Perspective Chapter: Role of Cancer-Associated Fibroblasts in Oncogenesis

Abstract

The tumor microenvironment consists of multiple types of cells, including endothelial cells, pericytes, neutrophil macrophage mast cells, lymphatic cells, basement membrane extracellular matrix, as well as fibroblasts. Fibroblasts populations found in cancers, also known as cancer-associated fibroblasts, have been implicated in the initiation, progression, and metastasis of tumors. This chapter will focus on the roles of cancer-associated fibroblasts in the progression of cancer and the studies of use of cancer-associated fibroblasts as a therapeutic target for cancer intervention.

1. Introduction

The tumor microenvironment (TME) is the environment in which tumor cells or cancer stem cells exist [1]. The TME consists of multiple types of cells, including endothelial cells, immune cells, and fibroblasts [1, 2, 3]. The TME also consists of components such as the extracellular matrix (ECM), soluble factors such as cytokines and growth factors, and physical properties such as pH and oxygen content [2]. The TME and the interactions between its components help to promote tumor growth and cancer progression (Figure 1) [3].

Figure 1.

Important interactions and mechanisms of the TME [1].

Fibroblasts are the most common type of cell in connective tissue, commonly defined as structural cells that specialize in depositing and remodeling the ECM [4]. Fibroblast populations found in primary and metastatic cancers, known as cancer-associated fibroblasts (CAFs), are implicated in tumor initiation, progression, and metastasis [5]. CAFs have wide varieties of cells-of-origin, heterogeneous phenotypes, and diverse functions, all of which are shared by other cells found in the TME [6]. This chapter will focus on CAFs and their potential use in cancer intervention.

2. Fibroblasts and cancer-associated fibroblasts

The precursors of CAFs are generally considered to be dormant tissue-resident fibroblasts and pancreatic and hepatic stellate cells, though different studies have also identified bone marrow-derived mesenchymal stem cells, endothelial cells, and adipocytes [5]. Fibroblasts play a prominent role in coordinating the wound repair response in skin; therefore, it is likely that key CAF traits correspond to the normal physiological role normal fibroblasts play [7]. Fibroblasts transform into CAFs through tumor-derived stimuli, including soluble factors secreted by the tumor, immune infiltrate, lysophosphatidic acid, fibroblast growth factor, interleukin-1 (IL-1), IL-6, and granulin [8]. Transforming growth factor β (TGFβ) and lysophosphatidic acid are well-established activating signals for fibroblasts, which promote the activity of SMAD transcription factors and serum response factors, respectively [7]. These fibroblast activating signals converge to drive expression of the fibroblast marker αSMA, as well as increase the activity of the contractile cytoskeleton [7]. Fibroblasts may become activated through Notch signaling when in direct contact with tumor cells [7, 8]. Other mechanisms that can activate normal fibroblasts to become CAFs are shown in Figure 2. In the TME, tumor cells secrete factors such as TGFβ, platelet-derived growth factor (PGDF), and fibroblast growth factor (FGF) to convert fibroblasts to CAFs [3]. A build-up of CAFs is often associated with poor prognosis in many cancer types [3].

Figure 2.

Mechanisms that activate normal fibroblasts to become CAFs. FGF, fibroblast growth factor; PDGF, platelet-derived growth factor; ROS, reactive oxygen species; RTK, receptor tyrosine kinase; TGFβ, transforming growth factor-β; TNF, tumor necrosis factor [7].

As shown in Figure 3 [10], TGFβ is a common factor in the conversion of many different cell types into fibroblasts and CAFs. There are many types of TGFβ. TGFβ-1 is one that is secreted by stromal and tumor cells and is the main factor in promoting the mobilization of residential fibroblasts and their activation into CAFs [10]. Through SMAD-dependent and SMAD-independent pathways, TGFβ-1 activates fibroblasts into CAFs, expressing alpha-smooth muscle actin, periostin, α-fibroblast activation protein, and fibroblast-specific protein-1 [10]. In addition, activated fibroblasts secrete TGFβ-1 [10], which could create a positive feedback loop, increasing fibroblast activation. TGFβ binds to the type 2 of TGFβ receptor (TGFBR2) on the surface of fibroblasts [11].

Figure 3.

Different origins of CAFs [9, 10].

2.1 CAFs in tumors

CAFs are a type of myofibroblast that enhance the malignancy and progression of cancer [12]. The presence of CAFs is identified in almost all solid tumors [13]. This suggests that CAFs are important to the formation of solid tumors. In an established tumor, the TME represents a changed part of the original normal tissue of the host [13]. Tumor cells mostly contribute to the change in their favor. [13]. The stromal transformation of TME is primarily dominated and maintained by CAFs [13]. The CAF component of the TME is the most critical in influencing most of the functions of the TME in real time [13]. CAFs alter the TME by directly interacting with cancer cells and regulatory paracrine signaling, control the immune response to neoplasia, deposit ECM components, stimulate angiogenesis, and provide a scaffold for tumor invasion and metastasis [14]. Additionally, CAFs can produce many growth factors and pro-inflammatory cytokines to promote angiogenesis and recruit immunosuppressive cells to the TME to evade the immune system [9].

While CAFs have historically been considered to be cancer-promoting components, recent studies have shown that CAFs could have tumor-restraining functions in certain circumstances [15]. The tumor-restraining actions of CAFs are likely dependent on stimulation of anticancer immunity, pro-inflammatory secretome, tumor inhibitory signaling, and the synthesis of ECM components as barriers to tumor cell invasion and dissemination [5]. A study in mice has shown that myofibroblast depletion leads to increased tumor invasion, which is associated with decreased survival [16]. This study suggests that CAFs have functions in restraining tumors. This paradoxical nature of CAFs can potentially be explained by the heterogeneity of CAFs [15].

2.2 Heterogeneity of CAFs

There is mounting evidence that CAFs are a heterogeneous population of cells [9]. This likely depends on the numerous precursors of CAFs [9]. CAFs can be recruited to the tumor from a distant source, such as bone marrow [14], or transdifferentiate from non-fibroblastic lineages, such as epithelial cells, blood vessels, adipocytes, pericytes, and smooth muscle cells [9]. Numerous precursors of CAFs are shown in Figure 3. The study of genetically modified mouse models (GEMMs) designed to limit the accumulation of CAFs in growing pancreatic tumors or to conditionally delete the vascular endothelial growth factors in breast CAFs revealed that there are distinct functional subtypes of CAFs [17].

2.3 Functions of CAFs

CAFs have both pro-tumor and antitumor tendencies [17]. Pro-tumorigenic functions of CAFs are generally driven by their altered secretive [17]. Paracrine signaling between cancer cells and CAFs leads to tumor progression by enhancing the survival, proliferation, stemness, and metastasis-initiating capacity of cancer cells, promoting cancer progression and enhancing resistance to therapy [17]. CAFs also have an indirect influence in promoting tumor growth due to their ability to remodel the ECM [17]. The stiffness of the tissue, which plays a critical role in tumorigenesis, is influenced by modifications in the ECM’s composition and cross-linking [18, 19]. CAFs express lysyl oxidase (LOX), an enzyme that cross-links and stiffens collagen fibers, promoting their stability [18]. CAFs also regulate the degradation of the ECM [18]. CAFs secrete cytokines and chemokines that regulate tumor immunity and the intratumoral vascular program [17]. Several studies have indicated that CAFs play an important role in chemoresistance via different mechanisms, including but not limited to increasing stem cancer cells, secreting cytokines, and secreting miRNAs [10]. miRNAs have been shown to inhibit tumor-repressor genes, thus promoting cell growth and invasion, metastasis, and tumorigenesis [20].

CAFs trigger tumor initiation and progression [18]. In vitro coculture and in vivo transplantation experiments have shown that human prostatic CAFs induced the proliferation and the ability to form tumors from immortalized nontumorigenic human prostatic epithelial cells [18]. This effect was not exhibited by normal fibroblasts. It is thought that CAFs’ secreted factors are what cause this tumor-initiating potential [18].

Numerous studies have shown that CAFs confer resistance to chemotherapy [6]. Some CAF-mediated resistant mechanisms include delivery of exosomes stimulating cancer cell survival, promoting cancer cell epithelial-mesenchymal transition, and thus decreasing expression of transporters responsible for drug uptake and scavenging chemo drug to reduce the amount of intratumoral chemotherapy drug [6]. CAFs also contribute to the resistance to targeted therapy [6]. Additionally, evidence suggests that CAFs contribute to immune evasion and immunotherapy resistance [6].

Antitumor functions of CAFs are predominantly associated with their functions as regulators of antitumor immunity [17]. Studies in mice have shown that fibroblast depletion leads to increased tumor invasion [16]. The use of defined gene promoter-driven expression of viral thymidine kinase proteins in GEMMs to study CAFs has allowed researchers to deplete populations of CAFs using ganciclovir, a substance that is toxic only to cells that express viral thymidine kinase [17]. A similar approach to deplete CAFs expressing αSMA suggested that αSMA+ stromal cells were predominantly acting to restrain cancer progression [17]. The depletion of these αSMA expressing CAFs yielded a more invasive tumor with enhanced intratumoral hypoxia [17]. A reduction in CAFs in GEMMs of pancreatic tumors with a deletion of sonic hedgehog (SHH) in the cancer cells also resulted in more aggressive tumors with increased cancer proliferation [17].

3. Targeting CAFs for cancer intervention

Numerous studies have proven CAFs’ significant role in cancer progression and subsequently the potential of CAFs as targets for effective cancer intervention. Traditionally, therapies involved targeting cancer cells directly [21]. Recent complementary efforts aim to disrupt the networks that promote cancer cell activity and behavior [21]. The depletion of CAFs and targeting of CAF-dependent pathways can indirectly result in malignant cell death through both immune-dependent and immune-independent mechanisms [21]. Most conventional cancer therapies, such as radiotherapy and chemotherapy, are likely to affect CAFs as well by preventing cellular division by inducing DNA damage, impeding DNA and RNA synthesis, and blocking the cytoskeleton remodel required for cell division [17]. However, the unintended impact of these therapeutic methods on the function and accumulation of CAFs is largely unknown [17].

As a result, research is being conducted to help target CAFs through alternative methods [21]. One approach involves targeting the regulatory pathways leading to fibroblast differentiation and activation [9, 17, 21]. For example, TGFβ is a common factor in the conversion of different cell types into CAFs. In a study, Mariathasan et al. found the two top scoring TGFβ pathway genes represent a ligand, TGFβ1, and receptor TGFβR2 [22]. In murine tumor models, blocking the TGFβ signaling by using the SM16 TGFβ receptor inhibitor or anti-TGFβ antibodies resulted in the recession of tumor growth [23]. By targeting these regulatory pathways, the activation of fibroblasts could be prevented, preventing CAFs from activating and functioning.

Another approach for targeting CAFs for cancer intervention is targeting CAF-secreted factors [11]. Numerous mitogens, chemokines, and matricellular proteins that CAFs release aid in the evolution of tumor progression and the development of drug resistance [11]. Targeting these CAF-secreted factors should prevent the promotion of tumor progression and drug resistance, making the tumor more susceptible to drugs. The heterogeneity of CAFs also proves as an advantage for cancer intervention via shifting the influence of pro- vs. antitumorigenic populations [21].

Currently, there are many drugs under trial as shown in Table 1. Of the potential targets identified in CAFs, fibroblast-activation protein (FAP) is the most studied. FAP has been neither detected in benign tumors nor in most normal quiescent adult stromal cells [35]. FAP is a type II integral membrane of the prolyl oligopeptidase family, or S9 family [36]. FAP is further classified into the dipeptidyl peptidase (DPP) subfamily (S9B) [36]. This class of enzymes is characterized by its capacity to cleave the pro-Xaa peptide link, where Xaa can be any amino acid. It has been demonstrated that this enzymatic activity contributes to the development of cancer by altering bioactive signaling peptides [36]. In vivo, FAP+ CAFs were successfully depleted by the FAP-depleting immunotoxin, and tumor models demonstrated strong tumor inhibitory effects. [6]. Other approaches in targeting FAP include DNA vaccine and chimeric antigen receptor (CAR) T cells [6].

Drugs	Target and mechanism	Cancer types	National Clinical Trial number	Status	Ref.
Sibrotuzumab	131I-labeled anti-FAP mAb	Colorectal, non-small cell lung, breast, or head and neck cancers	NCT02198274; NCT02209727	Phase I	[24]
Calcipotriol Paricalcitol	Vitamin D analogue	Early-stage skin cancer, breast cancer, pancreatic cancer	NCT03596073; NCT04617067; NCT02030860; NCT03138720; NCT04054362	Phase I/II	[25]
Pamrevlumab (FG-3019)	Anti-CTGF mAb	Pancreatic cancer	NCT03941093	Phase III	[26]
Plerixafor (AMD3100) BL-8040 (motixafortide)	CXCR4 receptor antagonist	Pancreatic cancer	NCT04177810; NCT02179970; NCT02826486; NCT03193190	Phase I/II	[27, 28]
IPI-926	Smoothened inhibitor	Pancreatic cancer	NCT01130142	Phase I	[29]
S-3304	MMP inhibitor	Advanced solid tumors	NCT00078390; NCT00033215	Phase I	[30]
131I-m81C6	131I-labeled anti-tenascin mAb	Brain tumors	NCT00002752; NCT00003461	Phase II	[31]
Imatinib	PDGFR inhibitor	Advanced solid tumors	NCT00161213; NCT00281996; NCT01048320; NCT00485485	Phase I/II	[32]
GS-6624 (simtuzumab)	LOXL2 mAb	Pancreatic cancer	NCT01472198; NCT01479465	Phase II	[33]
Tetrathiomolybdate	Copper; chelator, target LOX	Breast cancer, prostate cancer	NCT00195091; NCT00150995; NCT00405574	Phase II	[34]
Pegvorhyaluronidase alfa (PEGPH20; PVHA)	Recombinant human hyaluronidase	Lung cancer, pancreatic cancer	NCT01453153; NCT02563548; NCT01839487; NCT02715804	Phase I/II/III

Table 1.

Summary of various drugs’ efficacy against CAF-induced cancer progression in clinical and pre-clinical studies [6].

4. Conclusions

Heterogeneous populations of CAFs exist in the TME. The heterogeneous nature of CAFs likely comes from their different origins, and this heterogeneity is likely the cause of the paradoxical nature of CAFs having both pro-tumorigenic and antitumorigenic functions. CAFs have many functions in the TME. CAFs alter the TME. They produce growth factors and pro-inflammatory cytokines.

CAFs are a promising target for cancer intervention. They have many pro-tumorigenic functions. CAFs can be targeted through their activation pathway by blocking a step in the pathway. One method is by preventing FAP from being produced by introducing siRNAs that are complementary to the FAP mRNA.

Over time, our understanding of CAFs and their contribution to cancer progression has expanded greatly. We now have a better understanding of their heterogeneity and their functions in the TME. While the antitumorigenic functions may act as a roadblock to targeting CAFs for cancer intervention, it may be possible to develop a treatment that targets the pro-tumorigenic functions of CAFs without targeting the antitumorigenic functions of CAFs by targeting subpopulations of CAFs that express pro-tumorigenic genes.

While CAFs are a promising target for cancer intervention due to their pro-tumorigenic functions, their antitumorigenic functions may act as a roadblock. Further research would be required before targeting CAFs as a conventional method of cancer intervention.

However, there are issues with targeting CAFs for cancer intervention. Figure 4 shows the effects of targeting only tumor cells vs. targeting only the TME on a tumor. In both, there is a possibility that the tumor can grow back. In addition, studies in mice have shown that fibroblast depletion leads to increased tumor invasion [16]. In mouse models, deletion of SHH accelerated the progression of pancreatic ductal adenocarcinoma [37].

Figure 4.

A schematic diagram of targeting tumor cell or TME only and their potential resistance mechanisms. Left: Targeting tumor cells only (such as chemotherapies) kills majority tumor cells. However, the residue tumor cells may survive due to the TME, leading to tumor relapse. Right: Targeting the TME can inhibit the recruitment and activation of pro-tumor cells and enhance antitumor responses. However, the TME will be reconstituted by tumor cells via recruitment and programming of bone marrow derived cells or local resident stromal or immune cells [6].

Acknowledgments

Funding for the publication of this paper was made possible by a grant from The Assisi Foundation of Memphis. Brown, Chester, PhD (PI). We also thank Yirui Tang for drawing the figures used in this paper.

Conflict of interest

The authors have declared that no conflict of interest exists.