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Researching for your CAFS project

Cafs research planner.

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Research Methodology - Research Methods

• Open access
• Published: 11 October 2021

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

• Xiaoqi Mao 1 , 2 , 3 , 4   na1 ,
• Jin Xu 1 , 2 , 3 , 4   na1 ,
• Wei Wang 1 , 2 , 3 , 4   na1 ,
• Chen Liang 1 , 2 , 3 , 4 ,
• Jie Hua 1 , 2 , 3 , 4 ,
• Jiang Liu 1 , 2 , 3 , 4 ,
• Bo Zhang 1 , 2 , 3 , 4 ,
• Qingcai Meng 1 , 2 , 3 , 4 ,
• Xianjun Yu 1 , 2 , 3 , 4 &
• Si Shi   ORCID: orcid.org/0000-0002-6652-0629 1 , 2 , 3 , 4  

Molecular Cancer volume  20 , Article number:  131 ( 2021 ) Cite this article

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Cancer-associated fibroblasts (CAFs), a stromal cell population with cell-of-origin, phenotypic and functional heterogeneity, are the most essential components of the tumor microenvironment (TME). Through multiple pathways, activated CAFs can promote tumor growth, angiogenesis, invasion and metastasis, along with extracellular matrix (ECM) remodeling and even chemoresistance. Numerous previous studies have confirmed the critical role of the interaction between CAFs and tumor cells in tumorigenesis and development. However, recently, the mutual effects of CAFs and the tumor immune microenvironment (TIME) have been identified as another key factor in promoting tumor progression. The TIME mainly consists of distinct immune cell populations in tumor islets and is highly associated with the antitumor immunological state in the TME. CAFs interact with tumor-infiltrating immune cells as well as other immune components within the TIME via the secretion of various cytokines, growth factors, chemokines, exosomes and other effector molecules, consequently shaping an immunosuppressive TME that enables cancer cells to evade surveillance of the immune system. In-depth studies of CAFs and immune microenvironment interactions, particularly the complicated mechanisms connecting CAFs with immune cells, might provide novel strategies for subsequent targeted immunotherapies. Herein, we shed light on recent advances regarding the direct and indirect crosstalk between CAFs and infiltrating immune cells and further summarize the possible immunoinhibitory mechanisms induced by CAFs in the TME. In addition, we present current related CAF-targeting immunotherapies and briefly describe some future perspectives on CAF research in the end.

Introduction

In recent years, the tumor microenvironment (TME) has received increasing attention due to its crucial roles in tumor immune suppression, distant metastasis, local resistance and the targeted therapy response [ 1 , 2 , 3 , 4 ]. The TME is a highly complicated system mainly composed of tumor cells, infiltrating immune cells (such as macrophages, dendritic cells and lymphocytes), cancer-associated stromal cells (such as cancer-associated fibroblasts (CAFs)), endothelial cells and lipocytes, along with the extracellular matrix (ECM) and multiple signaling molecules [ 5 , 6 ]. As one of the most important stromal components in the TME, CAFs have shown biological heterogeneity in many aspects, including the cell of origin, phenotype and function [ 7 , 8 ]. Originating from a variety of cell types, CAFs are characterized by increased expression of markers such as alpha smooth muscle actin (α-SMA), fibroblast activation protein (FAP), fibroblast-specific protein 1 (FSP1), platelet-derived growth factor receptor (PDGFR)-α/β and vimentin [ 9 ]. Most of CAF subpopulations usually exhibit cancer-promoting effects, while the discovery of cancer-restraining CAFs (rCAFs), which are reported to exert inhibitory effects on tumor progression, indicates that some subsets are just the opposite [ 10 ]. Substantial previous reports have demonstrated that CAFs participate in multiple stages of tumor development through diverse pathways [ 11 , 12 , 13 ]. Through bidirectional signaling with tumor cells and other cells mediated by CAF-derived cytokines, chemokines, growth factors and exosomes within the TME, CAFs not only facilitate tumor proliferation but also induce immune evasion of cancer cells [ 14 , 15 , 16 ]. Moreover, CAFs are also able to degrade stromal ECM by releasing matrix metalloproteinases (MMPs) while synthesizing new matrix proteins to provide structural support for tumor invasion and angiogenesis [ 17 , 18 ]. Overall, more specific roles and detailed mechanisms of CAFs in cancer pathogenesis and progression remain to be further explored.

The tumor immune microenvironment (TIME) is a novel proposed concept that has been reported to be closely related to the clinical prognosis of patients with tumors [ 19 ]. Distinct immune cell populations, including innate and adaptive immune cells, such as myeloid cells and lymphocytes within the TME, comprise most of the TIME [ 20 , 21 ]. Notably, the TIME also determines the state of the immune response in the TME, which primarily depends on the composition and activity of infiltrated immune cells, as well as several correlated influencing factors, including the cell surface expression of immune checkpoint molecules and alterations in the associated matrix [ 20 ]. Currently, an increasing number of researchers have begun to focus on the immunosuppressive effect of CAFs that is achieved by interactions with TIME components, especially immune cells [ 22 , 23 , 24 ]. For instance, CAFs are capable of restricting the recruitment of immune effector cells such as CD8 + T cells into tumor tissues through the secretion of different chemokines [ 25 ]. Moreover, the proportions of immunosuppressive cells such as M2-type macrophages, regulatory T (Treg) cells and myeloid-derived suppressor cells (MDSCs), which are modified by CAFs, have been shown to be significantly increased in the TIME, thereby contributing to tumor immune suppression [ 26 , 27 , 28 ]. Additionally, some cytokines secreted by activated immune cells, such as interleukin (IL)-1β, can induce the transformation of normal fibroblasts into proinflammatory CAFs and further facilitate the recruitment of inhibitory immune cells and immune suppression in the TME [ 29 ]. Undeniably, a deep understanding of the multidimensional interactions between CAFs and infiltrating immune cells within the TME will help us better to determine the immunosuppressive mechanisms induced by CAFs, and further exploration of these interactions will probably identify more potential molecular targets for CAF-targeted therapy.

This review mainly focuses on recent advances in the crosstalk between CAFs and tumor-infiltrating immune cells, immune checkpoint molecules and related ECM alterations in the TME, along with the possible mechanisms of CAF-induced immune suppression according to these interactions. We also describe the current understanding of the origins, activators, heterogeneity and plasticity of CAFs. Finally, we introduce major CAF-based targeted immunotherapeutic strategies that may enhance antitumor immunity in the TME, and present some deficiencies of CAF studies currently existed and several promising research directions in the future.

Origins and activators of CAFs

Activated by diverse signaling pathways, CAFs are derived from multiple cells of origin (FIG. 1 ). The presence of various cellular precursors for CAFs, which might explain why CAFs are a heterogeneous cell population, has been confirmed by a large amount of evidence [ 8 , 30 , 31 ].

The origins and related activating pathways of cancer-associated fibroblasts (CAFs) in the tumor microenvironment (TME). CAFs are derived from multiple cell types through the following distinct mechanisms: A Tissue-resident fibroblasts and quiescent stellate cells are converted into CAFs by the stimulation of modulators including transforming growth factor-beta (TGF-β), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), fibroblast growth factor 2 (FGF-2), stromal-derived factor-1 (SDF-1), reactive oxygen species (ROS) and insulin-like growth factor 1 (IGF-1) as well as the deficiency of vitamin A; B The trans-differentiation progress of mesenchymal stem cells (MSCs) into CAFs contain epithelial-mesenchymal transition (EMT) along with the recruitment and activation induced by various stimulating molecules such as TGF-β1, C–C chemokine ligand 2 (CCL2), C–C chemokine ligand 5 (CCL5), C-X-C chemokine ligand 12 (CXCL12) and tumor-derived exosomes; C Adipocytes together with pericytes and smooth muscle cells can transdifferentiate into CAFs by TGF-β1 and Wnt3a; D Endothelial cells are transformed into CAFs through endothelial-to-mesenchymal transition (EndMT); E Epithelial cells are transformed into CAFs through epithelial-to-mesenchymal transition (EMT); F Monocytes are transformed into CAFs through monocyte-to-myofibroblast trans-differentiation (MMT)

Tissue-resident fibroblasts, also termed quiescent fibroblasts, are one of the major sources of CAFs [ 32 , 33 ]. Tissue-resident fibroblasts in distinct carcinomas are recruited and activated through the stimulation of different modulators [ 34 ], including transforming growth factor (TGF)-β [ 35 ], hepatocyte growth factor (HGF) [ 36 ], platelet-derived growth factor (PDGF) [ 37 ], fibroblast growth factor 2 (FGF-2) [ 37 ], stromal-derived factor-1 (SDF-1) [ 38 ] and reactive oxygen species (ROS) [ 39 , 40 ]. In some tumor types, stellate cells might be another source of CAFs. For instance, in liver and pancreatic cancer models, quiescent pancreatic stellate cells (PSCs) and hepatic stellate cells (HSCs) express CAF-like surface markers such as α-SMA upon activation with TGF-β and PDGF, which convert them into activated CAFs [ 41 , 42 ]. Moreover, vitamin A deficiency has been reported to be involved in the activation of PSCs and islet stellate cells (ISCs) [ 43 , 44 ]. Furthermore, a recent study revealed that the stimulation of insulin-like growth factor 1 (IGF-1) signaling is also critical for HSC activation [ 45 ].

A substantial number of reports have indicated that mesenchymal stem cells (MSCs) function as precursors of CAFs [ 46 , 47 ]. The transformation of bone marrow mesenchymal stem cells (BMSCs) into CAFs might be a multistep and complicated process involving epithelial-mesenchymal transition (EMT), bone marrow-derived progenitors, cell–cell communication and stimulation with various cytokines [ 48 , 49 , 50 ]. Effectors stimulating MSC trans-differentiation vary from cancer to cancer. In prostate carcinoma, MSCs transdifferentiate into CAFs through the activation of tumor cell- and stromal cell-secreted TGF-β1, as well as C-X-C chemokine ligand (CXCL) 16 [ 51 , 52 ]. In breast cancer, bone marrow-derived mesenchymal stem cells (BM-MSCs) are recruited and transformed into distinct CAF subsets through the TGF-β1-mediated osteopontin-myeloid zinc finger 1 (MZF1) pathway [ 53 ]. Subsequent studies further confirmed the importance of the TGF-β signaling pathway in the transformation of MSCs into CAFs; for example, the pathway is involved in their metabolic reprogramming toward increased glycolysis [ 54 ]. In addition, secreted C–C chemokine ligand (CCL) 2, CCL5 and CXCL12 in the TME are also involved in the recruitment and transformation of MSCs [ 46 , 55 ]. Notably, multiple internal mechanisms for MSC transformation might exist. For example, the differentiation of BM-MSCs into CAFs induced by cancer cells was reported to primarily depend on the Notch and Akt signaling pathways [ 56 ]. However, Peng et al. [ 57 ] discovered that tumor-derived GRP78, an endoplasmic reticulum (ER) chaperone, elicits the differentiation of BM-MSCs into CAFs in a TGF-β/SMAD-dependent manner.

Adipocytes, especially white adipocytes [ 58 , 59 ], are regarded as another cell type within CAF precursors. For instance, human adipose tissue-derived stem cells (HASCs) transdifferentiate into CAFs with a fibroblastic phenotype (α-SMA and tenascin-C expression) upon activation with TGF-β1 [ 60 ]. Moreover, research in breast cancer has found that Wnt3a produced by tumor cells mediates the β-catenin-dependent differentiation of adipocytes into adipocyte-derived fibroblasts (ADFs), which express one CAF marker, FSP-1, at high levels [ 61 , 62 ]. Furthermore, several other potential sources of CAFs have been identified, such as epithelial cells [ 63 , 64 ], pericytes [ 65 ], monocytes [ 66 ], endothelial cells [ 67 ] and smooth muscle cells [ 68 ]. These cells can be activated and differentiate into CAFs through various mechanisms. Through EMT and endothelial-to-mesenchymal transition (EndMT), most epithelial cells and endothelial cells can express a number of fibroblast markers, such as α-SMA and FAP [ 69 , 70 ]. Peritoneal mesothelial cells, one of the special cell types among epithelial cells, are reported to be converted into CAFs through TGF-β1-induced mesothelial-mesenchymal transition (MMT) [ 71 ]. Additionally, monocytes are able to transdifferentiate into myofibroblasts through a process termed monocyte-to-myofibroblast trans-differentiation (MMT), which is induced by ROS through the p38-mitogen-activated protein kinase (MAPK) signaling pathway [ 66 , 72 ].

During the generation of CAFs, various factors in the TME induce CAF activation by stimulating certain distinct signaling pathways (Table 1 ). In addition to the regulatory molecules described above, inflammatory mediators such as IL-1β and IL-6 act through the nuclear factor-kappa B (NF-κB) and Janus kinase (JAK)-signal transducer and activator of transcription 3 (STAT3) signaling pathways, respectively, to promote the malignant progression of CAFs [ 29 , 73 ]. Analogous to the classical activating mechanisms reported for normal fibroblasts, such as wound stimulation, CAFs respond to damage-associated molecular patterns (DAMPs) released by necrotic cancer cells, which subsequently induce the activation of the internal NOD-like receptor protein 3 (NLRP3) inflammasome signaling pathway and ultimately contribute to tumor growth and metastasis through the secretion of inflammasomes [ 74 ]. Furthermore, tumor-derived exosomes that contain different transmitters, such as the CD44v6/C1QBP complex, have exhibited significant facilitation of the activation of HSCs and thus direct CAFs to induce tumor metastasis as well as ECM remodeling [ 45 ]. Moreover, heat shock factor 1 (HSF1, a master regulator of the heat shock response) was also reported to primarily orchestrate concomitant stimulation of both the β-catenin and YAP/TAZ signaling pathways through Dickkopf-3 (DKK3, an HSF1 effector), consequently resulting in aggressive behaviors of CAFs [ 75 , 76 ]. Of note, epigenetic changes are capable of initiating and maintaining a proinvasive phenotype of CAFs [ 77 ]. Albrengues et al. [ 77 , 78 ] revealed that leukemia inhibitory factor (LIF) can induce a series of internal epigenetic modifications in fibroblasts, including alterations in STAT3 acetylation, phosphatase methylation of SH2-containing protein tyrosine phosphatase-1 (SHP-1) and JAK1 phosphorylation, ultimately stimulating the JAK1/STAT3 signaling pathway, which sustains the proinvasive activities of CAFs. Finally, CAF activation also depends on environmental stressors (ROS, matrix stiffening, etc.) [ 79 , 80 , 81 , 82 , 83 , 84 ] and DNA damage during radiation therapy [ 85 ].

Although researchers have recently employed advanced technology, such as genetic lineage tracing [ 86 ], the origins of CAFs among most cancer types remain elusive due to the lack of exclusive markers for normal fibroblasts and CAFs [ 87 ]. Lineage tracing methods combined with single-cell spatial analyses are urgently needed to identify the exact contribution of each cell type and illustrate the detailed mechanism of CAF activation during cancer development.

Heterogeneity and plasticity of CAFs

Due to the existence of multiple types of cellular precursors, the CAF population behaves as complex cells with various fibroblast phenotypes and distinct functions among many cancer types [ 88 ] (Table 2 ). During the past several years, several subtypes of CAFs in pancreatic ductal adenocarcinoma (PDAC) have been identified through the application of transcriptome analyses, but none of these subtypes was given a specific definition [ 89 , 90 ]. Ӧhlund and colleagues [ 91 ] first discovered and identified two spatially divided and totally opposite subsets of CAFs—myofibroblastic CAFs (myCAFs) and inflammatory CAFs (iCAFs). myCAFs are located in direct proximity to cancer cells and have high α-SMA expression; iCAFs are located far more distantly from neoplastic cells and express less α-SMA but secrete more IL-6 and other inflammatory factors (e.g., IL-8, IL-11 and LIF), and they might participate in immune suppression and tumor cachexia by stimulating the STAT3 signaling pathway. Subsequently, the presence of myCAFs and iCAFs in pancreatic cancer was confirmed through droplet-based single-cell transcriptomics technology [ 92 ]. Notably, the researchers also termed a newly discovered CAF subpopulation “antigen-presenting CAFs” (apCAFs), and these cells express MHC class II and CD74 instead of classical costimulatory molecules. Coincidentally, a subpopulation observed previously that was able to present antigens and contribute to the suppression of T cell-mediated antitumor responses was analogous to apCAFs [ 93 ]. Another study in PDAC reported similar subtypes of the CAF population described above. According to the results of single-cell RNA sequencing, fibroblast population 1 (FB1) and fibroblast population 3 (FB3) [ 94 ] might represent the previously described iCAF and myCAF populations, respectively. Interestingly, in that study, the researchers found that FB3 also processed and presented antigens by expressing numerous MHC-II-associated genes, indicating that FB3 might be a mixed population comprising myCAFs and apCAFs. Furthermore, a recent study further assessed the intertumor and intratumor heterogeneity of human PDAC-derived CAFs [ 95 ]. At least four subtypes of CAFs were characterized by different mRNA expression profiles, and periostin (POSTN), myosin-11 (MYH11), and podoplanin (PDPN) were selected as biomarkers for subtype A to C CAFs. Moreover, subtype A CAFs, which are located in the tumor invasive front, are associated with tumor capsule formation and metastatic progression. The subtype B population might be related to a poor prognosis, while subtype C CAFs appear to be related to a favorable clinical prognosis of patients with cancer. Various CAF subpopulations have been reported in human breast cancer. For example, four different CAF subsets (S1-S4) are classified based on their diverse expression of fibroblast markers (e.g., CD29, FAP, α-SMA, PDGFRβ, FSP1 and caveolin 1 (CAV1)) [ 96 ]. Both the CAF-S1 and CAF-S4 subsets exhibit protumorigenic properties, while the CAF-S1 subset enhances the differentiation, recruitment and activation of Treg cells, thereby facilitating immune suppression of tumors; the properties of this CAF-S1 subset are similar to those of the CAF-S1 subset observed in ovarian cancer [ 97 ]. Another study on axillary lymph nodes [ 98 ] further indicated that the CAF-S1 subset promotes cancer cell migration and EMT initiation mainly by secreting CXCL12 and TGF-β, while the CAF-S4 subset facilitates the migration and invasion of tumor cells through the NOTCH pathway. Additionally, the presence of myCAFs, iCAFs and apCAFs in breast cancer was recently documented, and these cells were previously identified in PDAC [ 99 ]. In addition, Bartoschek et al. [ 100 ] defined another four subpopulations of CAFs—vCAFs, mCAFs, cCAFs and dCAFs—according to their distinct cellular sources using single-cell RNA sequencing. vCAFs, mCAFs, and dCAFs appear to originate from perivascular cells, resident fibroblasts, and malignant cells, respectively. In addition, CAF subpopulations with different fibroblast phenotypes have also been detected in oral squamous cell carcinoma (OSCC) [ 101 ], colorectal cancer [ 102 ] and mesenchymal high-grade serous ovarian cancer (HGSOC) [ 97 ].

CAFs are composed of not only heterogeneous subsets with distinct phenotypes but also heterogeneous subpopulations with diverse functions [ 9 , 30 ]. Observations indicate that two functionally different populations of CAFs, cancer-promoting CAFs (pCAFs) and rCAFs, may exist [ 112 ]. Generally, most CAF subsets function as pCAFs rather than rCAFs. Studies have revealed that pCAFs mainly express FAP-α or α-SMA to suppress antitumor immunity through multiple pathways [ 38 , 93 , 96 , 103 , 113 ]. Modulators secreted by pCAFs, such as TGF-β, IL-6 and CXCL12, are able to promote the proliferation and invasion of cancer cells [ 114 ]. However, a recent study indicated that one of the CAF subsets in PDAC that expresses meflin (one potential marker) exerts antitumor effects on both mouse models and human cells, and this subset was subsequently identified as rCAFs [ 10 ]. Importantly, the presence of rCAFs is not limited to the context of PDAC [ 105 , 106 , 107 ]. Patel et al. [ 108 ] reported a myofibroblastic CAF subpopulation that inhibited cancer cell stemness by secreting bone morphogenetic protein 4 (BMP-4) in oral carcinoma. In other tumor types, including colon [ 109 ], bladder [ 110 ] and intestinal cancers [ 111 ], tumor-suppressive roles of CAFs have also been reported, suggesting a wide distribution of rCAFs across various types of cancer. However, considering the lack of in-depth phenotypic and functional characterization of CAFs, further explorations of CAF heterogeneity in most other cancer types are currently extremely difficult.

As CAFs contain multiple heterogeneous subpopulations, researchers have recently debated whether these diverse subtypes are able to interconvert, which would indirectly confirm the plasticity of CAFs. Several studies indicate that the answer to these questions is “yes”. For example, iCAFs in pancreatic cancer have been reported to be able to transform into myCAFs upon the activation of TGF-β signaling or the inhibition of the IL-1-induced JAK/STAT signaling pathway, suggesting potential plasticity between these two cellular subtypes [ 99 ]. Furthermore, research has also discovered an intermediate state between the iCAF and myCAF phenotypes termed α-SMA + p-STAT3 + cells, which might subsequently be a potential target for tumor immunotherapies [ 99 ]. Moreover, in colorectal cancer, CAF-A cells (one of the CAF subtypes) were reported to be capable of converting into CAF-B cells (another CAF subtype) during the transformation of normal fibroblasts into CAFs [ 102 ]. In addition to the research described above, few studies have recently reported CAF plasticity. Currently, in-depth research on many other reported important pathways, such as the epidermal growth factor receptor (EGFR), Wnt and Hippo signaling pathways [ 115 ], and improved recognition of the epigenetic regulation of CAF states are required to help improve our understanding of CAF plasticity [ 87 ].

Interaction between CAFs and the immune microenvironment in tumors

Based on accumulating evidence, CAFs in the TME play important roles in regulating the antitumor activities of tumor-infiltrating immune cells, including innate and adaptive immune cells, in the TIME [ 7 , 116 ]. In addition, they promote the expression of immune checkpoint molecules and ECM remodeling to indirectly influence the recruitment and activity of immune cells [ 116 ]. Through the secretion of cytokines, chemokines and other effector molecules, including TGF-β, CXCL2, collagens, MMPs and laminin, CAFs can prompt immune cells to participate in the occurrence and development of cancer, along with facilitating the degradation and remodeling of the ECM [ 117 , 118 ]. Of course, some noteworthy effects of several immune cells on CAFs have also been identified [ 29 , 119 ]. To date, many studies have shown that interactions between CAFs and immune cells as well as other immune components are capable of modulating the TIME and thus inhibiting the antitumor immune response (Fig.  2 ) [ 120 , 121 ].

Crosstalk between cancer-associated fibroblasts (CAFs) and immune components in the tumor immune microenvironment (TIME). CAFs can orchestrate an immunosuppressive TME via interacting with the immune microenvironment in tumor. Through the secretion of multiple chemokines, cytokines and other effector molecules such as transforming growth factor-beta (TGF-β), interleukin-6 (IL-6), C-X-C chemokine ligand 12 (CXCL12), C–C chemokine ligand 2 (CCL2), stromal-derived factor-1 (SDF-1), vascular endothelial growth factor (VEGF) along with indoleamine 2,3-dioxygenase (IDO) and prostaglandin E2 (PGE2), CAFs modulate immune cells-mediated antitumor immunity through the following pathways: Promoting the trans-differentiation or polarization of immune cells such as tumor-associated macrophages (TAMs), tumor-associated neutrophils (TANs), mast cells (MCs), dendritic cells (DCs) and T lymphocytes into certain protumorigenic cell subsets; Facilitating the activities of immune inhibitory cells in terms of recruitment, activation and immunosuppressive effects including M2-type TAMs, N2-type TANs, regulatory DCs (rDCs), regulatory T(Treg) cells and myeloid-derived suppressor cells (MDSCs); Restricting the cytotoxic activity and cytokines production of effector immune cells like natural killer (NK) cells and cytotoxic T lymphocytes (CTLs). Notably, several infiltrating immune cells such as TAMs, TANs, MCs and DCs can in turn exert promoting effect on CAFs activation and function, thereby contributing to the formation of immune suppressive loops. Moreover, CAFs can also upregulate the expression of immune checkpoint molecules such as programmed cell death protein 1 (PD-1)/programmed death receptor ligand-1 (PD-L1) and cytotoxic T lymphocyte associate protein-4 (CTLA4)/B7 in both themselves and other cells in the TME to induce T-cells dysfunction. Meanwhile, CAFs are able to remodel extracellular matrix (ECM) to facilitate immune suppression through the production of fibronectin, collagen and metalloproteinases (MMPs) as well as the activation focal adhesion kinase (FAK) signaling pathway. Finally, immune checkpoint molecule overexpression on CAF surface as well as matrix deposition around would inhibit CAF apoptosis and facilitate their activation and function

Interaction between CAFs and innate immune cells in the TME

Interaction between cafs and tumor-associated macrophages (tams).

Macrophages that infiltrate tumors, known as tumor-associated macrophages (TAMs), are classified into two distinct subsets that are activated by different polarizing cytokines, termed M1 (lipopolysaccharide (LPS) alone or with Th1 cytokines) and M2 (Th2 cytokines) [ 122 ]. M1-type macrophages mainly behave as an antitumor role in the TIME by mediating antibody-dependent cellular cytotoxicity and producing ROS and tumor necrosis factor (TNF) [ 123 ], whereas M2-type macrophages exhibit tumor-promoting activity by contributing to the activation of tumor angiogenesis, immune suppression, invasion and metastasis of cancer cells and remodeling of the ECM [ 124 , 125 ].

As a key component of the TIME, TAMs play critical roles in its modulation, especially in tumor immune suppression [ 126 , 127 ]. TAMs are the most prominent immune cells in the vicinity of CAF-populated areas, suggesting tight interactions between these two cell types [ 128 ]. High expression of both CAF and TAM markers, such as α-SMA, FAP and S100 calcium binding protein A4 (S100A4), along with CD163 and CD209, is related to worse clinical prognosis of patients with some tumors [ 128 , 129 ]. A substantial number of studies have shown that CAFs promote the recruitment of monocytes (macrophage precursors) and their differentiation into protumorigenic macrophage subsets (M2-type TAMs) via multiple regulatory molecules, thereby impairing responses from effector T cells and inducing immune suppression in the TME [ 130 ]. For example, in breast cancer, by secreting monocyte chemotactic protein-1 (MCP-1), SDF-1 and chitinase 3-like 1 (Chi3L1), CAFs are able to facilitate monocyte migration and enhance the tendency of these cells to polarize into the M2 phenotype [ 131 , 132 ]. Furthermore, a similar effect of CAFs on TAMs was discovered in prostate carcinoma [ 133 , 134 ]. Moreover, Mace et al. [ 135 ] documented the central role of CAF-derived macrophage colony-stimulating factor 1 (M-CSF1), IL-6, and CCL2 in monocyte recruitment and the increased M2/M1 macrophage ratio in pancreatic cancer. Other cytokines, including IL-8, IL-10, TGF-β and CCL2 (in skin tumors), secreted by CAFs have also been demonstrated to promote the recruitment of monocytes and their transformation into M2 macrophages [ 136 , 137 , 138 ]. In addition to facilitating macrophage recruitment and trans-differentiation, more importantly, CAFs are capable of inducing the immunoinhibitory properties of TAMs. Utilizing flow cytometry analysis, Gordon et al. [ 139 ] observed increased expression of programmed cell death protein 1 (PD-1) on the cell surface of CAF-induced M2-type TAMs. A high level of PD-1 expression in TAMs was proven to be involved in both innate and adaptive antitumor immune response suppression by subsequent studies, including decreasing their own phagocytic potency against tumor cells and inhibiting T-cell infiltration and proliferation [ 140 ]. In contrast to their stimulatory effect on TAMs, CAFs might also inhibit some aspects of TAM activities. Estrogen receptor alpha (ERα), for instance, whose expression on CAFs suppresses macrophage infiltration and restricts prostate cancer invasion, is mediated by decreased CCL5 and IL-6 expression. Mechanistically, Mazur et al. [ 141 ] revealed the importance of FAP (a CAF marker) in the communication between CAFs and TAMs. The authors found that FAP participated in the interaction between CAFs and SR-A (class A scavenger receptors) + TAMs mainly by cleaving type I collagen and increasing macrophage adhesion.

Reciprocally, TAMs with the M2 phenotype regulate CAF activation and progression as well [ 119 , 142 ]. In the study by Comito et al. [ 133 ], aside from confirming the promoting effect of CAFs on TAMs, M2-type macrophages were also able to enhance EMT progression to stimulate CAF activation by secreting soluble factors such as IL-6 and SDF-1. Moreover, TAMs were recently shown to influence the trans-differentiation and activity of MSCs, one of the cellular precursors of CAFs [ 48 ]. For instance, Zhang et al. [ 143 ] observed that macrophages could facilitate MSCs to acquire CAF-like properties and a proinflammatory phenotype to remodel the inflammatory microenvironment, which potentiated the oncogenic transformation of gastric epithelial cells. Additionally, in an in vitro coculture study, TAM-like macrophages were reported to induce both the proliferation and invasion of CAF-like BM-MSCs, thereby contributing to the progression of neuroblastoma [ 48 ]. Subsequently, activated CAFs induced by macrophages further enhance TAM activity, and consequently make up a positive loop that promotes cancer development and immune inhibition in the TME.

Recently, studies regarding the effect of CAFs on TAMs have been continuously reported, whereas the effect of macrophages on CAFs has not been comprehensively investigated and clarified. Further investigations of the mechanisms underlying CAF-TAM interactions in the TME are needed to advance current cancer-targeted therapies.

Interaction between CAFs and tumor-associated neutrophils (TANs)

Increasing evidence indicates that tumor-associated neutrophils (TANs), a significant component of the TIME, also exhibit phenotypic heterogeneity and functional versatility [ 144 , 145 ]. Analogous to the dichotomy of TAMs (M1 and M2), neutrophils can acquire an antitumor phenotype (N1) or a protumorigenic phenotype (N2) based on whether they are activated by TGF-β [ 146 , 147 , 148 ]. But unlike TAMs, the difference between N1 and N2 TAN phenotypes relies on the distinct degree of activation rather than different polarizing molecules [ 149 ].

Notably, CAFs might be able to modulate the polarization of TANs. As a recent study of hepatocellular carcinoma reported, CAF-derived cardiotrophin-like cytokine factor 1 (CLCF1) induces the polarization of N2-phenotype neutrophils by upregulating CXCL6 and TGF-β expression in tumor cells, thereby facilitating tumor progression [ 150 ]. More importantly, CAFs probably participate in all stages of the malignant progression of TANs and ultimately suppress the antitumor immune response in the TME. Through the secretion of SDF-1α, CAFs are able to recruit peripheral neutrophils to tumors [ 151 ]. Moreover, C-X-C chemokine receptor 2 (CXCR2), a cytokine receptor that is expressed by CAFs, was proven to be a primary factor participating in the recruitment of neutrophils in tumors, indicating that CAFs might enhance the migration of TANs in a CXCR2-dependent manner [ 152 , 153 ]. Next, CAF-derived IL-6 stimulates the STAT3 signaling pathway in TANs, consequently inhibiting the activity of T cells and inducing immune tolerance through the expression of PD-1/programmed death ligand 1 (PD-L1) [ 151 ]. In addition, Zhu et al. [ 154 ] discovered a bidirectional interaction between gastric cancer mesenchymal stem cells (GC-MSCs) and neutrophils. On the one hand, GC-MSCs can induce the chemotaxis and activation of neutrophils and sustain their survival through the IL-6-mediated STAT3-extracellular regulated protein kinases 1/2 (ERK1/2) axis. On the other hand, activated TANs, in turn, are capable of promoting the differentiation of MSCs into CAFs. Overall, the specific mechanisms underlying the mutual effects of CAFs and TANs on each other remain unclear due to the limited number of reports.

Interaction between CAFs and mast cells (MCs)

In recent decades, studies of mast cells (MCs) have placed more focus on their roles in cancer than on their roles in allergic diseases [ 155 , 156 ]. As a component of the TIME, interestingly, MCs exert dual effects on tumor progression—both promotion and inhibition of tumor growth—which depend on the specific MC localization, cancer type and the degree of tumor progression [ 157 , 158 , 159 , 160 , 161 ]. As cancer promoters, on the one hand, MCs contribute to the stimulation of angiogenesis and lymphangiogenesis along with the degradation of ECM by producing different pro-angiogenic molecules (vascular endothelial growth factor (VEGF)-A, VEGF-B, FGF-2, heparin, histamine and stem cell factor (SCF)) [ 162 , 163 , 164 , 165 , 166 ], lymphangiogenic molecules (VEGF-C and VEGF-D) [ 167 ], matrix metalloproteinase-9 (MMP-9) and proteases (tryptase and chymase) [ 168 , 169 ]. On the other hand, as antitumor effectors, MCs produce mediators (e.g., tryptase, chondroitin sulfate, TNF, IL-1 and IL-6) that increase antitumor inflammatory reactions, inducing tumor apoptosis and decreasing the invasiveness of cancer cells [ 170 , 171 ].

Excess numbers of CAFs and MCs in tumor islets are strongly associated with the aggressiveness of cancer, and their interactions directly contribute to tumor progression [ 172 , 173 ]. In prostate cancer, with the overexpression of estrogen inside, CAFs can potentiate MC proliferation, migration and inflammatory cytokine secretion and thus exhibit protumorigenic effects [ 174 ]. Meanwhile, CAF-derived CXCL12, induced by estrogen, was observed to be involved in the recruitment of MCs by combining with CXCR4 [ 174 ]. Furthermore, Ma et al. [ 175 ] discovered that PSCs could facilitate the activation and proliferation of MCs as well. This study also identified the stimulatory effect of MCs on CAFs. IL-13 and tryptase, which are released by MCs, conversely stimulate CAF proliferation in a TGF-β2-STAT6-independent manner [ 175 ]. Increasing CAFs subsequently resulted in the formation of a fibrotic TME and ultimately suppressed antitumor immunity and therapeutic responses [ 175 ]. Moreover, MCs in neurofibroma have also been reported to be capable of promoting CAF activity, such as enhancing the proliferation and secretion of CAFs through the TGF-β signaling pathway, thereby increasing the protumor effects of CAFs [ 173 ]. Additionally, a recent study in a microtissue model of prostate cancer revealed cooperation between MCs and CAFs, which induced the early malignant morphological transition of benign epithelial cells [ 176 ]. To date, research on the correlation between MCs and CAFs in tumors is still lacking. Considering the unique role of MCs and their mediators in the TIME, studies elucidating how CAF-MC interactions are implicated in tumor immunity are required to provide better immunotherapy and clinical services.

Interaction between CAFs and natural killer (NK) cells

Natural killer (NK) cells, members of the innate immune system, naturally respond to tumor cells [ 177 , 178 , 179 , 180 ]. The activity of NK cells depends on the expression and stimulation of activating or inhibitory receptors on the cell surface [ 181 ]. NK cell-activating receptors include NK group 2D (NKG2D), NKp30, NKp44, NKp46 and DNAX accessory molecule 1 (DNAM-1), while killer immunoglobulin-like receptors (KIRs) and CD94/NK group 2A (NKG2A) expressed on NK cells are inhibitory receptors [ 179 , 182 ]. In solid tumors, various soluble inhibitory factors and cell types, such as CAFs, comprise the immunosuppressive TME, contributing to the impaired functionality of infiltrating NK cells [ 183 , 184 ].

An increasing number of studies indicate that CAFs exert inhibitory effects on NK cells through multiple processes, including NK receptor activation, cytotoxic activity and cytokine production, in a direct or indirect manner [ 9 , 185 ]. For example, under the influence of melanoma-associated fibroblasts, both the expression of NKp30, NKp44 and DNAM-1 activating receptors on the cell surface and the formation of cytolytic granules in NK cells are suppressed, which mainly depends on the prostaglandin E2 (PGE2) released by CAFs [ 186 ]. In hepatocellular carcinoma, CAFs educate NK cells to transition into an inactivated phenotype through PGE2 and indoleamine 2,3-dioxygenase (IDO) and create an unresponsive state in antitumor immunity [ 187 ]. Interestingly, NK cells themselves can facilitate the formation of the suppressive loop induced by CAFs via promoting the secretion of PGE2 [ 188 ]. Certainly, CAFs can also restrict the activity and function of NK cells indirectly by modulating the expression of their activating receptor-associated ligands on tumor cells. For instance, according to Ziani et al. [ 189 ], CAFs in melanoma reduce the expression of MICA/B (two ligands of NK-activating receptors) on tumor cells, thereby suppressing NKG2D-dependent cytotoxic activity and IFN-γ secretion. Another study reported that a reduction in poliovirus receptor (PVR, a ligand of an NK-activating receptor) expression on the cell surface plays a critical role in the CAF-mediated suppression of NK cell killing activities [ 190 ]. In addition, macrophages induced by CAFs are reported to inhibit NK cell cytotoxicity and activation, which indicates that CAFs can regulate NK cells through other immune cells [ 137 ]. When cocultured with NK cells, higher PGE2 expression is detected on CAFs than on normal fibroblasts [ 188 ], suggesting that NK cells can influence the certain protein expression in CAFs as well. However, currently, only few studies have assessed the effect of NK cells on CAFs, and further investigations are needed to clarify this interacting progress.

The detailed mechanism of crosstalk between CAFs and NK cells is complicated and multiple effector molecules might participate in the interaction. TGF-β has been widely reported to be a key cytokine connecting CAFs with NK cells in tumors [ 191 ]. Substantial studies have proven that CAF-secreted TGF-β significantly inhibits the activation and cytotoxic activity of NK cells [ 192 ]. One of the possible mechanisms is that TGF-β reduces the production of interferon-γ (IFN-γ) and downregulates cell surface activating receptors, such as NKG2D [ 193 , 194 ]. For instance, TGF-β can inhibit DNAX-activation protein 12 (DAP12) transcription and reduce the expression of NKp30 and NKG2D by stimulating miR-183, thus silencing NK cells [ 195 ]. Moreover, Viel et al. [ 196 ] reported that TGF-β1 selectively downregulated NKp30, NKp46, NKG2D and DNAM-1 expression in vitro through the activation of the SMAD2/3-dependent signaling pathway. In addition to TGF-β, the exploration of other related molecules is still ongoing.

Interaction between CAFs and dendritic cells (DCs)

Tumor-infiltrating dendritic cells (DCs), a heterogeneous group consisting of diverse subpopulations, play a crucial role in the activation and regulation of innate and adaptive immune responses in the TIME through the high expression of class I and class II MHC complexes, adhesion molecules and costimulatory molecules [ 197 , 198 ]. In recent years, several investigations have illustrated that CAFs can drive immune evasion of tumor cells by blocking DC maturation, antigen presentation and their associated adaptive immune responses. However, their in-depth mechanisms remain unclear. By activating the IL-6-mediated STAT3 pathway, CAFs in hepatocellular carcinoma can recruit normal DCs and induce them to transdifferentiate into regulatory DCs (rDCs), disabled DCs that express costimulatory molecules at a low level and hardly present antigens, but secrete inhibitory cytokines such as IDO [ 199 ]. Further studies have revealed the importance of regulatory DC-derived IDO in the promotion of T cell anergy and Treg cell proliferation, which consequently results in the restriction of T cell-mediated immunity [ 200 ]. Another study of lung cancer indicated that both CAF-released IDO1 and tryptophan 2,3-dioxygenase (TDO2) induced by lung cancer-derived galectin-1 are responsible for the impaired differentiation and function of DCs through the degradation of tryptophan [ 201 , 202 ]. In addition, studies have demonstrated that VEGF produced by CAFs is involved in the abnormal differentiation and impaired antigen-presenting function of DCs via inhibiting the activation of NF-κB [ 203 , 204 ]. Meanwhile, VEGF is also able to facilitate immune tolerance by upregulating PD-L1 expression on the DC surface [ 205 ].

Interaction between CAFs and adaptive immune cells in the TME

Interaction between cafs and t lymphocytes.

T lymphocytes play a key role in modulating the adaptive immune response, and they comprise different subpopulations, such as Treg cells, helper T (Th) cells and cytotoxic T lymphocytes (CTLs) [ 206 ]. Numerous studies have illustrated the role of CAFs in modulating T cell activities and functions.

Treg cells with high Foxp3 expression are known to have crucial functions in the restriction of antitumor immunity [ 207 ]. Utilizing histochemical staining, Kinoshita et al. [ 27 ] confirmed that Treg cells are located adjacent to CAFs. Furthermore, the infiltration of both Foxp3 + Tregs and CAFs in the tumor stroma was correlated with a poor prognosis according to clinical data [ 27 ]. These results all indicate that potential crosstalk between CAFs and Treg cells might exist. Evidence of the interaction between CD70 + CAFs and naturally occurring Tregs has already been reported [ 208 ]. In a study of colorectal cancer, researchers revealed that CAFs stimulate the migratory activity of Treg cells and markedly increase their frequency in tumor sites [ 208 ]. Moreover, the recruitment of CD4 + CD25 + Treg cells to CAFs also depends on the chemokine CCL5 according to studies examining breast cancer [ 209 , 210 ]. Other molecules, such as VEGF-A, one of the growth factors released by CAFs, have been observed to directly or indirectly participate in Treg cell induction and maintenance [ 211 , 212 ]. In addition to promoting the recruitment and infiltration of Treg cells, CAFs also promote their transformation to ultimately induce immune suppression. As shown in the study by Chen et al. [ 213 ], CAF-derived TGF-β can facilitate the differentiation of naïve T cells into CD4 + CD25 + Treg cells by inducing the expression of the Foxp3 gene in T lymphocytes. Additionally, FAP + PDGFRβ + CAFs in breast cancer, also termed CAF-S1 cells (introduced earlier in the review), were proven to not only enhance the migration of CD4 + CD25 + T cells by releasing CXCL12 but also express CD73, dipeptidyl peptidase IV (DPP4) and B7H3, enabling them to transform CD4 + T cells into Foxp3 + Treg cells [ 96 ]. Recently, Zhao X and colleagues [ 214 ] discovered that downregulation of CD68 in CAFs facilitates the secretion of CCL17 and CCL22 from tumor cells and further indirectly increases the infiltration of Treg cells. However, interestingly, Özdemir et al. [ 105 ] obtained the opposite result from the experiment: the exhaustion of myofibroblasts in PDAC increases the proliferation of CD4 + Foxp3 + Tregs and subsequently inhibits immune surveillance, suggesting that a possible mixed and dual relationship might exist between CAFs and Treg cells.

Th cell subsets mainly include Th1, Th2, and Th17 cells, which are mostly differentiated from naïve CD4 + T cells [ 215 ]. By secreting various specific cytokines, Th1 and Th2 cells participate in cellular and humoral immunity, respectively [ 216 ]. Several reports have shown the great influence of CAF-associated activities on Th cell polarization, while their specific effects remain unclear. For example, when CAF activation proteins are targeted by a DNA vaccine, the polarization of the Th2 subset is suppressed at the same time, indicating that activated CAFs might promote the differentiation above [ 217 ]. Subsequently, De Monte et al. [ 218 ] found that thymic stromal lymphopoietin (TSLP) produced by activated CAFs in pancreatic cancer functions to promote Th2 polarization. In prostate cancer, in contrast, CAFs drive the polarization of naïve CD4 + T cells from the Th2 to Th1 phenotype by stimulating the miR21/Toll-like receptor 8 (TLR8) axis through the release of lactate [ 219 ]. In addition, by producing TGF-β1, CAFs can facilitate Th17 cell differentiation in vivo and disease development [ 220 ]. Altogether, CAFs modulate the transformation of most Th cells into immunoinhibitory subpopulations in tumors to create an immunosuppressive and cancer-adaptive TME and then exert a proinvasive effect on cancer cells.

CD8 + T cells, also called CTLs, mediate cytotoxic activities mainly by inducing the apoptosis of tumor cells, which is considered the most critical component of antitumor immunity [ 221 , 222 ]. A substantial number of studies have reported the interactions between CAFs and CD8 + T cells and documented the inhibitory effect of CAFs on CD8 + T cell infiltration, growth and antitumor immunity [ 223 ]. Multiple factors account for the decreased infiltration of CD8 + T cells in the TME. For instance, by secreting cytokines such as CXCL12, activated PSCs are able to facilitate the trafficking of CD8 + T cells away from the juxta-tumoral compartment and thus reduce the frequency of infiltrating CTLs in tumor islets [ 25 ]. Subsequently, the importance of the CXCL12 signaling pathway in the regulation of tumor-infiltrating CD8 + T cell migration induced by FAP + CAFs, has been confirmed in several reports [ 224 , 225 ]. Certainly, the physical barriers and hypoxia in the TME caused by CAF-mediated ECM modification are also responsible for T cell movement restriction [ 226 ]. CAFs release various angiogenic factors in response to hypoxia, such as VEGF, which leads to decreased cell adhesion molecule (e.g., intercellular cell adhesion molecule (ICAM)-1/2 and vascular cell adhesion molecule-1 (VCAM-1)) expression on endothelial cells [ 227 ]. Due to the lack of cell adhesion molecules, the extravasating progress of peripheral CD8 + T cells into tumor sites through the vasculature is hard to maintain [ 228 ]. In addition, CAFs can also reduce CD8 + T cell recruitment by releasing IL-6 and TGF-β, and inhibit their cytotoxic activities toward tumor cells as well [ 113 , 229 ]. Further related clinical trials have indicated that IL-6 blockade therapy effectively improves the function of T cells and the prognosis of patients with cancer [ 113 , 229 ]. According to the research of Goehrig et al. [ 230 ], CAFs can exert a direct suppressive effect on CD8 + T cell function, including their proliferation, activation and cytotoxic activity, through the secretion of βig-h3 (one ECM protein, also termed TGF-βi). Mechanistically, CAF-derived βig-h3 induces the combination of hydrogen peroxide inducible clone-5 (HIC-5) protein and Y505 phosphorylated Lck by binding to CD61 (one CD8 + T cell surface marker) and consequently decreases the transduction of T cell receptor (TCR) signaling [ 230 ]. Moreover, arginase II and galectin expressed in CAFs are also involved in the progression of suppressing CD8 + T cell proliferation and promoting T cell anergy [ 231 , 232 , 233 ]. Of note, as previously described, CAFs are capable of inhibiting CD8 + T cell cytotoxic function in indirect manners. CAFs not only blunt antigen presentation of DCs or NK cells by disturbing their normal differentiation [ 187 , 199 ], but also induce immunoinhibitory subsets (e.g., TAMs and Treg cells) and immune checkpoint expression to impair effector T cell antitumor responses [ 130 , 151 ]. Recently, in-depth research has revealed a possible suppressive mechanism by which CAFs in the TME might function in a similar manner to normal DCs, including participating in antigen sampling, processing and presentation and upregulating the expression of immune checkpoint molecules (factor associated suicide (FAS)/factor associated suicide ligand (FASL) and PD-1/programmed death ligand 2 (PD-L2)), thereby promoting a decrease in the number of CD8 + T cells and an increase in tumor cell viability [ 93 ]. Since CAFs can suppress the immune reaction in the TME by regulating the properties of various T cell subsets, targeted immunotherapies aimed at the CAF-T cell interaction might be effective at stimulating an impaired antitumor response.

In conclusion, CAFs facilitate the cancer-promoting phenotype transition of naïve T cells, enhancing immune inhibitory T lymphocyte function and suppressing the activity of effector T lymphocytes, thereby resulting in immune suppression in the TME. Currently, there is still a lack of studies reporting the effect of T lymphocytes on CAFs, which might be a novel potential direction for future research.

Interaction between CAFs and MDSCs

Originating from bone marrow, MDSCs are famous for their strong immunosuppressive activity in the TIME [ 234 ]. MDSCs mainly contain two cell subsets, termed polymorphonuclear MDSCs (PMN-MDSCs) and monocytic MDSCs (M-MDSCs), which are phenotypically and morphologically similar to neutrophils and monocytes, respectively [ 235 , 236 ]. In contrast to MDSCs that are activated by bacteria and viruses, MDSCs in the TME exhibit less phagocytic activity while continuously releasing anti-inflammatory cytokines, ROS and nitric oxide (NO), thereby contributing to the promotion of cancer angiogenesis, invasion, metastasis and immune tolerance [ 237 , 238 , 239 ].

Recently, a novel MDSC subset, named circulating fibrocytes, was reported to exhibit phenotypic and functional similarity to CAFs, suggesting a possible association between MDSCs and CAFs [ 240 ]. By releasing various cytokines and chemokines, CAFs can facilitate the infiltration and generation of MDSCs and consequently suppress effector T cell antitumor activity. Evidence indicates the essential role of CCL2 in the recruitment of both PMN-MDSCs and M-MDSCs [ 28 , 241 ]. As a major source of CCL2, CAFs might induce MDSCs to migrate into tumor sites by stimulating the STAT3 signaling pathway [ 225 ]. For example, CAFs in lung squamous cell carcinoma (LSCC) have been reported to promote peripheral C–C chemokine receptor (CCR)2 + monocyte migration via CCL2 and then reprogram them into M-MDSCs [ 242 ]. The accumulation of immunoinhibitory subpopulations (M-MDSCs) in the TME finally contributes to CD8 + T cell growth and IFN-γ production restriction [ 242 ]. Moreover, in hepatic carcinoma, Deng et al. [ 243 ] found that recruited monocytes can differentiate into M-MDSCs, and this transformation is induced by CAFs through IL-6 in a STAT3-dependent manner, which subsequently results in extensive inhibition of T cell proliferation and function. Another study described similar effects of CAF-secreted CXCL12 on monocytes in triple-negative (TN) breast cancers [ 244 ]. Recent research in esophageal squamous cell carcinoma confirmed the importance of CAF-secreted IL-6 in MDSC differentiation and observed that CAF-derived exosome-packed microRNA-21 (miR-21) is also responsible for the generation of M-MDSCs via activating STAT3 signaling [ 245 ]. In addition, under the inhibitory action of tranilast (a CAF suppressor), the expression of CAF-derived SDF-1, PGE2 and TGF-β1 is decreased, along with a low-level differentiation of original MDSCs [ 246 ]. These findings indicate that SDF-1, PGE2 and TGF-β1 probably participate in the differentiation and modulation of MDSCs [ 246 ]. Finally, CXCL1, a granulocytic chemokine produced by CAFs, might also be involved in PMN-MDSC recruitment [ 247 ].

Interaction between CAFs and other immune cells

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.

Interaction between CAFs and other immune components in the TME

Cafs upregulate the expression of immune checkpoint molecules on the cell surface to induce immunologic tolerance.

High expression of immune checkpoint molecules on the surface of T-cells and tumor cells has been identified as a main contributor to the dysfunction of T lymphocytes in the TME [ 248 , 249 , 250 , 251 ]. PD-L1 and PD-1, for example, are well-known checkpoint molecules. The binding of PD-L1 to its receptor PD-1 on activated T cells hampers antitumor immunity by counteracting T cell-activating signals [ 252 ].

CAFs themselves can express different ligands of immune checkpoint molecules on their cell surface, including PD-L1, PD-L2, B7-H3/H4, galectins and the enzyme IDO [ 93 , 253 , 254 , 255 , 256 ]. Studies have demonstrated that the overexpression of PD-L1 and PD-L2 on CAFs among colon tumors, melanoma, carcinomas and lung cancer substantially induces T cell exhaustion and deactivation [ 93 , 257 , 258 , 259 ]. Furthermore, α-SMA + CAFs expressing high levels of B7-H3 were recently shown to exhibit prolonged survival because of the antiapoptotic effect of this checkpoint molecule, and its presence also predicts a poor prognosis of gastric adenocarcinomas (GACs) [ 255 , 260 ].

In addition to the upregulation of molecules on their own surface, CAFs also produce various types of cytokines and exosomes to upregulate checkpoint molecules on other cells, such as tumor cells and immune cells in the TME, which indirectly exert inhibitory effects on T cell function and antitumor responses. For instance, CAFs in pancreatic cancer have been reported to upregulate the expression of certain immune checkpoint molecules, including PD-1, cytotoxic lymphocyte-associated antigen-4 (CTLA-4), T cell immunoglobulin, mucin-domain containing-3 (TIM-3) and lymphocyte-activation gene-3 (LAG-3), on both CD4 + and CD8 + T cell surfaces, which consequently inhibits proliferating T cells and their specific recognition of tumor cells [ 261 ]. During the regulation of immune checkpoints, CAF-derived βig-h3 might play a crucial role in promoting the expression of certain immune checkpoint molecules [ 230 ]. When applying βig-h3-targeted depleting Ab therapy, researchers observed the reduced expression of PD-1 and TIM-3 on the tumor-specific CD8 + T cell surface along with the recovery of their proliferation and activity [ 230 ]. Moreover, IL-6 secreted by CAFs, as described before, can induce PD-L1 expression on neutrophils by activating the STAT3 signaling pathway to restrict effector T cell function [ 151 ]. Interestingly, CAF-derived factors involved in the upregulation of PD-L1 in different tumor cell types are distinct. Through the secretion of soluble factors like CXCL2, α-SMA + CAFs can increase PD-L1 expression in lung adenocarcinoma cells, thereby influencing antitumor immunity [ 262 ]. In melanoma and colorectal carcinoma, Li et al. [ 263 ] found that CAF-derived CXCL5 was involved in the expression of PD-L1 on the tumor cell surface in a PI3K/AKT-dependent manner. Recent studies have revealed some detailed intracellular signaling mechanisms. As shown in the research by Zhang et al. [ 264 ], CAFs in colorectal cancer facilitate extracellular signal regulated kinase 5 (ERK5) expression and phosphorylation to increase the synthesis of PD-L1 protein. Additionally, in human breast cancer, studies recently confirmed that microRNA-92 in CAF-derived exosomes targets LATS2 (a target gene of miR-92) and enhances the nuclear translocation of yes-associated protein 1 (YAP1); in this way, YAP1 binds to the enhancer region of PD-L1 to promote its transcriptional activity [ 265 ]. However, less is currently known about CAF induction of immune checkpoint molecule expression on other cells in the TME.

Overall, CAFs not only induce endogenous overexpression of checkpoint molecule ligands but also upregulate the expression of immune checkpoint molecules on other cells in the TME, thereby contributing to the impaired function of tumor-infiltrating T lymphocytes and immunologic tolerance. Certainly, further studies are needed to clarify the deeper mechanisms of CAF-induced immune checkpoint molecule expression, which might be a potential target for CAF-specific immunotherapies.

CAFs remodel the extracellular matrix (ECM) to facilitate immune suppression

The extracellular matrix (ECM) is a complex network consisting of different macromolecules, including collagens, fibrin, glycoproteins and proteoglycans, responsible for maintaining the architecture, integrity, development and homeostasis of normal tissue [ 18 , 266 , 267 ]. ECM alteration in the TME is a common phenomenon in tumor tissues and is usually related to cancer progression [ 268 ]. Many studies have demonstrated the pivotal role of CAFs in remodeling the ECM [ 17 , 269 ]. By secreting multiple matrix proteins (e.g., fibronectin and type I collagen) and producing a variety of matrix metalloproteinases (MMPs), such as MMP-1 and MMP-3, CAFs can facilitate the degradation of normal ECM structure along with increasing matrix stiffness [ 270 , 271 , 272 , 273 , 274 ]. Moreover, CAFs also release the cytokine TGF-β1, a growth factor that is reported to be one of the most important regulators during ECM remodeling [ 275 , 276 ]. The modified ECM, in turn, exerts promoting effects on CAF activation and protumorigenic function. A positive feedback loop between CAFs and the ECM has been identified by Calvo et al. [ 81 ]. Through activated YAP, CAFs are capable of upregulating the expression of several cytoskeletal regulators (e.g., anillin (ANLN) and diaphanous-related formin-3 (DIAPH3)) to contribute to ECM stiffening [ 81 ]. When the matrix becomes stiffer in the ECM, isometric tension within CAFs significantly increases and further facilitates YAP activation by stimulating Src family kinases (SFKs), consequently maintaining the CAF phenotype and their cancer-promoting properties [ 81 ].

Based on accumulating evidence, the modified ECM induced by CAFs is associated with the migration and invasion of cancer cells [ 17 , 277 , 278 ]. More importantly, this modified matrix participates in the induction of immune suppression within the TME. The CAF-remodeled ECM protein network serves as a physical barrier for immune cells, especially T lymphocytes, thus inhibiting their recruitment into cancer sites and subsequently reducing their opportunities to participate in the immune response in the TME [ 279 , 280 ]. The collagen density of the ECM is able to determine the T cell distribution in the TME. Increased collagen deposition surrounding tumor cell clusters in lung tumors and pancreatic cancers was observed to restrict T lymphocyte access to contacting cancer cells [ 281 , 282 ]. In addition, the accumulation of numerous matrix proteins in the ECM also results in a chronic hypoxia state in the TME [[ 283 ]] . As previously described, some soluble factors such as VEGF induced by hypoxia can decrease the effusion rate of circulatory T cells through tumor vessels and then reduce their infiltration [ 227 , 228 ]. Further study revealed the critical role of focal adhesion kinases (FAKs, nonreceptor tyrosine kinases, including FAK1 and PYK2/FAK2), as fibrotic regulators, in the poor infiltration of CD8 + cytotoxic T cells induced by CAF-directed matrix deregulation [ 284 ]. The fibrous stroma of the ECM around tumor islets often blocks high-molecular-weight drugs and thus decreases the efficacy of cancer chemotherapy [ 285 ]. Recent studies have indicated that FAK-targeted inhibition can decrease the stromal density and consequently increase the responsiveness of tumors to chemotherapy and immunotherapy, suggesting that it might be a potential therapeutic target for tumor chemoresistance [ 286 ].

In addition, the CAF-modified ECM can modulate the activities of other immune cell populations as well. Abnormal cancerogenic collagenous matrix is involved in TAM recruitment and function [ 287 ]. For instance, the collagen-rich matrix induced by CAFs not only promotes monocyte migration and proliferation, but also shifts macrophage differentiation to M2 polarization (a protumorigenic cell subset) [ 288 , 289 , 290 , 291 ]. Reciprocally, TAM direct or indirect modulatory regulation of collagen deposition and geometrical organization gradually increase matrix rigidity and ultimately accelerate ECM remodeling progress [ 287 ]. Moreover, the ECM also facilitates the infiltration of other immunoinhibitory subpopulations. Increased collagen density or stiffness in the ECM leads to extensive FAK activation within cells, and activated FAKs subsequently drive the direct exhaustion of CD8 + T cells and enhance the recruitment of Tregs, MDSCs and TAMs, thereby contributing to the formation of an immunosuppressive TME [ 292 , 293 ]. Altogether, the ECM has been demonstrated to crosstalk with several immune cells to induce immune suppression, whereas the effect of the ECM on other cell types, such as DCs and TANs, remains unclear.

Therapeutic strategies for targeting CAFs to enhance the anticancer immune response

With in-depth research and an understanding of the immune response suppression driven by CAFs, these cells are becoming one of the most promising therapeutic targets for cancer intervention. In recent decades, the number of preclinical experiments that restore the anticancer immune response through CAF-targeted therapies has increased dramatically. Currently, there are three main strategies for CAF-based immunotherapy: direct CAF depletion, CAF activation and functional suppression along with CAF-induced ECM remodeling restriction (FIG. 3 ). Tables 3 & 4 briefly summarize the current therapeutic strategies against CAFs investigated in clinical and preclinical studies. In addition to immune checkpoint molecule-targeted inhibitors such as ipilimumab, pembrolizumab and nivolumab [ 294 ], CAF-specific therapies have been an essential complement to immunotherapies and have provided considerable clinical benefits for patients with tumors. However, due to the lack of specific markers for CAFs, as mentioned earlier [ 120 ], current CAF-targeting therapies have to address the intractable problem of how to improve the antitumor effect and decrease systematic side effects at the same time, and this issue might explain why only a few CAF-targeted therapies have been translated into the clinic. To discover more specific and efficient molecular targets for CAFs, further in-depth investigations on these cells are still required in the future.

Major CAF-targeted immunotherapeutic strategies. There are three main approaches against cancer-associated fibroblasts (CAFs) and their associated molecules for immunotherapy: A Through the immunotherapies or transgenic technologies that targeting CAF markers such as fibroblast activation protein (FAP), alpha-smooth muscle actin (α-SMA) and platelet-derived growth factor receptors (PDGFR), CAFs can be directly depleted and consequently enhance immune response in the tumor microenvironment (TME); B CAF activation and function can be suppressed by inhibiting their crucial effector molecule or signaling pathways such as vitamin A, transforming growth factor-beta (TGF-β), interleukin-6 (IL-6) together with Janus kinase-signal transducer and activator of transcription 3 (JAK-STAT3) signaling pathway, C–C chemokine ligand 2 (CCL2)-C–C chemokine receptor (CCR2) signaling axis and C-X-C chemokine ligand 12 (CXCL12), thereby restricting the immune suppression induced by CAFs in the TME; C CAF-derived matrix proteins such as tenascin-C (TNC), hyaluronan (HA) and matrix metalloproteinases (MMPs) as well as related fibrosis-activated signaling pathways, like focal adhesion kinase (FAK) signaling pathways, are the ideal targets to effectively restrict extracellular matrix (ECM) remodeling

Depleting CAFs directly by targeting surface markers

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.

Suppressing CAF activation and function by targeting associated effector molecules

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 , 347 , 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 , 351 , 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 , 354 , 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 ].

Restricting CAF-induced ECM remodeling in the TME

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 ].

Challenges and directions

Considering a large number of CAF characteristics might change with the culture environment alteration (in vivo to in vitro), some questions have continuously arisen and need to be solved. First, to retain the CAF phenotype in in vitro culture as much as possible, researchers have tried various culture conditions and found that lower serum concentrations and matrices with more physiological mechanical properties might be preferable to keep the CAF original phenotype [ 87 ]. Recent studies have identified that several inhibitors of CAF activating molecules, such as TGF-β inhibitors, can effectively restrict the transformation of the CAF phenotype in vitro culture [ 365 ], which indicates that adding some CAF activator suppressors into in vivo culture medium might be a novel strategy to accurately preserve the in vivo phenotype of CAFs. Certainly, more in-depth studies are required to investigate more suitable in vitro culture conditions for CAFs.

Second, currently, single-cell transcriptome analyses have been a useful method to understand the characteristics and heterogeneity of CAFs. Aside from single-cell transcriptome analyses, researchers usually utilize immunoassay technology, such as high-quality antibodies against CAF marker proteins, to detect CAFs in tissue. However, due to the heterogeneity of CAFs, antibodies against certain CAF subpopulation markers require complex optimization, which hampers their adoption in laboratories. Recently, the technology of multiplexed mRNA probes has been rapidly developed, and thus accurate quantitative methods for the detection of CAFs, in the long term, are promising [ 87 ]. Further investigations should be performed to explore and develop more universal, stable, standardized and accurate quantitative methods for CAF detection in the future.

In addition, while multiple methods have been recently developed for the detection of CAF phenotype expression, as introduced above, such as specific antibodies, mRNA probes and transcriptome analyses, there is still a lack of a method to identify CAF phenotype changes in a timely and precise manner during the cultivation process.

Finally, it is necessary to deepen the understanding regarding the origins and subpopulations of CAFs, especially the time and stage heterogeneity of CAFs [ 366 ], by investigating CAFs in different experimental stages and different clinical stages. According to a previous study [ 367 ], for example, researchers can perform a longitudinal study of whole CAF populations in certain cancer animal models by utilizing whole transcriptome analysis in FACS-sorted fibroblasts from early to late stages or distinct pathological grades, and ultimately observe alterations in the CAF transcriptome and phenotype. Furthermore, a longitudinal study of certain fibroblast cell lines in different culture stages can also be conducted, including primary, early isolation, long-term passage and immortalization cells, through techniques such as single-cell transcriptome analyses.

Additionally, to date, the origins and subtypes of some CAFs, especially the anticancer subpopulation (rCAFs), are still unknown, and a deep understanding of rCAFs may become a future research direction. Moreover, the number of studies on CAF-immune cell interactions is far from sufficient, and most of the studies mentioned in our review have not completely illustrated the detailed cell-internal mechanisms by which CAFs affect immune cells. These factors should be considered in further subsequent investigations. Therefore, a thorough exploration of the crosstalk between CAFs and the TIME is required in the future to enable us to identify the basis of the impaired immune response induced by CAFs and might identify an essential method to restimulate the antitumor response which is distinct from strategies that directly restrict and eliminate cancer cells.

Furthermore, although an increasing number of CAF-targeting therapeutic strategies are being developed, the lack of more specific markers and the low number of large-scale randomized clinical trials are still two huge challenges facing CAF-targeting treatment.

Conclusions

Since it acts as a crucial role in tumor initiation and progression in the TME, CAFs have received increasing attention in the past decade. CAF populations exhibit extensive heterogeneity in terms of cell origin and phenotype, which leads to their distinct behaviors during cancer development: most CAF subtypes (pCAFs) function as tumor facilitators; however, some other subtypes (rCAFs) exert tumor-inhibiting effects. Additionally, the intertransformation of several subpopulations partly indicates the plasticity of CAFs, while more investigations are needed to confirm this. Recently, studies have confirmed the importance of the interaction between CAFs and the immune microenvironment in the TME during tumor progression. Meanwhile, an increasing number of research regarding the effect of CAFs on the immune components of the TIME have gradually clarified the mechanisms by which CAFs orchestrate an immunosuppressive TME, and the results facilitate the translation of related CAF-based therapeutic targets into clinical trials.

In this review, we describe the interaction between CAFs and immune cells infiltrating the TME in detail and propose a possible immune inhibitory mechanism by which CAFs not only directly influence the activities of immune cells but also indirectly result in immune effector cell dysfunctions by upregulating immune checkpoint molecule expression on the cell surface and remodeling the ECM within the TME. By secreting various chemokines, cytokines and other effector molecules, CAFs directly inhibit immune cell-mediated antitumor immunity mainly through three main mechanisms, as listed below: (i) to drive the abnormal polarization or trans-differentiation of immune cells such as TAMs, TANs, MCs, DCs and T lymphocytes into certain procancerogenic cell subsets; (ii) to promote the activities of immune inhibitory cells, including M2-type TAMs, N2-type TANs, rDCs, Treg cells and MDSCs, in terms of their recruitment, infiltration, activation and immunosuppressive behaviors; and (iii) to reduce the cytotoxic activities and cytokine secretion of immune effector cells like NK cells and CTLs. Notably, some infiltrating immune cells, such as TAMs, TANs, MCs and DCs, are capable of enhancing the activation and function of CAFs. These interactions constitute immunoinhibitory loops that further heighten immune suppression in the TME. Moreover, CAFs have also been reported to modulate anticancer immunity through indirect means: (i) to upregulate the expression of immune checkpoint molecules such as PD-1/PD-L1 in both themselves and other cells in the TME to induce T cell dysfunction and immunologic tolerance; (ii) to degrade and remodel the ECM through the production of fibronectin, collagen and MMPs and the activation of FAK to restrict effector immune cell infiltration while increasing the recruitment of inhibitory immune cells, such as Tregs, MDSCs and TAMs, and consequently block the initiation of the immune response. Certainly, excess expression of immune checkpoints in CAFs and surrounding matrix deposition would in turn prolong CAF survival, stimulating their activation and maintaining their protumor properties. In view of the diverse immune suppressive effects of CAFs, current CAF-targeted therapeutic strategies that target CAF surface markers, associated effector molecules and their relevant signaling pathways along with restricted ECM remodeling have been developed to enhance antitumor immunity, which has produced considerable clinical benefits. More importantly, in combination with checkpoint blockade immunotherapies or chemotherapies, CAF-targeted treatment might hold promise for the treatment of tumors with a fibroblast-rich TME.

Availability of data and materials

Not applicable.

Abbreviations

• Cancer-associated fibroblasts
• Tumor microenvironment

Extracellular matrix

• Tumor immune microenvironment

Alpha smooth muscle actin

Fibroblast activation protein

Fibroblast-specific protein 1

Platelet-derived growth factor receptor

Cluster of differentiation

Matrix metalloproteinase

T regulatory

Myeloid-derived suppressor cells

Interleukin

Transforming growth factor

Hepatocyte growth factor

Platelet-derived growth factor

Fibroblast growth factor 2

Stromal-derived factor-1

Reactive oxygen species

Pancreatic stellate cells

Hepatic stellate cells

Islet stellate cells

Insulin-like growth factor-1

Mesenchymal stem cells

Bone marrow mesenchymal stem cells

Epithelial-mesenchymal transition

C-X-C chemokine ligand

Myeloid zinc finger 1

C–C chemokine ligand

Endoplasmic reticulum

Human adipose tissue-derived stem cells

Adipocyte-derived fibroblasts

Endothelial-to-mesenchymal transition

Mesothelial-mesenchymal transition

Monocyte-to-myofibroblast trans-differentiation

P38-mitogen-activated protein kinase

Janus kinase

Signal transducer and activator of transcription 3

Damage-associated molecular patterns

NOD-like receptor protein 3

Heat shock factor 1

Yes-associated protein

Leukemia inhibitory factor

SH2-containing protein tyrosine phosphatase-1

Pancreatic ductal adenocarcinoma

Myofibroblastic CAFs

Inflammatory CAFs

Antigen-presenting CAFs

Major histocompatibility complex

Fibroblast population

Oral squamous cell carcinoma

High-grade serous ovarian cancer

Cancer-promoting CAFs

Cancer-restraining CAFs

Bone morphogenetic protein 4

Epidermal growth factor receptor

Tumor-associated macrophages

Lipopolysaccharide

Tumor necrosis factor

S100 calcium binding protein A4

Monocyte chemotactic protein-1

Chitinase 3-like 1

Programmed cell death protein 1

Macrophage colony-stimulating factor 1

Estrogen receptor alpha

Class A scavenger receptors

Tumor-associated neutrophils

CAF-derived cardiotrophin-like cytokine factor 1

Programmed death ligand 1

C-X-C chemokine receptor

Gastric cancer-mesenchymal stem cells

Extracellular regulated protein kinases 1/2

Vascular endothelial growth factor

Stem cell factor

Natural killer

DNAX accessory molecule 1

Killer immunoglobulin-like receptors

Prostaglandin E2

Indoleamine 2,3-dioxygenase

DNAX-activation protein 12

Interferon-γ

MHC class I chain-related gene A/B

Poliovirus receptor

Dendritic cells

Regulatory DCs

Tryptophan 2,3-dioxygenase

Cytotoxic T lymphocytes

Dipeptidyl peptidase IV

Thymic stromal lymphopoietin

Toll-like receptor 8

Intercellular cell adhesion molecule

Vascular cell adhesion molecule-1

Hydrogen peroxide inducible clone-5

T cell receptor

Factor associated suicide

Factor associated suicide ligand

Programmed death ligand 2

Polymorphonuclear MDSCs

Monocytic MDSCs

Nitric oxide

Lung squamous cell carcinoma

C–C chemokine receptor

Triple-negative

Gastric adenocarcinomas

Cytotoxic lymphocyte-associated antigen-4

Mucin-domain containing-3

Lymphocyte-activation gene-3

Phosphatidylinositol 3-kinase

Protein kinase B

Extracellular signal regulated kinase 5

Diaphanous-related formin-3

Src family kinases

Focal adhesion kinase

Cyclophosphamide

A20-specific shRNA

Tyrosine-related protein 2

Adenoviral vector of chimpanzee serotype 68

Chimeric antigen receptor

Single-chain variable fragments

Transforming growth factor beta receptor

Anti-tenascin C dsRNA

Immune checkpoint blockade

Connective tissue growth factor

Endothelin-1

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Acknowledgements

This study was jointly funded by the National Natural Science Foundation of China (No. 81802352, 81772555 and 81902428), the National Science Foundation for Distinguished Young Scholars of China (No. 81625016), the Shanghai Sailing Program (No. 19YF1409400 and 20YF1409000), the Shanghai Rising-Star Program (No. 20QA1402100), the Shanghai Anticancer Association Young Eagle Program (No. SACA-CY19A06), the Clinical and Scientific Innovation Project of Shanghai Hospital Development Center (No. SHDC12018109 and SHDC12019109) and the Scientific Innovation Project of Shanghai Education Committee (No. 2019–01-07–00-07-E00057).

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Xiaoqi Mao, Jin Xu and Wei Wang contributed equally to this work and shared the first authorship.

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Department of Pancreatic Surgery, Fudan University Shanghai Cancer Center, No. 270 Dong’An Road, Xuhui District, Shanghai, 200032, China

Xiaoqi Mao, Jin Xu, Wei Wang, Chen Liang, Jie Hua, Jiang Liu, Bo Zhang, Qingcai Meng, Xianjun Yu & Si Shi

Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, 200032, China

Shanghai Pancreatic Cancer Institute, Shanghai, 200032, China

Pancreatic Cancer Institute, Fudan University, Shanghai, 200032, China

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XQM, JX and WW collected the related studies and drafted the manuscript. CL, JH, JL and BZ participated in the design of the review. SS, XJY and QCM initiated the study and revised the manuscript. All authors read and approved the final manuscript.

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Correspondence to Qingcai Meng , Xianjun Yu or Si Shi .

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Mao, X., Xu, J., Wang, W. et al. Crosstalk between cancer-associated fibroblasts and immune cells in the tumor microenvironment: new findings and future perspectives. Mol Cancer 20 , 131 (2021). https://doi.org/10.1186/s12943-021-01428-1

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Characterising cancer-associated fibroblast heterogeneity in non-small cell lung cancer: a systematic review and meta-analysis

• Andrew F. Irvine 1   nAff3 ,
• Sara Waise 1 ,
• Edward W. Green 2 ,
• Beth Stuart 4 &
• Gareth J. Thomas 1  

Scientific Reports volume  11 , Article number:  3727 ( 2021 ) Cite this article

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• Cancer microenvironment
• Non-small-cell lung cancer
• Prognostic markers

Cancer-associated fibroblasts (CAFs) are a key component of the tumour microenvironment with evidence suggesting they represent a heterogeneous population. This study summarises the prognostic role of all proteins characterised in CAFs with immunohistochemistry in non-small cell lung cancer thus far. The functions of these proteins in cellular processes crucial to CAFs are also analysed. Five databases were searched to extract survival outcomes from published studies and statistical techniques, including a novel method, used to capture missing values from the literature. A total of 26 proteins were identified, 21 of which were combined into 7 common cellular processes key to CAFs. Quality assessments for sensitivity analyses were carried out for each study using the REMARK criteria whilst publication bias was assessed using funnel plots. Random effects models consistently identified the expression of podoplanin (Overall Survival (OS)/Disease-specific Survival (DSS), univariate analysis HR 2.25, 95% CIs 1.80–2.82) and α-SMA (OS/DSS, univariate analysis HR 2.11, 95% CIs 1.18–3.77) in CAFs as highly prognostic regardless of outcome measure or analysis method. Moreover, proteins involved in maintaining and generating the CAF phenotype (α-SMA, TGF-β and p-Smad2) proved highly significant after sensitivity analysis (HR 2.74, 95% CIs 1.74–4.33) supporting attempts at targeting this pathway for therapeutic benefit.

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Cancer-associated fibroblasts expressing fibroblast activation protein and podoplanin in non-small cell lung cancer predict poor clinical outcome

Single-cell analysis reveals prognostic fibroblast subpopulations linked to molecular and immunological subtypes of lung cancer

Clinical and therapeutic relevance of cancer-associated fibroblasts

Introduction.

Non-small cell lung cancer (NSCLC) remains the leading cause of cancer death worldwide 1 , 2 , 3 . Despite more recent therapeutic advances, outcomes remain poor, with a 10-year survival rate of only 5% 4 . NSCLC shows a relatively low degree of tumour cell purity compared to other tumours, with high infiltration by immune and stromal cell populations 5 .

Fibroblasts are the most common stromal cell type in a range of solid tumours 6 , 7 , 8 , 9 , where they are referred to as cancer-associated fibroblasts (CAFs). CAFs are most commonly described as having an α-SMA-positive, “myofibroblastic” phenotype, analogous to that observed in wound healing 10 . These cells are associated with a number of the hallmarks of malignancy, including promotion of tumour invasion and metastasis, angiogenesis and immune evasion 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 . Unsurprisingly, this population correlates with poor prognosis in a range of malignancies 9 , 19 , 20 , 21 . However, there is increasing evidence that CAFs are in fact a heterogeneous cell type, with a range of distinct phenotypes and functions 22 , 23 , 24 . For example, an inflammatory CAF sub-group has been described in a number of different tumours including pancreatic cancer 25 . Nevertheless, the relative contribution of specific populations is likely to vary by tumour type and has yet to be defined fully and for some cancer types, including NSCLC, the impact of CAFs on patient outcomes is less clear.

α-SMA is the most commonly used CAF marker 26 , but is also expressed by smooth muscle cells 27 and pericytes 28 and no single marker has been shown to reliably identify the entire CAF population. Indeed, FAP, another commonly-used CAF marker has been shown to identify both inflammatory and myofibroblastic CAFs in pancreatic ductal adenocarcinoma 17 and breast cancer 18 . Other frequently used CAF markers include podoplanin and fibroblast-specific protein-1 (FSP-1) 9 , 29 with CAFs expressing the latter known to have immunomodulatory functions 30 , 31 , 32 . However, for others, the downstream functional pathways are yet to be characterised.

CAFs are an attractive therapeutic target, but despite promising data from pre-clinical models, the results of clinical trials targeting CAFs have been mixed 33 , 34 . Characterisation of CAF phenotypes and their impact on outcomes has gained increasing interest in recent years, and there are now multiple studies profiling CAF heterogeneity at single-cell resolution 22 , 23 , 24 . To date, there have been many individual reports describing the prognostic effect of single CAF markers in NSCLC. The impact of CAFs seems to vary by marker and, in some cases, is contradictory (e.g. FAP 35 , 36 ). This may be explained, at least in part, by the known heterogeneity within this population, where common markers can be expressed by functionally distinct subgroups.

Although meta-analyses examining the relationship between protein marker expression and outcomes in NSCLC have been performed previously 37 , 38 , these studies only focused on a small number of pre-determined markers and did not use methods to extract hazard ratios from studies which failed to quote them, leading to possible publication bias. Moreover, the number of studies published in the intervening period has increased significantly reflecting the increased interest in CAFs. Here, we perform a systematic review and meta-analysis of the literature assessing the prognostic effect of all CAF markers in NSCLC characterised thus far, as well as the cellular processes they are involved in. In addition, we also use several statistical methods, including a newly-published method 39 which improves on the accuracy of extracted HRs when not quoted in the original studies. Assessing the prognostic significance of these markers is important in characterising the heterogeneity now widely accepted in CAFs whilst determining the most prognostic cellular pathways might help inform stromal targeting strategies in NSCLC.

Study characteristics

Of the 13,797 articles identified, 290 were eligible after screening titles and abstracts. Of these, 44 were included based on the full-text article, representing a total population of 7582 patients (Fig.  1 ). Cross-checking of previously published reviews on the roles of fibroblasts in lung cancer yielded one additional study that was not detected within the search strategy 40 . Of the 246 studies excluded, 196 described protein expression within the wider microenvironment, rather than specifically by fibroblasts which was the most common reason for study exclusion. A further 37 studies did not include survival statistics or a Kaplan–Meier (KM) plot from which these could be calculated. Twenty-six protein markers were identified from the 44 included articles. Five of these (podoplanin, carbonic anhydrase IX (CAIX), α-SMA, periostin and FAP) appeared in at least 2 separate cohorts, rendering them suitable for meta-analysis. Of all included markers, 21 (81%) were identified as a component of at least one common cellular process that defines, or is a hallmark, of cancer-associated fibroblasts 41 .

Flow chart describing steps carried out in selecting articles.

The main characteristics of the included studies are shown in Supplementary Table S1 . The earliest included study was published in 1998. However, the majority (30/44, 68%) were published within the last 5 years, likely reflecting the increased interest in CAFs. The median cohort size was 129 (range 52–729). Many studies reported cohorts focusing on lung adenocarcinoma (19/44, 43%) or a combination of adenocarcinoma and squamous cell carcinoma (17/44, 39%). In terms of treatment, 19 studies (43%) failed to report information on neo- or adjuvant therapy whilst 13 studies (30%) excluded patients who had received neo-adjuvant therapy. Almost half of studies (21/44, 48%) reported overall survival (OS) as the only survival outcome. The majority of studies reported a mix of univariate and multivariate hazard ratios (HR) (24/44, 55%) with 21/44 studies (48%) reporting a KM plot but no associated HR. To extract these missing HRs, we used a set of statistical techniques depending on the available information, including the Nlopt method 39 , a novel algorithm based on non-linear optimisation (see “ Methods ” section for more details). In these cases, the Nlopt method was used most frequently (14/21, 67%), with the Parmar 42 (3/21, 14%) and Guyot 43 method (4/21, 19%) also required in several instances. In total, the 44 included studies yielded 96 survival outcome measures.

Quality assessment

Assessment of study quality was determined by calculating a score based on the REMARK criteria 44 , 45 (summarised in Supplementary Figure S1 ; raw data given in Supplementary Table S2 ). The mean score was 14 (range 9.5–17) with most studies scoring moderately well against all domains of the REMARK criteria. The exception was the “data” domain in which all but one study 46 registered low- to medium-quality scores. The data domain describes the flow of patients through the original study, as well as the relationship of the tumour marker to standard prognostic variables. In total, 3 studies had overall REMARK scores ≤ 50%, all of which were included in subsequent sensitivity analyses. Although the REMARK criteria were first published in 2005, in the studies included in this systematic review, there has not been a significant increase in scores over this time (R 2  = − 0.017, P  = 0.603; Supplementary Figure S2 ).

Individual marker results

Podoplanin, α-SMA, FAP, periostin and CAIX all had at least two HRs calculated using similar outcome measures and assessed using either univariate or multivariate analysis deeming them eligible for meta-analysis. Calculating these separately was recommended in guidance published on carrying out meta-analyses on prognostic factors 47 . This approach resulted in twelve separate outcome measures of pooled HRs analysed using a random effects model as represented in the network tree (Fig.  2 A). Example forest plots for univariate analysis of the OS/DSS outcome group for each marker are shown in Fig.  2 B; full results are summarised in Table 1 .

Analysis of individual markers. ( A ) Tree network showing number of studies for each marker per outcome group and analysis method. Figure generated using the vtree package in R (version 3.5.2). ( B ) Random-effect forest plots of individual markers from the OS/DSS outcome group and univariate analysis method.

Podoplanin and α-SMA were the most frequently reported of the five markers and were consistently associated with statistically significant poorer survival outcomes, regardless of outcome measure or analysis method (Table 1 ). However, significant test heterogeneity was found in a subset of these measures. As expected, HRs for RFS/DFS were always higher than OS/DSS although no other trends emerged when comparing survival outcomes from different groups. In contrast, CAIX (HR 1.40, 95% CI 0.71–2.76) and periostin (HR 1.55, 95% CI 0.95–2.53) did not show a significant correlation with survival. FAP expression was only associated with a statistically significant poor prognostic impact in the multivariate analysis from the OS/DSS outcome group (HR 2.25, 95% CI 1.39–3.63).

In all eight podoplanin and one α-SMA random effects models, there were a sufficient number of studies to carry out sub-group analysis based on histological subtype (Fig.  3 A,B, Table 2 ). In the case of podoplanin, all were significantly associated with a poorer survival outcome, with the exception of multivariate analysis of the OS/DSS outcome group in adenocarcinoma and univariate analysis of the OS/DSS outcome group in squamous cell carcinoma. The univariate analysis of α-SMA in a cohort of patients with only adenocarcinoma was statistically significant (HR 5.91, 95% CI 3.49–10.00).

Sub-group analysis of individual markers based on histological subtype. ( A ) Tree network showing number of studies for each marker per outcome group, analysis method and histological subtype. Figure generated using the vtree package in R (version 3.5.2). ( B ) Random-effect forest plots of individual markers from the OS/DSS outcome group and univariate analysis method with histological subtype indicated.

After sensitivity analysis excluding studies with low REMARK scores, two scores for podoplanin (OS/DSS > Univariate > All histology and OS/DSS > Univariate > adenocarcinoma only) remained unchanged (Supplementary Table S3 ).

CAF markers in cellular processes

Our next aim was to assess the prognostic significance of the cellular processes known to be crucial in the function of CAFs. CAFs are known to have a variety of functions which influence cancer progression and which have been summarised in a number of recent reviews 26 , 41 , 48 . We established a consensus of functions from these reviews, creating a table of what are currently considered the most important functions or hallmarks of CAFs (Supplementary Table S4 ). Next, to determine the function of each of the identified markers, a separate literature search was performed focusing on studies which had identified a functional role of the marker specifically in CAFs. In several cases, this came from the functional studies published in the original paper, for example, c-Met 49 , GFAT2 50 and IGF-II 51 . Such studies generally included co-culture experiments in vitro or more complex mouse models in vivo or a combination of the two (Supplementary Table S5 ). Each of the markers was then assigned to one or more of the functions (Table 3 ). In two cases, CD34 and irisin have no clear role in CAFs currently so were excluded from this step of the analysis. In addition, three other markers, CD90, HSF-1, CD200 did not have outcome measures in the OS/DSS group so were also excluded from the analysis (see “ Methods ” section for more detail). In total, this resulted in 21/26 of the markers being attributed to at least one of 7 common processes. Analysis of the pooled HR for each process showed all were in fact associated with poorer survival (Table 4 , Fig.  4 ) although generation and maintenance of the CAF phenotype (HR 2.25, 1.27–4.00 95% CIs) and enhancing the proliferation and survival of tumour cells (HR 2.06, 1.25–3.40 95% CIs) were the only processes with a HR above 2. A significant level of heterogeneity was again detected in 4 of the 7 cellular processes but in the case of the CAF phenotype, this was non-significant after sensitivity analysis excluding poor quality studies (Supplementary Table S3 ) and the pooled HR in fact increased to 2.74 (1.74–4.33 95% CIs).

Ferris wheel plot summarising random-effect model HRs for each cellular process in CAFs. The height of each bar represents the HR for each process with the width of each bar indicating the % weight that each marker contributed to the random-effects model. The random-effect model HRs and 95% CIs are stated below each cellular process. Figure generated using Adobe Illustrator, 2020 (version 24.2). Icons representing each cellular process are from BioRender.com.

As with all meta-analyses, small-study effects should be examined to determine the extent of any publication bias. We produced funnel plots and tested asymmetry with linear regression in any meta-analysis with 7 or more studies (Supplementary Figure S3 ) due to the low power of these tests 52 . In the case of funnel plots for the univariate analysis of α-SMA in the OS/DSS outcome group and the invasion and proliferation meta-analysis (Supplementary Figure S3 B, D, E), the plots were clearly asymmetrical but likely due in part to the heterogeneity that was also detected (Tables 1 , 4 ). Only one of the plots was deemed to significantly deviate from a symmetrical distribution (cellular processes: invasion, P  = 0.02) although this is in part likely due to the heterogeneity mentioned above. However, visual assessment of this funnel plot suggested some values missing where small studies with larger standard errors would be expected suggesting an element of publication bias in this particular random effects model.

CAFs are a key component of the tumour microenvironment. Growing evidence suggests they are a heterogeneous population with respect to function 53 and expression of both RNA and protein 22 , 54 . We therefore performed a meta-analysis of all published protein markers of CAFs in NSCLC in an attempt to better characterise this heterogeneity by determining their prognostic significance. We implemented a search strategy focused on sensitivity, resulting in a number needed to read of 313. In addition, we calculated HRs from studies when these were not directly quoted using the most up-to-date extraction methods. In total, this yielded 26 different protein markers. These included well known markers of CAFs including α-SMA and FAP 26 , but also new potential markers identified from omic-type screens such as CD200 55 and GFAT2 50 . To ensure that this analysis was fibroblast-specific, we excluded studies which did not explicitly state if the protein marker was expressed by fibroblasts or just more generally within the stroma or tumour microenvironment.

Podoplanin is the best characterised of the identified markers, and in keeping with previous data 37 , was often associated with poor survival in this study. Podoplanin is a 44-kDa glycoprotein that was initially characterised as a platelet-aggregation factor on cancer cells from colorectal tumours 56 . It is also expressed by both lymphatic endothelium 57 and inflammatory macrophages 58 . Functionally, podoplanin-positive fibroblasts in cancer have been shown to enhance the invasive properties of carcinoma cells 59 , play an important role in re-modelling of the ECM 60 , 61 , 62 , as well as promoting an immunosuppressive microenvironment 63 . Although podoplanin was consistently associated with poor survival in this study, a significant test heterogeneity was detected for the OS/DSS outcome group in multivariate analysis but when sub-grouping based on histological variant was carried out this became non-significant. The same analysis also revealed that squamous but not adenocarcinoma tumours were significantly associated with survival suggesting a possible difference in prognostic effect based on NSCLC histological subtype, explaining the heterogeneity originally identified. However, this trend was not observed in the RFS/DFS outcome measure suggesting further comparisons of podoplanin-positive fibroblasts in squamous and adenocarcinoma tumours are warranted.

α-SMA was also commonly reported and associated with a poorer survival. α-SMA is a member of a highly conserved group of proteins that regulate the cell cytoskeleton 64 . In fibroblasts, this protein is crucial in regulating the contractility of the cell which is itself required to both generate and maintain the CAF phenotype 65 . Although α-SMA was associated with poor survival, it tended to result in pooled HRs with larger variances. For example, in the case of analysing univariate HRs from OS/DSS outcomes, HRs ranged from 0.9 for Kilvaer et al . 35 to over 7 for Qiu et al . 66 whilst contributing similar weights to the random effects model. In comparing these two studies at either extreme, both featured cohorts of patients with stage I-IIIA NSCLC, excluding those treated with neo-adjuvant therapy and scored well on the REMARK criteria. Qiu et al . 66 focused solely on adenocarcinoma cases with Kilvaer et al . 35 considering both squamous cell carcinoma and adenocarcinoma. However, both studies used different immunohistochemistry scoring systems: Kilvaer calculated the dominant staining intensity in positive cells whereas the Qiu study used an index combining both staining intensity and extent. Such a difference in scoring might explain, at least in part, the variation seen in these two studies. Indeed, the use of different scoring criteria in biomarker research and subsequent difficulty in comparing studies is well known 47 , leading for calls to ensure scoring for markers is standardised 67 , and validates the need for a meta-analysis.

Three other markers were also suitable for meta-analysis in this study, CAIX, periostin and FAP. Unlike for α-SMA and podoplanin, fewer studies have been carried out on these markers so there was only one combination of outcome measures and forms of analysis for CAIX and periostin and two for FAP.

CAIX is a member of the carbonic anhydrase family, the expression of which is induced under hypoxic conditions by HIF-1 68 . Whilst hypoxia is clearly an important aspect of tumourigenesis 69 with the tumour microenvironment (including CAFs) playing an important role in its regulation 70 , CAIX expression in CAFs was not associated with reduced survival in this meta-analysis.

Periostin, an ECM protein produced by fibroblasts 71 , has previously been shown to enhance the proliferation and invasive potential of tumour cells 72 but was also not associated with poor survival in NSCLC. In the case of FAP, meta-analysis produced conflicting results.

FAP is a type II integral membrane serine protease shown to be involved in ECM re-modelling and tumour cell migration 73 and has been used as a marker of activated fibroblasts in a number of studies. In this meta-analysis, random effects models showed univariate analysis had no significant effect on prognosis whilst multivariate analysis did prove to be statistically significant. Notably, one of the univariate studies actually showed that increased CAF FAP expression was associated with improved survival in NSCLC 46 . Whilst not statistically significant in a mixed cohort, sub-group analysis of only squamous carcinoma cases was statistically significant, in contrast to findings of Chen et al . 74 In what is emerging as a common theme, these two studies used different antibodies, grading systems and scored either whole slides or tissue micro-arrays; this may account for the discordant results in these studies. Such discrepancies are concerning though as FAP is currently regarded as a CAF target for molecular-based imaging 75 and therapeutic targeting 76 . Without a clear understanding of its role in CAF biology, such trials might produce inconsistent results.

Some CAF markers were only analysed in a single study or did not share a common outcome group/form of analysis and were therefore excluded from the meta-analysis. These included several interesting studies; Chen et al. 51 showed high expression of IGF-II in CAFs in a cohort of 80 patients resulted in a HR of 19.15 (95% CIs 6.32–58) for overall survival. In this study, CAFs from primary tumours were shown to promote stemness characteristics of lung cancer-stem cells (expression of Nanog and Oct3/4), an effect which was shown to be partly dependent on the expression of IGF-II. IGF-II in CAFs is known to accelerate tumour growth in cholangiocarcinoma xenograft models 77 and promotes proliferation of anal squamous cell carcinoma cells 78 . Moreover, expression of IGF-II has been shown to promote differentiation of fibroblasts into myofibroblasts in idiopathic pulmonary fibrosis and scleroderma/systemic sclerosis-associated pulmonary fibrosis 79 . Together, this result suggests further examination of IGF-II expression in CAFs in NSCLC could lead to key biological pathways being elucidated or identification of additional sets of patients with poor survival.

Cav-1, a scaffold protein crucial to caveolae 80 was also excluded from meta-analysis as the two studies which analysed its prognostic role only calculated either a univariate or multivariate hazard ratio. Along with FAP, Cav-1 was the only other marker that resulted in statistically significant outcome measures with opposite effects on survival. In the study by Shimizu et al . 81 , high expression of Cav-1 was associated with a decrease in overall survival (HR 2.78) whilst the study by Onion et al . 82 showed high expression of Cav-1 was associated with improved survival (HR 0.64). The studies used different antibodies and scoring systems whilst the cohort used in the Shimizu study was larger and consisted of only patients with Stage I adenocarcinoma. The Onion study featured a cohort with Stage I–III NSCLC but did not state the histological classification of the tumours included. Given this, it is possible that the differences are due to histological subtype if the cohort in the Onion study was mainly composed of cases of squamous NSCLC. Loss of Cav-1 has previously been shown to correlate with poor survival in other cancers, for example prostate 83 and breast 84 , in agreement with the Onion study but given the discrepancies identified in this analysis, further studies assessing the prognostic role of Cav-1 in CAFs in NSCLC would be warranted to clarify its prognostic role in lung cancer.

A variety of functions have been attributed to CAFs in recent years, leading to the question of whether all CAFs perform these functions, or whether there exist subsets of CAFs with different functions.

CAFs are increasingly recognised as a heterogeneous cell type. Recent studies have described transcriptomically-distinct CAF phenotypes in NSCLC, which may correspond to discrete functional subsets 22 , 23 . Our aim was to therefore determine whether a set of protein markers, grouped together by function, would show prognostic differences. This in turn may suggest a subset of CAFs with specific functions leads to poorer survival outcomes. CAFs are crucial in depositing and re-modelling the ECM within a tumour 41 . Intrinsic to this is their ability to secrete growth factors and matrix proteases, promoting and enabling tumour cell migration and invasion 85 , 86 . CAFs also promote angiogenesis 87 , the proliferation and survival of tumour cells 88 , and an immunosuppressive microenvironment by reducing T cell responses 89 .

Analysing the prognostic effect of these processes in CAFs showed all were in fact correlated with a poor survival outcome. However, proteins involved in the generation and maintenance of the CAF phenotype were most prognostic with a HR approaching 3 after sensitivity analysis. This suggests that although different functional subsets of CAFs might exist, conversion of a fibroblast into a CAF is a uniting feature, creating a population of cells which ultimately contribute to poor survival outcomes in NSCLC. Targeting of this process might thus prove an effective treatment strategy. Indeed, such an approach is currently a significant area of research with a recent study showing pharmacological inhibition of NOX4, a protein important in this conversion, reduced tumour growth in mouse xenograft models 90 . In addition, a number of clinical trials targeting proteins which are also important in CAF activation such as FGFR 91 and TGF-β 92 , 93 are currently underway with their results awaited. Other attempts at targeting molecules, such as the vitamin D receptor, which aim to revert CAFs to a more normal state are also ongoing 94 . Thus, in the case of NSCLC, the results of this meta-analysis are in keeping with treatments targeting pathways important in generating and maintaining the CAF phenotype.

Although several significant survival associations were observed in this analysis, there are a number of limitations. Some issues common in research carried out on prognostic factors 44 have already been mentioned, such as the use of different scoring methods and cut-off values for the same marker. In addition, a number of studies did not report whether patients received neoadjuvant or adjuvant therapy and those that did failed to report sub-group outcome analyses. Since adjuvant therapy is now commonplace in treating eligible patients with lung adenocarcinoma 95 , it is feasible that CAFs could exhibit both prognostic and predictive effects. Indeed, CAFs are known to mediate increased tissue tension, a factor known to affect drug delivery 96 . Thus, future studies should include outcome measures based on therapy where possible. Similarly, several studies examined cohorts with mixed histology, generally squamous and adenocarcinoma. In this meta-analysis, there was some evidence of outcome differences between histological subtypes suggesting that subtle trends may exist, which can only be identified with subtype analysis.

A further issue with the analysis of survival data was the adjustment factors used in calculating a multivariate HR. Such adjusted values are crucial in determining the independent effects of prognostic markers 47 but whilst many studies described these factors and the model they used, there was a significant variation in the final adjustment factors. As suggested in guidance published on reporting prognostic studies 47 , analyses could include multivariate HRs with a core, agreed set of factors alongside other models facilitating more direct comparisons for studies such as this one. In general, scoring each study against the REMARK criteria captured elements of the limitations described above, further validating the approach in conducting sensitivity analyses. Interestingly, although the REMARK criteria have been in place since 2005, there has been no increase in these scores in the intervening years. This suggests that authors should still be encouraged to comply as fully as possible with these criteria, to ensure consistent publication of high-quality studies.

Another issue with reporting of survival outcomes was that many studies published a KM plot but no associated HR. Although we used well-established methods to extract these missing values, including a novel algorithm 39 recently published which improves upon existing methods when the number at risk is not included below the KM plot, such techniques are still associated with varying degrees of error 39 , 97 . However, if no attempt is made to obtain such values, a number of studies would have been excluded and in several cases resulted in non-significant values being ignored leading to publication bias, a significant concern in any meta-analysis 47 . We assessed this using funnel plots and an asymmetry was clear in three cases and significant in one (cellular process: invasion) but this was likely due to the associated heterogeneity identified in all cases, which is another well-known cause of funnel plot asymmetry 52 . Use of extraction methods would certainly reduce publication bias this but would only apply for univariate HRs as such methods require KM plots which are not generated in a multivariate analysis. On balance, although a degree of publication bias was present in one of our random effects model, this was not the case in the remaining models and so we do not believe publication bias was prevalent in this meta-analysis.

In conclusion, the aim of this study was to address the now widely accepted hypothesis that CAFs are a heterogeneous population 41 which is therefore likely to mean distinct functional sub-groups of CAFs represented by different proteins/markers. The study was designed in such a way as to address both of these issues by: (1) summarising the prognostic significance of every protein so far examined in CAFs in NSCLC and (2) linking each of these proteins to a cellular process that is currently believed to be crucial in CAF function. This approach is based on the fact that proteins which are prognostically important might represent key proteins that are crucial to CAF biology as well as identifying functional sub-groups within CAFs generally. An additional approach as previously mentioned is the use of scRNA sequencing experiments to identify transcriptomically-different sub-populations of CAFs. Such experiments are already yielding exciting results 22 , 23 and the combination of these analyses whilst also assessing the prognostic effect of any identified proteins, as in this study, has the potential to further our understanding of CAF biology and in particular, its heterogeneity.

Notwithstanding, the current results from this study show that, despite the limitations common in prognostic research and inherent to meta-analyses, CAF expression of podoplanin or α-SMA was consistently associated with poor survival in NSCLC. Moreover, the proteins and pathways required to generate and maintain the CAF phenotype might represent potential therapeutic targets in anti-cancer treatments in NSCLC.

This review was prospectively registered with PROSPERO (CRD42019130307), an International prospective register of systematic reviews ( https://www.crd.york.ac.uk/prospero/ ). Guidelines for carrying out systematic reviews and meta-analysis of prognostic factor studies 47 were followed where possible.

Search strategy

Literature was retrieved using Medline, Embase, Scopus, Web of Science, and Cochrane databases on the 29th January, 2020 with no date restriction. All results were then updated again with a search on 24th July, 2020. The full search strategy for each database is available in Supplementary Table S6 .

Screening and selection of studies

All identified articles were exported into Rayyan 98 , a web-based application for carrying out systematic reviews. All titles, abstracts and full-text articles were independently screened by AI and SW with discrepancies resolved by consensus. The following P(atient) E(xposure) C(omparator) O(utcome), PECO was used to select articles: Patients: Individuals diagnosed with NSCLC (histological subtypes to include squamous, adenocarcinoma and large cell) who underwent surgical resection, treated with or without neoadjuvant or adjuvant therapy. Exposure: Tumour resections analysed for the presence of CAFs stained with antibodies against any protein marker using immunohistochemistry. For the definition of CAFs, an explicit statement in the methods or results section that fibroblasts, myofibroblasts, cells with a spindle-shaped morphology or similar were scored was required. Statements equivalent to positive staining within the tumour stroma or tumour microenvironment were not sufficient and such studies excluded. Comparator: Comparison of expression profiles (e.g. low/high, negative/positive) of the reported protein markers. Outcomes: The following survival outcomes were all considered for inclusion: overall survival (OS), disease-specific survival (DSS), progression-free survival (PFS), recurrence-free survival (RFS), and disease-free survival (DFS). Studies which failed to define the survival outcome were excluded.

Data extraction

Data extraction was carried out by AI and SW with the following information for every study collected: first author; year of publication; journal; protein marker; staging, histological subtype, size and treatment details for each cohort; scoring and cut-off criteria; survival outcome, HR including associated 95% confidence intervals (CI) and P value. If outcome measures were related to the absence and not presence of the identified marker, the HR and associated CIs were inverted. If different studies used the same or overlapping cohorts, the largest cohort was used for the random-effects models. In the case where a KM plot was included but no associated HR was quoted, three statistical methods were used to infer the HR value.

Methods to extract HRs from KM plots

Several methods exist to infer HRs from KM plots where they were not quoted within the article 97 . Here, we used the Parmar 42 and Guyot 43 methods as well as a new method, known as Nlopt 39 , based on the mathematical technique, non-linear optimisation. All three are associated with varying degrees of error 97 , but the Nlopt method is the most accurate when the number at risk (found at the bottom of a KM plot) are not included but a P value is; whereas the Guyot method is more accurate when the number at risk are included. The Parmar method was used when both the number at risk and P value were not included. All three methods rely on extracting a sufficient number of points from each KM plot. To carry this out, digitized KM plots were loaded into the Fiji distribution of Image J (version 1.52p; NIH, USA) and the axes calibrated using the Figure Calibration Plugin (Frederic V. Hessman, University of Gottingen). The specific guidance for extracting points for each method was then followed resulting in a number of X,Y points. In the case of the Parmar et al . method, we followed guidance from Tierney et al. 99 to determine the minimum and maximum follow-up times for each study, as these values are crucial in extracting accurate HRs from KM plots when using this method. HRs and standard errors (SEs) for the Parmar et al . method were calculated in Excel, whilst we used the R script published with the Guyot et al . and Nlopt method to determine these HRs and SEs. The SE of the HRs were increased by 5 and 10% respectively for the Guyot/Nlopt method and Parmar method, reflecting the known error associated with each method 39 , 97 .

Study quality assessment

To assess the quality of a study, a score from the REporting recommendations for tumour MARKer prognostic studies (REMARK) criteria was calculated for each included study by AI and SW. Discrepancies were resolved by consensus. Although checklists for assessing the quality of prognostic studies do exist (e.g. the QUIPS checklist 100 ), the REMARK criteria are specific to tumour marker studies and have previously been used in meta-analyses of tumour markers 101 . The REMARK criteria is composed of twenty items split into several domains: introduction, patients, specimen characteristics, assay methods, study design, statistical analysis methods, data, and analysis, and discussion. Each article was scored 1 point per item, with a score of 0.5 for items where the study fulfilled some but not all of the criteria. Cut-offs for each domain were used to represent a low-, medium- and high-quality score. For assessment of overall quality, cut-offs for low, medium and high were ≤ 10, ≤ 15 and > 15 respectively. In the case of a random effects model including a low-quality study, sensitivity analysis was used to exclude these studies and the model re-analysed. The traffic light plot in Supplementary Figure S1 was produced using the robvis package 102 in R. The relationship between year of publication and REMARK score was assessed using a linear model in R and plotted using the R package ggplot2 103 .

Defining the cellular processes key to CAF function

Cancer-associated fibroblasts have a wide range of functions which influence cancer progression and have been summarised in a number of recent reviews 26 , 41 , 48 , 104 , 105 , 106 . These reviews were used as the basis to create a set of common cellular processes/functions crucial to CAF function (Supplementary Table S4 ).

Assigning individual markers to each cellular process

To determine the proposed function of an individual CAF marker, the literature was reviewed for functional studies which investigated the role of that particular protein in some aspect of cancer progression. The following strategy was used to search Medline as a way of identifying relevant articles:

(name of marker) AND (cancer OR tumour OR tumor) AND (fibroblast OR stroma)

Titles and abstracts were initially screened and the full-text reviewed if relevant. This strategy was used in preference to the alternative option of a bioinformatics approach using a database such as DAVID 107 as the function recorded for each protein would not be specific for CAFs. Since there were only 26 identified markers, the decision to manually annotate the functional role of each marker was instead chosen as way of increasing the specificity of the highlighted functional process whilst accepting a potential loss of sensitivity. Functional studies that investigated the role of each marker in NSCLC were favoured but where these did not exist, other tumour types were used. Functional studies were occasionally determined in the same paper that also analysed the prognostic role of the particular protein in NSCLC. Functional studies generally included co-culture experiments with CAFs and tumour cells in tissue culture as well as mouse models whether these were  injection studies or genetically-engineered strains. Each marker was then placed into the relevant cellular process as identified in the Methods described above. If no relevant functional process was identified, these proteins were excluded from the analysis.

After extraction of all relevant data, we first combined similar survival outcomes resulting in three groups: OS/DSS, RFS/DFS and PFS. However, we considered HRs derived from either univariate or multivariate analysis separately, as recommended by Riley et al . 47 . In the case of an individual marker with at least 2 distinct cohorts based on the same outcome group and analysis method, a random effects model using the inverse variance method was used to create weighted HRs with 95% CIs and P value. A variable tree for the individual markers was generated using the vtree package 108 in R. Heterogeneity was assessed by calculating I 2 and Ʈ 2 values with a P value generated to assess the statistical significance of the heterogeneity. The aggregate HRs for the cellular processes were calculated in the same manner but to ensure as many of the markers could be included in the analysis as possible we used HRs from the OS/DSS outcome group and combined multivariate and univariate HRs with the former used in preference to the latter where available. The ferris wheel plot was generated using ggplot2 103 in R. The random effects model were carried out using the meta package 109 in R. The icons representing the cellular processes in Fig.  4 are from BioRender.com. A P value of ≤ 0.05 was considered statistically significant for all tests carried out.

Unless otherwise stated, all analysis and figures were generated in RStudio (Version 1.3.959) with version 3.5.2 of R. Panels of figures were assembled using Adobe Illustrator 2020 (Version 24.2).

Ethics statement

No animals or humans were used in generating data for this study.

Data availability

Any of the data generated in this study are available from the corresponding author on reasonable request.

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Acknowledgements

AI was supported by the University of Southampton National Institute of Health Research (NIHR) Academic Foundation Programme and a University of Leeds NIHR Academic Clinical Fellowship. SW was supported by Cancer Research UK, the Medical Research Council Clinical Research Training Fellowships (MR/R001286/1) and a Pathological Society Trainees’ Small grant.  This work was additionally funded by support from Cancer Research UK (grant nos. A20256, A27989) to GT.  AI would like to thank Professor Tony Kendrick for help with designing the study protocol and his support throughout. We thank Paula Sands of the Health Sciences Library at the University of Southampton for invaluable help in generating the database search queries.

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Andrew F. Irvine

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Andrew F. Irvine, Sara Waise & Gareth J. Thomas

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A.I. and G.T. developed the idea for the study. A.I. and B.S. developed the study protocol which this study is based on. A.I. and S.W. carried out the acquisition and analysis of data. E.G. helped design some of the figures. B.S. provided statistical help throughout. All authors were involved in writing the paper and had final approval of the submitted version.

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Irvine, A.F., Waise, S., Green, E.W. et al. Characterising cancer-associated fibroblast heterogeneity in non-small cell lung cancer: a systematic review and meta-analysis. Sci Rep 11 , 3727 (2021). https://doi.org/10.1038/s41598-021-81796-2

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Paige Clark

LITERATURE REVIEW

This literature review is designed to investigate previous research that has been undertaken to help aid in the creation of my IRP: “to what extend does the media influence the formation of youth’s identity.” By analysing different forms of literature in depth, this has allowed myself to gain a deeper understanding into the topic that I have chosen, to assist in the further completion of my IRP.

According to Essena O’Niell/ www.letsbegamechangers.com (Date: 11/2015) states that she doesn’t agree with social media as it currently stands.

Society currently creates validation among our community based on “likes” and “views.”

Numbers on a screen shouldn’t dictate the quality of our work, our success and our value

Social media should be based on quality, not social approval. There needs to be a website created where content is not based upon how many “likes” or “views” a person gets, but based upon the quality of their content.

Exterior motives such as gaining social approval are just “ideas of success.”

Social media can become detrimental to human health and ability.

The author concludes that there should be a form of media created that isn’t based on views, likes and followers. Teenagers seek validation and therefore insecurity through likes and followers.

Another idea, by Raising Children Network/ http://raisingchildren.net.au (date 26/6/15) states that media influences do play a part in shaping the behaviour of teenagers. Once the individual is more aware of media influence, they’ll be able to handle media pressure better.

Media influence on teenagers can be deliberate, for example, a large proportion of advertising is directed at children and teenagers. The result of this is that youth in general are becoming gradually more aware about brands and images.

Other types of influential media upon youth appear to be more indirect. In recent years; advertising, magazines, music videos and television have portrayed to their audience unrealistic images of women, with their bodies being advertised as “sexy,” which is a recent trend in the over sexualisation of content.

Indirect media influences are suggestive in ways that they portray “standard” ways to look and behave. For example; suggesting to teenagers that violence in video games and coarse language in song lyrics is a “normal” way to act.

Body image is influenced by many factors – such as peer attitudes, the fashion industry, family environment, ability or disability and one of the most influential forms - mass media (e.g.: social media and advertising.)

When unrealistic “thin” body types are portrayed in the media, the impact upon teenagers can be influential, affecting their own body image and dieting patterns. This is even more prominent when there is no one around to tell the individual that they disagree with the messages that media portrays, such as “thin is beautiful.”

The ideal body image portrayed by the media has subjected teenagers to the idea of plastic surgery. For example, there has been an increased rise in teenage girls wanting laser hair removal and breast implants, while boys are becoming increasingly interested in muscle enhancers (soft tissue fillers.)

Media isn’t all negative, it can be a positive influence on teenagers. For example, young people can also pick up vital health messages that the media advertises; such as healthy eating, exercising and preventing youth depression and suicide.

An influential celebrity role model who displays a positive lifestyle to their audience can be a powerful media influence. A role model who educates about their hard work and success can provide encouragement, particularly if their lifestyle or behaviour is respectable.

Teenagers should learn to interpret media by asking themselves some questions about it, to understand what they are influenced by. These could be questions such as:

≥ Who’s behind the image?

≥ What’s their motivation?

≥ What do they want from you?

≥ How does the advertisement make you feel and why?

The author concludes that even though there are solid links between media content that results in harmful teenage behaviour, teenagers don’t necessarily take to mind everything the media portrays to their audience – in fact, teenagers can actually be savvy users of media messages. Adult responsibility should involve helping young adolescents understand that exposure to media influences is part of present life, but there are vital ways in helping youth handle media influences.

In relation, research conducted by Deakin University Researcher Dr Michiko Weinmann/ http://www.deakin.edu.au/news/latest-media-releases/2015-media-releases-archives/unmasking-perceptions-of-cosmetic-surgery-among-young-women (Date: 10/11/15) is to use her new project, ‘Operation Belle,’ to research whether an increasing number of young women have turned to cosmetic surgery, due to the rise in celebrity and “selfie” culture.

Cosmetic surgery which is defined as ‘elective surgery for aesthetic purposes,’ is a topic in which teachers, parents and even peers can find very complex.

A young person’s cultural background, school involvement, friends and appearance are a list of major factors that contribute to the development of finding and defining a young person’s identity, which is a vital pathway to growing up.

Social media, magazines, advertisements, reality television and videos are some of the mediums that constantly remind young people every day that we live in a surgical society.

The list of cosmetic surgeries is forever gradually expanding, with young Australians being affected more than ever before.

Advertisements linked to interviews with cosmetic surgeons, clinics and even ‘before and after’ pics, constantly fill magazines with articles that are linked to cosmetic surgeries.

The Australasian College of Cosmetic Surgery has revealed that the beauty industry in Australia is a big business, with Australians each year spending around $1 billion on cosmetic procedures.

The author concludes that there are increasing numbers of women who are turning to cosmetic surgery in the pursuit for individuality, attractiveness and a sense of identity.

In addition, Kids Helpline/ http://www.kidshelpline.com.au/grownups/news-research/hot-topics/body-image.php (Date: 10/2014) suggests that body image has the ability to be separate from the physical appearance of one’s own body, with the example being that an individual who fits within an average weight range and who is classed as “healthy,” may distinguish themselves as being overweight.

Body image can be defined as “one’s sensitivity, opinions and outlooks about how they view their body”, including how outside individuals may perceive their figure, and how they believe their body fits in with ‘society’s expectations.’

To have a positive body image, one will feel confident in their appearance and comfortable in their own skin, while a negative body image results in the individual viewing their body as undesirable.

When an individual has a positive body image and a healthy state of mind, this leads to an increased freedom and confidence in their interaction with others to indulge in life’s experiences and challenges. Having a negative body image may hold a person back, as there is a strong link between poor body image and low self-esteem.

Factors that can affect body discontent can include:

≥ Skin appearance – skin colour, birth marks, pigmentation, acne and scaring

≥ Hair – colour and/or style

≥ Physical Defects

≥ Disabilities

≥ Cultural diversity that could be linked to a particular style of clothing and/ or appearance

Young children will intake substantial messages about body image and eating habits from adults of the family that they are growing up in, as young people have not yet had the exposure to different viewpoints for them to form their own judgement, therefore making these receiving messages significant.

Direct comments about a child’s physical image are passed down through parents and/or carers, linking to how these adults model their attitudes and values about physical appearance.

Mass media has led to unrealistic expectations in regards to the “ideal female body,” which in Western society is often characterised as “being thin.” Due to this, a large percentage of a female’s body image problems are more likely to lead towards losing weight, while for males, the “ideal body” focuses on being lean and muscular which leads to males focusing on developing a more well-built upper body.

In mass media, young adolescents are often seeking an “ideal image” which is unrealistic, due to professional photographers using certain techniques to make models appear “more beautiful,” through the use of lighting and effects. Other tricks include but are not limited to the use of Photoshop and printing techniques to portray a person in having perfect skin, teeth, hair, body etc.  

Australian data dictates that up to 1 in 3 young Australians have body image issues, particularly amongst adolescent females and young adults.

Issues regarding body image are more likely to arise in males around the time of early adolescence, while in females these concerns occur in mid to late adolescence.

Males with a poor body image are divided between those who want to lose weight and those who wish to gain weight to increase muscle.

Poor self-esteem in relation to issues surrounding body image can be linked to depression, anxiety, mood swings or all-round negative behaviour.

The author concludes that it is considered “normal” for a young person to be thoroughly aware of their body image, due to the arrival of puberty and the physical body images that can begin to attract the attention of others. However, when the urge to “fit in” becomes the prime purpose, this can lead to severe unhappiness that has the potential to result in disorders such as substance abuse, binge eating, anorexia and bulimia.

Another idea by Cosima Marriner/ http://www.essentialkids.com.au/life/technology/impact-of-social-media-on-teen-girls-20130722-2qe3x is that teenage girls are more likely to develop low self-esteem due to being displeased with their bodies, as a result of spending more time on social media. In an online world where everything’s a “competition,” for example, the number of likes an individual get on a Facebook picture, or through the act of posting “weight loss selfies” to Instagram, teenage girls are vulnerable to be caught up in their virtual headspace.

When interviewing over 1000 high school girls, Flinder’s University researchers found that due to social media, conversations about an individual’s physical appearance are “exaggerated,” due to the involvement of peers.

90% of girls in Years 10 and 11 who were interviewed had a Facebook account and were regularly uploading pictures of themselves to it, with an average of 475 “friends.” The average time spent on social media was roughly 2 and a half hours per day.

46% of girls who were surveyed stated that they were displeased with how much they currently weigh, even though 80% very considered to be within the healthy weight range. These negative feelings have resulted from the overuse of social media websites, according to Amy Slater from Flinders University's School Of Psychology.

According to Social Media Commentator Melinda Tankard Reist, negative health outcomes in teenage girls can be the result of seeking self-affirmation on social media, as often there is a constant feeling of the need to be “on display.” Today’s society provides affirmation to exhibitionism as the population is constantly judged on their physical appearance.

Ms Tankard Reist also states that television is a prime influence on how media can influence young adolescences. ‘Australia’s Next Top Model’ conducted a “selfie competition” where contenders as young as 13 were invited to post a selfie using the hashtag #antmselfie to win a VIP Pass to the show’s final and feature in the ‘Cosmopolitan’ magazine. This is a leading example of how media these days prioritises looks, with over 37000 girls entering this competition.

Dr Slater states that seeking self – affirmation and positive comments from peers are of great significance as a lot of conversations lead to the talk of one’s physical appearance.

By teenage girl’s copying what others are doing in regards to media, it leads to the individual always trying to look at themselves through the eyes of others, creating a situation where self-worth is less valued.

The author concludes that social media can have a stronger and more authoritative influence on teenage girls more so than traditional media, due to the key elements being so influential and allowing for more open channels of communication.

In relation, The National Eating Disorders Collaboration/ http://www.nedc.com.au/body-image (Date: 24/04/15) is that a poor body image begins through an internal process but can become further prominent due to the influence of external factors such as family, friends, teachers and mass media, resulting in a drastic impact into how the individual feels about themselves mentally and physically

There are four aspects of body image:

≥ PERCEPTUAL – How an individual “sees” themselves in physical form, but might not be correct to what their body actually looks like. A common example is someone viewing themselves as overweight, when in fact they are actually underweight.

≥ AFFECTIVE – The level of satisfaction or dissatisfaction an individual feels about their weight, size and shape of their singular body parts.

≥ COGNITIVE – How an individual thinks about their body which in some cases cause a person to develop a “fixation” with their weight and the physical appearance of their body. An example is when an individual believes that they would have an increased level of self - confidence if they were slimmer or well-built.

≥ BEHAVIOURABLE – Behaviour patterns in individuals which reflect how they feel about their body. For example, an individual who feels guilty about the way they look, may choose to cut off themselves from others in society due them feeling discontented with their physical form. However, severe cases can result in an individual developing destructive behaviours to try and change their appearance. For example, engaging in excessive exercising and dieting.

Having a positive body image is vital as it allows an individual to build a barrier in protecting themselves against eating disorders, as by having the self – confidence in their own body, they will mentally appear as more “resilient,” as they are able to accept, appreciate and admire their own body.

A positive body image will help improve one’s:

≥ SELF - ESTEEM – Prescribes how an individual feels about themselves which can contribute to multiple aspects of life to promote all aspects of wellbeing and general happiness.

≥ SELF – ACCEPTANCE – Makes a person feels content with their physical form which results in them being less likely to be affected by the unrealistic images that the media portrays, which convey that individuals in society have to portray a certain “look.”

≥ HEALTHY OUTLOOK AND BEHAVIOURS – When an individual has a substantial knowledge of what is needed to adequately fuel their body, they will find it easier to lead a healthier lifestyle by indulging in balanced practices and attitudes relating to the consumption of food.

A higher risk of poor body image can occur when an individual is surrounded in an environment obsessed by appearance, or when negative feedback about their exterior is made known to them.

All members of society are subjected to unrealistic and inaccessible images which are portrayed by different forms of media such as the internet, TV, magazines and advertising. Often these images are deemed “unobtainable” due to the fact that they are frequently fabricated by art teams, stylists and edited through Photoshop methods. Poor body image often occurs when individuals feel as though they can’t physically “compare” to the people they see blasted through mass media, resulting in destruction to the individual’s psychological and physical wellbeing. Due to this, the media is a major external contributor to poor body image among individuals.

Influences that can link to certain individuals developing a poor body image:

 ≥ LOW   self-esteem and/or depression

 ≥ TEASING – Individuals who are teased about their weight and/or appearance

≥ BODY SIZE – In a society where being “skinny” is seen as a body ideal, often people who are seen as “larger” may be at risk of developing a poor body image.

≥ FRIENDS AND FAMILY – Individuals who are exposed to role models in their life voicing body image anxieties and showcasing damaging weight loss behaviours, regardless of what their physical state may be.

≥ GENDER – On average, adolescent females are more prominent to develop body dissatisfaction than adolescent males. But there is evidence that males are imminently approaching a similar level of body dissatisfaction to match those who are female.

AGE – Late childhood and adolescence are usually the prime time where an individual’s feelings regarding their body are developed. In contrast though, feelings of body dissatisfaction have the ability to affect anyone, no matter what age the person is.

The author concludes that it is vital to understand that everyone is different in regards to their weight, shape, size and physical appearance. While some aspects of the body cannot be changed due to genetics, for example a person’s height, muscle build-up and bone structure, it is important for an individual to learn to be more accepting of the body that they have been given, as this is a key step to challenging “beauty ideals.”

Another idea by The Better Health Channel/ https://www.betterhealth.vic.gov.au/health/healthyliving/body-image-and-diets (Date: 04/13) states that an individual who develops a poor body image is likely to participate in over exercising and dieting, as well as being at risk to developing eating disorders such as bulimia nervosa, anorexia nervosa and a binge eating disorder, which can be linked to other mental health issues such as poor self-esteem, depression and anxiety. 

Australian’s each day spend up to $1 000 000 on “fad” diets which not only can be nutritionally unsafe, but can sometimes have very little effect on the person’s actual weight.

Dieting frequently can lead to:

≥ Over-excising                                                        

≥ Poor health

≥ Developing depression and anxiety

≥ developing an eating disorder

≥ Binge eating

≥ Not in taking enough nutrients per day due to the restriction of food intake

≥ Purging food or abusing laxatives

45% of women 23% of men who fit within a healthy weight range think they are overweight

More than 20% of women who are actually underweight, exploit dieting as a weight loss method with the belief that they are in fact overweight

There are links between culture and body image. An example being that research has shown that Asian women who have migrated to Australia, are more likely to take on dieting and body image habits that are not common in Asia.

Research has proven that nearly every young woman and almost half of all middle aged women have tried dieting at least once in the attempt to lose weight.

Men in today’s society are under a growing amount of pressure to have the “ideal” body that is portrayed in the media. Statistics show:

≥ 17% of men are currently undertaking a “fad” diet

≥ More men today than ever are undergoing cosmetic surgery

≥ A rapidly increasing amount of men are purchasing grooming and cosmetic products

Frequent dieting will not remove all the fat that is on a women’s hips and thighs, as it is considered “normal” to have a sizeable amount of fat on those parts, as it is vital for:

≥ Healthy skin, nails, eyes and hair

≥ Fertility and breastfeeding

≥ To prevent osteoporosis

The author concludes that Dieting (as long as the person understands healthy eating behaviours) can be beneficial to those who want to remain within the healthy weight range by engaging in practices to eat a nutritional diet. However, constant dieting can be damaging to a person’s physical and mental wellbeing and can lead to depression, especially if the individual already had a poor body image to start off with.

CONCLUSION OF LITERATURE REVIEW:

Throughout my secondary research, I have been able to complete in depth further study to allow myself to gain a deeper insight into the topic that I have chosen for my IRP “to what extend does the media influence the formation of youth’s identity.” While I acknowledge that the topic chosen is very broad, my literature review has allowed me to draw out the main components which are heavily researched in my area of study.

SOCIAL ACCEPTANCE: Throughout my secondary research, it became clearly evident that due to the rise in social media, the urge to be “socially accepted” has become more prominent than ever. Two of the sources of information that I studied confirmed my suspicion that the more time spent specifically on social media, the more likely the individual is to develop issues surround self-esteem. According to the article written by Cosima Marriner , she writes that social media every day is having a more drastic effect on individuals in society, particularly those who are younger and more vulnerable. She further explains that research undertaken by researchers at Flinder’s University reveals that a large majority of every day conversations held by these adolescent girls are as the result of social media. These conversations can consist of anything, from the amount of “likes” on Facebook that a fellow peer has, to scrutinizing the physical appearance of one of their “friends” in the photo that she posted on Instagram. In a school yard filled with some particularly “catty” young adolescent girls who are spending on average, roughly 2 and a half hours per day on social media, there is a constant feeling going around of the neediness to feel “accepted,” as adolescents today are growing up in a society where individuals are rewarded for exhibitionism, and one’s physical appearance is “exaggerated” in conversation, due to the prominence of social media. This results in adolescents very easily becoming vulnerable as they are caught up in their online presence of social media. However, not all adolescents in today’s society are willing to keep engaging in the online pressures of today’s world. Another form of literature that I studied followed an 18 year old female named ‘Essena O’Niell’ from the Sunshine Coast, who quit social media and therefore, quit her career as a life-style blogger and model. Before she quit, O’Niell had accumulated over 500 000 Instagram followers, more than 250 000 YouTube subscribers and was signed with a modelling agency while she promoted well-known brands. Since making the decision to switch off social media, she has launched her own website and personal blogging platform where she reveals the truth behind her previous social media life, revealing that in one photo she “took over 100 photos in similar poses trying to make her stomach look good.” Are these the levels where teenagers today feel as though they have to go above and beyond to be accepted as “beautiful” within our society? But this is where it stopped for O’Niell, as she explained in a tearful video that she was addicted to the attention and the money that she gained from her “hidden advertising” on Instagram, where she promoted bikini’s, cosmetic products and clothing, resulting in her living her life “through a screen” and that she didn’t know who she truly was anymore. Since deleting all her social media networks, the launch of her website and blogging platform has allowed her to write and create visual content that isn’t dictated by “likes” or “views” as there is no place for this form of communication to occur on her new platform. Any video content that is now created through her website is completely unedited and “raw,” which allows her to get her message across that an adolescent’s value these days shouldn’t be based off numbers on a screen, but instead the quality of the content created should speak for itself. By removing herself from all her social media platforms, the creation of her new website allows her to still create written and audio content, but without having to have that “social” interaction with others, as there is no way for an audience to communicate to her throughout her website. She has continually advised all her viewers to step back from social media, as it can hinder human health and capability and trying to gain social approval is a false idea of success.

COSMETIC SURGERY: Another reoccurring issue in the battle between the media and its influences on different aspects of youth was the rising talk of cosmetic surgery. In multiple sources of literature that I studied, this controversial topic came up multiple times throughout my research, catching my attention as it became obvious that more and more young individuals are choosing to go under the knife to alter their physical appearance. An article by ‘The Raising Children Network’ stated that because the media has constantly portrayed to young adolescents the “ideal body” for males and females, with this exposing to teenagers the “want” and the “need” to participate in cosmetic practices to achieve that ideal figure, suggesting that there has been a rapid increase in the amount of teenage girls wanting breast implants and laser hair removal. But now there has even been a growing trend in teenage boys wanting to alter their appearance, with the media placing an importance on biceps and big muscles, resulting in the increased interest of muscle enhancers. Another source of information conducted by Deakin University Researcher Dr Michiko Weinmann, suggests that because media platforms every day are glamorizing cosmetic surgery through advertisements, social media and reality television, young adolescents are constantly reminded that we do live in a surgical society that celebrates physical appearance over self-worth. Advertising in magazines has become a great platform for companies positing “before and after” cosmetic surgery pictures, linking to the rise in “celebrity” and “selfie” culture, to assist individuals in their pursuit for attractiveness and to help develop their sense of identity.

BODY IMAGE: One of the major components of my secondary research has been finding out more information about how the media can influence an adolescent’s body image. While many of my resources listed the facts about how having a poor body image can seriously affect an individual’s physical and mental wellbeing, I was surprised to learn the extent of the damage that can occur, or the fact that an increasingly alarming number of males are participating in fad diets as they feel that the media portrays men in a more “masculine” fashion – to look lean and muscular to develop a well-built upper body. The statement that “only females feel self-conscious” is a complete myth, as throughout my reading, I have studied the statistics of men who are suffering from a low self-esteem in regards to their body. One of my resources ‘The Better Health Channel’ gave me a great insight into the facts that I didn’t know previously about body image issues in males. The statistics show that 17% of men are currently undertaking a diet that is considered a “fad,” while buying grooming and cosmetic products, and even considering utilizing the service of plastic surgery. But why though? One of the main culprits lowering men’s self-esteem is the media, especially the use of advertising to make men feel pressured to have to confine to physically look a certain way.  However, even though there is evidence to show that an increasing number of males these days are feeling the pressures of “society’s beauty ideals,” there has been more research conducted surrounding the topic of body image issues in females. Could this be because of the existing stigma that males don’t ever feel self-conscious about their body? Referring back to the research conducted by ‘The Better Health Channel,’ the statistics surrounding body image issues in females is alarming, as research has shown that almost all young women have tried dieting at least once in an attempt to alter their weight. Not only that, but up to 45% of all women who fit within a perfectly healthy weight range believe that they are overweight, putting these individuals at risk of developing poor self-esteem, depression, anxiety and even eating disorders like bulimia nervosa. Another worrying fact is that many of these women haven’t been educated on appropriate eating and exercising behaviors that can be used to adequately fuel their body, and because of that, are participating in excessive dieting and exercising habits. This can have a detrimental effect on their physical and mental wellbeing. In both sexes ‘The National Eating Disorders Foundation’ discusses the effects of mass media being one of the biggest external contributors of body dissatisfaction, through the use of excessive advertising, magazines, TV shows and the internet. Unfortunately, what many young people don’t realize is that these images are often fabricated though the use of technology to utilize editing programs and lighting effects, to make a person appear as though they have the “ideal body,” regardless of if they are male or female. While the media keeps blasting unobtainable images to young, vulnerable people in our society, there will continue to be a climb in the rate of adolescents who are experiencing destructive negative body image behaviours, impacting how the individual feels about themselves, mentally and physically.

Role of cancer-associated fibroblasts in oral squamous cell carcinomas, surgical margins, and verrucous carcinomas: An immunohistochemical study

Affiliations.

• 1 Department of Oral Pathology and Microbiology, Bharati Vidyapeeth (Deemed to be University) Dental College and Hospital, Sangli, Maharashtra, India.
• 2 Department of Oral Pathology and Microbiology, KLE VK Institute of Dental Sciences, KLE Academy of Higher Education and Research (KLE Deemed to be University), Belagavi, Karnataka, India.
• 3 Department of Oral Pathology and Microbiology, Dr. D. Y. Patil Dental College and Hospital, Dr. D. Y. Patil Vidyapeeth, Pimpri, Pune, Maharastra, India.
• PMID: 35261929
• PMCID: PMC8898639

Background and aim: Cancer-associated fibroblasts (CAFs) are among the key tumor microenvironment components that determine tumor invasion, progression, and resistance to cancer therapeutics. Histologically normal mucosa adjacent to oral squamous cell carcinoma (OSCC) has been shown to harbor CAFs which aid in the loco-regional recurrence of the lesion. Verrucous carcinoma (VC), a low-grade variant of squamous cell carcinoma, has a better clinical outcome. However, few VCs show an aggressive biological course and necessitate wide excision with strict follow-up. Scarce literature is available regarding the role of CAFs in VCs. Thus, our study aimed to evaluate the frequency of CAFs in OSCC, normal mucosa adjacent to OSCC, and VC.

Methods: Thirty cases of squamous cell carcinoma, normal mucosa adjacent to OSCC, and VC each were included in the study. The sections were stained with an antibody against alpha-smooth muscle actin protein and CAF frequency was evaluated.

Results: The CAF frequency was highest in squamous cell carcinoma, followed by VC, and least in normal mucosa adjacent to OSCC ( P <0.001).

Conclusion: CAF frequency progressively increases with an increase in the grade or biological behavior of the lesion. Thus, screening CAF frequency in these benign and malignant oral lesions is necessary for better treatment outcomes.

Relevance for patients: The immunohistochemical screening for CAFs in OSCC and VC can serve as an integrated approach for the development of a directed treatment plan that leads to a better patient prognosis. Routine assessment of CAF frequency in surgical margins can serve as an adjunct in determining clear margins and possible locoregional recurrence. Furthermore, target therapy for CAFs can be used to minimize possible recurrence and distant metastasis.

Keywords: cancer-associated fibroblasts; squamous cell carcinoma; tumor margins; tumor microenvironment; verrucous carcinoma.

Copyright: © Whioce Publishing Pte. Ltd.

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CAFS: Literature Review

• Thread starter jasmineerrosee
• Start date Oct 30, 2017

jasmineerrosee

Please read my literature review and provide feedback please! ​ Literature Review The purpose of my literature review is to examine and analyse secondary information that is currently available with relation to my research topic. My following piece of literature reviews includes website articles, an interview and a print resource. These secondary resources show an relation to my research topic. #1 Title: Study confirms HSC Exams source of major stress to adolescent. Source: The Conversation Date Published: September 11 2015 Date Accessed: 11th October 2017 Authors: Ben North, Miraca Gross, Susen Smith Link: https://theconversation.com/study-confirms-hsc-exams-source-of-major-stress-to-adolescents-46812 This article examines the cause and effects of academic pressure, along with researched statistics that support research on students with stress symptoms, and practical skills students can develop to manage stress. This article focuses on why students may experience high level stress, along with describing main causes of pressure, and how students are coping with the pressure and stress. It describes symptoms of anxiety both emotionally and physically, and finally gives advice to students on how to take care of their physical health and the importance of it, building resilience, relaxation and yoga, effective coping strategies, learning to identify when stress and anxiety become problematic, as well as the usual lessons in study skills and organisation. This article also provides extensive statistical detail regarding stress and anxiety symptoms and, along with pressure students' experience. This can be viewed as extremely reliable as it is sustained with statistics and examples. This article is also an extremely reliable source as the author Ben North holds a PhD Candidate in Education; Head Teacher (Secondary). Miraca Gross is an Emeritus Professor and Susen Smith a Senior Lecturer in Gifted & Special Education, GERRIC Senior Research Fellow. This article contains extremely useful information in regards to my topic as it provided an extensive amount of professional information, which was reliable and relevant. #2 Title: How the education system is making kids stressed and sick. Source: ABC Date Published: July 17th 2016 Date Accessed: 11th October 2017 Authors: Hayley Gleeson and Lucy Clarke Link: http://www.abc.net.au/news/2016-07-17/beautiful-failures-education-making-kids-sick/7589084 This article examines the pressure on children to achieve high levels of academic success is overriding the joys of education and making kids anxious and depressed, what is going so wrong with education in Australia, and what can be done to fix it from a personal point of view. This interview and article focuses on the changes of HSC pressure and stress on students, and how those pressures are affecting students' mental health. The interviewee of this article is a mother of a daughter who experienced the HSC with large amounts of stress and anxiety, and shares her point of view of the effects of stress created by HSC on students emotional and mental wellbeing. Lucy Clarke (the interviewee) explains her daughter's situation on the mentality behind stress from HSC and gives an array of opinions on the education system, and the effects it may have on not just her daughter but students from Australia’s population. This article lacks any specific statistical data, and could have shown a more-in-depth response in regards to the emotional and mental side of stress from the HSC. This article is only partly reliable and valid, with some personal experience. It is necessary to treat with caution as the subject may be lying or exaggerating. In saying that, this article is useful to my topic as it gave a personal experience and point of view on emotional and mental effects of stress on students wellbeing. Although statistical data could have been sustained, it was still relevant to my topic. #3 Title: The ultimate test, David VS The Avalanche of Exams Source: Headspace Date Published: 24th April 2012 Date Accessed: 11th October 2017 Authors: Melbourne creative agency Draftfcb Link: https://i.pinimg.com/236x/cd/94/0e/cd940e8afac002b728ea2fc9322e9c06.jpg This print resource focuses on the amount of exams and other practical school work rather than the impacts of school stress. This print resource could be aimed at people who know they’re affected by HSC stress and seek advice, and treatment available. This print resource was apart of a campaign by National Youth Mental Health Foundation Headspace to raise an awareness on mental health which will be reached to young people of 12-25 who are eligible to use the foundation for support. This print resource is part of a total of 6 other print resources, but David VS the avalanche of exams is most relevant to my research topic. In saying that, this print resource is useful to my topic as it gave me a focus on the physical part of HSC that students may experience rather than an emotional and mental aspect.  

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