Cancer‐associated fibroblasts in nonsmall cell lung cancer: From molecular mechanisms to clinical implications

Abstract Lung cancer is the common and leading cause of cancer death worldwide. The tumor microenvironment has been recognized to be instrumental in tumorigenesis. To have a deep understanding of the molecular mechanism of nonsmall cell lung carcinoma (NSCLC), cancer‐associated fibroblasts (CAFs) have gained increasing research interests. CAFs belong to the crucial and dominant cell population in the tumor microenvironment to support the cancer cells. The interplay and partnership between cancer cells and CAFs contribute to each stage of tumorigenesis. CAFs exhibit prominent heterogeneity and secrete different kinds of cytokines and chemokines, growth factors and extracellular matrix proteins involved in cancer cell proliferation, invasion, metastasis and chemoresistance. Many studies focused on the protumorigenic functions of CAFs, yet many challenges about the heterogeneity of CAFS remain unresolved. This review comprehensively summarized the tumor‐promoting role and molecular mechanisms of CAFs in NSCLC, including their origin, phenotypic changes and heterogeneity and their functional roles in carcinogenesis. Meanwhile, we also highlighted the updated molecular classifications based on the molecular features and functional roles of CAFs. With the development of cutting‐edge platforms and further investigations of CAFs, novel therapeutic strategies for accurately targeting CAFs in NSCLC may be developed based on the increased understanding of the relevant molecular mechanisms.

With the development of cutting-edge platforms and further investigations of CAFs, novel therapeutic strategies for accurately targeting CAFs in NSCLC may be developed based on the increased understanding of the relevant molecular mechanisms.

K E Y W O R D S
cancer-associated fibroblast, heterogeneity, nonsmall-cell lung cancer, tumor microenvironment

| INTRODUCTION
Lung cancer is one of the most common malignancies worldwide, particularly in men. 1 The American Cancer Society reported approximately 2.2 million new cases of lung cancer and approximately 1.8 million new deaths in 2020. 1 Lung cancer is histologically classified as small-cell lung carcinoma (SCLC) and nonsmall-cell lung carcinoma (NSCLC). NSCLC represents approximately 80% of all lung cancer 2 and is mainly divided into squamous cell carcinoma, adenocarcinoma and large cell carcinoma. These subtypes have unique histological and biological features. 3,4 Enhancing insight into the genome alterations revealed various oncogenic driver mutations in NSCLC. 5,6 To understand the biological perspectives of lung cancer, researchers have mainly focused on malignant cells, such as various signaling pathways. 7 However, these just represent one of the hallmarks of cancer. Cancers are not simply composed of cells with deranged signaling pathways but include a complex tumor microenvironment (TME). [8][9][10] Like the theory of "Seed and Soil" 11 which was proposed by Dr Stephen Paget, cancers cells seed in congenial soil, the TME, where they grow and expand. The TME is an ecosystem composed of multicellular and noncellular components. 12,13 Four major components of the TME are: (1) the tumor immune microenvironment (TIME) consists of immune cells such as natural killer (NK) and T cells; (2) vascular components include lymphatic endothelial cells (LECs) and pericytes; (3) the extracellular matrix (ECM) is comprised of diverse collagen, glycoproteins and proteoglycans; (4) stromal components consist of mesenchymal STEM cells (MSCs) and cancer-associated fibroblasts (CAFs). 14 These cells in the TME interact with the malignant cells closely, which promote the whole tumorigenesis process, from tumor initiation to progression.
CAFs are one of the well-known and critical components in the tumor stroma. CAFs are worthy of mention since they are conducive to all aspects of tumorigenesis in different stages and many cancer types, including tumor proliferation, tumor invasion and metastasis and interfacing with the immune system. 14,15 Given the multifaceted functions of CAFs, many studies attempted to "switch off" the function of CAFs to target tumors more effectively. Controversially, some investigations have demonstrated that some CAFs have an antitumorigenic role. 16,17 More importantly, how can CAFs transit from a tumor defender into a tumor supporter? For example, tumor-associated exosomes have been identified recently as an essential cellular interchange mechanism between tumor cells and CAFs. 18,19 Isolated exosomes from tumor cells and CAFs are implicated in multiple steps of CAFs evolution, such as normal fibroblasts (NFs) differentiation into CAFs, CAF-like state maintenance and promotion of CAFs' oncogenic properties. [20][21][22][23] Extracellular vesicles produced by tumor cells can activate normal fibroblasts to a CAF-like state, which in turn produces a secretome to modulate the tumor microenvironment. 24,25 In this review, we summarized recent studies on the roles of CAFs and, particularly in NSCLC, where scar formation and fibrosis are common phenomena.

| Fibroblasts
Fibroblasts were first identified in the 1850s as connective tissue cells responsible for synthesizing collagen. 26 Fibroblasts in normal tissue are generally considered quiescent, that is, in a resting state. Fibroblasts can be challenging to define because of a lack of unique markers expressed exclusively and by all fibroblasts. 27 Some markers such as vimentin, platelet-derived growth factor receptor-α (PDGFRα) and fibroblast specific protein 1 (FSP1) can be used as markers for quiescent fibroblasts. [28][29][30][31] However, these markers are not only expressed in fibroblasts. Thus, the tissue location and morphology are always required for their identification. Quiescent fibroblasts are the major component of ECM under physiological conditions. They are activated by tissue repair and regeneration in response to tissue damage. As observed in wound healing, 32 fibroblasts accumulate at the damaged site and transform into myofibroblasts, and subsequently promote angiogenesis and deposition of ECM. Myofibroblasts produce many kinds of cytokines and chemokines. 15,33 They are also a significant source of ECMdegrading proteases, maintaining ECM homeostasis by regulation of ECM turnover, 34 and promoting angiogenesis with increased production of vascular endothelial growth factor A (VEGFA). 35 Myofibroblasts secrete transforming growth factor-beta (TGF-β) and express α-smooth muscle actin (α-SMA) at closing wounds, 36 and are critical for maintaining the homeostasis of adjacent epithelial cells by growth factors (GFs) secretions and by direct mesenchymal-epithelial cell interactions. 37 When the wound is healed, myofibroblasts are restored to their quiescent status or are removed by apoptosis. 38 Such reversibility is a hallmark feature of fibroblasts associated with tissue repair.

| Activation of fibroblasts into CAFs
Tumors may be considered as "wounds that do not heal." 26 In a normal situation, fibroblasts have an antitumorigenic activity that suppresses tumor growth. For example, fibroblasts in lymph nodes transport potential antigens and contribute to leukocytes' migration, resulting in effective immune responses. 17 However, cancer is an advancing and unabated injurious stimulus which initiates fibroblast activation. Fibroblasts are then transformed into irreversible cancer-associated fibroblasts (CAFs), which behave like myofibroblasts in some aspects. 39 They are not removed by apoptosis. This process is called cancer fibrosis.
To acquire tumor-promoting phenotypes, the quiescent fibroblasts are activated via diverse mechanisms ( Figure 1A). First, epithelial cancer cells secrete growth factors into the surrounding microenvironment, stimulating the recruitment and activation of fibroblasts. Among these factors, transforming growth factor-beta (TGF-β), platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) are critical regulators. In lung cancer, TGF-β facilitates invasion of cancer cells through tumor-stromal interactions. 40,41 TGF-β orchestrates tumor stroma development and promotes angiogenesis, immune evasion and remodeling of the ECM. 42,43 In microarray gene expression analysis, the gene signatures related to TGF-β signaling are enriched in CAFs isolated from NSCLC tissues compared to the normal tissue. 44 PDGF is one of the profibrotic growth factors secreted by cancer and stromal cells, inducing CAFs activation. 45,46 Cancer cells secrete PDGF to act on the stromal cells, especially endothelial cells and fibroblasts in vivo. 47 In contrast to TGF-β, the primary functions of PDGF are enhancing fibroblasts' growth and proliferation through MAPK downstream signaling pathways 48-50 without causing their differentiation into myofibroblasts. 51 PDGF is also a crucial factor in neo-angiogenesis and establishing protumorigenic stroma. 45,52 FGF, an angiogenic endothelial cell mitogen, is a pleiotropic molecule that functions on epithelial and mesenchymal cells in an intracrine, autocrine and paracrine manner. 53,54 Most studies focused on FGF-2, which describes how it changes the phenotype of fibroblasts, leading to cell activation. 53 Besides growth factors, lung cancer cells produce different inflammatory modulators such as the interleukin family (IL-6, IL-8, IL-17, IL-22), tumor necrosis factor-α (TNF-α) and VEGF to promote their progression, invasion and angiogenesis. 55 Many studies found that these inflammatory cytokines are related to fibroblast activation in lung cancer. Leukemia inhibitory factor (LIF), an IL-6 class proinflammatory cytokine, is an example. It mediates ECM remodeling and TGF-β-dependent actomyosin-contractility via crosstalk between the JAK1/STAT3 and RhoA/ROCK/MLC2 signaling pathways, which results in carcinoma cell invasion in vitro and in vivo. 56 Actomyosin contractility generates mechanical force to remodel the ECM for cell migration, which is caused by CAFs. 57 The roles of STAT3 and SMAD are also implicated in lung fibrosis. 58 In NSCLC, the oxygen level is deficient (0.7-46 mm Hg), 59 thus hypoxia is a characteristic of the lung cancer microenvironment. This remodels the composition of TME, and induces the expression of HIF-1α in fibroblasts. [60][61][62][63] The expression of HIF-1α in fibroblasts also induces the conversion of normal fibroblasts into CAFs, and CAFs activation can be inhibited effectively by HIF-1α-specific inhibitors or HIF-1α knockout. 64 Moreover, p62, an autophagy regulator, is highly expressed in NSCLC under hypoxia. 65 This induces autophagy, the nuclear factor erythroid 2-related factor 2 (Nrf2)-related antioxidant signaling, and the activating transcription factor 6 (ATF6)-related ER-stress response, causing (C) Subpopulation of lung cancer CAFs Characterized by therapeutic profiling:

| The origin of CAFs
Emerging evidence suggests that CAFs are a highly heterogeneous population of cells. 16 Such heterogeneity might be due to the numerous potential cellular sources and precursors of CAFs ( Figure 1B). 88 Studies also suggested that pericytes, 99,100 smooth muscle cells surrounding small border vessels can transdifferentiate into CAFs. Pericytes have been considered an essential source of myofibroblasts. 101,102 The process starts with pericyte detachment from endothelial cells, followed by migration into the lung interstitium and then activation to become myofibroblast via TGF-β/Smad2/3 and PDGFβ/Erk signaling pathways. [103][104][105] Here, the transforming growth factor-β receptor (TGF-βR) and platelet-derived growth factor-β receptor (PDGF-βR) are modified by core fucosylation (CF). α1,6-fucosyltransferase (FUT8) is the only known enzyme that catalyzes CF. 106,107 In FUT8 knockdown cells, CF is out of function, and this inhibits TGF-β/Smad2/3 and PDGFβ/Erk signaling pathways. 108 Some studies suggested that Sonic Hedgehog (SHh) is also involved in CAFs activation. 109,110 SHh contributes to branching morphogenesis lung specification in the developing lung. 111 In normal conditions, Hedgehog (Hh) activity is low. In the context of bleomycin injury, lung damage induces Hh pathway activity, and SHh overexpression increases fibrotic collagen deposition. 112 In idiopathic pulmonary fibrosis, Hh activity can promote multiple profibrotic processes, including enhanced sensitivity to TGFβ and PDGF, leading to increased migration, contractility and survival in human lung fibroblasts. 113,114 CAFs can also derive from epithelial cells (ECs). 115 ECs differentiate into functional CAFs, which express FSP-1 and αFAP via TGF-β-mediated epithelial to mesenchymal transition (EMT). 116,117 Endothelial cells contribute to the pool of CAFs through endothelial-to-mesenchymal transition (EndMT) in cancer, mainly via TGF-β and SMAD signaling. 118

| Subpopulation of CAFs in NSCLC and other cancers
Determination of subtypes of CAFs has met significant obstacles due to the heterogeneity of their origin, phenotype and function among different individuals in different tumor types. Based on different classification methods, there are different names for different subtypes of CAFs, as shown in Table 1 and Figure  In breast cancer, CAFs subtypes can also be defined by their biomarkers, such as CD146 + CAFs and CD146 À CAFs. Compared to CD146 + CAFs, CD146 À CAFs have higher metastasis and invasion ability and lead to a poorer prognosis. 125 In pancreatic cancer, the subtypes of CAFs can be characterized by their phenotypes, namely the myofibroblastic phenotype (myCAFs) and inflammatory phenotype (iCAFs). 126 MyCAFs are highly expressed in αSMA and located adjacent to cancer cells, while iCAFs secrete inflammatory mediators such as interleukin-6 (IL-6) and are located far away from cancer cells. 126 They are related to chemoresistance and poor survival in breast and lung cancer patients. 130 Hu et al also found three functional subtypes identified by lung cancer therapeutic profiling. 131

| The main molecular markers of CAFs
Due to the heterogeneity of CAFs, no marker can be used as a universal and specific marker for all CAFs in different types of cancers. 136 In addition, there are different subsets of CAFs in the tumor, increasing the difficulty in defining the appropriate markers for CAFs. 137 In lung cancer, the most used CAF markers include, but are not limited to, alpha-smooth muscle actin (α-SMA) and fibroblast activation protein-1 (FAP-1). The reported markers are summarized in

| THE PROMOTING ROLE OF CAFs ON CANCER CELLS
CAFs play a pivotal role in tumorigenesis and are involved in different oncogenic pathways (Figure 3). Increasing evidence has supported the protumorigenic roles of CAFs, which are summarized in Table 3.

| Invasion and metastasis
CAFs are a vital component in the TME and can act as a bridge between the TME and cancer cells. CAFs facilitate cancer cell crosstalk within the TME. 219 CAFs stimulate invasion and metastasis through two main aspects, which include EMT 220 and ECM remodeling ( Figure 3B). 221 CAFs induce EMT by secreting soluble factors. 222,223 In lung cancer, CAF-secreted IL-6 induces EMT programming and modulate metastasisrelated genes through the JAK2/STAT3 signaling pathway in vitro and in vivo. 168

| Angiogenesis
Angiogenesis plays an important role in tumor growth and metastasis. [234][235][236][237] The process requires several regulatory molecules such as VEGF receptors (VEGFR), 238 bFGF, 239 type I collagen 240 and fibronectin. 241,242 CAFs express these regulatory molecules to initiate angiogenesis ( Figure 3C). There is also evidence that in NSCLC

| Immune escaping
To achieve immune evasion, CAFs are involved in shaping the immunosuppressive TME ( Figure 3D). 244 However, the mechanism and crosstalk between the CAFs and immune cells are still to be fully eluci- To promote an immunosuppressive environment, CAFs diminish the antitumorigenic activity of natural killer cells (NK cells) 249 To  252 The above studies suggested that CAFs play a critical role in immune checkpoint biology. However, the interplay between CAFs and cancer cells remains elusive and insufficiently delineated.

| THE ROLE OF CAFs IN CHEMORESISTANCE
The two primary mechanisms used by CAFs to help cancer cells evade therapy have been demonstrated, including the physical barrier method and interplay of CAFs and lung cancer ( Figure 3E). The final manuscript has been approved by all authors. The work reported in the paper has been performed by the authors, unless clearly specified in the text.

ACKNOWLEDGMENTS
The figures in the manuscript were partly generated from BioRender

CONFLICT OF INTEREST
The authors declare that they have no conflict of interest.