Crosstalk between cancer‐associated fibroblasts and immune cells in cancer

Abstract Multiple studies have shown that cancer‐associated fibroblasts (CAFs) play an important role in tumour progression, including carcinogenesis, invasion, metastasis and the chemoresistance of cancer cells. Immune cells, including macrophages, natural killer cells, dendritic cells and T cells, play a dual role in the tumour microenvironment. Although increasing research has focused on studying interactions between distinct cells in the tumour microenvironment, the complex relationships between CAFs and immune cells remain unclear and need further study. Here, we summarize our current understanding of crosstalk between CAFs and immune cells, which may help clarify their diagnostic and therapeutic value in tumour progression.

immunosuppressive microenvironment in head and neck squamous cell carcinoma. 19 Other studies showed that CAFs secreted macrophage colony-stimulating factor to induce an M2 macrophage phenotype, which further promoted pancreatic tumour progression. 20 Because of complex compositions of the TME, the relationship between CAFs and immune cells needs further study and analysis. The present review discusses interactions between CAFs and different immune cell types, their impact on tumour progression and potential therapeutic targets.  22 A large proportion of CAFs appear to originate from the activation of resident tissue fibroblasts, which is constitutive and persistent. 23 Several studies showed that the activation of fibroblasts is a reversible process. 23, 24 Ren et al reported that the inhibitor of miR-21, AC1MMYR2 (AMR), could reprogramme the breast cancer-associated fibroblasts (BCAFs) into NFs. 24 Furthermore, the transdifferentiation of pericytes, endothelial cells and epithelial cells can also produce a CAF-like hybrid cell population that undergoes an endothelial-mesenchymal transformation 25 and epithelial-mesenchymal transformation. 26 Moreover, studies have reported that MSCs could recruit and proliferate into myofibroblasts in the TME. 27 Mesenchymal stem cells that derived from bone marrow acquired activated CAF phenotypes only in a conditioned medium of tumour cells. 28 29 Notably, that there is no precise molecular definition of CAFs, and CAFs tend to be a cellular state rather than a cell type. 30 Importantly, CAFs need to be distinguished from normal fibroblasts. 31 These F I G U R E 1 Origins of cancer-associated fibroblasts and its roles in cancer progression. CAFs can originate from resident tissue fibroblasts, bone marrow-derived mesenchymal stem cells, hematopoietic stem cells, epithelial cells, endothelial cells, vascular smooth muscle cells and pericytes. In the TME, CAFs can regulate cancer growth and proliferation, invasion and metastasis, angiogenesis, chemoresistance and metabolic reprogramming of the TME activated fibroblasts can be identified according to molecular markers. Some commonly used markers, such as α-SMA, fibroblast-specific protein-1 (FSP-1 or S1001A4) and FAP 1,3 ( Figure 2). Studies have shown that quiescent fibroblasts express vimentin as a molecular marker. However, the most widely used marker in CAFs is α-SMA, which may be because more myofibroblasts are in the tumour matrix. 32 In fact, myofibroblasts are thought to be the same as CAFs, but not all CAFs express SMA. Thus, myofibroblasts are considered one subtype of CAF. Another common marker of CAFs is FAP, which is also a marker of myofibroblasts. 33 However, the tissue distribution and function of FAP-α is not limited to stromal fibroblasts, and expression can also be detected in epithelial malignant cells. 34 Other studies have shown that tenascin-C, 35 periostin, 36 glial cell antigen-2 (NG-2), 21 desmin, platelet-derived growth factor-α and β (PDGFR-α and -β), thy-1 (CD90) and podoplanin can also be considered markers of CAFs. 37 However, these markers are not necessarily specific to CAFs; they can also be expressed in other cells. For example, α-SMA is also expressed in vascular myocytes. NG2 and PDGFR-β are also expressed in normal pericytes, and podoplanin is expressed in lymphatic endothelial cells. 38 Furthermore, Cannon et al found that paladin, a highly expressed actin binding protein in cancer, was expressed earlier than α-SMA when activated in CAFs. 39 Cytokeratin and CD31 are regarded as negative markers because CAFs had no epithelial or endothelial characteristics. 37,40 Because of the lack of specific CAF markers, combinations of markers may help identify CAFs. Combinations of morphological and markers are the most reliable way to identify CAFs.

| CROSS TALK B E T WEEN C AN CER-A SSO CIATED FIB ROB L A S TS AND IMMUNE CELL S
Inflammation is considered a marker of cancer and closely related to the reactivity of matrix fibroblasts. The relationship between inflammation and immune cells is inseparable. CAFs interact with immune cells and cancer cells via secreting cytokines, chemokines and other factors, such as collagens, MMPs, laminin, CXCL2, VEGF and TGF-β. 41 A large number of immune cells in cancer tissue have typically been associated with a better prognosis. However, accumulating evidence indicates that immune cells in cancer tissue do not play an anti-tumour role but rather contribute to the occurrence and development of cancer. 42 However, CAFs also play an important role in the TME Several studies have shown that immune cells interact with CAFs to regulate the TME 41,43,44 (Figure 3).

| Interaction between cancer-associated fibroblasts and macrophages
Numerous studies have shown that CAFs and macrophages interact to promote the progression of cancer. Some studies reported that accumulation of macrophages in TME correlated with poor prognosis of patients. 45,46 Mazur et al reported that type I collagen cleaved by FAP from activated fibroblasts, a post-prolyl peptidase, could act as the substrate of macrophages which recognized by macrophages class A scavenger receptors (SR-A); therefore, it increased the macrophages adhesion in cancer. 47 Tumour-associated macrophages (TAMs) consist of two groups with different phenotypes. M1 macrophages play an anti-tumour role by activating the immune system and producing reactive oxygen species, nitric oxide and TNF. M2 macrophages perform immunosuppressive functions, promoting tumour progression 48 and angiogenesis and degrading extracellular matrix. 49 Accumulating evidence indicates that CAFs drive epithelial-mesenchymal transformation, maintain the growth of cancer cells and interact with M2 macrophages to promote the occurrence and progression of malignant tumours. 50 Interestingly, TAMs and CAFs are often detected in the same area in tumour tissues.
However, the proportions of these two cells are different in distinct tumours. In gastrointestinal cancer, lung cancer, pancreatic cancer and prostatic cancer tissues, CAFs expressed higher density than TAMs. 51 However, in brain cancer, lymphoma, kidney cancer and hepatocellular carcinoma tissues showed higher density of TAMs. 51 Gok et al reported that compared to NFs, CAFs F I G U R E 2 Markers of CAFs could not only recruit monocytes via monocyte chemotactic protein-1 (MCP-1) and stromal cell-derived factor-1 (SDF-1), but also differentiated monocytes into M2 macrophages with higher expression of IL-10, therefore exerting immunosuppressive roles in breast cancer. 52 In prostate cancer, CAFs are beneficial factors for monocyte recruitment to cancer cells, mainly by transferring stromal derived growth factor-1 and promoting the transformation of macrophages into the M2 phenotype. 53 This complex interaction between tumour cells, CAFs and M2 macrophages enhances the motility of tumour cells, thereby promoting escape from primary tumours, metastatic diffusion and the angiogenesis of endothelial cells. High levels of ERα expression in CAFs inhibited the invasion and migration of prostate cancer by affecting TAM infiltration in vitro and in vivo. 54 ERα decreased the expression of chemokine (C-C motif) ligand 5 (CCL5) and IL-6 in CAFs and macrophages that were co-cultured with CAFs in conditioned medium. These data suggest that ERα + CAFs can be used as prognostic markers to predict the progression of prostate cancer. Kock et al found that the expression of prostaglandin E2 (PGE2), which was mainly secreted by CAFs, was blocked by the small-molecular inhibitor compound III (CIII), which regulated the activity of microsomal prostaglandin E synthase-1 (mPGES-1). CIII inhibited tumour progression by shifting the M1/M2 ratio in neuroblastoma. 55 Immunohistochemical staining and flow cytometry showed that CIII decreased the expression of CD206 and increased the ratio of M1/M2. 55 Cancer-associated fibroblasts regulate macrophages by secreting various cytokines. Macrophages can also regulate the status of CAFs. M2 macrophages affect the epithelial-mesenchymal transformation of fibroblasts. Fibroblasts that were activated by macrophages dynamically stimulated prostatic cancer, which were mediated by IL-6 and SDF-1. 53 Zhang et al found that in a co-culture with macrophages, MSCs that were derived from the human umbilical cord differentiated into CAFs, and promoted gastric epithelium cell malignancy via epithelial-mesenchymal transition (EMT). 56 However, research that has focused on the influence of macrophages on CAFs has been very limited, and further studies are needed.

| Interaction between cancer-associated fibroblasts and mast cells
Studies of mast cells initially focused on asthma and allergic diseases. However, in recent decades, more studies have found that mast cells also participate in innate and adaptive immune-related diseases, including cancer. [57][58][59] As a hormone-dependent tumour, CAFs in prostate tumours have a higher oestrogen receptor/androgen receptor (ER/AR) ratio compared with NFs. Ellem et al found that oestrogen-induced CAF expression recruited mast cells to the TME by secreting CXCL12 in prostate cancer. 60 Therefore, estrogens play an important role in mediating the interaction between CAFs and mast cells in prostate cancer.
Pereira et al found that mast cells secreted tryptase to promote the CAF-induced transformation of prostate epithelia cell morphology in a micro-tissue model. 61 In ameloblastomas, the expression of myofibroblasts and mast cells was positively correlated, and this positive correlation was also correlated with the aggressiveness of cancer cells. 62 This study showed that mast cells induced the differentiation fibroblasts into myofibroblasts and promoted myofibroblast proliferation. 62 Yang et al found that mast cells and fibroblasts interact with each other to regulate the tumour phenotype in neurofibromatosis type 1 (NF1). 63 NF+/− mast cells secreted TGF-β, which promoted the proliferation of fibroblasts. 63  Compared to the normal endometrial fibroblasts (NEFs), the expression of poliovirus receptor (PVR) was downregulated in CAFs. 66 PVR is expressed on the cell surface of NEFs and CAFs, which is a ligand of the NK activating receptor DNAX accessory molecule-1 (DNAM-1). Therefore, CAFs could inhibit the killing activity of NK cells through downregulation PVR on the cell surface, which further promoted cancer development. 66 Additionally, Zhang et al reported that CAFs secreted IL-8 to attract monocytes and promoted M2 macrophages polarization. 67 Furthermore, the synergetic effects of CAFs and TAMs, which induced by CAFs increasing the suppression of NK cells functions in colorectal cancer. 67 In addition, CAFs significantly suppressed the cytotoxicity function of NK cells via releasing PGE2 in melanoma. 68 In the presence of NK cells, the CAF-induced expression of PGE2 was higher than fibroblasts, 64 suggesting that NK cells can also influence the cytokine expression of CAFs. However, studies of the influence of NK cells on CAFs are limited, and further studies are needed.

| Interaction between cancer-associated fibroblasts and dendritic cells
Dendritic cells (DCs) are effective antigen-expressing cells that can stimulate the primary immune response by expressing class I and class II MHC complexes, co-stimulatory molecules and adhesion molecules, which can stimulate the immature T cell population. 69 Cancer-associated fibroblasts play a key role in immune regulation, but their role in regulating dendritic cells remains unclear.
Cheng et al reported that CAFs that were derived from hepatocellular carcinoma promoted the production of regulatory DCs, which was associated with low co-stimulatory molecule expression, high suppressive cytokines production and the enhancement of regulation of the immune response, including the proliferation of Treg cells through the upregulation of indoleamine-2,3-dioxygenase (IDO). 70 This research also showed that IL-6 that was derived from CAFs was necessary for the generation of IDO. Additionally, IDO inhibitors, anti-signal transducer and activator of transcription 3 (STAT3) and anti-IL-6 antibodies reversed the regulatory effect of CAFs on DCs. 70 Several studies have shown that IDO is highly expressed in regulatory DCs in tumour areas, which can inhibit the anti-tumour immune response. [70][71][72] IDO participates in the immune tolerance and suppression of tumour cells by inducing T cell anaemia and Treg cell proliferation. Therefore, the interaction between CAFs and DCs is complex and highly correlated with the T cell immune response. Vinit et al found that CSF1R inhibition decreased TAMs, which was correlated with the recruitment and accumulation of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) in tumours, and an increase in the secretion of CXCL-1 by CAFs. 76 This demonstrated a potential correlation between CAFs and MDSCs. Further studies showed that the inhibition of CXCR2 (a major CXCL-1 receptor that is expressed by granulocytes), combined with the inhibition of CSF1R reduced both TAMs and PMN-MDSCs in tumours, which significantly inhibited tumour growth. 76 Moreover, such inhibition further enhanced the tumour inhibition ability of PD-1 antibody.

| Interaction between cancer-associated fibroblasts and myeloid-derived suppressor cells
Circulating fibrocytes were reported to be a subset of MDSCs that are involved in regulating tumour immune escape. 77 However, CAFs appear to be similar to circulating fibrocytes, with few differences in molecular expression. Fibrocytes were shown to express both MHC-II and CD80/86, whereas CAF only expressed MHC-II.
CD11b was reported to be expressed by murine and human MDSCs, and CAFs were also mildly positive for CD11b/c. 77 This suggests that CAFs might have a similar function to MDSCs.

| Interaction between cancer-associated fibroblasts and tumour-associated neutrophils
Studies that have focused on tumour-associated neutrophils (TANs) are very limited. In hepatocellular carcinoma (HCC), CAFs promoted the activation and survival of neutrophils through the IL6/STAT3/ programmed death ligand 1(PDL1) signalling pathway. 78 In co-culture with CAFs, TANs expressed more PDL1, IL-8, CCL2 and TNF-α. 78 Neutrophils that were activated by CAFs regulated the STAT3-PDL1 pathway to impair the immunity function of T cells in HCC. 78

| Interaction between cancer-associated fibroblasts and T lymphocytes
Several studies have shown that Tregs are more abundant in stroma than in the cancer nest. Patients with higher Tregs expression had a worse prognosis than patients with lower Tregs expression. 80 Berna et al reported that the number of CD4 + Foxp3 + T-regulatory cells (Tregs) was higher in myofibroblast-depleted mice with pancreatic cancer, which exhibited the suppression of angiogenesis and promotion of EMT. 81 However, the depletion of myofibroblasts promoted tumour invasion, thus leading to a decrease in overall survival time in mice. Worse survival and poorly differentiated tumours were correlated with lower SMA + expression in patients with pancreatic ductal adenocarcinoma (PDAC). 81 In contrast to the traditional view that CAFs promote tumour progression, this study found that the presence of myofibroblasts was correlated with immunotherapy and a better prognosis of PDAC patients. Additionally, CAFs in adenocarcinomas with higher Tregs expression had higher TGF-β and VEGF expression. 80 Histochemistry confirmed that most TILs, including Tregs cells, were located in the cancer matrix and adjacent to CAFs. These findings suggested that these two cells play a role in the TME. 80 Immunoregulatory cytokines expression in CAFs may induce Tregs in the matrix, create a tumour-promoting microenvironment in lung adenocarcinoma, and result in a worse prognosis. 80 Interestingly, Costa et al found that because of the heterogeneity of CAFs in breast cancer, CAF-S1 and CAF-S4 represented two subsets of myofibroblasts. 82 This study found that CAF-S1 was correlated with CD25 + FOXP3 + lymphocytes and promoted the attraction of CD4 + CD25 + T lymphocytes via the secretion of CXCL12, thus promoting formation of an immunosuppressive environment. 82 Importantly, CAF-S1 induced Tregs differentiation and activity, but CAF-S4 did not exhibit these properties. 82  in CAFs could reactivate cytotoxic T cells. 85 Kato et al reported that co-cultured colon cancer cells with CAFs increased the expression of FoxP3 + TILs (regulatory T cells), while decreased the expression of CD8 + TILs (cytotoxic T cells). 86 This was due to CAFs secreted high levels of IL-6. The blockade of IL-6 could not only inhibit tumour growth, but also promote the accumulation of CD8 + TILs in tumour.

| REG UL ATI ON OF C AN CER-A SSO CIATED FIB ROB L A S TS IN C AN CER
Cancer-associated fibroblasts play an important role in cancer. The crosstalk between cancer cells and TME, especially stroma cells is widely discussed, which might be the major cause of tumour progression. 87 Fu et al showed CAFs could secrete and produce the energy metabolites, such as pyruvate and ketone bodies, in order to supply cancer cells. 88 Cancer-associated fibroblasts stimulate the growth and proliferation of tumour cells. Matthew et al found that CAFs activated an immune checkpoint to suppress the function of T cells, which was mediated by the engagement of PDL2 and FASL Therefore, CAFs contribute to the suppression of anti-tumour T cell response by regulating immune cells, such as promoting antigenspecific T cells death. 85 Cancer-associated fibroblasts can also promote the invasion and metastasis of tumour cells. Cancer-associated fibroblasts produce the tryptophan metabolite kynurenine and were shown to inhibit the differentiation of DCs and promote tumour cell growth and migration in lung cancer. 9 Galectin-1 upregulated tryptophan 2,3-dioxygenase (TDO2) expression in CAFs. The inhibition of TDO2 decreased tumour metastasis by promoting the T cell response in vivo. 9 Moreover, metalloproteinases that were secreted by CAFs promoted the release of Ras-related C3 botulinum toxin substrate (Rac1b)/cyclooxygenase 2 (COX-2)-mediated reactive oxygen species in cancer cells, which is essential for EMT, cell stemness and metastasis. 89  Finally, CAFs regulated metabolic reprogramming of the TME. By targeting nuclear transcription factors, p62 repressed tumour progression by regulating metabolic reprogramming. 91 In fact, p62 deficiency in stroma promoted the upregulation of ATF4, thus further promoting tumour proliferation by generating asparagine, which served as a source of nitrogen for cancer. 91 In addition, several researches have shown that exosomes from CAFs could promote the tumour progression. 92,93 Richards et al found that exosomes from CAFs significantly increased the chemoresistance to gemcitabine in pancreatic ductal adenocarcinomas (PDACs). 92 Li et al reported that exosomes from CAFs increased the expression of TGF-β1, which further enhanced the migration and invasion ability via SMAD signalling pathway in ovarian cancer. 93

| ANTI -C AN CER-A SSO CIATED FIB ROB L A S T THER APIE S
Fibroblasts from different parts of the body and organs have different characteristics, including their susceptibility to acquire CAFs phenotypes and interactions with adjacent epithelial cells and immune cells. 94 In contrast to cancer epithelial cells, genetic changes in CAFs (eg changes in copy number or mutations of oncogenes or tumour suppressor genes) are extremely rare. 30,95 Therefore, the promotion of tumours by CAFs does not appear to result from genetic alterations. Anti-CAFs therapies, including many drugs that target FAP, have been shown to exert significant anti-tumour effects in pre-clinical models. 96 High concentrations of curcumin were shown to have cytotoxic effects on CAFs. However, a low concentration of curcumin had few effects on the proliferation of CAFs but decreased the expression of α-SMA and vimentin, suggesting that curcumin at low concentrations can reverse the activation of fibroblasts. 97 Conditioned medium with CAFs increased the migration and invasion ability of pancreatic cancer cells, and conditioned medium with curcumin-treated CAFs had minimal effects on the migration ability of pancreatic cells. 97 105 Gottschalk et al developed a new compound DC vaccine (DC-shA20-FAP-TRP2) that targeted FAP and tumour antigen tyrosine-related protein 2 (TRP-2). 106 The vaccine enhanced the tumour infiltration of CD8 + T cells and induced antigen diffusion, leading to effective anti-tumour activity. 106 Mice that were treated with anti-CAF exhibited lower TGF-β expression in the TME. In mice that were immunized with the DC vaccine and anti-CAF, the expression of TGF-β was significantly reduced, which effectively reduced Tregs. 107 Additionally, reducing SDF-1 expression by inhibiting CAFs may be related to a decrease in Tregs migration in the TME, in which blockade of the CXCR4-CXCL12 axis prevented Tregs from migrating to the TME. 108 When combined with anti-CAF therapy, the efficacy of the DC vaccine increased, which contributed to the effective inhibition of tumour growth and reduced the level of immunosuppressive cytokines in tumour tissues. Furthermore, Yasuhiko et al reported that the inhibition of CAFs in SCID mice did not influence tumour growth but modulated the immune function in the TME. 107 In addition to basic research, some clinical studies have targeted CAFs. The activation of hedgehog (Hh) signalling is important for CAF function. In a phase I clinical trial (NCT02027376), Aurélie et al used docetaxel with smoothened inhibitors (SMOi), which inhibited hedgehog signalling in CAFs for the treatment of TNBC patients and improved outcomes. 109 Some patients even presented a complete clinical response (ie disappearance of the tumour lesions without new lesions). 109 In a phase I clinical trial, the antibody targeting of FAP with repeated infusions of sibrotuzumab was shown to be useful for the treatment of advanced FAP + cancer patients. 110 It is important for clinicians to find the antigens which specifically expressed in tumour cells or tumour stroma with minimum expressed in normal tissues.
Fibroblast activation protein (FAP) is a type II membrane-bound glycoprotein, which expressed on the activated CAFs in tumour stroma instead of normal tissues. 111 The blockade of FAP could inhibit the ability of CAFs in promoting cancer cells invasion and metastasis. Sibrotuzumab is the humanized antibody targeting FAP By using [ 131 I]sibrotuzumab, researchers found that the antibody mainly uptakes in the tumour sites instead of normal tissue sites. 110 Therefore, the advantage of targeting FAP is sibrotuzumab could successfully uptake in tumour sites because of FAP is not widely expressed in normal tissues antigen pool.
Thus, sibrotuzumab had high tumour specificity. 110  PDAC cells produce SHH ligands that bind to receptor patched-2 on CAFs. 112 However, in a phase II clinical trial (NCT01064622), the combination of gemcitabine with vismodegib (ie an inhibitor of the hedgehog pathway) did not improve overall survival or progressionfree survival in pancreatic cancer patients. 113 This treatment failure may be attributable to an inappropriate therapeutic combination.
Several studies showed that the inhibition of Hh is caused by IPI-296, which is also known as saridegib. 114 As an anti-FAP monoclonal antibody, unconjugated sibrotuzumab BIBH1 may be a potentially useful immunological-related antibody to target FAP for the treatment of colon cancer, 115 but no complete or partial response was observed in this study. Only two of 17 patients presented stable status in advanced metastasis colorectal cancer. This treatment failure may be attributable to the possibility that the unconjugated antibodies could not achieve a sufficient effect against solid tumours. 116 Narra et al found that as the Val-boroPro (Talabostat; an inhibitor of FAP enzymatic activity) inhibited tumour progression in metastatic colorectal cancer in a phase II trial. 96

E TH I C A L A PPROVA L A N D CO N S E NT TO PA RTI CI PATE
This review does not contain any studies with human participants or animals performed by any of the authors.

CO N FLI C T S O F I NTE R E S T S
The authors have declared that no competing interest exists.

AUTH O R S' CO NTR I B UTI O N S
Qing Yang designed the review. Yuanyuan An wrote the manuscript. All the figures were prepared by Yuanyuan An and revised by Qing Yang. Fengtian Liu and Ying Chen contributed to revise the manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used to support the findings of this study are included within the article.