Crosstalk between cancer-associated fibroblasts and immune cells in the tumor microenvironment: new findings and future perspectives

Certainly, other immune cells, such as monocytes and B cells, can crosstalk with CAFs as well. As we described above, CAFs are able to facilitate monocyte migration and trans-differentiation into M2-type TAMs [131, 132]. For B cells, only CXCL13 secreted by CAFs has been reported to enhance the recruitment of B cells [116]. Moreover, no other study has reported CAF-B cell interactions.

Direct CAF-depleting therapeutic strategies mainly depend on surface markers of CAFs, such as FAP, α-SMA and PDGFR. Therefore, CAF marker-based inhibitors are currently the major type of CAF-depleting therapies (Table 3). As one of the most viable CAF markers for potential clinical application, FAP has been prominent in recent studies of CAF-targeted therapy [324, 325]. The elimination of CAFs by FAP-targeting therapy can enhance the anticancer immune response mediated by high levels of certain inflammatory mediators, such as IFN-γ and TNF-α, and facilitate the toxic effects and metabolism of CD8 + T cells with a decreased desmoplastic stroma in the TME [224, 300, 301, 326]. Current FAP-targeting therapies mainly include diverse types of tumor vaccines and other immunotherapeutic approaches, such as adoptive T cell therapy, all of which can eliminate FAP + cells [327, 328].

FAP-based DNA vaccines are one of the principal types of cancer vaccines [315, 329]. The first DNA vaccine against the cancer stromal antigen FAP was developed by Loeffler et al. [330] in multidrug-resistant colon and breast carcinoma murine models. This vaccine is capable of eliminating CAFs by stimulating a CD8 + T cell-mediated immune response and further inhibit tumor growth and metastasis [330]. Recently, with advances in DNA vaccine studies, a novel type of vaccine termed the SynCon FAP DNA vaccine has been shown to not only disrupt immune tolerance and promote the antitumor immunity of both CD8 + and CD4 + T cells but also enhance the effects of other relevant tumor antigen-specific DNA vaccines [295]. Since the anticancer therapeutic effect of a single DNA vaccine targeting FAP is extremely limited [331], subsequent studies identified a novel therapeutic strategy that combines cyclophosphamide (CY) with a DNA vaccine that significantly increases the tumor inhibition rate by overcoming the tumor-stromal blockade and enhancing the nonspecific toxic effects of CY on tumor cells [296, 297].

In addition, DC vaccines are regarded as an effective strategy that induces a strong tumor immune response by replacing the role of impaired DCs in the TME [332]. Specifically, DC vaccines can enhance tumor antigen presentation by increasing costimulatory molecule and proinflammatory cytokine expression, thereby heightening cancer-specific T cell responses [333]. To improve the finite therapeutic effect of the previously established A20-silenced DC vaccine, Gottschalk et al. [298] developed a compound DC vaccine (DC-shA20-FAP-TRP2) that cotargets both tumor cells and FAP-positive CAFs. This vaccine was reported to elicit broad T cell responses and potent antitumor activity [298]. Moreover, when cooperating with other anti-CAF therapies, DC-based vaccines have been shown to reduce the level of TGF-β and consequently inhibit the migration of Treg cells into tumors [299]. Recently, studies have demonstrated that the fusion of DCs and CAFs contributes to a strong CTL response against CAFs, suggesting that it might be a potential method for improving the anticancer effect of current DC vaccine strategies [334]. Adenoviral vector vaccines are another type of FAP-targeting vaccine [335]. Similar to DNA vaccines, adenoviral vector vaccines such as the adenoviral vector of chimpanzee serotype 68 (AdC68)-mFAP vaccine can also induce T cell recruitment and enhance the function of melanoma-specific effector CD8 + T cells, thereby destroying FAP + stromal cells within the TME [300]. Additionally, other tumor vaccines contain whole-cell vaccines [312, 336] and peptide immunization vaccines [337].

FAP is also an important target for adoptive T cell therapy, especially for chimeric antigen receptor (CAR) therapy [338]. FAP-specific CAR T cells function to deplete most FAP + cells and restrict tumor stroma generation, along with promoting the uptake and antitumor effects of chemotherapeutic drugs, such as gemcitabine [301]. Notably, several studies have observed that the elimination of FAP + cells by CAR T cells causes severe side effects, such as significant bone marrow toxicity and cachexia [339, 340]. Considering that CAR T cells usually deplete FAP-overexpressing cells (e.g., CAFs) rather than normal cells with basal FAP levels, there might exist a different window of therapeutic opportunity for differential single-chain variable fragments (scFv) of CAR constructs [326]. In view of the possible toxicity of FAP-targeted adoptive T cell therapy, scientists try to develop prodrugs that are activated only by FAP through unique postprolyl endopeptidase activity, and these prodrugs have been proven to induce less systemic toxicity and have greater therapeutic potential [341, 342]. For instance, an in vivo and in vitro study in breast and prostate cancer illustrated that a FAP-activated prodrug contributed to the selective death of stromal cells and exerted a significant anticancer effect [302]. Finally, other FAP-targeting treatments, including FAP-inhibiting small molecules (talabostat and cisplatin) [303, 304], antibodies [305], immunotoxins [306, 307] and FAP-targeted liposomes [308, 343], also provide therapeutic benefits.

α-SMA has been identified as another surface marker of CAFs [324]. Current studies of therapies targeting α-SMA remain stagnant because of their dual effects on tumor progression. First, the depletion of α-SMA + CAFs was proven to suppress the metastasis of cancer cells as well as tumor angiogenesis in breast cancer and PDAC models [309, 310]. However, more importantly, targeting α-SMA was also reported to induce disease aggression and progression by enhancing the infiltration of CD3 + Foxp3 + Treg cells in the TME [310]. For other CAF markers, such as PDGFR, the associated clinical trials are still ongoing [311, 344].

As discussed before, neither FAP nor α-SMA is exclusively expressed on CAFs, suggesting that more highly selective markers are required to improve the precision of CAF-based therapies. Recently, Su et al. [104] identified two novel specific surface proteins for a CAF subpopulation (CD10 and GPR77), which might be promising targets for inhibiting tumorigenesis and tumor chemoresistance.

Considering the crucial role of interactions between CAFs and other cells, particularly the crosstalk between CAFs and the TIME, in the immune suppression induction of the TME, it seems more feasible to restrict CAF activation and their interacting progress by targeting CAF-associated crucial effector molecules such as growth factors, cytokines and chemokines as well as signaling pathways (Table 4). For example, vitamin A deficiency is a main contributor to the activation of PSCs [43]. Therefore, by restoring retinol levels in PSCs, all-trans retinoic acid (ATRA) can reset them to the inactive state [43]. In a parallel study, ATRA treatment of a PDAC model exerted substantial antitumor effects, including remarkably increasing the numbers of CD8 + T cells in juxta-tumoral compartments and limiting tumor cell invasion [312]. TGF-β plays an important role in the activation of CAFs and the interaction between CAFs and immune cells, as previously described, indicating that TGF-β inhibition therapy might be capable of restoring impaired immune responses in the TME [30, 191, 213]. Currently, multiple preclinical and clinical studies of TGF-β-based immunotherapies are ongoing [192]. Galunisertib (LY21577299), for example, is a small-molecule inhibitor of transforming growth factor beta receptor 1 (TGF-βR1) with discernable cardiac toxicities rarely reported during treatment [345]. Phase II clinical trials for pancreatic cancer and hepatocellular carcinoma have exhibited the significant therapeutic activity of galunisertib against tumors, whether administered in combination with gemcitabine or as monotherapy [313, 314]. Additional reports have documented that the combination of a treatment targeting CAF-derived TGF-β with checkpoint inhibitors such as anti-PD-L1 antibodies exerts greater immunological effects on tumors than the respective monotherapies [346‐348]. Therefore, Ravi et al. [349] attempted to engineer anti-CTLA4 or anti-PD-L1 antibodies fused with the TGF-βR2 extracellular domain, resulting in anti-CTLA4-TGF-βR2 and anti-PDL1-TGF-βR2 chimeras. Compared with ipilimumab (a type of anti-CTLA-4 antibody) monotherapy, the anti-CTLA4-TGF-βR2 molecule presents more effective at decreasing tumor-infiltrating Treg cells and suppressing tumor progression [315]. In addition, previous studies have demonstrated that IL-6 together with the JAK/STAT3 signaling pathway in the TME participate in processes that strongly suppress immune effector cell function and facilitate tumor progression induced by CAFs [122, 243, 350‐352]. Tocilizumab, a humanized anti-IL-6R monoclonal antibody, has exhibited extensive antitumor and anti-chemoresistance effects on multiple cancer types in preclinical studies [353‐355]. In a phase I clinical trial, high-dose tocilizumab was observed to stimulate CD8 + T cell activation and increase the levels of antitumor-associated effectors such as IFN-γ and TNF-α, thereby enhancing anticancer immunity [316]. Moreover, preclinical evidence indicates that therapy targeting IL-6/JAK/STAT3 signaling might also augment the antitumor efficacy of immune checkpoint-inhibiting monoclonal antibodies [356, 357]. Since the CCL2-CCR2 signaling axis plays an essential role in MDSC-induced immune suppression, therapies suppressing the CCL2-CCR2 signaling pathway might be effective in blunting MDSC immunoinhibitory effects [242]. Clinical trials have previously reported that CCR2 inhibition in combination with FOLFIRINOX (fluorouracil[5-FU], leucovorin, irinotecan and oxaliplatin) can significantly reduce the numbers of tumor-infiltrating macrophages and Treg cells while increasing the number of effector T lymphocytes in the TME, consequently enhancing antitumor immunity in pancreatic cancer [358]. Recently, a treatment combining CCX872 (a CCR2-specific antagonist) and FOLFIRINOX was reported to achieve a better therapeutic effect and clinical prognosis with less M-MDSC infiltration [317, 318]. Another essential chemokine, SDF-1 (also termed CXCL12), is also involved in the activation and immune suppression of CAFs. Via blocking the combination of SDF-1 and its receptor CXCR4, AMD3100 (a CXCR4 inhibitor) is able to rapidly promote the accumulation of T cells and effectively eliminate cancer cells by synergizing with an anti-PD-L1 antibody [114].

CAF-targeted treatments are also being designed to block fibrosis progression, including therapies targeting fibrosis-activated signaling pathways and their fibrosis products (Table 4), which ultimately restrict CAF-induced ECM remodeling. The altered ECM after treatment partly alleviates the suppression of immune effector cell recruitment into tumors in the TME, thus enhancing anticancer immunity [281, 282, 359].

As mentioned before, the FAK signaling pathway is an important fibrosis-activated signaling pathway of CAFs involved in matrix stiffness and immune suppression [284]. A specific FAK inhibitor (VS-4718) was reported to inhibit immunosuppressive cell infiltration, such as TAMs, MDSCs and Treg cells, in the TME, and significantly improved overall survival (OS) in a PDAC model [284]. Moreover, as FAK inhibitors might heighten the antitumor effect of immune checkpoint inhibitors, associated phase I clinical trials have been set up to assess their therapy responses [320].

Therapies against CAF-derived ECM proteins, such as tenascin C (TNC), HA and MMPs, might also be capable of inhibiting desmoplastic reactions and consequently reducing the immunosuppressive effect of ECM on immune cells. The ECM protein TNC appears to be an appealing target for antitumor treatment due to its high expression in cancer tissues and functional association with tumor cell adhesion, migration, invasion and proliferation along with immune evasion [323]. Several antibodies have been established to specifically target TNC in order to improve the delivery of effector molecules into TNC-rich tumor tissue [360]. For example, the antibody F16 shows good specificity for TNC and is designed in complex with IL-2 to promote the recruitment of immune cells in the TME [361]. In a breast cancer model, when in combination with F16-IL-2, cytotoxic drugs such as paclitaxel or doxorubicin induce a more obvious restriction of tumor growth than chemotherapeutic agents alone [319]. Furthermore, anti-TNC dsRNA (ATN-RNA), which has sequence homology to TNC mRNA and is developed using RNA-based technologies, has produced substantial improvements in the clinical prognosis of patients with glioblastoma multiforme (GBM) [321]. Recent research in autophagy-deficient TN breast cancer revealed that TNC suppression sensitizes T cell-mediated cell killing and enhances the anticancer effects of single anti-PD1/PD-L1 therapy, indicating a potential therapeutic strategy that links TNC inhibitors and immune checkpoint blockade (ICB) for TN breast cancer [362]. Excessive tumor-stromal HA together with collagen usually results in substantial vessel compression, which blocks the delivery of peripheral immune cells and drugs into tumor vessels [363]. PEGPH20, a PEGylated human recombinant PH20 hyaluronidase, functions to deplete HA and then potentiate chemotherapeutic efficiency by improving vascular patency. Further clinical trials confirmed that PEGPH20 along with gemcitabine and nab-paclitaxel combination therapy could suppress tumor growth and significantly increase patient survival [322]. Moreover, losartan (an angiotensin inhibitor) also exhibits the ability to reduce the production of stromal collagen and HA by inhibiting TGF-β1, connective tissue growth factor (CTGF) and endothelin-1 (ET-1) profibrotic signals [323]. Finally, regarding MMP therapies, the disappointing antitumor effects of current MMP inhibitors have been gradually reported, but several novel types are being translated into early clinical trials [364].