Dysregulated tumor-associated macrophages in carcinogenesis, progression and targeted therapy of gynecological and breast cancers

Cancer cells are thought to divide out of control and proliferate indefinitely, making them difficult to be removed and destroying the surrounding normal tissues. Different studies have shown that TAMs secrete IL-6 [78, 79], which is a cytokine belonging to the interleukin family associated with tumorigenesis. It has been proved that IL-6 played a vital role through IL-6-JAK-STAT signal transduction [80‐82], and activation of STAT3 was an important factor affecting cell proliferation, differentiation and survival [83]. STAT3 protein level and phosphorylation state were significantly induced in HPV-positive cervical cancer cells compared with HPV-negative ones, which has been demonstrated to be mediated by HPV E6 oncoprotein [84]. Meanwhile, Luan et al. observed a synchronous increase in IL-6R expression of cervical cancer cells, which appears to be detrimental to patients’ prognosis [85]. This suggests that IL-6 secreted by TAMs may affect tumor proliferation, migration and adhesion through up-regulated IL-6R. However, the activation of IL6/STAT3 pathway which was found in a brand-new study promoted the metastasis of ER⁺ intraductal breast cancer in vivo, and it strikingly constituted another oncogenic pathway which was relatively independent of ER pathway [86].

Another member of interleukin family, CXCL8 (also known as IL-8), has been widely concerned in tumorigenesis due to its pluripotency including angiogenesis and mitogenic effects [87]. CXCL8 is mainly secreted by mononuclear macrophages. It has been reported that TAMs down-regulated the expression of ERα in endometrial carcinoma tissues by over-secretion of CXCL8 via homeodomain transcription factor HOXB13, which is associated with tumor invasion and poor prognosis [88]. The parallel results were found in ERα⁺ breast cancer. Up-regulated HOXB13 accelerated cell proliferation and metastasis both in vitro and in vivo through direct suppression of ERα, which could be inhibited by reducing the expression of mTOR [89]. In the meantime, one research found that the expression of ERα in cervical cancer decreased by more than 15 times as cervical cancer progressed from normal to cancer [90]. However, the relationship between ERα reduction and TAMs in cervical cancer cells has not been reported.

Moreover, it has been demonstrated that estrogen, IL-6 and CXCL8 are capable of interacting with each other to regulate the growth of epithelial ovarian cancer through cascade amplification effect via an ERα pathway [91, 92].

The increasing division and growth of cancer cells indicate that they need more nutrients from the blood. Therefore, tumor angiogenesis plays a pivotal role in solid tumor growth, invasion and metastasis. Recently, TAMs have been shown to tight tie with angiogenesis and lymphangiogenesis through the secretion of VEGF-A, VEGF -C, VEGF-D, EGF, placental growth factor (PlGF), TGF-β, TNF-α, IL-1β, IL-8, CCL2, CXCL8 and CXCL12 [93]. In breast cancer, TAM infiltration is closely associated with VEGF expression and microvascular density (MVD), which jointly affects the prognosis of patients [94]. In addition, previous studies have confirmed that VEGF-C, VEGF-D and their receptor VEGFR-3 were positively correlated with different stages of cervical lesions, allowing the development of lymphangion [95, 96]. Interestingly, the expression of VEGF-C and VEGF-D increased significantly after the disease progressed to CIN3 stage [96]. However, the opposite conclusion has been drawn in endometrial cancer [95]. One study reported that lower level of VEGF-C was detected in endometrial cancer samples [95]. We suspect tha t the char acteristics of different tumors and the limited sample size may account for the controversy.

On the other hand, TAMs expressed HIF-1α to active angiogenesis [97]. In breast cancer, HIF-1α-stabilizing long noncoding RNA (HISLA) was proved to prevent the inactivation of HIF-1α from hydroxylation by prolyl hydroxylase domain 2 (PHD2) [98]. Moreover, HISLA produced by extracellular vesicles released by TAMs can inhibit the interaction between HIF-1 α and PHD2 and promote the stability of HIF-1α in TME [97].

In the study of transcoelomic metastasis of ovarian cancer, TAMs in tumor clusters (spheroids) motivated EGF expression at early stage of metastasis which was integrated with activated tumor EGFR, further supporting tumor cell proliferation and migration by up-regulating VEGF/VEGFR signaling in surrounding tumor cells [99]. EGF was also observed to increase αMβ2 integrin on TAMs and ICAM-1 on tumor cells, promoting the adhesion between TAMs and tumor cells [99]. Ishihara et al. discovered that Wiskott–Aldrich syndrome protein (WASp) allowed TAMs to secrete EGF and approach CSF-1-expressing breast cancer cells chemotactically to regulate tumor metastasis [100].

EMT and extracellular matrix remodeling constitute remarkable links in promoting tumor metastasis. Jing et al. revealed that under the action of ERα agonists, ERα⁺ M2 macrophages significantly up-regulated CCL18 and activated the mTOR/KIF5B signaling pathway in endometrial cancer cells to promote EMT [101].

Matrix metalloproteinases (MMPs), which are widely presented in TME, can not only facilitate EMT, but also promote lymphangiogenesis in conjunction with the VEGF [93]. Recently, one study demonstrated that M2-like macrophages have been found to secrete large amounts of MMP-9 that promoted tumor progression both in breast cancer and in colon cancer, which was considered as prognostic markers and therapeutic targets [102, 103]. In ER⁺ breast cancer, MMP-9 secreted by TAMs was associated with worse overall survival, but not in ER-negative breast cancer or triple-negative breast cancer (TNBC) [104]. Wen et al. reported that MMP-7 elevation in macrophages which was co-cultured with ovarian cancer cells occurred through the p38 pathway and promoted the infiltration of macrophages to form TME [105]. At the same time, the majority of the studies supported the view that increased MMP-9 can also promote the development of cervical cancer [106, 107], although a few articles have suggested that MMP-9 was helpful for cervical cancer prognosis [103]. Therefore, taking into account the differences in researches, the effect of MMP on cervical cancer needs to be further optimized.

Moreover, TAMs also exert an enormous function in promoting tumor cell metastasis by releasing urokinase-type plasminogen activator (uPA) and up-regulating the expression of uPA receptor (uPAR), which belongs to the fibrinolysis system [108]. The combination of uPA and uPAR initiates the reactions to reshape the extracellular matrix by degrading the relative components and thereafter leads to the distant migration of tumor cells from the primary site [109]. Additionally, increased uPAR can also induce macrophage aggregation to tumor tissue, forming a positive feedback [110]. In breast ductal carcinoma in situ and invasive breast carcinomas, blood monocytes with higher uPAR levels might be selectively recruited into cancer tissues, which induced elevated uPAR levels in TAMs through paracrine action as well [110].

As a member of transforming growth factors, TGF-β is a multifunctional cytokine. It acts as a potent tumor suppressor in the early stage of cancers, while later these cytostatic effects become resistant and TGF-β is involved in inducing EMT [111]. With regard to high-grade serous ovarian cancer, ascites with high levels of TAMs and TGF-β1 facilitated cancer cell migration [112]. In cervical cancer, GDF15 which belongs to TGF-β superfamily phosphorylated PI3K/AKT and MAPK/ERK signaling pathways via ErbB2, a member of EGFR family, to achieve the purpose of promoting tumor progression [113].

Tumor cells escape from recognition and attack by the body's immune system through multitudinous mechanisms. IL-10 is a multicellular cytokine with immunosuppressive effects and can inhibit antigen delivery by the mononuclear phagocyte system [114, 115]. One previous study found that IL-10 promoted the transformation of macrophages into M2-like macrophages in TME, and polarized M2-like macrophages in turn highly secrete IL-10 [116]. In cervical lesions, it has been reported that the amount of IL-10 in TME was associated with the type of HPV infection and the degree of progression from lesions to cervical cancer. Moreover, with the significantly increase in IL-10, the expression of HPV E6 and E7 was detected to augment [117]. Besides, IL-10 has been proven to play a role in anti-tumor T cells [119, 120]. In breast cancer, macrophage-derived IL-10 overexpression resulted in tumor development via inefficiency of CD8⁺ T cell-dependent responses to chemotherapy [121]. As Chu et al. reported, IL-10 was also an independent factor for adverse prognosis in patients with ER-negative breast cancer [122].

In ovarian cancer tissues, Qu et al. discovered the increased expression of PD-L1 on the surface of TAMs, which stimulated apoptosis of T cells to suppress immunity [123]. Up-to-date study has found that overexpression of human epididymis protein 4 (HE4) in ovarian cancer cells may be one of the reasons for the elevated PD-L1 by macrophages [124]. In the meantime, TAMs as well as IFN-γ, TNF-α, IL-10 and especially IL-6 secreted by TAMs induced the expression of PD-L1 both in the cell membrane and in the cytoplasm of ovarian cancer cells, which inhibited the function of CD8⁺ T cells and promoted tumor growth [125]. The same inhibitory effect on T cells was also detected in HPV16-associated cervical cancer through M2 macrophages [119]. Recently, in studies of progression of stromal tumors, the inhibition of CD8⁺ T cells by PD-1/PD-L1 may be realized through the PI3K/Akt/mTOR signaling pathway [126]. Additionally, hypoxic macrophages promote the release of exosomes containing miR-223. It can be transferred to co-cultured epithelial ovarian cancer cells to enhance drug resistance through the PTENPI3K/AKT pathway both in vivo and in vitro [127]. As mentioned above, accompanied by TME hypoxia, VEGF is overexpressed in macrophages. It not only drives angiogenesis, but also suppresses immune cells from playing their normal role [128]. For T cells, VEGF blocks the differentiation of T progenitor cells into CD4⁺ and CD8⁺ lymphocytes, weakens the immune function of T cells. On the other hand, VEGF increases the infiltration of TAMs into tumor sites which forms a positive feedback from VEGF to TAMs [129].

Taken together, TAMs are involved in the secretion of cytokines and growth factors to influence tumor growth, metastasis and progression. Different types of gynecological and breast cancers have similar or specific characteristics of TAMs (Fig. 4). Comprehensive studies need to be carried out to find more factors secreted by TAMs which take part in tumor regulation, providing more insight into targeted treatments toward TAMs.

The polarization of TAMs is regulated by a variety of microenvironmental cytokines, growth factors and signals from both tumor and stromal cells, which is a process of mutual conversion between M1 and M2 [14]. Considering the extensive role of TAMs in TME, controlling the number of TAMs, in other words, suppressing the polarization of M2 macrophages or guiding macrophages to differentiate into M1-like have wide interest as a new treatment strategy.

In terms of reducing the number of TAMs, clodronate has emerged as the primary choice for drugs to deplete macrophages [130]. In cervical cancer, Lepique et al. injected clodronate-containing liposomes (CL) in a mouse model to consume macrophages by accumulating methylene-containing ATP analogues in the cytoplasm. Although CL did not directly regulate the cytoactive of cancer cells, the reduced number of macrophages inhibited tumor growth in vivo and contribute to the infiltration of tumors by HPV16 E7(49–57)-specific CD8 lymphocytes [119]. However, the therapy still needs further optimization to meet the clinical needs, for example, how to focus on TAMs without interfering with other macrophages in normal body tissues.

In another way, impeding the recruitment of TAMs into tumor tissue has broad application prospects in the future by preventing TAMs recruitment through cytokines. Engeletin (ENG, dihydrokaempferol 3-rhamnoside) is a substance which has anti-inflammatory properties [131, 132]. In cervical cancer, one research found that ENG significantly reduced the secretion of CCL2 via blocking NF-κB signaling pathway, which could be considered as an inhibitor of NF-κB [133]. Due to the help of ENG, monocytes in peripheral blood were less recruited to TME. CSF1/CSF1R inhibitors such as MCS110, Pexidartinib (PLX3397, PLX108-01) and Emactuzumab (RG7155) are being studied in clinical trials in solid tumors including ovarian and endometrial cancers [43, 134]. Heusinkveld et al. reported that a certain concentration of PGE2 and IL-6 could induce CD14⁺ macrophages to differentiate into CD163⁺ M2 macrophages [34]. Fortunately, the effect of PGE and IL-6 was completely blocked by the cox-oxidase inhibitor and IL-6 antibodies [34]. Similarly, the use of inhibitors of various pro-factors such as IL-4, IL-10, TGF-β and IL-13 may reduce the migration of TAMs toward the tumor microenvironment to a certain extent [135‐137].

In cervical cancer, compared with HPV preventive vaccine which has been already universally in use as a countermeasure of HPV-associated cervical cancer [138], the therapeutic HPV vaccine is still in development. In a vaccine trial involving the HPV16 E7 43–77 peptide, a decrease was observed in the number of M2 macrophages and the expression of M2 macrophage-related cytokines, such as IL10, TGF-β, MMP-2, MMP-9 and VEGF in the vaccine group [139], while M1 macrophage-related chemokines (such as CXCL-9 and CXCL-10) were up-regulated [139].

As mentioned above, low M1/M2 rate often predicts poor patients’ prognosis [140]. Therefore, trying to increase the number of M1 or improve the ratio of M1/M2 is a feasible way for possible treatment [141]. Indoleamine-2,3-dioxygenase (IDO1) is a key enzyme to degrade amino acid tryptophan through the kynurenine pathway [142], which can regulate macrophages and inhibit the immune function of T cells in a variety of diseases and cancers [142‐145]. In cervical cancer, existing studies have shown that IFN-γ activated autophagy of cervical cancer cells and macrophage phagocytosis reactivation through IDO1 overexpression and kynurenine metabolism [146, 147]. In addition to cervical cancer, Feng et al. confirmed that high expression of IDO1 showed better survival prognosis in breast and ovarian cancer [148]. Although these studies revealed that up-regulation of IDO1 was beneficial to the suppression of cancer to some degree, more studies indicated that overexpressed IDO1 was related to immunosuppression and played an adverse role in the process of tumor development [149, 150].

Cancer cells express CD47 to protect cells from phagocytosis by binding and activating the receptor SIRPα on macrophages to inhibit macrophage phagocytosis. Targeting CD47-SIRPα axis has become a potential therapeutic mechanism in various preclinical models by stimulating the phagocytosis of cancer cells in vitro and in vivo [151, 152]. Up-regulated expression of CD47 was found in almost all types of tumor cells, which was associated with poor survival and prognosis [153‐155]. Huang et al. constructed a novel oncolytic adenovirus carrying a signal regulatory protein-α (SIRPα)-IgG1 Fc fusion gene (termed SG635-SF), which significantly inhibited the growth of ovarian cancer cells in vivo [156]. Samanta et al. reported that treatment of TNBC cells with carboplatin, doxorubicin, gemcitabine or paclitaxel promoted the enrichment of CD47⁺CD73⁺PDL1⁺ TNBC cells and promoted tumor immune evasion, which could be eliminated by inhibiting HIF [157]. Based on the crucial role of CD47-SIRPα axis in tumor immune escape, clinical trials of anti-CD47 antibodies are under way, involving drugs such as Hu5F9-G4, HX009 and AK117. A breast cancer study found that CD47 and HER2 activated each other at the transcriptional level [158]. Double antibody blocking CD47 and HER2 not only inhibited tumor clone formation, but also enhanced macrophage-mediated attack [158]. In TNBC, anti-CD47 antibody inhibited the differentiation of cancer stem cells (CSC) and down-regulated the expression of EGFR [159]. In addition, immunoliposome (ILips) combined with anti-CD47 and paclitaxel also produced a synergistic anti-tumor effect on converting M2 to M1 and enhancing systemic T cell immune response [160].

High-mobility group box 1 (HMGB1) is a non-histone chromatin-binding protein that exists widely in the nucleus and cytoplasm, which has been proven to up-regulate Treg cells and M2 macrophages in basal-like breast cancer cells [161]. Moreover, this effect could be blocked by HMGB1 inhibitors A Box, RAP, or EP, resulting in a reduced proportion of Treg cells and an up-regulated M1/M2 ratio [161]. Similarly, in the study of traditional Chinese medicine (TCM) treatment for ovarian cancer, astragaloside IV was found to weaken the polarization of M2 macrophages by inhibiting HMGB1-TLR4 signal transduction [162].

Toll-like receptors (TLRs) refer to conserved membrane proteins that recognize pathogens [163]. A study verified that TLR7 bound with TGF-β receptor I inhibitors SB431542 (SB4x) jointly provoked the polarization of M1 and CD4⁺, CD8⁺ and CD19⁺ cells [164]. Travers et al. found that DNMTi 5-azacytidine (5AZA-C) activated type I interferon signaling in ovarian cancer, catalyzed the number of IFN-γ⁺ immune cells and lowered the percentage of macrophages in TME [165]. Furthermore, 5AZA-C combined with the ornithine decarboxylase inhibitor α-difluoromethylchlorine (DFMO) significantly reduced the M2 macrophages and increased the M1 macrophages [165]. Zhao et al. discovered that colorectal cancer treated with Cetuximab repolarized TAMs from M2 to M1 macrophages in an IL6- dependent manner [166].

In the above description of biomarkers for TAMs, we mentioned that stimulating macropha ges with different factors can affect the expression of receptors (Tyro3, Axl and MerTK receptor) on the surface of macrophages. Sun et al. proved that the expressions of Axl and its ligands Gas6 were abnormal in highly differentiated endometrial cancer, which may be related to tumor progression and inhibition of apoptosis [168]. However, Goyette et al. found that high AxL protein levels were associated with tumor metastases in HER2⁺ breast cancer, which was independent of Gas6 [169]. Taking into account that TAMs receptors mediate tumor growth, invasion and metastasis, the researches on receptor inhibitors are booming [167]. Several studies found that pan TAMs kinase inhibitor BMS-777607 could inhibit the activation of TAMs receptor, further block the PD-L1 signaling pathway and suppress tumor progression [170], while selective Axl inhibitor BGB324 appears to induce apoptosis by inhibiting the PI3K pathway [171]. In addition, the function of AVB-S6-500 has been confirmed in clinical trials as an anti-Axl Fc fusion protein in epithelial ovarian cancer, primary peritoneal cancer and fallopian tube cancer [172].

Additionally, Zhao et al. demonstrated that promoting the expression of long noncoding RNA (lncRNA)-Xist in M1 macrophages and down-regulating microRNA (miR)-101 expression in M2 macrophages could facilitate M2-to-M1 macrophage-type conversion, which might play a beneficial role in inhibiting the proliferation and migration of breast and ovarian cancers [173].

As previously stated, macrophages promote the growth and metastasis of tumor cells by secreting cytokines and growth factors. Therefore, blocking the corresponding signal transduction to interfere with cytokines is another method to restrict the role of TAMs.

Recent studies have shown that in cervical cancer, Poly(I:C), an adjuvant for cancer vaccine, may act through the NF-κB signaling pathway to promote the expression of M1-type cytokines (such as IL-1β and IL-6) and inhibit the expression of M2-type cytokines (such as IL-10 and CCL22) [174]. Another study showed that TAMs autocrine VEGF also acted through NF-κB pathway [175]. Blocking the transmission of TAMs internal NF-κB pathway can weaken the pro-angiogenesis effect of VEGF as well. Besides, in MMP-12 knockout macrophages, the expression and phosphorylation of p38 and ERK1/2 were reduced, and the secretions of IL-1β, IL-6, TNF-α, CXCL1 and CXCL3 were down-regulated [176]. Three novel selective inhibitors of hydroxy ester MMP-12 (CGA, CGA-1 and AGA) and two 99mTc radioactive tracers derived from AGA [99mTc]-AGA-1 and [99mTc]-AGA-2 have been reported in recent studies [177]. In addition, a broad-spectrum MMP inhibitor CP-471/474 and a dual MMP-9/MMP-12 inhibitor (AZ11557272) showed well inhibition of MMP [178, 179]. However, further clinical trials are needed for selective MMP inhibitors to reduce drug toxicity. As for uPA, its inhibitor, 4-iodo benzo [b]thiophene-2-carboxamidine (B-428), has been shown to hinder the growth and metastasis of breast cancer in combination with tamoxifen back since the 1990s, which was superior to their separative effects [180].

Based on the important role of TAMs-derived IL-6 in cancer, therapeutic strategies targeting the IL-6 pathway are in active development. Siltuximab pertains to monoclonal antibodies (Mabs) that block IL-6 receptor [181]. In Siltuximab-treated ovarian cancer and ERα-positive breast cancer, cancer cells were found to reduce IL-6 production, which inhibited tumor growth, TAMs invasion and angiogenesis [182‐184]. Analogously, Tocilizumab was effective in ovarian cancer, cervical cancer, endometrial cancer and breast cancer [185‐188]. Besides, one study discovered that Cetuximab could also enhance the anti-tumor function of macrophages through IL-6 pathway [166].

To deal with up-regulated TGF-β, the researchers used antibodies to block both the receptor and the ligand, such as Fresolimumab (GC1008) and LY3022859 [189]. Altering the structure or composition of the ligand or the receptor to affect their binding is also helpful [189]. TGF-β targeting agents, such as LY2109761 and 1D11, have been proven effective in inhibiting metastasis in metastatic breast cancer [190].

IL-10 is an attractive therapeutic target due to its immunosuppression function and participation in tumorigenesis. One previous experiment showed that IL-10 receptor-blocking mAb (αIL-10R; clone 1B1.3A) increased the therapeutic effect of chemotherapy in breast cancer model in a CD8⁺ T cell-dependent manner [121]. Additionally, Alotaibi et al. used RNAi to reduce secretion of IL-10, resulting in accelerating the apoptosis of breast cancer cells with a suppression in PI3K/Akt pathway [136].

To sum up, therapy targeting cytokines and growth factors can be performed in the following ways. Firstly, at the gene level, RNA interference technology can influence molecule expression. Besides, the expression of genes can be blocked by interfering the normal signal transducing pathway. In addition, we can also use a variety of methods to block the binding of receptors and ligands and further block the role of ligands.

In recent years, chimeric antigen receptor (CAR) T cell therapy has developed rapidly in the treatment of hematological tumors. At the same time, human macrophages, which are modified with edited specific CAR to target antigen (such as CD19 and HER2) in order to recognize tumor cells and improve the ability of phagocytosis and antigen presentations, are in development as an immunotherapy [191]. The application of CAR-M therapy in solid tumors was first reported by Klichinsky et al. in 2020 [192]. The study has shown that CAR-M targeting HER2 significantly reduced tumor load, inhibited lung metastasis and prolonged the survival time in ovarian cancer mouse model [192]. Moreover, the expression of CAR structure can also promote TAMs polarization and even reverse M2 macrophages to transform into M1 macrophages [192, 193]. In addition, CAR-expressing macrophage cells (CAR-iMac) based on induced pluripotent stem cells (iPSCs) showed the ability to inhibit tumor cell growth both in hematological and in ovarian cancer model with lift expression of M1 pro-inflammatory cytokines [193].

As a novel strategy, tumor immune vaccines based on macrophages can reshape the TME and prevent tumor recurrence after operation. Wang et al. designed a biologically fused cytomembrane (FM) which contained both M1 macrophage and 4T1 breast tumor cell cytomembrane [194]. With the assistance of vaccine adjuvant cytosine-phosphate-guanosine oligodeoxynucleotides (CpG ODNs), the vaccine successfully increased the number of pro-inflammatory M1 macrophages, promoted the maturation of dendritic cells and activated memory T cells [194]. On the other hand, the combined vaccine which consists of therapeutic vaccine and TAMs receptor inhibitor provides a new way to treat cancer by inhibiting the up-regulated TAM receptors after vaccine treatment [191].

Nanobiotechnology targeting TAMs is another promising immunotherapy. Nanoparticles loaded with TLR agonists, cytokines, monoclonal antibodies and metal oxides have achieved preferable results in cancer therapy [195, 196]. CAR-M combined with nanocarrier has been proved to be effective in solid tumors [197]. Kang et al. treated monocytes or macrophages with genes encoding CAR and IFN-γ through polymer nanocarriers, which successfully led to the phenotypic transition from M2 to M1 and the enhancement of macrophage phagocytosis [197]. In breast cancer model, TAMs were modified by iron-based metal organic framework (Fe-MOF) nanoparticles with ferroptosis-inducing agents, which allowed iron ions released in TAMs and further enhanced ferroptosis-associated stress in macrophages to induce effective M1 phenotypic polarization [195]. TAM-membrane-coated nanoparticles with conjugated photosensitizer (NPR@TAMM) and CSFR not only consumed tumor-derived CSF1, but also induced M2 macrophages transform into M1 via photodynamic immunotherapy [198]. Besides, nanoparticles loaded with TLR-7/TLR-8 agonist resiquimod (R848) and doxorubicin (DOX), which induced immunogenic cell death (ICD) or loaded with curcumin (Cur) and baicalin (Bai), had satisfactory tumor cytotoxicity in TME and significantly improved the efficacy of immunotherapy [199, 200].