DCsis necessary for effective noregulatory properties, such as regulatory DC, B cells PD1 blockade therapy [140]. or MSCs, is still under investigation. Although there are Synergy of TLR-mediated activation of DCs and ICB very few reports about the role of Bregs in human can- can be further improved by FLT3L-mediated expansion cer, some preclinical studies have targeted them in a vari- of DC populations [141]. ety of cancer models [133]. Mitogen/extracellular signal Further evidence that cross-priming is the critical func- regulated kinase (MEK) is an intermediary component of tion mediated by cDC1 in this context has come from the MAPK pathway, and its inhibition affects tumors in WDFY4-deficient mice, which fail to reject immunogenic which MAPK is activated, alone or in combination with tumors due to a defect in a vesicular transport pathway other therapies. Mechanistically, MEK inhibition may needed for cross-presentation [142]. down regulate the expression of surface molecules asso- Wdfy4 −/− mice failed to prime virus-specific C ­ D8+ T ciated with suppressive functions [134–136]. cells in vivo or induce tumor rejection, revealing a critical Das and colleagues investigated Bruton’s tyrosine role for cross-presentation in anti-viral and anti-tumor kinase (BTK) as a potential modulator of Breg differen- immunity [142]. tiation and the immunosuppressive function [137] and Enhancing DC functionality may improve and/or found that tirabrutinib, a BTK inhibitor, suppressed Breg broaden responsiveness to ICB regimens. cGAS and differentiation as well as IL-10 and IL-35 production STING are both necessary for intrinsic anti-tumor in vitro. immunity and efficient responses to anti-PDL1, which is These studies have confirmed that the inhibition of at least partially mediated by DCs [143]. Breg may help block cancer progression. Further research Targeting type I interferons to activate cDC1s also is needed to develop a Breg-targeting therapeutic regi- improves anti-PDL1 treatment, suggesting that tumor men for cancer. DCs may require activation to support ICB-induced Though immunotherapy strategies, such as DC-based effector T cell activity. cancer vaccination, have been developed that are based Increasing DC chemokine production, may also on the ability of DCs to coordinate innate and adap- increase responsiveness to ICB [144]. In turn, ICB pro- tive immune responses [19], the role of TolDC, which is motes DC accumulation within the TME. Combining related to the effect of immunotherapy in cancer, remains pembrolizumab (anti-PD1) treatment with TLR9 ago- poorly studied. nists is associated with an elevated tumor-infiltrating DC In the context of the tumor microenvironment, tumor signature and an initial clinical benefit [145]. cells can promote DCs with regulatory features [138]. Mesenchymal stem cells (MSCs) are multipotent stro- Several approaches have been pursued that aim to acti- mal cells that can differentiate into various cell types. A vate DCs to suppress cancer progression, among them large number of studies have shown the beneficial effects immunotherapy, which can affect DCs, promoting their of MSC-based therapies in treating various pathologies differentiation functions. [146]. However, the therapeutic potential of MSCs in The anti-tumoral immune response induced by DCs cancer is still controversial. Some studies indicate that can be amplified using monoclonal antibodies against they may contribute to cancer pathogenesis: MSCs can coinhibitory receptors (such as the PD1–PDL1 axis) migrate to chronic inflammatory sites such as cancer, or antibodies that potentiate the activation of costim- where they contribute to metastasis by secreting TGF, ulatory receptors (such as CD137) on T cells. Experi- which promotes EMT [147]. MSCs inhibit the prolif- mental melanomas with stabilized β-catenin signaling eration of T and B cells [148], suppress the activation are associated with reduced cDC1 tumor infiltration of natural killer cells [149], and prevent generation and and nonresponsiveness to immune checkpoint block- maturation of monocyte-derived dendritic cells [150]. ade (ICB) therapy. Indeed, vaccination with natu- Furthermore, MSCs can promote the generation of reg- rally occurring cDC1s loaded with immunogenic cell ulatory T cells [151], which exert immunosuppressive effects. However, the unmodified MSCs have been shown Iglesias‑Escudero et al. Molecular Cancer (2023) 22:26 Page 11 of 15 to have anti-tumor effects in vitro and in several mouse of these regulatory cells. When an immunosuppressive models of cancer [152]. tumor microenvironment is already established, it will be The unique ability of MSCs to home to tumors and to more difficult to overcome and the immunotherapy will directly transport anti-cancer agents to neoplastic niches be less effective. renders them potential therapeutic vehicles for lung Although there are some current indications of the use of cancer. Genetic engineering is one of the most common ICIs as first-line treatments, one of the main difficulties in strategies used to produce MSCs delivering tumor-sup- clinical trials is that they were carried out in patients with pressing agents into cancer cells. Several studies provide advanced-stage tumors that were refractory to chemo- compelling evidence that MSCs can be genetically engi- therapy. Therefore, in tumors with low immunotherapeutic neered to deliver anti-tumor drugs (PTX, DOX) and efficacy, it is plausible to consider that their failure to reach immunomodulatory factors (IL-12, IL-24, IFN-ϒ, IFN-β, clinical end-points was due to the trial design. TRAIL, PEDF, apotin, CDA/UPRT and CX3CL1) to tar- Another challenge is that, in order to have validated get cells, thereby conferring anti-tumor/anti-metastatic drugs, it is necessary to study the possible anti-target actions [153]. effects of these molecules. The improvement in the Aside from their anti-cancer effects, MSCs are of design of clinical trials and the deep knowledge of the special relevance for personalized cell-based therapies target and off-target effects of the treatments could lead because they can be easily obtained by minimally inva- to improved toxicity profiles and more selective drugs sive procedures and rapidly scaled up [154]. To date, 25 with less severe side-effects. clinical trials that aim to use MSCs under various cancer The discovery of new molecular targets could also help conditions have been registered on ClinicalTrials.gov. predict the tumors that are most immunogenic and most Fourteen of these trials are using MSCs as a therapeutic likely to respond to treatment, thereby making it possible agent to treat cancer directly. The use of MSCs as Trojan to detect responders to immunotherapy. horses to deliver therapeutic factors represents an impor- In tumors characterized by a highly immunosuppres- tant step forward in the application of MSC-based thera- sive environment, the development of new treatment pies to provide more efficient cancer treatment [155]. options should certainly explore the efficacy of different The effect of immunotherapy in other cells with immu- drug combinations. noregulatory properties, such as CD8 and NK regula- Numerous ongoing studies and clinical trials are tory cells remains to be determined. In this regard, in exploring chemo-immunotherapeutic combinations spite of the lack of studies regarding these regulatory that aim not only to eradicate the tumor mass but also cells in human cancer, it is likely that strategies devel- to neutralize tumor-induced immunosuppression, oped to target other regulatory cells will also affect these thereby facilitating the effect of concurrent immuno- subpopulations. therapy. Results obtained from these studies will shed light on the field and enable established approaches to Conclusions be modified in order to better treat cancer patients in The extent of the immune infiltrate can be a good prog- the next few years. nostic indicator in some cancers, but the anti-tumor immune response is, in most cases, insufficient to pre- Abbreviations vent disease progression. Several mechanisms and cell Tregs Regulatory T cells Mregs Regulatory macrophages types are involved in negatively regulating the immune MDSCs Myeloid suppressor cells response, and the infiltration of large numbers of regu- tolDCs Tolerogenic dendritic cells latory cells into tumor tissues is associated with poor Bregs Regulatory B cells MSCs Mesenchymal stem cells prognosis. ICIs immune checkpoint inhibitors In inflamed tumors, negative immune regulatory fac- CTLA-4 cytotoxic T-lymphocyte antigen 4 tors tend to dominate due to the chronic nature of the PD-1 programmed cell death protein 1 PBMCs peripheral blood mononuclear cells immune infiltrate. In cold tumors, it has been postulated TILs tumor-infiltrating lymphocytes that resistance to treatment may be due to low antigenic- mAbs monoclonal antibodies ity, which causes less lymphocytic infiltration and is ATG​ anti-thymocyte globulin MAPK mitogen-activated protein kinase associated with an increase in the frequency of immuno- AMPK mitogen-activated protein kinase pathway suppressive cells and other factors. RLI recipient leukocyte infusion Therapeutic success may depend on the phase and state TAMs tumor-associated macrophages of anti-tumor immune responses as the timing of the treatments seems to be critical to the differential control Iglesias‑Escudero et al. Molecular Cancer (2023) 22:26 Page 12 of 15 Acknowledgements 10. Fleming V, Hu X, Weber R, Nagibin V, Groth C, Altevogt P, et al. Targeting The authors would like to thank Dr. Climent and Dr. Fondelli for providing myeloid-derived suppressor cells to bypass tumor-induced immuno‑ scientific consultation. suppression. 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