Functions of CAFs in microenvironment of non-small cell lung cancer: based on updated hallmarks of cancer

Origin of CAFs

CAFs in NSCLC can be transformed from various stromal cells [16, 17]. Lung resident fibroblasts are one of the essential sources of CAFs. Quiescent lung resident fibroblasts are activated in response to stimuli such as tissue trauma, inflammation, and fibrosis, which promote tissue repair and regeneration [18]. NSCLC tumor cells can activate fibroblasts for epigenetic reprogramming to obtain a tumor-promoting phenotype [19]. Another important source of CAFs is mesenchymal stem cells, which can differentiate into CAFs after being stimulated by tumor signaling [20]. The high expression of α-1 antitrypsin (A1AT) in NSCLC promotes the Epithelial-to-Mesenchymal Transition (EMT) in epithelial cells and forms a pro-tumor CAFs phenotype [21]. Adipose mesenchymal stem cells (MSCs) can be induced by tumor cells, secrete high levels of interleukin-6 (IL-6) family cytokines, adipokines, and adhesion molecules that activate the signal transducer and activator of transcription 3 (STAT3) signaling pathway, increase the expression of matrix metalloproteinases in various cancer types (like bladder cancer), and induce EMT, exhibiting a pro-tumor CAFs phenotype [22]. Bone marrow mesenchymal stem cells can be recruited to tissue injury or inflammation sites through endocrine signals to exert their tissue repair function. In contrast, the tissue regeneration function of MSCs may be faulty in the tumor microenvironment of NSCLC and differentiate into CAFs [23]. Recent studies have found that macrophages and endothelial cells are also a source of CAFs in the NSCLC microenvironment [24, 25]. Although distributed in different locations in the tumor microenvironment, smooth muscle cells and pericytes exhibit similar phenotypes and functions and also might be another origin of CAFs through transdifferentiation [14].

Biomarkers of CAFs

Common CAFs markers include fibroblast activation protein (FAP), alpha-smooth muscle actin (α-SMA), platelet‐derived growth factor receptor (PDGFR), PDPN (podoplanin), vimentin, etc. FAP is a type II membrane protein that has been implicated in tissue repair, fibrosis, and degradation of the extracellular matrix in fibroblasts, and the expression of FAP on fibroblasts in NSCLC has been linked to its prognosis and survival [26]. α-SMA, a member of the actin family, induces myofibroblast contraction and promotes granulation tissue contraction through microfilament bundle and stress fiber regulation. Increased expression of α-SMA is associated with poor prognosis in tumors of NSCLC patients [27]. PDGFR is a universal marker for fibroblasts, and multiple studies have observed that PDGFR expression in CAFs is closely related to the prognosis of NSCLC. PDPN is a glycoprotein, also known as a lymphangitic marker, and multiple studies have found that PDPN⁺ CAFs have tumor-promoting functions and are associated with a poorer prognosis in patients with LUSC [28]. Vimentin is an intermediate filament protein (type III) that plays a vital role in organelle structure, cell migration, and adhesion in mesenchymal cells. Fibroblasts are characterized by their mesenchymal phenotype, and vimentin is highly expressed in NSCLC interstitial CAFs and is involved in CAFs motility to promote tumor cell invasion and metastasis [29]. In addition, the membrane-bound extracellular enzyme γ-glutamyltransferase (GGT) hydrolyzes γ-glutamyl bonds, and high expression of GGT in CAFs predicts poor survival in patients with LUAD [30]. CAFs generally have a pro-tumor phenotype, but FAP-positive CAFs have also been shown to be associated with a better prognosis in NSCLC patients with high levels of CD3 and CD8 double positive T cells, suggesting a tumor-suppressive effect of CAFs. Still, in most studies, CAFs have played a tumor-promoting role [31].

Subtypes of CAFs in NSCLC

The expression of CAFs-related markers has been reported to help analyze CAFs subsets [32]. However, their low specificity and heterogeneity pose significant challenges to typing CAFs. CAF-related markers are expressed in a variety of tumors. FAP is highly expressed in CAFs of malignant tumors such as NSCLC, pancreatic cancer, and breast cancer. It is also closely related to the progression of chronic inflammatory diseases [31], especially playing a pivotal role in the occurrence and development of interstitial lung diseases [33]. The myofibroblast phenotype with high expression of α-SMA can secrete ECM and cause protein deposition, which plays an important role in the occurrence and development of lung cancer and idiopathic pulmonary fibrosis [27]. Hu et al. established a gene pool of CAFs, including NSCLC-related CAFs. They determined the heterogeneity of CAFs by analyzing the differences in the expression levels of CAFs genes and then identified three functional subtypes: (1) hepatocyte growth factor (HGF) and fibroblast growth factor 7 (FGF-7) with a strong protective effect on tumors, (2) moderately protective tumors and highly expressed FGF-7 isoforms, and (3) functional subtypes that provide minimal protection [34].

Other new techniques, such as single-cell imaging mass cytometry (IMC), could also be applied for categorying CAFs. This functional classification of CAFs is related to the patient’s clinical response to targeted therapy and the tumor immune microenvironment and thus can provide a pathway for guiding personalized treatment [35].

Promote tumor cell growth and maintain cell stemness

Tumor cells can activate CAFs through abnormal signaling and interact with CAFs to secrete growth factors and cytokines that have pro-tumor effects, silence tumor growth inhibitory signals, and maintain stem cell properties. It has been demonstrated that the hypoxic microenvironment of NSCLC can enhance the expression of hypoxia-inducible factor-1α (HIF-1α) in fibroblasts and induce the conversion of normal fibroblasts to CAFs. At the same time, the expression of HIF-1α in fibroblasts can activate the NF-κB signaling pathway and enhance C-C chemokine ligand 5 (CCL5) secretion, thereby promoting tumor growth [36]. Another study has shown that IL-22 secreted by CAFs reduces apoptosis and significantly promotes the proliferation of NSCLC cells by activating the phosphatidylinositol 3-kinase (PI3K)-Akt-mTOR signaling pathway [37]. The down-regulation of MiR-101-3p in CAFs can activate CAFs and promote tumor growth [38]. On the other hand, CAFs could silence inhibition signals of tumor cell growth. Disruption of the retinoblastoma protein (pRb) pathway releases the E2F transcription factor, allowing cells to enter the proliferative cycle. Bouchard et al. compared the phenotypic differences of CAFs (invasive margins and tumor centers) at different sites of LUAD and found that different CAFs with abnormal glucose metabolism can have different activation signaling pathways, including cyclin-dependent kinase 4 (CDK4) and phosphorylated retinoblastoma protein axis (CDK4-pRb axis), which indirectly regulate the invasion characteristics of cancer cells [39]. CAFs also could maintain tumor cell stemness. Several studies have shown that CAFs in NSCLC can regulate the expression of Nanog in tumor cells through paracrine signaling, which in turn regulates the plasticity of cancer stem cells (CSCs) [31]. The mechanism by which CAFs contribute to the occurrence and progression of NSCLC is very complex, and although many studies have been conducted, there are still many gaps in the understanding of this field.

Promote angiogenesis

Tumor angiogenesis is one of the important features of most tumors, and CAFs promote the formation of NSCLC angiogenesis through a variety of mechanisms, thereby promoting tumor progression. CAFs can promote the growth and infiltration of NSCLC through their pro-angiogenic effects. It has been found that CAFs are one of the essential sources of vascular endothelial growth factor A (VEGFA) in the NSCLC microenvironment, and the low expression of miR-101-3p in NSCLC-related CAFs can promote VEGFA secretion and thus angiogenesis [38]. NSCLC cell-derived exosomal mi-210 increases the expression of pro-angiogenic factors matrix metalloproteinase-9 (MMP9), fibroblast growth factor 2 (FGF2), and VEGFA by regulating the JAK2/STAT3 signaling pathway and promoting angiogenesis [40]. In LUAD models, CAFs overexpress transforming growth factor-β (TGFβ), MMP7, FGF9, and FGF2, synthesize more collagen, and promote tumor angiogenesis [41]. In some NSCLCs, the interaction between tumor cells and CAFs in the tumor microenvironment activates the paracrine Hedgehog/GLI signaling pathway, which promotes VEGF-dependent endothelial cell activation and tumor angiogenesis, thereby promoting tumor progression [42]. CAFs can also provide nutrients to tumor cells by promoting vascular mimicry (VM). Through in vitro experiments, Tsai et al. showed that CAFs can induce lung cancer cells to form capillary-like structures, provide a channel for tumor cells and tumor-promoting neutrophils, and alter the ability of tumors to form VM networks through Notch2-Jagged1-mediated direct contact [43].

Enhance invasion and metastasis

CAFs can promote tumor metastasis to distant organs by promoting increased tumor aggressiveness, which is also one of the crucial mechanisms of NSCLC progression. Studies have shown that CAFs can promote metastasis by secreting cytokines, and the stromal cell-derived factor-1 (SDF-1) secreted by CAFs enhances the expression of C-X-C chemokine receptor type 4 (CXCR4), β-catenin, and peroxisome proliferator-activated receptor delta (PPARδ) in LUAD cells, enhancing their aggressiveness [44]. RAS-related C3 botulinum toxin substrate 1 (RAC1), a small molecule G protein, is a signaling molecule that regulates various cellular activities and gene expression. T-cell lymphoma invasion and metastasis 2 (TIAM2), which is highly expressed in CAFs, can promote the invasion and migration of NSCLC cells by activating RAC1 [45]. CAFs-specific miR-196a promotes increased tumor aggressiveness by activating annexin A1 (ANXA1) and secreting CCL2 [46].

CAFs could also promote tumor metastasis through autocrine. CAFs promote NSCLC invasion by activating HMGB1/NF-κB while maintaining tumor cell viability through the autocrine high-mobility protein B1 (HMGB1) circuit [47]. In addition, CAFs can also promote lymphatic metastasis. LUAD cells co-cultured with PDPN⁺ CAFs have been found to have a higher risk of lymph node metastasis [28].

Promote chronic inflammation

The human immune system can recognize and respond to tumor cells, and inappropriate and sustained activation of the immune response can lead to a chronic inflammatory state, thereby increasing the risk of tumorigenesis and promoting tumor progression, in which CAFs play an important role. CAFs can promote tumor inflammatory states by secreting pro-inflammatory cytokines. CAFs produce large amounts of CXCL12, which enhances colony-forming capacity and promotes the production of pro-inflammatory chemokine CCL20 in NSCLC [48]. The expression of genes related to the inflammatory response pathway in CAFs was up-regulated, and the cardiotrophin-like cytokine factor 1/ciliary neurotrophic factor receptor (CLCF1/CNTFR) and IL-6/IL-6R signaling pathways played an important role in promoting the growth of NSCLC [49]. Conventional chemotherapy mainly targets actively-cycling cancer cells, while cancer progression in NSCLC patients may result from a small number of slow-cycling cancer cells with a quiescent-like phenotype. It has been found that slow-cycling cancer cells can form a pro-inflammatory microenvironment through activating transcription factor 6 (ATF6)-mediated transcriptional upregulation of various pro-inflammatory cytokines and synergistically recruit fibroblasts in a paracrine manner. Activated fibroblasts are up-regulated by COX2 and Col-I transcriptionally, triggering the transition of PGE2 and integrin/Src signal-mediated slow-cycling cancer cells (SCCs) to actively cycling cancer cells (ACCs) [50].

Promote immune escape

The human immune surveillance system is the line of defense against tumorigenesis, and CAFs can promote cancer progression by forming an immunosuppressive microenvironment. CAFs can promote the formation of immune microenvironment by secreting cytokines and chemokines to recruit immunosuppressive cells. CAFs recruit CCR2-positive monocytes by secreting CCL2 and induce their differentiation into a myeloid-derived suppressor cell (MDSC) phenotype in LUSC [51]. NSCLC-derived CAFs form an immunosuppressive microenvironment by decreasing NK cell proliferation rate and cytotoxic capacity [52]. Tumor-associated macrophages (TAMs) can change from an anti-tumor phenotype (M1 type) to a pro-tumor phenotype (M2 type) under the influence of TME [53]. Monocytes from NSCLC tumors were isolated and co-cultured with CAFs, and it was found that CAFs promoted the differentiation of macrophages to M2 type and inhibited the pro-inflammatory properties of M1 type macrophages [54]. Several studies have found that the expression of TGF-β1 in PDPN-positive CAFs in LUSC is up-regulated. TGFβ could inhibit the activation and proliferation of effector T cell subsets, including Th1, Th2 cells, and cytotoxic T lymphocytes, and promote the differentiation of naïve CD4 positive T cells into Treg cells, suggesting that PDPN positive CAFs are associated with immunosuppressive TME [31]. CAFs could also inhibit the infiltration and migration of immune cells by remodeling the extracellular matrix (ECM) and preventing the binding of immune checkpoint antibodies to their targets, thereby forming immune escape. T cells typically accumulate in fibrous loose areas of the tumor interstitium, and the dense ECM acts as a barrier to hinder the contact between T cells and tumor cells while inhibiting T cells’ binding to PD-1 inhibitors, thereby promoting resistance to immune checkpoint inhibitors [55].

Regulate tumor cell metabolism

Tumor cells adjust their metabolic patterns to maintain high proliferation rates in a hypoxic and low nutrient-concentration environment. CAFs are thought to be the primary regulators of tumor metabolism. The Warburg effect is currently the most widely accepted mode of tumor metabolism, the metabolic mode in which tumor cells are inclined to aerobic glycolysis. Pyruvate kinase M2 (PKM2) expression is up-regulated in NSCLC cells under hypoxia, and exosomes of tumor cells can deliver PKM2 to CAFs. Conversely, the PKM2 high CAFs release pyruvate, lactate, ketone bodies, and glutamine through autophagy, creating a favorable microenvironment that supports tumor growth [56]. As knowledge of tumor metabolomics continues to grow, it has been discovered that tumor cells can also be powered by the anti-Warburg effect. It has been found that CAFs can provide energy support to NSCLC cells by downregulating the expression of glycolytic enzymes in tumor cells and increasing the ability of CAFs to release lactate [57]. In vitro co-culture studies suggest that CAFs increase the glycolytic capacity of tumor cells, while tumor cells increase the mitochondrial function of CAFs, and metabolic reprogramming is associated with reactive oxygen species (ROS) and TGFβ signaling pathways [58]. Metabolic crosstalk between CAFs and tumor cells can lead to the progression of tumors.

Promote resistance to anti-tumor therapies

Despite the continuous development of therapeutic approaches, tumor resistance is inevitable, with CAFs mediating fundamental mechanisms of NSCLC resistance to chemotherapy, targeted therapy, immunotherapy, and radiation therapy [59]. CAFs could promote chemotherapy resistance in NSCLC by mediating EMT, remodeling the ECM, maintaining stemness of CSCs, and modulating metabolic reprogramming, making NSCLC cells resistant to tyrosine kinase inhibitors by promoting tumor EMT and forming a hypoxic microenvironment, and promote immune escape mainly by remodeling the ECM and preventing immune checkpoint inhibitors from binding to their targets [23]. CAFs can mediate resistance to NSCLC through a variety of paracrine signals. The study found that CAFs can promote the chemoresistance of NSCLC to cisplatin through the IL-11/IL-11R/STAT3 signaling pathway, mediate drug resistance through the IGF2/AKT/Sox2/ABCB1 signaling pathway, and secrete chondroitin sulfate proteoglycan serglycin (SRGN) and tumor cell receptor CD44 interactions promote a malignant phenotype [31]. CAFs after radiotherapy can promote radiotherapy resistance of NSCLC cells through the JAK/STAT pathway, and the apoptosis of CAFs can be specifically induced by the Foxo4-p53 interfering peptide FOXO4-DRI, which has a significant radiosensitizing effect on NSCLC cells and has a significant therapeutic impact on alleviating radiation-induced pulmonary fibrosis [59]. The resistance mechanism of CAFs to treatment also requires the involvement of other components of the TME. Studies have shown that CAFs and the ECMs they produce can act as physical barriers, thereby preventing efficient drug delivery and inhibiting apoptosis of tumor cells. TME changes caused by anti-tumor therapy will promote NSCLC treatment resistance through CAFs. Tumor necrosis factor superfamily member 4 (TNFSF4) was significantly up-regulated in CAFs under the stressful environment of chemotherapy, radiotherapy, and anti-angiogenic therapy, and TNFSF4 inhibited apoptosis of LUAD cells by enhancing the activity of the NF-κB/Bcl-X_L pathway in LUAD cells, thereby promoting chemoresistance in LUAD cells [60]. In addition, the balance between apoptosis and autophagy of cancer cells could be modulated by CAFs through exosome-transferred non-coding RNAs and paracrine signaling (such as IL-6), which might be engaged in resistance to cancer treatment [15].

Unlock the phenotypic plasticity of NSCLC cells

Cell differentiation results in cell inhibition of proliferative activity in most cases, and unlocking tumor cell phenotypic plasticity is an important feature of tumor progression. CAFs can unlock phenotypic plasticity in tumor cells by promoting differentiation and metastasis. The expression of SOX2/SOX9 and NKX2-1 (TTF1) in the lung epithelium is critical in determining lung progenitor cells’ lineage properties and cell fate. In the TME co-culture system composed of epithelial cells in LUSC, CAFs, and extracellular mesenchymal cells, Chen et al. found that under the influence of the microenvironment, epithelial cells changed from proliferation and dysplasia to invasion and phenotypic transition of stem cells, during which SOX2 overexpression mediated the transition from hyperplasia to dysplasia, while CAFs mediated invasive changes [61]. Netrin-1 is up-regulated in most human tumors, and interference with Netrin-1 is associated with tumor growth and metastasis inhibition. It has been found that the secretion of Netrin-1 by CAFs during co-culture with cancer cells is associated with an increase in CSCs, and the Netrin-1 inhibitor Net-1-mab inhibits the plasticity of NSCLC cells by regulating the expression of CAFs-mediated cytokines such as IL-6 and inhibiting cell-to-cell signaling between CAFs and cancer cells [62]. In the NSCLC model, CAFs can also activate the IGF-1R signaling pathway through the secreted insulin-like growth factor (IGF), induce the expression of stem cell-related gene Nanog, and thus convert tumor cells to CSCs.