Enhancement of PD-L1-attenuated CAR-T cell function through breast cancer-associated fibroblasts-derived IL-6 signaling via STAT3/AKT pathways

Breast cancer (BCA) is the most common cancer of women worldwide [1]. The 2020 global cancer statistics estimate 2.3 million new cases in women [2]. BCA is clustered into four subtypes, including Luminal A, Luminal B, HER2-positive, and triple-negative BCA (TNBC) [3]. Although increased early screening and understanding of molecular mechanisms of metastasis leads to more effective prognosis and treatment, some patients have relapses and chemoresistance, especially in the TNBC [4].

The tumor microenvironment (TME) consists of tumor cells, cancer-associated fibroblasts (CAFs), endothelial cells, extracellular matrix, and immune cells [5]. CAFs, the most abundant cells in TME, secrete substances such as transforming growth factor-β (TGF-β), vascular endothelial growth factor (VEGF), and interleukin-6 (IL-6) which can promote cancer progression [6‐9]. CAF-derived substances regulate epithelial-mesenchymal transition and promote drug resistance by secreting IL-6 [10, 11]. In addition, fibroblast activation protein (FAP) α1-positive CAFs drive immunosuppression [12‐14]. These facts declare the importance to investigate CAF-mediated immunosuppression and the involved mechanisms.

IL-6 is released by various cells including cancer cells, CAFs, and immune cells [15]. IL-6/IL-6 receptor (IL-6R) interaction activates Janus kinase (JAK) tyrosine kinases leading to phosphorylating the signal transducer and activator of transcription 3 (STAT3) [15]. This interaction can phosphorylate mitogen-activated protein kinases (MAPK)/ERK leading to enhance cell growth [16]. The phosphoinositol-3 kinase (PI3K)/AKT pathway is induced by IL-6 following JAK phosphorylation. The PI3K/AKT phosphorylation induces gene expression by activating NF-κB to promote anti-apoptosis [17, 18].

Programmed death ligand 1 (PD-L1), the ligand for programmed death 1 (PD-1), is overexpressed in cancer cells after being induced by cytokines [19]. PD-L1 expression in cancer cells leads to immune escape, drug resistance, and cancer metastasis [20]. The PD-L1/PD-1 interaction attenuates T cell function via T cell apoptosis [21]. Patient-derived hepatocellular carcinoma (HCC)-CAFs secrete IL-6 which induces PD-L1 expression on neutrophils via the STAT3 pathway [22]. The PD-1/PD-L1 immune checkpoint inhibitor was approved by the US Food and Drug Administration (FDA) to treat advanced TNBC tumor-expressed PD-L1 [23]. Immunoregulatory roles of CAF-derived IL-6 in promoting drug resistance and inducing T cell dysfunction through IL-6-mediated PD-L1 expression have not been well-addressed in BCA.

In this study, the IL-6 ELISA assay of BCA-derived CAFs was performed to identify IL-6 levels as a major component in CAFs. The roles of IL-6 signaling to exert doxorubicin (Dox) resistance and response to folate receptor (FR)α-chimeric antigen receptor (CAR)-T cell killing actions were evaluated in parental and IL-6R -transient knocked down TNBC cell lines, with or without specific inhibitors against signaling molecules. The findings highlight the role of CAFs-derived IL-6 in mediating Dox resistance in MDA-MB-231 and HCC70 TNBC cell lines. It can also reduce FRα-CAR T cell function via the upregulation of PD-L1 through the STAT3/AKT pathway. Interestingly, using either blocking IL-6/IL-6R signaling or PD-1/PDL1 inhibitors may serve as a therapeutic target for sensitizing cancer cells to CAR-T cell treatment and restoring BCA cells to Dox treatment.

CAFs produce many substances such as IL-6, IL-8, and TGF-β impacting cancer progression and immunosuppression [6‐9, 14]. TGF-β activated CAFs to secrete IL-6 resulting in the enhancement of CRC metastasis [28]. IL-6 is a major factor of BCA-CAF secretion that can induce resistance to anti-estrogens [29]. In HCC, CAFs were the major source of IL-6 [30]. High IL-6 expression CAFs greatly decreased CD8⁺ T cell infiltration in tumors [30]. CAFs-derived IL-6 induced pancreatic cancer cell migration and invasion [31]. CAF-secreted IL-6 reduced NK cells' function in CRC cells by promoting monocyte differentiation into M2 macrophages and recruiting to the tumor [32, 33]. No investigation, however, has assessed the role of CAF-derived IL-6 in T cell function regulation via induction of PD-L1 in BCA. In this current study, CAF-derived IL-6 exhibited a significantly higher level among other cytokines. IL-6 releases into TME and may be involved in cancer progression, immunosuppression, and the responses of cancer cells to chemotherapy and immunotherapy.

CAFs were positive for vimentin (VIM) and alpha-smooth muscle actin (ASMA), but negative for cytokeratin (CK) expression ensuring that CAFs culture is not contaminated by cancer cells [13]. The immunosuppressive role of FAP⁺ CAFs has been extremely studied in various types of cancers such as head and neck, pancreatic, breast, lung, and liver cancers [13]. In head and neck cancer, FAP⁺ CAFs inhibited CD8⁺ T cell proliferation and promoted regulatory T cell (Treg) recruitment by secreting TGF-β and IL-6 [34]. In the BCA mouse model, the elimination of FAP⁺ CAFs showed an increasing CD8⁺ T cell population [35]. IL-6 from CD10⁺ GPR77⁺ CAFs promoted tumor formation in lung cancer [27, 36]. CD10⁺GPR77 ⁺ CAFs enriched a survival niche for cancer stem cells (CSCs), enhanced tumor formation, and induced cancer chemoresistance by secreting IL-6 leading to driving the activation of NF-κB via p65 phosphorylation and acetylation, which maintained by complement signaling via GPR77 [27]. Targeting the CD10⁺GPR77⁺CAFs subset retards tumor formation and reverses chemoresistance by destroying the CSC niches [27, 36]. CD10⁺ GPR77⁺ FAP⁺ CAFs were associated with chemoresistance and overall survival of advanced gastric cancer patients who underwent neoadjuvant chemotherapy (NCT) [37]. FAP increasing drug resistance are various, such as promoting immunosuppression and producing chemokine [37]. CD10 and GPR77 have been proven to promote cancer formation and chemoresistance by providing a survival niche for CSCs and promoting an epithelial-mesenchymal transition [37]. In addition, CAFs affect the chemotherapy through an increased interstitial fluid pressure forming a barrier to the transcapillary transport of drugs from blood vessels to cancer cells [38]. All mentioned evidence supports the present results that high IL-6-secreting CAFs were positive for CD10⁺ GPR77⁺ FAP⁺ which may correlate with drug resistance and may regulate anti-tumor immune responses.

In addition to the IL-6 derived from CAFs, IL-6 can be secreted from BCA cells which then promotes cancer cell metastasis in an autocrine effect [39, 40]. Cancer cell-derived IL-6 acted to promote tumorigenesis in BCA, HCC, and lung cancer [41, 42]. Secreted IL-6 binding to membrane IL-6R induces STAT3 expression which is aberrantly active in BCA to promote cancer proliferation and anti-apoptosis [43, 44]. Moreover, STAT3 cooperates with NF-κB in IL-6 induction and the IL-6/STAT3 pathways synergize in the induction of c-MYC leading to promoting cancer cell survival [45].

In contrast to the tumor-promoting effect of IL-6 through the classical IL-6 signaling via membrane IL-6R and glycoprotein 130 (gp130) subunit, in some cancers, IL-6 trans-signaling which is a complex of IL-6 and the soluble IL-6R and membrane gp130 [46], but not IL-6 classic signaling, is mandatory for the development of liver carcinogenesis [46]. Therefore, specific inhibition of IL-6 trans-signaling, rather than total inhibition of IL-6 signaling, is sufficient to blunt tumor initiation and impair tumor progression without compromising IL-6 classic signaling-driven protective immune responses.

CAFs contribute to chemoresistance and immunotherapeutic resistance by upregulating immune checkpoint molecules [22, 23]. The CAF-derived IL-6 promoted PD-L1 expression via STAT3 and AKT pathways and specific inhibitors against pSTAT3 and pAKT attenuated PD-L1 expression induction by IL-6-derived CAF-CM. HCC-CAFs-induced PD-L1⁺ neutrophils through the IL6-STAT3 pathway involved in immunosuppression were previously reported [22]. Moreover, the activation of the STAT3-PI3K/AKT pathway leads to high expression of PD-L1 in various types of cancers [47]. PD-L1 expression is transcriptionally regulated by STAT3 and MYC [48]. The pSTAT3 dimers directly bind on the PD-L1 gene promoter inducing its expression [49] which has been detected in non-small cell lung cancer [50] and head and neck squamous cell carcinoma [51]. The immunosuppressive PD-1/PD-L1 pathway participates in cancer immune escape via the abnormal activation of the tumor-intrinsic STAT3-PI3K/AKT pathway leading to high expression of PD-L1 resulting in attenuation of anti-tumor T cell responses. Interestingly, IL-6 pathway inhibitors are currently tested in combination with chemotherapy in patients with BCA (NCT03135171), pancreatic cancer (NCT04258150, NCT02767557), or liver cancer (NCT04338685), who displayed potent anti-cancer activity with a low incidence of IL-6 drug toxicity [6]. The PD-L1 expression decreased by hesperidin through inhibition of AKT and NF-κB signaling pathways in the MDA-MB-231 cell line [19]. These reports confirm present findings that IL-6 can inhibit antitumor immunity by promoting PD-L1 expression via STAT3 and AKT pathways.

It is suggested that targeting the IL-6/IL-6R/STAT3 axis is expected to become an important strategy for BCA treatment [52]. IL-6 acts directly on cancer cells to induce the expression of STAT3-encoding proteins driving cancer proliferation, i.e., cyclin D1 and survival, i.e., BCL2-like protein 1 (BCL-xL). Early-phase trials exploring the safety and effectiveness of tocilizumab (anti-IL-6RmmAb) in BCA patients are currently ongoing (NCT03135171) [52]. Some high IL-6-containing-CMs, however, did not alter PD-L1 expression. These may be explained by the possibility that CAF secreted various substances which may affect IL-6 secretion by CAFs such as CXCL10 [6, 31]. Current evidence points to chemotherapy-induced DNA damage with nanoparticle albumin bound-paclitaxel (Nab-PTX) influencing the repertoire of CAF-secreted factors after treatment [31]. Nab-PTX treatment increases CXCL-10 expression in pancreatic cancer cells leading to reduced secretion of CAF-derived IL-6 subsequently impairing cancer migration and invasion [31].

DNA damage during chemotherapy is sensed by cyclic GMP-AMP synthase, a stimulator of interferon genes (STING). Dox-mediated DNA damage induced STING-mediated NF-κB activation in TNBC cell lines resulting in the induction of IL-6 secretion which could activate pSTAT3 leading to cancer cell survival enhancement and an immune-suppressive mechanism by PD-L1 expression induction [53]. The cancer cells containing DNA double-strand breaks increased IL-6 secretion by DNA damage response proteins such as ataxia-telangiectasia-mutated during oncogene-induced senescence [54]. Moreover, IL-6 could induce ATM kinase leading to DNA damage repair [55].

Moreover, CAFs reprogramming by vitamin A and vitamin D were shown to inhibit tumor-supportive secretomes [6, 56]. Vitamin A and vitamin D reprogramming strategies are both associated with TGF-β signaling pathway inhibition. Indeed, vitamin D decreases fibroblast activation [57], whereas vitamin A inhibits the fibroblasts' capacity to release active TGF-β, thus impeding CAF-secreted factors that are associated with the chemo- and immunotherapeutic resistance [58]. Additional research with a better understanding of the mechanisms of CAF secreted-substances that affect IL-6 secretion is needed to confirm.

Importantly, the previous studies of IL-6 in animal models support our findings that IL-6 mediated drug resistance and induced PD-L1 expression. Activation of IL-6 inflammatory loop-induced trastuzumab resistance in breast cancer mouse xenografts [59]. Using a Bcl-2 antagonist (sabutoclax) could inhibit the IL-6/STAT3 signaling to resensitize Dox-resistant breast cancer in vitro and in vivo models [60]. Blocking of STAT3 can prolong the survival time of tumor-bearing mice by suppressing tumor growth and increasing Dox sensitivity in osteosarcoma [61]. Moreover, the combination of IL-6 blockades and anti-PD-L1 antibodies reduced cancer progression in mouse pancreatic cancer [62].

CAR-T cell immunotherapy has emerged as a cancer therapeutic strategy [63]. FRα expression is associated with poor prognosis in BCA patients [64]. Several FRα-targeting immunotherapies, such as monoclonal antibody [65] and adoptive T cell therapy [66], have been developed. In the present study, FRα expression was found in TNBC cell lines which is consistent with a prior study that reported the high prevalence of FRα in TNBC [67]. It was proven that FRα-CAR-T cells specifically lysed FRα-expressing cells in vitro [25]. These current findings indicate that CAF-derived IL-6 increases PD-L1-attenuated CAR-T cell cytotoxicity (Fig. 6C). Targeting CAF-derived cytokines and chemokines in combination with immunotherapies can improve anticancer efficiency such as when targeting the CXCL12-CXCR4 axis reverses FAP + CAF-mediated immunosuppression and synergizes with anti-PD-L1 immunotherapy in pancreatic cancer [68]. In conclusion, therapeutics aim at interfering with the IL6/IL-6R and PD-1/PD-L1 pathways may be combined to improve the response to chemotherapy and cell therapy in TNBC patients.