Cancer Stemness Meets Immunity:From Mechanism To Therapy(Part 2)

To learn more info plz contact david.wan@wecistanche.com

In multiple cancer types, macrophage and microglia pathways and/or factors involved in pro-tumor polarization include STAT3, nuclear factor (NF)-kB, and PI3Ky pathways(Kaneda et al., 2016; Qian and Pollard,2010). Among these, the STAT3 transcription factor plays a prominent role. Macrophage STAT3 is activated by CSC-derived IL-6, L-10, and/or exosome cargo, resulting in the upregulation of genes promoting-tumor programming and inhibition of genes encoding anti-tumor cytokines(Malyshev and Malyshev,2015). Moreover, STAT3 inhibition abolishes CSC-induced pro-tumor macrophage polarization in GBM(Gabru-siewicz et al.,2018; Wu et al.,2010; Yao et al,2016), bladder cancer (Kobatake et al,2020), and breast cancer (Weng et al.,2019). The NF-kB pathway is essential for CSC-induced pro-tumor macrophage polarization in ovarian cancer (Deng et al。2015). In summary, the study of CSC-induced macrophage polarization has identified prominent and therapeutically actionable macrophage pathways in specific cancers (Table 1).

Click here to learn more about Cistanche

TAMs Promote Cancer Cell Stemness and the CSC Niche Mirroring CSC actions, TAMs can support CSC stemness and the CSC niche (Figure 3). The niche is particularly important in the maintenance of CSC self-renewal, repopulation potential, and tumor initiation. This supportive microenvironment is composed of cancer cells, immune cells, mesenchymal stem cells (MSCs), fibroblasts, endothelial cells, and extracellular matrix (ECM) components (Figure 3; Plaks et al,2015). Paracrine factors derived from these diverse stromal cell types play prominent roles in promoting CSC stemness in the niche. Specifically, in breast and colon cancers, MSCs contribute to CSC niche formation by secreting prostaglandin E2(PGE2), IL-6, IL-8, and CXCL1(Liet al.2012). Fibroblasts can induce metastatic niche for breast cancer CSCs via secretion of POSTN (Malanchi et al, 2011). TAMs produce factors to enable"differentiated" cancer cells to acquire CSC-like features and to maintain CSC stemness in breast cancer(Lu et al,2014), oral squamous cell carcinoma (Li et al.,2019), renal cell carcinoma (Yang et al.,2016), hepatocellular carcinoma (HCC)(Wang et al.,2016), and pancreatic cancer (Mitchem et al,2013). Correspondingly, depletion of TAMs via inhibition of colony-stimulating factor 1 receptor (CSF1R) and C-C motif chemokine receptor 2 (CCR2) diminishes the tumor-initiating properties of CSCs in mouse models (Mitchem et al,2013).In GBM, TAM support of the CSC niche depends on its pro-tumor phenotype, and reprogramming to an anti-tumor phenotype(using amphotericin Bor vitamin B3) attenuates cancer cell stemness and tumorigenicity in vitro and in vivo and sensitizes these tumors to chemotherapy (Sarkar et al,2014,2020). These findings highlight the therapeutic potential of disrupting the CSC niche via reprogramming TAMs toward an anti-tumor phenotype.

The importance of TAMs in CSC biology is reinforced by a growing list of TAM-derived factors implicated in the maintenance of CSC stemness. Table 2 summarizes such factors and their purported mechanisms in different cancer models. EMT is an important process that enables cancer cells to acquire CSC-like features and maintain CSC stemness(Biddle and Mackenzie,2012). In breast cancer cells, EMT is associated with the upregulation of CD90 and EphA4, which mediate physical interactions between CSCs and TAMs(Lu et al,2014). As a result, TAMs can further accelerate breast cancer cell EMT, thus inducing a positive feedback loop to reinforce CSC stemness via secreting a panel of CSC-supporting cytokines, such as IL-6, IL-8, and IL-1β(Guo et al.,2019a; Let al.,2014; Valeta-Magara et al,2019). Similarly, accumulating evidence shows that TAMs in GBM (Hide et al,2018), HCC (Fan et al, 2014; Wan et al,2014), pancreatic cancer(Nomura et al, 2018; Saint al.,2015; Zhang et al.,2019a), and ovarian cancer (Raghavan et al.,2019)can promote cancer cell EMT and/or secrete a variety of CSC-supporting cytokines (including IL-1β, IL-6, and TGF-), thus promoting CSC stemness, tumor progression, and therapy resistance. In addition to cytokines, TAMs can specifically produce unique factors to support CSC stemness. For example, CCL5, pleiotrophin (PTN),globule-epidermal growth factor-Val (MFG-E8), and CCL8 are preferentially expressed and secreted by TAMs in prostate cancer (Huang et al,2020), lymphoma(Wei et al,2019b), colorectal cancer (CRC)(Jinushi et al,2011), and GBM (Zhang et al,2020), respectively, where they promote CSC stemness and tumor progression. Finally, in addition to soluble factors, TAMs can pro-mote CSC stemness via direct interactions. Specifically, in breast cancer, liver, and lymph node sinusoidal endothelial cell C-type, lectin is a transmembrane protein highly expressed on TAMs that interacts with hydrophilic subfamily 3 member A3 receptor on cancer cells to enhance stemness(Liu et al.,2019). TAM-derived factors and TAM-cancer cell physical interactions activate several pathways in cancer cells that are pivotal to the maintenance of stemness. These key CSC pathways include STAT3, SHH, and NOTCH(Han et al,2015; Hirata et al.,2014; Zhang et al.,2019b), as well as PI3K/AKT, WNT/β-catenin, and NANOG(Morgan et al,2018; Wang et al, 2010b,2019a; Wei et al,2013; Zhang et al,2019c). Available evidence supports the view that TAM-derived factors activate these pathways to enhance or maintain CSC stemness (Table 2). Among them, STAT3 appears most important as a result of its potent upregulation of stemness-related genes and activation of stemness-promoting pathways, such as NF-kB(Galoczova et al。2018). Accordingly, STAT3/NF-kB inhibition abolishes TAM-promoted stemness in breast cancer(Lu et al.,2014; Val-eta-Magara et al,2019; Yang et al,2013), HCC (Li et al, 2017; Wan et al,2014), prostate cancer (Huang et al,2020), pancreatic cancer (Mitchem et al,2013; Nomura et al.,2018), and CRC (Jinushi et al,2011). The WNT/β-catenin and SHH pathways can also promote CSC stemness in some settings. Aberrant activation of WNT signaling, common in many tumor types, often defines the CSC state and maintenance of CSC biology (de Sousa E Melo and Vermeulen,2016). Cell culture and mouse model systems demonstrate that TAMs activate the WNT/β-catenin pathway in CSCs and inhibition of this pathway impairs TAM-induced upregulation of CSC stemness in HCC(Chen et al.,2019b), prostate cancer(Huang et al., 2020), and lymphoma (Wei et al,2019b). Similarly, the SHH pathway has been implicated in regulating CSC stemness either directly or through interaction with other stemness-related pathways, such as TGF-β(Takebe et al.,2015). With respect to TAM-supported CSC stemness, CRC relies on the SHH pathway (Jinushi et al,2011), pancreatic cancer on the TGF-β1/SMAD2/3/NANOG pathway (Zhang et al,2019a), HCC on the NOTCH pathway (Wang et al。2016), breast cancer on the v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog (avian) (SRC) pathway (Lu et al,2014), and glioma on the extra-cellular regulated kinase 1/2 (ERK1/2) pathway (Zhang et al。2020). Collectively, these findings highlight STAT3/NF-kB and WNT/β-catenin as key pathways responsible for TAM-induced CSC stemness. However, the diversity of pathways across many cancers underscores the need to develop context-specific strategies to target them.

CSC-MDSC Crosstalk

The Impact of CSCs on MDSC Biology

MDSCs are a heterogeneous population of myeloid cells that include granulocytic or polymorphonuclear(PMN-MDSC) and monocytic (M-MDSC) subgroups(Gabrilovich and Nagaraj 2009).PMN-MDSCs account for more than 80% of all MDSCs, and M-MDSCs can differentiate into TAMs (Gabrilovich,2017; Kumar et al.,2016).MDSCs are generated in the bone marrow and recruited into tumors by tumor-derived chemokines, such as CCL2 and CCL5. Similar to TAMs, MDSCs play an important role in the regulation of tumor angiogenesis, growth, metastasis, and immune suppression (Gabrilovich,2017; Kumar et al 2016). Increasing evidence also has revealed symbiotic interactions between CSCs and MDSCs in the TME, where CSCs contribute to MDSC infiltration, expansion, and activation via secretion of soluble factors and exosomes in different cancer types (Figure 4; Table 3). In SCCHN, compared with CD44cells, CD44* CSCs secrete higher levels of IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF), and TGF-βand induce larger populations of MDSCs when they are co-cultured with peripheral blood mononuclear cells(PBMCs) (Chika-matsu et al.,2011).In GBM, GSCsnot only promotes the differentiation of PBMCs into M-MDSCs via secretion of exosomes(Do-menis et al.,2017) but also activate MDSCs to suppress immune responses via secretion of macrophage migration inhibitory factor (MIF) (Otvos et al,2016). In melanoma, the expression of miR-92 in CD133+ CSCs is reduced when compared to CD133～ cells, which upregulates the expression of TGF-β via the x5 integrin/SMAD2 pathway, resulting in more PMN-MDSCs in the TME (Shidal et al,2019). MDSCs Promote CSC Stemness

Once MDSCs infiltrate into the TME, they can reciprocally pro-mote CSC stemness via distinct mechanisms in a number of cancer types(Figure 4; Table 3). In ovarian cancer, MDSCs induce miR-101 and GM-CSF expression in cancer cells, which increases stemness via upregulation of the corepressor gene C-terminal binding protein-2 (CtBP2)(Quiet al,2013) and activation of the STAT3 pathway (Li et al.,2020), respectively. In multiple myeloma, PMN-MDSCs trigger the expression of piwi-inter-acting RNA piRNA-823 in cancer cells, which promotes stemness via activation of DNA methyltransferase 3B (DNMT3B)to facilitate DNA methylation(Ai et al.,2019). Although these data highlight the essential role of MDSCs in promoting cancer cell stemness, the identities of the MDSC-derived factors in these cancer types are still emerging. Of note, STAT3 again appears to be one of the key pathways responsible for stemness. In breast and pancreatic cancers, MDSCs secrete IL-6(in both cancer types) and nitric oxide (in breast cancer) to activate STAT3 and NOTCH pathways to promote stemness (Panini et al,2014; Peng et al,2016). In breast cancer, activated STAT3 promotes activation of NOTCH, which in turn facilitates persistent STAT3 activation, thus creating a feedback loop to reinforce stemness (Peng et al.,2016). In addition to stemness, M-MDSC-derived NOS2 can also promote EMT via activation of the STAT3 pathway, which drives cancer cell dissemination and metastasis (Ouzounova et al,2017).In CRC, PMN-MDSCs can secrete exosomal S100A9 to promote cancer cell stemness via activation of the STAT3 and NF-kB pathways, which is further amplified under hypoxic conditions (Wang et al.,2019b),

establishing NF-kB in MDSC-induced stemness. Indeed, MDSCs are the major source of PGE, in several types of cancer (e.g., ovarian, cervical, and endometrial cancers; Komura et al, 2020; Kuroda et al.,2018; Yokoi et al.,2019), which can foster cancer cell stemness by activating NF-B via E-type prostanoid receptor 4 (EP4)-Pl3K and EP4-mitogen-activated protein kinase (MAPK) pathways (Wang et al,2015). Thus, a number of factors and pathways underlie MDSC-CSC interactions, and STAT3 and NF-kB are again very prominent.

CSC-T Cell Crosstalk

Impact of CSCs on T Cell Biology

Computational analyses have also revealed correlations between cancer cell stemness and CD8t T cells in a broad range of cancer types(Miranda et al.,2019).In SCCHN, GBM, and melanoma, high stemness correlates with low expression of cancer-associated antigens and immune-stimulatory molecules (e.g., CD86, CD40, major histocompatibility complex [MHC] Il, transporter associated with antigen processing, histocompatibility leukocyte antigen [HLA]-A2, melanoma antigen recognized by T-cells 1, melanoma inhibitor of apoptosis, New York esophageal squamous cell carcinoma 1, and melanoma-associated antigen-A) and high expression of immune checkpoint inhibitors (e.g., PD-L1; Chikamatsu et al.,2011; Schatton et al.,2010; Wei et al.,2010). Consistent with these correlates, CSCs regulate the composition and function of T cells via several experimentally validated mechanisms(Figure 4; Table 4). First, in GBM, GSCs produce TGF-β, CCL2, and galectin-3, which suppress CD8+and CD4+ T cell activation and proliferation (Wei et al,2010). Second, GSC exosome tenascin-C (TNC) engages x5β1 and a5β6 integrins on T cells to downregulate AKT/mTOR signaling and inhibit T cell activation and proliferation (Domenis et al, 2017; Mizaei et al,2018). Finally, in prostate cancer, CSCs secrete TNC to inhibit the activation and proliferation of CD8+and CD4+Tcells via interaction with x5β1 integrin on T cells(Ja-chetti et al.,2015).

T-reg cells are an immunosuppressive subset of CD4+ T cells characterized by expression of forkhead box P3(FoxP3) and by tumor promotion via inhibition of effector T cells (Togashi et al, 2019).CSCs attract and activate T-reg cells via various soluble factors(Figure 4; Table 4), including, most notably, TGF-β, which controls T-reg cell recruitment and expansion. For example, CSCs can produce high levels of TGF-βin SCCHN (Chikamatsu et al.,2011), GBM(Wei et al.,2010), and melanoma(Shidal et al, 2019), which in turn promotes T-reg cell recruitment and expansion via activation of the a5 integrin/mothers against decapentaplegic homolog 2 (SMAD2) pathway, thus inducing T cell apoptosis and inhibiting T cell proliferation and activation. In addition, several C-C chemokine family members, such as CCL1 (Xu et al,2017a), CCL2 (Wei et al,2010), and CCL5 (You et al.,2018), have been shown to be highly produced by CSCs in distinct types of cancer, where they can stimulate the infiltration of T-reg cells into the TME. Together, CSC-secreted TGF-β and specific chemokines play key roles in T-reg cell recruitment and expansion in the TME. T Cells Regulate CSC Stemness

Emerging evidence demonstrates that different subsets of T cells can regulate CSC stemness(Figure 4; Table 4). In GBM, a T-cell-conditioned medium inhibits GSC self-renewal via secretion of TNF-αand interferon(IFN)-r (Mirzaeiet al,2018).InNSCLC, CD8+Tcells are the main sources of IFN-Y, where low levels of IFN-γ promote CSC stemness via activation of the PI3K/AKT/NOTCH1 pathway and high levels of IFN-y induce cancer cell apoptosis via activation of the Janus kinase 1 (JAK1)/STAT1/caspase pathway(Song et al.,2019).In pancreatic cancer, infiltrating Th2 cells produce cytokines-4 and IL-13 to activate the JAK1/STAT6 pathway in cancer cells, which in turn increases MYC-driven glycolysis (Dey et al,2020), an anabolic process that supports CSC stemness(Chen et al,2020b; Sancho et al。,2015). In addition to the secretion of soluble factors, T cells can regulate CSC stemness via a direct cell-to-cell contact mechanism in breast cancer where cognate non-lytic interactions between CD8+T cells and cancer cells can promote cancer cell stemness (Stein et al.,2019).

In addition to effector T cells, T-reg cells and Th17 cells can also regulate stemness. For example, in AML, T-reg cells secrete IL-10 to promote the stemness of leukemic stem cells via activation of the PI3K/AKT/OCT4/NANOG pathway (Xu et al.,2017b). The stemness-promoting effect of T-reg cells is also observed in breast cancer, where unknown-reg cell soluble factors upregulate stemness-related pathways: SOX2; NANOG; and OCT4 (Xu et al,2017a).In HCC, T-reg cells secrete TGF-β to support CSC stemness by promoting EMT(Shi et al,2018a; Xu et al, 2009), whereas, in CRC, T-reg-cell-derived TGF-β drives cancer cell dedifferentiation (Nakano et al,2019), suggesting context-specific actions of TGF-β in CSCs. These observations are consistent with the known highly contextual functions of TGF-βin cancer(Massagué,2008). Although Th17 is a subset ofT help-er cells that mediate anti-tumor immune responses(Guery and Hugues,2015), IL-17 from Th17 cells or CD4+Tcells also pro-mote CSC stemness through activation of NF-kB and p38 MAPK pathways in ovarian and pancreatic cancers (Kiang et al.,2015; Zhang et al,2018) and STAT3 pathway in gastric cancer(Jiang et al。,2017). In addition, IL-17 can be upregulated in T-reg cells under hypoxic conditions, which in turn fosters CSC stemness in CRC(Yang et al., 2011). Thus, different T cell subsets contribute to the maintenance of CSCs via a variety of mechanisms involving soluble factors and cell-to-cell contact.

Therapeutic Potential of Intercepting CSC-Immune Cell Crosstalk

Targeting CSC-TAM Crosstalk

Major preclinical and clinical efforts have sought to target the distinct biologic characteristics and crucial signaling pathways of CSCs and TAMs, as reviewed previously (Agliano et al., 2017; Pathria et al.,2019; Zhao et al.,2018). As summarized in Figure 5, clinical trials that can target CSC biology include inhibitors of the SHH, NOTCH, WNT/β-catenin, STAT3, and NANOG pathways (Agliano et al., 2017; Zhao et al., 2018) and anti-CD44 antibodies (Menke-van der Houven van Oordt et al., 2016). To date, the SHH inhibitor, vismodegib, has been approved for metastatic or locally advanced basal cell carcinoma (Sekulic et al., 2012), and clinical trials are underway for agents targeting macrophage recruitment (CR2, CXCR4, integrin subunit alpha 4 [ITGA4] and ITGA5 inhibitors), polarization (toll-like receptor 4 [TLR4], TLR7, TLR9, and CD40 activators and PI3Ky inhibitor), and survival (CSF1R inhibitors; Gregoire et al., 2020; Pathria et al., 2019). However, despite the appeal of CSC-targeting agents, dramatic responses have not been observed, perhaps owing to a lack of truly specific CSC targets (Agliano et al.,2017; Turdo et al.,2019) as well as the high plasticity of CSC, which enables loss and reacquisition of stemness

under varying TME conditions (Agliano et al.,2017;Müller et al, 2020; Plakset al.,2015).Similarly, TAM-targeted therapies, such as CSF1R inhibition, have shown meager anti-tumor responses in GBM due in part to resistance conferred by activated Pl3K signaling in glioma cells (Quail et al,2016).

It is tempting to speculate that targeting more deliberately the entwined co-dependencies of CSCs and TAMs and their plasticity in specific contexts could yield more robust responses. For example, IL-6/STAT3 and Pl3K are essential for the regulation of both CSCstemness and macrophage pro-tumor polarization in many tumor types; inhibition of these pathways has shown anti-tumor activity (Agliano et al.,2017; Kobatake et al.,2020; Pathria et al,2019; Weng et al,2019). Moreover, selecting patients with high CSC and TAM signatures could enhance responses to agents targeting these pathways (Agliano et al, 2017; Pathria et al,2019). Such trials could benefit further from pharmacodynamic assessment of whether these agents can modulate these pathways and their associated tumor biology(Figure 5). Finally, given the plasticity of this system, these pharmacodynamic studies should be complemented by integrated omic analyses of the adaptive responses to these therapeutic interventions, which may further inform combination trials of synergistic agents.

An appealing strategy to disrupt CSC-TAM crosstalk may include blockade of the CD47-signal regulatory protein alpha (SIRPa) pathway. CD47 is a transmembrane protein expressed on CSCs and cancer cells that functions as a"don't eat me" signal (Cioffi et al。2015); interaction of CD47 with SIRPa on macrophages results in inhibition of phagocytosis by TAMs(Matozaki et al,2009). Anti-CD47 therapy increases CSC phagocytosis in vitro and decreases tumor burden in vivo(Chan et al,2009; Cioffi et al.,2015; Majeti et al.,2009), which is further augmented when this therapy is combined with chemotherapy (Cioffi et al,2015). Several clinical trials testing monoclonal anti-CD47 antibodies(Hu5F9-G4, SFR231, CC-90002, and IBlI188) and small-molecule inhibitors (TTI-621 and ALX148) are underway (Figure 5; Gregoire et al.,2020; Pathria et al,2019). Another opportunity to disrupt CSC-TAM crosstalk centers on targeting soluble factors that reciprocally support each cell type. For example, inhibition of the CSC-specific POSTN and its related pathway (Zhou et al,2015) or TAM-derived CCL5(Huang et al,2020) have been shown to interrupt CSC-TAM crosstalk, suppress tumor growth, and extend survival in mouse models of GBM and prostate cancer.

On a more conventional note, the standard of care chemotherapies have shown limited promise for advanced metastatic disease due to severe toxicity and rapid development of resistance. As CSCs and TAMs play critical roles in the development of drug resistance (Agliano et al.,2017; De Palma and Lewis,2013), disruption of CSC-TAM crosstalk could improve its response to chemotherapy. Along these lines, CSCs in chemoresistant tumors secrete cytokines to create a pro-tumorigenic microenvironment by skewing macrophages toward an apron-tumor phenotype (Yama-shiniest al.,2014). Indeed, depletion of TAMsreduces CSCstem-ness inhibits metastasis and improves chemotherapeutic responsiveness in pancreatic cancer(Mitchem et al,2013). Mechanistically, TAMs promote CSC stemness and chemoresistance via the release of MFG-E8, which can trigger activation of STAT3 and SHH pathways in CSCs of CRC(Jinushi et al, 2011). Thus, mounting evidence points to the potential of targeting the CSC-TAM circuits for novel cancer treatments as well as for enhancement of chemotherapy effectiveness.

Targeting CSC-MDSC Crosstalk

The importance of MDSCs in promoting tumor growth, metastasis, angiogenesis, CSC stemness, and immune suppression has motivated the testing agents that inhibit MDSCs(Fleming et al, 2018). MDSC inhibition strategies target recruitment (e.g., inhibition of CCR5 and CXCR2), promote depletion (e.g., tyrosine-kinase inhibitors and chemotherapeutic agents), and block immunosuppressive activity (e.g, inhibition of STAT3, phosphodiesterase-5, and class I histone deacetylases; Fleming et al.,2018). However, the development of MDSC-targeted therapies is hampered by the heterogeneity of MDSCs and the lack of cellular markers(Lu et al.,2019). That is, MDSCs are heterogeneous immature myeloid cells composed of PMN-MDSCs and M-MDSCs, which possess distinct biological functions. Current MDSC-targeted agents may target both MDSC subgroups and other cell types in the TME. In addition, the lack of specific markers for human MDSCs and identification of the equivalent murine MDSCs has impeded translational research. Finally, MDSC density and activation states can change dynamically in the TME.

Notwithstanding these challenges, several strategies targeting CSC-MDSC crosstalk are worth considering. One strategy would be to target STAT3, which is dually essential for CSC maintenance and MDSC infiltration/activation, and inhibition of STAT3 has demonstrated potent anti-tumor activity associated with impaired CSC stemness and MDSC infiltration/activation in cancer mouse models (Fleming et al,2018; Peng et al, 2016). A second strategy would be to target soluble factors fostering CSC-MDSC crosstalk. For example, inhibition of CSC-derived MIF or MDSC-derived IL-6 extends survival in mouse models of GBM (Otvos et al,2016) and breast cancer (Peng et al.,2016). A third strategy would involve the neutralization of exosome-stimulated CSC-MDSC symbiosis. Specifically, knockdown of S100A9 in MDSC exosomes impairs STAT3 activation and inhibits tumor growth in mouse models of CRC (Wang et al,2019b). Together, these various mechanistic insights point to disruption of the IL-6/STAT3 pathway as a strategy to interfere with CSC-MDSC crosstalk and inhibit tumor growth. Targeting CSC-T Cell Crosstalk

The symbiotic interactions between CSCs and T cells may also offer several precision therapeutic strategies. First, in breast cancer, blockade of CSC-derived T-reg cell supporting factors, such as CCL1, has been shown to significantly inhibit tumor growth and T-reg cell infiltration (Xu et al,2017a). A similar anti-tumor effect has been observed in a mouse model of pancreatic cancer by inhibition of T-cell-derived stemness sup-porting factors, such as Th17 cell-derived IL-17, which dramatically impaired tumor growth and CSC stemness (Xiang et al, 2015). The second approach is to harness the potential of T-cell-based immunotherapies, especially immune checkpoint inhibition (ICI). The anti-tumor effectiveness of ICls relates to the expression of immune checkpoint molecules, such as PD-L1, in the TME (Ravindran et al.,2019). Following activation of the STAT3 and NOTCH3/mTOR pathways(Lee et al,2016; Mansour et al., 2020), CSCs express higher PD-L1 levels compared to non-CSCs in many cancer types, including GBM, melanoma, SCCHN, CRC, breast cancer, gastric cancer, and ovarian cancer, in which PD-L1 can further promote CSC stemness, thus inducing a positive feedback loop (Gao et al,2019; Gupta et al.,2016; Ravindran et al.,2019; Wei et al.,2019a).In addition, the PD-L1 levels can be amplified following symbiotic CSC-immune cell interactions. For example, MDSCs can promote stemness and upregulate PD-L1 in CSCs via activation of the PI3K/AKT/mTOR pathway (Komura et al,2020). Consequently, CSCs secrete exosomes to upregulate PD-L1 in macrophages via activation of the STAT3 pathway(Gabrusie-wicket al,2018). Together, these findings point to the potential utility of IClagents, a concept supported by the anti-PD1 therapy enhancement of the anti-tumor activity of a CSC vaccine in a mouse model of bladder cancer (Shi et al.,2018b). The third approach would be the development of combination therapies targeting CSC-immune cell crosstalk and immune checkpoints. ICUs produce remarkable responses in some cancer patients; however, the majority of patients do not have responses. Mechanistic studies have shown that the effectiveness of ICUs is highly dependent on the TME(Murciano-Goroff et al.,2020). TAMs, MDSCs, and T-reg cells are the most prominent immune cells in the TME, where they form symbiotic interactions with CSCs, interact with each other, and suppress T cell function (Figure 4). Mechanistically, TAMs and MDSCs can suppress T-cell-mediated anti-tumor immune responses by high expression of immune checkpoint molecules (e.g., PD-L1, PD-L2, CD80, and CD86), production of immunosuppressive cytokines (e.g., IL-10 and TGF-β), and recruitment of immunosuppressive T-reg cells into the TME (Engblom et al.,2016; Kumar et al.,2016; Manto-vani et al,2017). These studies highlight the promise of TAM-or MDSC-targeted therapies for improved ICl effectiveness. Indeed, a growing body of evidence demonstrates that macro-phage-targeted therapies, such as activation of macrophage phagocytosis (Lian et al.,2019; Liu et al.,2018) or reprogram-ming of TAMs from pro- to anti-tumor phenotype (Baer et al, 2016; Guerriero et al,2017; Kaneda et al.,2016; Zhu et al., 2014), synergize with ICls in multiple cancer mouse models. Similarly, MDSC-targeted therapies, by inhibiting MDSC infiltration(Flores-Toro et al.,2020; Highfillet al.,2014; Liao et al.,2019; Zhao et al.,2020) or block MDSC activation (Davis et al, 2017; Liu et al,2020; Lu et al.,2017), show robust synergy with ICUs in mouse models. These preclinical studies have prompted combination therapy trials for many cancer types (Hou et al., 2020; Pathria et al., 2019).

Concluding Remarks and Future Perspectives

The genetic paradigm has dominated our approach to cancer therapy, generating many agents targeting driver oncogenic events in cancer cells. In recent years, the success of targeting immunity and angiogenesis has heightened interest in targets operating within the TME ecosystem. This review specifically has cataloged the molecular circuitry underlying reciprocal inter-actions between CSCs and immune cells, including TAMs, MDSCs, and T cells, in tumor maintenance. This bidirectional crosstalk is manifested on several levels, including CSC-directed immune cell recruitment and activation and the role of these immune cells in promoting cancer cell stemness and establishing a supportive CSC niche. The molecular characterization of CSC-immune cell symbiosis has uncovered potential therapeutic strategies, including dual targeting of vital pathways activated in both CSCs and immune cells (e.g., STAT3 and PI3K, disrupting the molecules responsible for physical CSC-immune cell interactions (e.g., CD47-SIRPa), and neutralizing soluble fac-tors that reciprocally support both CSCs and immune cells (e.g., IL-6). Collectively, elucidation of these symbiotic CSC-immune cell interactions has also revealed the centrality of these novel molecular mechanisms in driving tumorigenesis, metastasis, and chemotherapy resistance. Thus, targeting this molecular circuit has the potential to disrupt CSC-immune cell co-depend-denies and enhance the effectiveness of conventional therapies.

Although our knowledge of CSC-immune cell crosstalk is maturing, multiple questions will need to be answered in order to effectively and systematically convert mechanistic insights into new therapeutic interventions. First, many studies investigating CSC-immune cell crosstalk have relied on cell line co-culture models or isolated cells from tumor tissues, which highlights the need for complementary studies using in vivo models, genetic validation, and dynamic analyses of the TME using lineage tracing and live micro positron emission tomography (microPET)/computed tomography (CT) imaging technologies. Such in vivo models could be complemented by organoid cultures, which appear to more faithfully recapitulate the features of their source tissues (Baker, 2018). That is, cancer cell organoids and immune cell co-cultures could serve as more robust and higher throughput model systems to study the dynamic and reciprocal interactions between CSCs and immune cells and to test therapeutic agents targeting CSC-immune cell crosstalk. Second, the remarkable plasticity and heterogeneity of both CSCs (transitioning between stem versus non-stem states) and immune cells (including the transitions within and across cell types, such as the transition across the phenotypic spectrum in TAMs, differentiation of MDSC to PMN-MDSC and M-MDSC subgroups, and differentiation of M-MDSCs to TAMs) highlight the challenges in identifying the context-specific nature of distinct critical CSC-immune cell circuits at different tumor stages and in different cancer types, as well as changes in CSC-immune cell interactions resulting from therapeutic interventions. Thus, harnessing the full potential of targeting CSC-immune cell crosstalk will require extensive use of siRNA-seq to identify new subpopulations and define the physiological states of CSCs and immune cells, as well as their crosstalk in specific tumor contexts and under exposure to certain therapies. Such single-cell auditing must be complemented by functional and genetic analyses using in vivo model systems to identify and validate targets and mechanisms governing CSC-immune cell co-depend-denies. Given the number of factors involved, bispecific anti-bodies dually targeting key factors acting in concert in the CSC-immune cell circuit should be considered. Third, across many tumor types, the IL-6/IL-6R/STAT3 pathway appears to be the most prominent and important driver of CSC-immune cell cross-talk, as evidenced by the finding that pharmacological inhibition of the IL-6R/STAT3 pathway impairs tumor progression and re-duces CSC stemness, TAMs, and MDSCs in bladder cancer, breast cancer, and HCC mouse models (Kobatake et al., 2020; Peng et al., 2016; Wan et al., 2014). However, a more detailed investigation of the actions of these drugs is needed, as they also target other stromal cells in the TME. That is, the anti-tumor actions may not relate to CSC-immune cell crosstalk and/or may target stromal cells with opposing actions to CSCs, TAMs, and MDSCs. In this regard, genetically engineered mouse models would be useful to dissect the myriad roles of the IL-6/L-6R/STAT3 pathway specifically in CSCs, TAMs, MDSCs, and/or T-cells versus other cells within the TME ecosystem. Finally, in addition to TAMs, MDSCs, and T cells, unbiased analyses on TGGA datasets demonstrated that high cancer cell stemness is associated with reduced NK cells (Miranda et al,2019), suggesting potential CSC-NKcell crosstalk. CSCs are generally susceptible to killing by activated NK cells; however, a growing body of evidence shows that CSCs may be resistant to NK cells in some cancer types, such as GBM, AML, and breast cancer (Sultan et al,2017). Emerging evidence demonstrates that the anti-CSC activity of NK cells is largely dependent on the TME and that NK cell activation can be suppressed by TAMs, MDSCs, and T-reg cells (Bruno et al, 2019; Ghiringhel et al,2006; Kmeta et al, 2017). In addition to NK cells, very limited evidence demonstrates that cancer cell stemness is related to the presence of dendritic cells (DCS), B-cells(Hsuet al.,2018; Miranda et al.,2019), and neutrophils(Hira et al,2015; Hwang et al.,2019). However, the nature of the crosstalk between CSCs and these four types of immune cells (NK cells, B cells, DCs, and neutrophils) is largely unknown. Therefore, further studies characterizing such crosstalk, as well as the relationship of these four cell types with other immune cells, including TAMs, MDSCs, and T cells, will pave the way for developing novel and more effective immunotherapies.

In summary, we have presented mounting evidence implicating CSC-immune cell interactions as drivers of tumor development involving many hallmarks of cancer and as modulators of the response to therapeutic interventions. Harnessing the therapeutic potential of these interactions will require rigorous validation of the targets and mechanisms underlying this symbiotic relationship as well as a deeper understanding of the specific biological contexts in which they play essential rate-limiting roles in tumor maintenance. Successful achievement of this goal would greatly benefit cancer patients.