Cancer-Associated Fibroblasts (CAFs)

• Cancer-associated fibroblasts (CAFs) are a major part of the tumor microenvironment (TME) in both primary and metastatic tumors.
• CAFs influence cancer cell behavior.
• They are involved in cancer progression through interactions with cancer cells and other stromal cells.
• CAFs have versatility and plasticity, allowing cancer cells to "educate" them, leading to changes in stromal fibroblast populations.
• Understanding the phenotypic and functional heterogeneity of CAFs is therefore important

CAF Origins and Heterogeneity

• Researchers are exploring the origins and diversity of CAFs, as well as the molecular mechanisms that regulate CAF subpopulations
• The focus being on selectively targeting tumor-promoting CAFs, since this provides insights for future research and clinical studies on stromal targeting

Tumor Microenvironment (TME)

• The TME concept was first proposed in 1889, but has gained traction recently.
• Evidence shows that various cell types within a tumor interact to create a supportive environment for cancer cell survival, growth, and escape from the immune system.
• Cancer behavior depends on cancer cell-intrinsic defects and cancer cell-extrinsic factors, particularly the intricate TME.
• The TME is a dynamic niche with cellular and molecular components that interacts with cancer cells to meet their needs.
• Diverse cell types contribute to TME formation, including immune cells, CAFs, and normal epithelial cells, collectively termed stromal cells.
• Soluble factors like growth factors and cytokines, capillaries, and the extracellular matrix (ECM) form the complex network of tumor stromal signaling.
• CAFs, or activated fibroblasts, are central to the reactive stroma within the TME.
• CAFs interact with cancer cells directly through secreted molecules or cell-cell adhesion, and indirectly through ECM remodeling and immune cell infiltration.
• Studies show CAFs have a tumor-promoting function.
• It has been noted CAFs may restrain cancer progression as a host defense against neoplasia.
• This contradictory function is due to the heterogeneity and plasticity of CAFs, which depends on CAF precursor origin, cancer type, and tumor progression stage.
• Distinct CAF subtypes with specific markers have been identified: myofibroblast-like CAFs (myCAFs), inflammatory CAFs (iCAFs), and antigen-presenting CAFs (apCAFs).
• These subtypes have different and sometimes contradictory roles in tumorigenesis.
• Single-cell RNA-sequencing and proteomic technology have further divided CAF subpopulations based on distinct transcriptional profiles and modifications of stromal myofibroblast populations.
• Variations in stromal composition impact intratumoral architecture and contribute to functional changes in tumor cell behavior, highlighting the importance of CAF assessment when considering treatment options.

Fibroblasts and Wound Healing

• Fibroblasts play a central role in tissue repair.
• Tissue fibroblasts respond to injury and become reversibly activated to initiate regenerative repair.
• Activated fibroblasts, or myofibroblasts, express α-smooth muscle actin (α-SMA), have enhanced contractility, ECM production, and inflammatory mediator secretion, which initiates wound healing responses.
• Myofibroblasts contribute to ECM turnover at the late stage of tissue repair, synthesizing ECM-degrading proteases like matrix metalloproteinases (MMPs) and urokinase-type plasminogen activator (uPA).
• Once the repair process is complete, these activated fibroblasts undergo apoptosis or reprogramming to the resting state.

Fibroblasts and Chronic Wound Healing/Cancer

• Acute wound healing is a natural physiological reaction to acute tissue injury.
• Chronic or repetitive injury often results in continuous activation of fibroblasts and excessive ECM component deposition, leading to pathological tissue fibrosis with impaired organ function.
• One key mechanism underlying tissue repair versus irreversible fibrosis is that fibroblasts are transiently activated in acute wound repair.
• Durining repetitive damage, fibroblasts become resistant to apoptosis or have a limited ability to reacquire a quiescent phenotype.
• Cancer, especially solid tumors, shares features with tissue fibrosis, such as activated fibroblasts and increased stiffness of the ECM
• CAFs remain perpetually activated with a high capacity for ECM synthesis and microenvironmental remodeling, leading to stromal desmoplasia
• CAFs share basic characteristics with fibroblasts found in nonmalignant tissue fibrosis, such as a secretory phenotype and capacity to synthesize ECM components.
• Classic markers found in fibroblasts, including α-SMA, vimentin, desmin, fibroblast-specific protein 1 (FSP1; also known as S100A4) and fibroblast activation protein (FAP), have been used to distinguish CAFs in recent years.
• CAFs remodel the TME and influence cancer cell behavior, CAFs are directly or indirectly reprogrammed by cancer cells and other stromal cells, leading to distinct epigenetic and transcriptional profiles correlated with proliferative and invasive properties.
• Surface markers, such as platelet-derived growth factor receptor-α/β (PDGFRα/PDGFRβ), discoidin domain-containing receptor 2 (DDR2) and integrin α11β1, have emerged to identify CAFs in specific TMEs.
• It is suggested that the same marker at different expression levels may define the CAF subsets associated with specific stages of cancer development.
• Loss of caveolin1 (CAV1) is found in metabolically reprogrammed CAFs that promote tumorigenesis.
• High CAV1-expressing CAFs contribute to invasion and metastasis in breast cancer.
• None of these markers is exclusively expressed by CAFs, suggesting the possibility of diverse cellular origins of CAFs, necessitating biological deciphering of CAF evolution.
• Single-cell RNA-sequencing (scRNA-seq) analysis indicates that no single marker appears capable of discriminating CAFs from all other cell types and or even discriminating CAF subtypes.
• Combinations of two or more biomarkers are emerging to differentiate and isolate all CAFs across distinct cancer types.

Cellular Origins of CAFs

• CAFs are derived from tissue resident fibroblasts or nonfibroblast lineages.
• CAFs are highly heterogeneous populations that can originate from disparate precursors.
• The precise cellular origins of CAFs remain elusive owing to their substantial heterogeneity and a lack of definitive biomarkers for each subset.
• The most direct source of CAFs is normal resident fibroblasts or quiescent stellate cells.
• CAFs in the pancreas and liver are traditionally thought to originate from pancreatic stellate cells (PSCs) and hepatic stellate cells (HSCs), respectively, under the influence of tumor-derived stimuli.
• Cancer cell-derived chemokines, cytokines and microRNAs activate PSCs or HSCs, enabling them to gain myofibroblast-like features and transcriptional signatures associated with CAFs in pancreatic ductal adenocarcinoma (PDAC) or hepatocellular carcinoma.
• Activated PSCs and HSCs maintain their activity with enhanced synthetic and secretory capacities via autocrine loops and contribute to desmoplasia.
• The contribution of PSCs to PDAC CAFs in vivo in the context of tumorigenesis remains elusive.
• It was found that, contrary to expectations, PSCs only give rise to a small minority of CAFs in PDAC, suggesting the existence of diverse CAF progenitors and raising an important question that needs to be addressed regarding the additional cellular origins of PDAC CAFs.
• Normal resident fibroblasts reside around tumor cells that have been found to be activated via the tumor cell-derived signaling pathway and give rise to a subset of CAFs in PDAC, gastrointestinal cancer and breast cancer
• Current studies attempt to trace specific lineages of fibroblasts and delineate their contribution to stroma formation.
• Gli1, but not Hoxb6, specifically contributes to a portion of PDAC CAFs.
• A CAF subset marked by melanoma cell adhesion molecule (MCAM) is derived predominantly from intestinal pericryptal leptin receptor (Lepr) lineage cells, suggesting that inherent fibroblast heterogeneity may be linked to a specific subpopulation of CAFs.
• CAFs have been found to originate from multiple nonfibroblast lineage cells, including epithelial and endothelial cells, through epithelial/endothelial-to-mesenchymal transition (EMT/EndMT).
• Other suggested CAF precursors, although less common, include adipocytes, pericytes, mesothelial cells, and smooth muscle cells.

CAFs Derived from Recruited Bone Marrow Cells

• Lineage tracing has revealed the potential of bone marrow contribution to the CAF pool in several neoplasias, including rectal adenoma, gastric cancer, hepatocellular carcinoma, PDAC and breast cancer.
• Mesenchymal stem cells (MSCs) recruited from the bone marrow can differentiate into a subpopulation of CAFs under tumor-derived TGF-β, WNT, and IL-6/STAT3 signaling.
• Studies suggest the possibility that other bone marrow-derived cells may serve as CAF precursors.
• Bone marrow macrophages/monocytes can convert into CAFs in PDAC and Lewis lung carcinoma (LLC) via macrophage-myofibroblast transition (MMT).

CAF Heterogeneity and Plasticity

• A collection of diverse subpopulations of CAFs from different progenitors coexist in distinct tumor types and coevolve with epithelial genetic events during the development of cancer.
• It is tempting but also challenging to investigate the full CAF reservoir and the mechanisms governing the transformation from normal precursors to CAF subtypes during cancer evolution to gain an in-depth understanding of the tumor-associated stroma for targeted anticancer therapy.
• Functional categorization was first recapitulated in a coculture system of PDAC organoids and murine PSCs, which identified two mutually exclusive subtypes of CAFs, termed aSMA high IL-6 low myofibroblasts (myCAFs) and aSMA low IL-6 high inflammatory CAFs (iCAFs)
• myCAFs are activated by direct contact with neoplastic cells and reside adjacent to tumor foci.
• iCAFs are induced by cancer cell-derived factors such as IL-1a and TNFα, and are located more distant from tumor cells.
• iCAFs are generally tumor-promoting via the secretion of inflammatory cytokines and growth factors.
• myCAFs exhibit dual tumor-restraining and tumor-promoting roles, depending on the stage of the tumor and the complex context of the surrounding TME.
• Activation of fibroblasts may be a host defense mechanism acting as a dense barrier limiting tumor spread.
• The cues emanating from the TME may contribute to the opposing effects of myCAFs at different stages of tumor development.
• The distinct subsets of CAFs are not permanent but interconvertible via manipulation of specific signaling, as evidenced by conversion of iCAFs to myCAFs via the TGFβ signaling pathway, supporting the notion that CAF subpopulations have high potential for plasticity
• Antigen-presenting CAFs (apCAFs) suggesting an immunomodulatory function of CAFs.
• ApCAFs are considered to be immunosuppressive by inducing Treg cell formation in breast and pancreatic tumors
• Inter- or intratumoral heterogeneity has been more evident by single-cell sequencing and multiomics approaches.
• New spatially and functionally distinct CAF subpopulations have been increasingly identified in different cancer types, including vascular CAFs (vCAFs), cycling CAFs (cCAFs), and developmental CAFs (dCAFs)
• PIGF could be a key regulator of the CAF balance between quiescence and activation states in ICC.
• A stem-like 'universal' type of fibroblast cell, marked by expression of peptidase inhibitor 16 (Pi16) and Col15a, found in the steady state across tissues, serves as a reservoir to yield specialized fibroblasts where they undergo transition into highly activated fibroblasts

Mechanisms Regulating CAF Heterogeneity

• In general, CAF heterogeneity could arise from cancer cell-derived genetic evolution, epigenetic modulation or metabolic reprogramming.
• Tumor cells educate CAFs, as evidenced by the difference in signature genes expressed by fibroblasts cocultured with different tumor cells.
• Genetic Heterogeneity is manipulated by extrinsic or intrinsic factors by numerous tumor cell-derived growth factors
• Genetic Heterogeneity is manipulated by activating TGF-β, EGF, PDGF, FGF, interleukin 6 (IL-6), and interleukin 1β (IL-1β), skewing CAFs toward specific subsets via activation of key regulatory pathways
• Epigenetic modulation is seen CAFs that harbor been genetic aberrations have changes, as evidenced by genome-wide DNA methylation profiles of CAFs, which were found to be enriched at regulatory regions of the genome and key transcription factor-binding sites
• Metabolic reprogramming occurs through the "reverse Warburg effect" where researchers have found the tumor stroma relies on the "reverse Warburg effect" to feed adjacent cancer cells where aerobic glycolysis occurs in CAFs and generates energy-rich metabolites

Novel Therapeutic Strategies Against Tumor-Promoting CAFs

• Over the past decade, Direct depletion of CAFs through genetic manipulation, pharmacological targeting or specific antibodies, enhanced tumor growth and aggressiveness
• Targeted ablation of Tumor-Promoting CAFs relies on the identification of specific and convenient markers
• Specific CAF subpopulations has pointed to fine-tuning, which is based on their inherent plasticity seems to be more feasible and less challenging for therapeutic interventions

Inhibition of Progenitor Cell Differentiation

• Elucidation of the molecular mechanisms underlying CAF subset formation has made it possible to inhibit the transition from precursor cells toward tumor-promoting CAFs, such as IL-1 and TGF-β
• Epigenetic reprogramming provides dynamic and reversible modulation of stromal cells, targeting the epigenome via regulation of DNA methylation or histone modification has been investigated in preclinical models
• Targeting CAF activity: Researchers also elucidate the crosstalk between cancer cells and CAFs to inhibit the tumor-promoting activity of CAFs