Cancer-Associated Fibroblasts: Roles in Multidisciplinary Cancer Therapy and Targeted Strategies

1. Introduction

Cancer remains a major challenge in life science and clinical medicine. With the continuous development of oncology research, our understanding of tumor mechanisms has shifted from focusing solely on tumor cells to the microenvironmental regulatory system. Research confirms that tumors are dynamic biological systems with high complexity and heterogeneity, maintained not only by intrinsic tumor cell characteristics but also by interactions with the tumor microenvironment (TME).

The TME is a complex microecosystem composed of non-tumor stromal cells, extracellular matrix (ECM), and various soluble factors. Among TME components, cancer-associated fibroblasts (CAFs) are the most central cells. In most solid tumors such as breast cancer, CAF abundance can account for up to 80% of total tumor volume [1]. CAFs are characterized by high phenotypic heterogeneity and functional diversity, with their origins, biomarkers, and functions exhibiting significant tissue specificity [2]. During tumor progression, CAFs promote proliferation, invasion, and metastasis through secreting factors, activating pro-tumor pathways, and remodeling the ECM [3].

Beyond regulating tumor progression, CAFs play crucial roles in inhibiting tumor treatment. They mediate treatment resistance through multiple mechanisms: secreting cytokines and remodeling ECM to form dense barriers hindering drug penetration; regulating local tumor metabolism and inducing epigenetic changes to reduce treatment sensitivity; and expressing immune checkpoint ligands to suppress immune function [4]. As CAFs’ regulatory role in tumor treatment is progressively elucidated, therapeutic strategies targeting CAFs have become a research hotspot [5], offering new solutions for clinical tumor treatment [6]. This review comprehensively summarizes CAFs’ origins, heterogeneous classification, and core biological functions, explores their critical role in treatment resistance and clinical applications, and systematically categorizes targeting strategies, aiming to provide theoretical basis for multidisciplinary precision tumor management.

2. Cancer-Associated Fibroblasts

2.1. Background and Origin of Cancer-Associated Fibroblasts

Fibroblasts are terminally differentiated stromal cells originating from embryonic mesenchyme and are the most abundant cellular component in loose connective tissue. In a quiescent state, they typically appear elongated or fusiform with clear boundaries and moderate nucleus-to-cytoplasm ratio [7]. Their core functions include synthesizing and remodeling ECM and secreting bioactive molecules to regulate tissue homeostasis and immune responses [8]. However, abnormal persistent activation can lead to pathological transformation into CAFs.

Under normal tissue homeostasis, fibroblasts remain relatively quiescent [7]. In the 1970s, researchers first discovered special fibroblasts with wound-healing-like functions in the TME, subsequently defined as CAFs [9]. CAF origins are diverse, with the most direct sources being resident fibroblasts and mesenchymal stem cells (MSCs) [10,11]. Additional sources include smooth muscle cells, epithelial-derived cells, endothelial cells, and adipose-derived stem cells [10,11]. Some researchers speculate CAFs might also originate from activated hepatic stellate cells [12]. Furthermore, epithelial-mesenchymal transition (EMT) involving normal epithelial cells adjacent to tumor cells may also promote CAF formation [13]. Studies marking renal tubular epithelial cells found up to 30% of activated fibroblasts in renal fibrosis originated from EMT [14,15].

The complete molecular mechanisms underlying fibroblast transformation into CAFs remain unclear. However, in vitro studies confirm that transforming growth factor-β (TGF-β) can effectively induce normal fibroblasts to acquire CAF characteristics, and this factor mediates fibroblast activation during wound healing and organ fibrosis [7]. Besides TGF-β, tumor-derived exosomes can mediate CAF and MSC recruitment and activation. Concurrently, HIF-1 induced by hypoxia can promote CAF transformation through decreased prolyl hydroxylase (PHD) activity under hypoxic conditions, reducing HIF-1 degradation and leading to its accumulation [16].

2.2. Classification and Function of Cancer-Associated Fibroblasts

CAFs exhibit significant heterogeneity due to diverse cellular origins and transformation pathways [17,18]. Researchers have systematically analyzed CAF heterogeneous characteristics using single-cell RNA sequencing (scRNA-seq) and multiplex imaging techniques [19]. Based on functional specificity, main CAF subtype classifications include:

Subtype	CoreFunction	ImpactonTumor	KeyBiomarkers	MainTumorTypesPresent	References
iCAFs	Secrete a variety of related inflammatory cytokines; recruit immune cells to remodel the local inflammatory microenvironment.	Promote tumor growth, proliferation and immunosuppression; induce epithelial-mesenchymal transition (EMT) in tumor cells; accelerate tumor cell invasion and metastasis.	IL-6, IL-8, CXCL1, CXCL12	Pancreatic ductal adenocarcinoma, gastric cancer, colorectal cancer, lung adenocarcinoma	[10,19]
myCAFs	Secrete collagen, fibronectin and lysyl oxidase; promote extracellular matrix (ECM) remodeling.	Enhance tumor cell migration and invasion; mediate chemoresistance.	α-SMA, ANTXR1 ITGA11, SPARC	Breast cancer, pancreatic cancer, bladder cancer, colorectal cancer	[20,21]
dCAFS	Carry developmental or progenitor cell-like expression programs; express genes related to development and stemness.	Promote tumor cell proliferation, differentiation and matrix remodeling.	FOXL1, GREM1	Breast cancer, non-small cell lung cancer	[22,23]
vCAFs	Interact with endothelial cells; promote angiogenesis and vascular remodeling.	Regulate the formation of new blood vessels and hemodynamics in the tumor microenvironment (TME).	VEGFA, FOXL1	Non-small cell lung cancer (mainly lung squamous cell carcinoma)	[24,25]
apCAFs	Express MHC class II molecules; directly interact with T cells.	Induce tumor cell immune evasion and immunosuppression; inhibit tumor-related immune responses.	MHC class IImolecules, CD74	Peritoneal metastatic cancer, renal clear cell carcinoma, pancreatic cancer, colorectal cancer	[19,26]
ifnCAFs	Secrete interferon-related factors; regulate immune cell infiltration; modulate immune responses.	Regulate TME homeostasis; maintain tumor cell stemness; induce tumor cell immune evasion.	IFNB1, CXCL9	Non-small cell lung cancer (mainly lung adenocarcinoma)	[27]
rCAFs	Regulate immune responses; secrete chemokines to recruit specific immune cells; participate in immune regulation.	Modulate tumor cell proliferation and differentiation; mediate crosstalk with external signaling pathways.	PD-L1, High expression of CXCL12	Breast cancer	[28,29]
TGF-β-secreting CAFs	Secrete large amounts of TGF-β; induce epithelial-mesenchymal transition.	Enhance tumor cell invasiveness; participate in the construction of fibrotic stroma; mediate tumor chemoresistance.	TGF-β, FAP	Breast cancer, cervical cancer	[27,28]
Tumor-suppressive CAFs	Maintain ECM stability; secrete anti-tumor factors; promote immune drug infiltration.	Inhibit tumor cell growth.	Low expression of α-SMA	Breast cancer	[27]

1. Inflammatory CAFs (iCAFs): Characterized by high secretion of cytokines and chemokines (e.g., CXCL1, IL-6, IL-8), regulating inflammatory responses to promote tumor growth and immunosuppression [19]. Typically located at tumor periphery, they recruit immune cells and alter the local inflammatory environment [10,19]. They can induce EMT in tumor cells and secrete CXCL12 to form physicochemical barriers, helping tumor cells resist immunotherapy.

2. Myofibroblast CAFs (myCAFs): Characterized by high α-SMA expression and specific localization near tumor cells. Driven by TGF-β signaling, they secrete collagen, fibronectin, and lysyl oxidase to promote ECM remodeling and induce desmoplastic reactions. This increases tissue stiffness, forms physicochemical barriers supporting tumor invasion, impedes drug delivery, and influences mechanotransduction pathways promoting metastasis and immunosuppression [20,21].

3. Developmental CAFs (dCAFs): Closely related to embryonic development, carrying developmental or progenitor-like expression programs. After EMT, they express genes associated with development and stemness [22], playing crucial roles in tumor cell proliferation, differentiation, and stromal remodeling [23].

4. Vascular CAFs (vCAFs): Originating from perivascular stromal cells [24], participating in angiogenesis and vascular remodeling, interacting with remodeling epithelial cells to influence new blood vessel formation and blood flow dynamics within the TME [25].

5. Antigen-presenting CAFs (apCAFs): Possessing antigen-presenting potential, expressing MHC-II molecules that can directly interact with T cells [26]. They typically induce immunosuppression and inhibit immune responses by recruiting T cells [19].

6. Interferon-type CAFs (ifnCAFs): Capable of secreting interferon-like cytokines, participating in immune responses and regulation, maintaining tumor cell stemness and inducing immune evasion [27].

7. Regulatory CAFs (rCAFs): Can influence tumor cell behavior through profound immune suppression, including proliferation, differentiation, and external signaling pathway connections [28,29].

8. TGF-β-type CAFs: Primarily transformed via TGF-β signaling pathway. After activation, they secrete large amounts of TGF-β factors, activating EMT-related pathways and inducing ECM transformation, endowing tumor cells with enhanced proliferative and invasive capabilities. They also synthesize collagen and fibronectin, forming dense physicochemical barriers hindering drug penetration and leading to drug resistance [27,28].

9. Tumor-suppressive CAFs: Unlike most pro-cancer CAFs, their primary function is maintaining ECM balanced stability. They can inhibit tumor cell growth and migration by secreting anti-tumor factors and promote immune drug infiltration [27].

Despite significant functional heterogeneity, different CAF subtypes possess similar biological characteristics. Besides characteristic markers mentioned above, there are non-specific markers such as FAP and α-SMA [30,31]. CAFs possess powerful proliferative and migratory potential, allowing them to survive and maintain biological activity during large-scale tumor treatment interventions [32]malignant tumor cell behavior through multiple pathways, including accelerating proliferation, driving angiogenesis, promoting metastasis and invasion, and mediating treatment resistance [17]. Different CAF subtypes regulate the TME through specific molecular mechanisms, playing both pro-cancer and anti-cancer regulatory roles.

2.3. Cancer-Associated Fibroblasts and Organoids

In the process of tumor treatment, traditional two-dimensional tumor cell line cultures, although widely used in tumor molecular mechanism research and drug efficacy evaluation, have the problem that they cannot fully replicate the complex cell interaction network within the in vivo TME, making it difficult to restore the true in vivo tumor growth environment, and the treatment effects are usually suboptimal. Therefore, researchers have developed a co-culture system of CAFs and tumor organoids. This system, by precisely preserving the biological characteristics and tumor cell heterogeneity of the patient’s in vivo tumor, achieves in vitro biomimetic reconstruction of the TME and relevant cellular components, and has now become an emerging hot spot in the field of tumor mechanism research and drug screening [33,34].

2.3.1. Organoid Construction

Organoids refer to a class of three-dimensional cell clusters derived from human pluripotent stem cells or adult stem cells, capable of mimicking the cellular heterogeneity, structure, and function of human organs, and carrying genetic information [35]. The core steps for constructing CAF organoids are:Acquisition and isolation of tumor tissue. Typically, tumor tissue from the inner margin of the same patient’s tumor is extracted to ensure that all tumor components have identical genetic characteristics. The obtained tumor tissue is digested using relevant enzymes (e.g., trypsin, collagenase). The digested tumor tissue is then separated using methods such as density gradient centrifugation, magnetic bead sorting, and flow cytometry, referencing CAF-specific markers, to obtain CAFs and other tumor components [36]. Construction of the co-culture model. The isolated tumor components are cut into pieces approximately 1 mm in diameter, and a tissue suspension is obtained by filtering through a mesh. This tissue suspension serves as a biomimetic scaffold, with Matrigel matrix gel implanted underneath, and specific culture medium added for organoid culture [37]. Subsequently, the cultured CAFs and organoids are mixed to complete the construction of the co-culture system. Construction methods for mixing include: chamber systems, direct mixing of CAFs and organoids, and others. Among these, direct mixing of CAFs and organoids involves co-culturing the two cell types directly in a Matrigel matrix, achieving sufficient physical contact and contact-dependent signal transduction, which can realistically simulate the in vivo microenvironment and cell interaction network. A chamber system, on the other hand, separates CAFs and organoids using a semi-permeable membrane, allowing for paracrine signaling between them. Its core advantage lies in the ability to quantify specific factors and study relevant signaling pathways [38,39].Here is the translation:

Currently, there are multiple examples of CAF and organoid co-culture models being constructed. In colorectal cancer (CRC), researchers established a co-culture organoid model involving conventional organoids and CAFs. This team utilized a chamber system to construct the co-culture. The method involved culturing other tumor components in cell culture inserts with porous membranes, while CAFs were cultured on the carrier plate. This model allowed for free exchange of culture medium, but cells could not pass through. Gene expression profile analysis of both groups (co-culture and conventional organoids) and the original tumor tissue revealed that some cell proliferation and anti-apoptotic genes (such as the REG family and dual oxidase) showed decreased expression in conventional organoids, while CAF co-culture organoids could restore their expression levels. This indicates that the CAF co-culture system is more similar to the original tumor, demonstrating the ability of the CRC-CAF organoid co-culture system to replicate the TME [40]. In pancreatic ductal adenocarcinoma (PDAC), researchers directly mixed CAFs with PDAC organoids for construction. After culture, it was found that the CAF co-culture organoid system exhibited rapid proliferation, reduced chemotherapy-induced cell death, and simultaneously induced a pro-inflammatory phenotype and EMT, indicating a high similarity between the co-culture system and the TME. Researchers used the real-time imaging drug detection method DeathPro to evaluate the response of the CAF co-culture system to some first-line chemotherapeutic drugs, and explored its underlying mechanisms using methods such as scRNA-seq, ultimately elucidating tumor-stroma interactions at the single-cell level. This research provides feasibility for investigating the role of CAFs in vivo in patients and for tumor drug screening [39]. Notably, researchers established CAF-associated organoids in vitro from liver cancer patients, using a common method within chamber systems: Transwell co-culture. This strategy involved seeding CAFs in a Transwell insert, separated from other components but sharing the culture medium. After culture, comparison with conventional organoids and analysis of their gene expression characteristics demonstrated the pro-tumor and drug resistance effects of CAFs [41]. In summary, CAF-associated organoid systems facilitate the study of relevant components within the TME and the screening of anti-tumor drugs.

2.3.2. Clinical Applications of Organoids

CAFs-related organoids, by simulating the real TME, possess significant utility in clinical applications. In terms of drug screening, CAFs organoid co-culture models can highly recapitulate the TME, thereby more precisely simulating drug responses and improving the accuracy of drug screening. For example, in a study on CRC and CAFs organoids, researchers, by analyzing CAFs-related organoids, validated the inhibitory properties of CAFs against 5-fluorouracil, SN-38 (the active metabolite of irinotecan), oxaliplatin, and the EGFR inhibitor cetuximab [42]. In the field of clinical prediction, CAFs organoid systems can, by simulating anti-tumor treatments within the TME and studying changes in relevant cells and components, predict patient treatment responses. A clinical trial in advanced rectal cancer demonstrated that organoid simulation can achieve prediction of clinical radiotherapy and chemotherapy responses [43]. Furthermore, in gastrointestinal cancers (such as colorectal cancer, gastroesophageal cancer, etc.), researchers analyzed organoid models and real clinical data, finding that organoids had a positive predictive value of 88% and a negative predictive value as high as 100% for targeted therapy or chemotherapy [44]. In summary, by constructing CAFs organoid systems, relevant anti-tumor drugs can be screened, and simultaneously, based on drug screening and clinical prediction, personalized treatment plans can be provided to patients, aiding the progress of cancer treatment.

3. How Do Cancer-Associated Fibroblasts Affect Chemotherapy, Radiotherapy, Targeted Therapy, and Immunotherapy?

3.1. How Do Cancer-Associated Fibroblasts Affect Chemotherapy?

As a critical TME component, CAFs inhibit chemotherapeutic drug uptake by tumor cells through multiple pathways, leading to chemotherapy resistance.

3.1.1. CAFs Secrete Cytokines or Collagen

CAFs directly mediate drug resistance by secreting relevant cytokines. In melanoma, CXCL12 secreted by iCAFs promotes tumor progression and mediates gemcitabine resistance by upregulating SATB-1 expression [45]. In colorectal cancer (CRC), chemotherapy induces CAF secretion of IL-17A, enhancing cancer-initiating cells’ (CICs) self-renewal and invasive capabilities [46]. Multiple studies confirm significant positive correlation between thrombospondin 2 (THBS2) and chemotherapy resistance [47]. THBS2 originates from CAF subtypes and can interact with tumor cells via the collagen pathway in CRC, promoting oxaliplatin resistance [48]. Mechanistically, THBS2-CAFs secrete Collagen type VIII alpha 1 chain (COL8A1), which interacts with Integrin β1 receptors on drug-resistant tumor cells, activating PI3K-AKT signaling, promoting EMT, and ultimately leading to oxaliplatin resistance [49].

3.1.2. CAFs Remodel Tumor Cell Metabolism

CAFs mediate chemotherapy resistance by remodeling tumor cell metabolism, upregulating metabolic levels and enhancing resistance. In osteosarcoma and clinical samples, CAFs regulate tumor cell lipid oxidation pathways, prompting lactate secretion under chemotherapy stress, providing energy sources [10]. CAFs undergo aerobic glycolysis, producing large amounts of pyruvate and lactate. These metabolites, once taken up by tumor cells, participate in the tricarboxylic acid cycle and energy production, significantly increasing ATP synthesis [36]. Sufficient ATP provides necessary energy for drug efflux and DNA damage repair, ultimately reducing chemotherapeutic drug cytotoxicity [27].

3.1.3. CAFs Form Physicochemical Barriers

CAFs participate in ECM remodeling, forming dense physicochemical barriers impeding chemotherapeutic drug action. They construct fibrotic matrices providing natural physicochemical barriers, inhibiting drug penetration and leading to chemotherapy resistance [27]. MyCAFs, driven by TGF-β signaling, secrete collagen, fibronectin, and lysyl oxidase, causing tissue hardening and forming dense physical barriers obstructing effective drug penetration, inducing primary drug resistance [20]. CAFs stimulated by PDGF promote integrin-ECM interactions, causing tumor matrix contraction and increasing interstitial fluid pressure (IFP). Elevated IFP balanced with moderate microvascular pressure (MMP) significantly inhibits large molecule drug extravasation and uniform distribution, severely affecting chemotherapy progress [50].

3.1.4. Epigenetic Modification and Paracrine Signaling

CAFs activate DNA methyltransferases (DNMTs) and histone deacetylases (HDACs) within tumor cells by secreting factors like TGF-β, altering DNA methylation and histone acetylation, promoting tumor gene transcription, significantly reducing drug-induced DNA damage, and weakening chemotherapy efficacy [51]. Additionally, CAF-derived paracrine signaling pathways are crucial in mediating non-cell-autonomous chemotherapy resistance. Stromal cells including CAFs secrete cytokines, growth factors, and soluble ligands that transmit pro-survival signals to adjacent tumor cells via paracrine pathways, conferring drug resistance [52].

3.2. How Do Cancer-Associated Fibroblasts Affect Radiotherapy?

Radiotherapy kills tumor cells through high-energy ionizing radiation, primarily inducing DNA damage via direct and indirect actions. Direct action involves high-energy rays directly causing molecular ionization within tumor cells, damaging biomolecules like DNA. Indirect action involves ionizing radiation acting on water molecules, producing free radicals that attack DNA [53]. Increasing research confirms radiotherapy impacts not only tumor cells but also the entire TME, with CAFs playing crucial roles.

3.2.1. Core Changes and Overall Effects of Irradiated CAFs

During radiotherapy, CAF biological characteristics undergo significant changes after irradiation. These altered CAFs promote aggressive tumor growth and reduce radiosensitivity through various pathways. CAFs survive radiotherapy due to their unique radiation-resistant phenotype [54]. Importantly, irradiation induces CAF senescence and triggers specific secretory phenotypes [55]. Radiotherapy regulates CAF biological behavior and secretomes, causing them to secrete large quantities of pro-angiogenic factors, immunomodulatory factors, ECM regulatory factors, and inflammatory cytokines driven by persistent DNA damage signals [54]. These active factors directly interfere with radiotherapy, significantly weakening efficacy.

Irradiated CAFs promote epithelial malignancy growth and metastasis through tumor cell interactions. They secrete various soluble factors, upregulating c-Met phosphorylation and MAPK activity [56], enhancing primary cancer cell strain (PCC) invasive capacity [57]. They also promote tumor cell proliferation and invasion through senescence or bystander effects [58]. Irradiation induces DNA double-strand breaks in CAFs, causing permanent proliferative arrest and exhibiting typical senescence characteristics. Morphologically, cell volume significantly increases with flattened, irregular shapes. Functionally, they activate the Senescence-associated secretory phenotype (SASP). Senescent CAFs become highly active, secreting large amounts of inflammatory factors like IL-6 and IL-8, regulating surrounding tumor cell biological behavior, ultimately promoting tumor growth, invasion, and radioresistance [59].

3.2.2. Core Mechanisms of CAF-Mediated Radioresistance

Irradiated CAFs reduce tumor cell radiosensitivity through various pathways:

Post-radiation CAFs secrete soluble factors (e.g., HGF/c-Met), inhibiting tumor cell apoptosis and promoting proliferation, survival, and enhanced invasiveness. HGF binds to cMet receptors on tumor cell surfaces, activating downstream PI3K/Akt pathways, upregulating anti-apoptotic proteins like BCL-2 while inhibiting pro-apoptotic protein bim [60].

CAFs secrete TGF-β, enhancing tumor cell activity and significantly increasing radiotherapy resistance. Radiation-stimulated CAFs secrete large amounts of TGF-β, promoting EMT, making tumor cells more migratory and enhancing DNA repair capabilities, exhibiting radiation tolerance [20].

CAFs increase β1 integrin expression, enhancing tumor cell survival after radiotherapy. β1 integrin mediates cell adhesion to matrix components like fibronectin. Post-radiation CAFs highly express β1 integrin, promoting cell adhesion, activating downstream FAK/Src pathways, forming anti-apoptotic feedback loops, weakening radiotherapy effects [27].

CAFs interact with cancer cells, secreting growth factors and reducing radiation-responsive gene expression, evading radiation-induced cell death. Post-radiation CAFs release natural EGF ligands, activating tyrosine kinases within tumor cells and interfering with DNA damage pathways. This global gene expression remodeling enables rapid radiotherapy adaptation, reducing radiosensitivity [27].

CAF-secreted exosomes interact with tumor cells, regulating tumor suppressor gene expression and lifting downstream signaling pathway inhibition, enhancing radiotherapy resistance. Exosomes also deliver antioxidant enzymes, reducing tumor cell damage and leading to radiation therapy resistance [55].

3.3. How Do Cancer-Associated Fibroblasts Affect Targeted Therapy?

Targeted therapy achieves precise tumor cell elimination by identifying and attacking specific cellular molecules, becoming a modern precision cancer therapy hotspot. During targeted therapy, CAFs significantly interfere, mainly disrupting targeted drug delivery and limiting tumor cell impact through cytokine secretion, ECM remodeling, and paracrine signaling pathways [27].

3.3.1. CAFs Form Physicochemical Barriers

CAFs participate in constructing physicochemical barriers on tumor cell surfaces, limiting targeted drug penetration and effect. TGF-β secreted by CAFs contributes to fibrotic matrix formation, leading to highly fibrotic tumor tissue creating dense physicochemical barriers [61] restricting targeted drug penetration and diffusion [28,62]. CAFs promote ECM remodeling, including matrix deposition and degradation. They promote ECM synthesis by secreting deposited matrix molecules like fibronectin and collagen [61], increasing TME stiffness. Increased integrin expression on CAFs induces existing collagen and fibronectin fiber structure remodeling, further exacerbating ECM stiffness [62]. This altered tissue microstructure leads to increased physical stress-induced vascular compression and elevated interstitial fluid pressure, hindering targeted drugs from reaching tumor sites. Increased mechanical tension within the matrix can induce EMT in tumor cells via mechanosensitive YAP/TAZ and/or TWIST1 signaling axes, enhancing tumor cell invasiveness, dissemination, and therapeutic resistance [62,63].

3.3.2. CAFs Regulate Signaling Pathways

CAFs activate oncogenic pathways inhibited by targeted therapy through paracrine signaling, reducing targeted treatment efficacy. In NSCLC treatment, TKIs like gefitinib or erlotinib target and inhibit EGFR [64]. CAFs not only promote DNA repair in NSCLC cells but also induce tumor cells to arrest in treatment-resistant S-phase. CAFs upregulate and stabilize cMyc expression, activate Wnt/β-catenin signaling, mediating transcriptional activation of key glycolytic enzyme HK2, mitigating DNA damage impact on NSCLC cells [65]. When targeted drugs inhibit EGFR pathway, it leads to abnormal CAF-mediated Wnt/β-catenin pathway activation, maintaining tumor cell survival and ultimately inhibiting targeted therapy efficacy [66]. CAFs can promote tumor cell EMT by activating PDGF-C signaling, mediating tumor cell escape from VEGF treatment [11].

3.3.3. CAFs Alter Metabolic Patterns

CAF metabolic products and EMT can render tumor cells resistant to targeted therapy. CAFs have close metabolic relationships with tumor cells within the TME. Under these conditions, CAFs produce large amounts of high-energy metabolites like lactate and pyruvate through aerobic glycolysis [67].These metabolites can be efficiently utilized by tumor cells, enabling survival under targeted drug pressure [20]. CAFs promote tumor cell dissemination and invasion by inducing EMT, allowing tumor cells to evade targeted therapy [68]. CAFs induce EMT by secreting cytokines like TGF-β and HGF, leading to tumor cell transformation into mesenchymal phenotype. Tumor cells with this phenotype can survive targeted therapy, significantly weakening effectiveness [64,68].

3.4. How Do Cancer-Associated Fibroblasts Affect Immunotherapy?

Immunotherapy has evolved from cutting-edge exploration into a cancer treatment pillar [69]. Its core function is employing appropriate technical means to induce and activate patients’ own immune systems to precisely identify and eliminate tumor cells [70]. During immunotherapy, CAFs primarily hinder treatment by directly mediating T-cell functional suppression and recruiting suppressive cells.

3.4.1. CAFs Affect T-cell Function and Remodel ECM

CAFs secrete large amounts of inhibitory immune checkpoint molecule ligands (e.g., PD-L1, PD-L2) [70], which bind to programmed death receptors (e.g., PD-1, PD-2) on T cell surfaces, transmitting immunosuppressive signals, ultimately leading to immunosuppression [71]. CAFs influence the immune system by secreting inhibitory cytokines (e.g., IL-6, IL-10, TGF-β), suppressing T-cell function and causing immunosuppression [72]. CAFs remodel the ECM, participating in physicochemical barrier construction, causing ECM tissue fibrosis and stiffening, ultimately impeding immune cell infiltration [71,72], leading to immunosuppression.

3.4.2. CAFs Recruit Suppressive Immune Cells

CAFs actively recruit immunosuppressive cells and promote their proliferation and activation by secreting specific chemokines. Chemokines like CXCL12 secreted by CAFs efficiently recruit regulatory T cells (Tregs) to the TME [72]. Tregs suppress immune responses by consuming T-cell growth factors (IL-2) and secreting inhibitory cytokines, interfering with immunotherapy efficacy [72]. CXCL12 can bind to CXCR4 receptors on T cells, causing CXCR4-expressing T cells to be retained in stromal regions, preventing migration within the TME and exerting anti-tumor functions, ultimately decreasing immunotherapy effectiveness [71,73]. CAFs can alter glutamine, lactate, and other substance metabolism within the TME, affecting its internal metabolic state and limiting effective T-cell infiltration, thereby reducing immunotherapy efficacy [66].

3.4.3. Specific Effects of Specialized CAFs

Given CAF high heterogeneity, specialized CAFs exhibit significant specificity in mediating immunotherapy regulation. For instance, apCAFs express MHC-II molecules, directly interacting with T cells to induce immunosuppression and recruit T cells, suppressing immune responses [27]; ifnCAFs secrete interferon-like cytokines, enhancing antigen presentation while inhibiting immune cell infiltration [74]; iCAFs regulate TME inflammatory responses by secreting inflammation-related cytokines (IL-6, IL-8), promoting immune suppression, recruiting immune cells, inducing EMT, and promoting invasion and metastasis. Additionally, iCAFs form physicochemical and chemotactic barriers by secreting CXCL12, resisting immune responses; myCAFs, driven by TGF-β signaling, secrete collagen, fibronectin, and lysyl oxidase, promoting ECM remodeling, increasing tissue stiffness, forming physicochemical barriers supporting tumor invasion and hindering drug delivery. This remodeling participates in regulating tumor cell mechanotransduction, ultimately promoting metastasis and immunosuppression [20,21].

4. Role of Cancer-Associated Fibroblasts in Tumor Diagnosis, Prognosis, and Prediction of Antitumor Treatment Response

4.1. Role of Cancer-Associated Fibroblasts in Tumor Diagnosis

With scRNA-seq and multiplex imaging technology development, numerous studies reveal CAFs are highly heterogeneous cell populations, with different subtypes exhibiting distinct functions. The relative abundance and spatial distribution of various CAF types collectively constitute TME biological characteristics [30,75]. In cancer treatment, CAFs often suppress antitumor therapy efficacy and promote tumor growth and migration. CAF abundance typically correlates with tumor cells [76]. Therefore, CAFs demonstrate significant diagnostic potential in precise tumor diagnosis and treatment. In benign versus malignant tumor differential diagnosis, identifying CAF subtypes can achieve more precise diagnostic determination compared to traditional morphological assessment [77]. By detecting CAF abundance, tumor current biological behavior can be accurately predicted [78]. CAF application in tumor diagnosis can be categorized into qualitative and quantitative diagnosis.

4.1.1. Qualitative Diagnosis

The core of CAF qualitative diagnosis lies in precise subtype identification. Current CAF subtyping research primarily distinguishes and defines them based on functional characteristics. The fundamental theoretical basis for achieving CAF subtype identification is their inherent high heterogeneity, reflected in different CAF subtypes possessing specific molecular markers and exhibiting significant differences at DNA, RNA expression profile, and specific antibody recognition levels.

Traditional qualitative diagnostic methods include:

1. Histopathological observation and TSR analysis: HE staining reveals CAFs exhibit typical spindle or stellate morphology with elongated oval or rod-shaped nuclei and loosely packed chromatin [79]. TSR is closely associated with specific CAF subtype spatial distribution. In pancreatic cancer, iCAFs are mainly enriched in dense stromal regions with low TSR, not directly contacting tumor cells; while myCAFs, in close proximity to cancer cells, are primarily distributed in high TSR regions with less stromal content [80].

2. Immunohistochemistry (IHC) and Immunofluorescence (IF): Using single antibodies to target specific CAF subtype biomarkers for staining, allowing CAF abundance and distribution identification and quantitative analysis [81]. Multiplex staining techniques simultaneously label multiple antibodies on the same section, achieving more precise CAF subtyping. In breast cancer, researchers successfully distinguished four CAF subtypes by developing multiplex staining protocols including six biomarkers: CD29, FAP, α-SMA, PDGFRβ, FSP1, and CAV1[82]

3. Western blot (WB): Used to detect CAF surface markers [83].However, due to CAF high heterogeneity, no single biomarker can define all CAFs, and traditional qualitative diagnosis has limitations. CAF subtypes may undergo dynamic conversion under different signaling stimuli [84]. With technological advancements, more cutting-edge and precise identification methods have emerged:

4. Single-cell RNA sequencing (scRNA-seq): Comprehensively identifies CAF subtypes and demonstrates conversion processes by characterizing gene transcription and expression at single-cell level with high resolution [85]. Specific identification involves analyzing characteristic expression features of genes in different CAF subtypes and reconstructing CAF state transition processes through time-series analysis [84].

5. Spatial transcriptomics (ST): Captures in situ mRNA from CAF-related tissue sections, preserving tissue structure while obtaining high-throughput gene expression data. By combining gene expression profiles with spatial tissue locations, it yields spatial distribution data for different CAF subpopulations [86]. This technology retains gene expression profile and cell spatial distribution information, directly reflecting cell-cell interactions, overcoming single-cell sequencing’s limitation in determining cell location [87].

6. FAP-targeted PET/CT imaging: Based on FAP being highly expressed on CAF surfaces [87], researchers developed FAP-targeted radioactive tracers combined with PET/CT technology. The procedure involves intravenous injection of FAP radioactive tracers (e.g., 68Ga-FAPI), which specifically bind to CAF surface FAP, followed by PET/CT scanning to obtain specific CAF characteristics [88]. This technology enables non-invasive, whole-body dynamic and precise CAF visualization, providing excellent imaging guidance for CAF-related cancer treatments.

4.1.2. Quantitative Diagnosis

Quantitative diagnosis core is determining substance expression levels, thereby assessing relative CAF content. This assists in clinically distinguishing benign versus malignant tumors, evaluating invasive potential, and predicting prognosis and treatment response. Main strategies include:

1. Flow Cytometry: Achieves precise CAF annotation and quantitative analysis through multi-parameter, quantitative analysis of single-cell suspensions from dissociated tumor tissue [89], clarifying various CAF subtype proportions and distribution characteristics.

2. Liquid Biopsy: Non-invasive circulating technology providing tumor diagnosis insights by analyzing tumor components in body fluids like blood [90]. Main detection targets include circulating CAFs (cCAFs) and exosomes. Under specific pathological conditions, CAFs can detach from TME and enter peripheral blood circulation, forming cCAFs. Precise cCAF identification in blood can be achieved through specific biomarker combinations [91]. A breast cancer study showed combined detection of cCAFs and CTCs significantly improved ability to distinguish cancer patients from healthy individuals [92]. Circulating substances like CAF-secreted exosomes are rich in proteins, DNA, and RNA. Systematic analysis of these components can intuitively reflect CAF functional state, making them potential liquid biopsy biomarkers [93].

3. Magnetic Resonance Imaging (MRI): Conventional MRI cannot directly identify CAFs, but they can be indirectly assessed through specific imaging sequences, with T2-weighted imaging (T2WI) most commonly used. T2WI signals primarily depend on tissue water content. In CAF-enriched regions, ECM is dense with physicochemical barriers, leading to reduced local water content, often appearing as low signals [94]. Additionally, dynamic contrast-enhanced MRI (DCE-MRI) can be employed. Since CAFs promote tumor angiogenesis and contribute to dense ECM formation, they significantly affect contrast agent penetration after intravenous injection. DCE-MRI indirectly reveals CAF characteristics through quantitative perfusion parameters and dynamic enhancement features, enabling non-invasive assessment of CAF distribution, abundance, and activity [95].

4. Quantitative Real-time PCR (qRT-PCR): Used to quantify specific gene expression levels. The principle involves using total RNA or mRNA extracted from CAFs as template to synthesize cDNA with reverse transcriptase, then using cDNA as template for real-time fluorescent quantitative PCR amplification, during which fluorescent dyes or probes detect amplified product amounts [96]. This method can measure and compare changes in specific CAF-related marker gene expression products (e.g., FAP, IL-6, CXCL12) [97].

4.1.3. Clinical Significance

Qualitative and quantitative assessment of CAF status and abundance assists clinicians in distinguishing benign versus malignant tumors, evaluating invasive potential, and predicting prognosis and treatment response. FAP is not expressed in benign ovarian epithelial tumors but is stably expressed in borderline and malignant tumors [98]. Breast cancer research confirms α-SMA expression level in ECM is positively correlated with tumor malignancy [99]. CAF density in tumor tissue is significantly positively correlated with tumor invasion depth and lymph node metastasis risk [100]. A CRC study revealed patients with high CAF burden had significantly shorter disease-free survival (DFS), suggesting high CAF levels are independent markers of poor tumor prognosis [101]. Integrating CAF qualitative and quantitative diagnosis with existing clinical diagnostic and treatment systems is expected to further enhance predictive accuracy of tumor prognosis and treatment response.

4.2. Prognostic Impact of Cancer-Associated Fibroblasts on Tumors

Tumor prognosis refers to scientific prediction of disease progression trajectory, clinical outcomes, and patient physiological state after tumor onset [102]. Tumor prognosis assessment is a complex, multi-factorial regulatory process, primarily influenced by patient characteristics, tumor type, treatment regimen, and microenvironment [103]. With scRNA-seq and other new technologies, CAFs’ important role in tumor progression has been increasingly elucidated, demonstrating profound impact on tumor prognosis, with the core basis being CAF high heterogeneity [104]. Ongoing research confirms CAF prognostic value exhibits clear cancer-type and subtype specificity, with significant differences in prognostic regulatory effects of various CAF subtypes across different cancer types [105].

Representative examples include:

Pancreatic Ductal Adenocarcinoma (PDAC): iCAFs and myCAFs are primary CAF subtypes [106]. When iCAF proportion is high, PDAC patient prognosis is relatively better; conversely, when myCAF proportion increases, patient prognosis is worse [106]. apCAFs possess unique immunomodulatory phenotype, expressing MHC-II molecules and acquiring antigen-presenting functions [107]. Additionally, PDAC harbors pCAFs driving perineural invasion (PNI). pCAFs are characterized by high glycolytic activity, secreting large amounts of lactate, promoting histone H3K18 lactylation modification and inducing epigenetic modifications activating nerve invasion-related genes (e.g., L1CAM and SLIT1), ultimately driving PNI and accelerating tumor invasion and progression [108]. PNI has been confirmed as critical adverse prognostic factor for PDAC patients. Statistical analysis based on recurrence risk showed PNI-positive patients had significantly higher tumor recurrence risk than PNI-negative patients (HR=2.714, 95% CI 1.885-3.906, P=0.000) [109].

Breast Cancer: myCAFs are core subpopulation mediating ECM remodeling and closely related to TME immune regulation. MyCAFs participate in constructing dense tumor stroma during ECM remodeling, forming physicochemical barriers. Multiple studies confirm stromal desmoplasia degree in breast cancer is significantly associated with poor patient prognosis [110]. Clinical analysis on TSR prognostic value showed patients with high stromal content (low TSR) had OS-HR=1.28 and DFS-HR=1.75 compared to patients with low stromal content (high TSR) [111]. Stroma-rich tumors are often accompanied by significant CAF density increase, suggesting worse clinical prognosis [112].

Colorectal Cancer: myCAFs, iCAFs, and apCAFs are common CAF subpopulations [113]. High CAF infiltration levels in CRC tissue are closely associated with poor patient prognosis [114]. A cohort study showed high-CAF group patients were generally associated with poorer prognosis (HR=3.63, 95% CI 2.24-5.88, P＜0.001) [115]. Beyond infiltration abundance, CAF spatial distribution characteristics are significantly linked to cancer patient prognosis. Specifically, co-localization status of different CAF subtypes with immunosuppressive cells is highly correlated with prognostic outcome after ICB therapy [115]. When CAFs and tumor cells are highly co-localized spatially, the former can exert pro-tumor effects, significantly promoting tumor cell proliferation and migration, leading to worse clinical prognosis [116].

4.3. The Role of CAFs in Predicting Anti-tumor Treatment Response

CAFs play important roles in predicting anti-tumor treatment. The core principle lies in CAF high heterogeneity, diverse biomarkers, and multifaceted functional properties across therapeutic modalities [117]. As CAF heterogeneity research deepens, this characteristic has become important basis for predicting anti-tumor treatment efficacy [118].

Predicting Chemotherapy Response: In breast cancer, α-SMA+CAF subpopulation density is significantly associated with reduced objective response rate and shorter PFS [119]. In PDAC, a Phase IIa clinical study demonstrated treating metastatic PDAC with CAF inhibitor Motixafortide combined with pembrolizumab and chemotherapy achieved 32% objective response rate and 77% disease control rate, with median response duration of 7.8 months [120]. By analyzing CAF action mechanisms on chemotherapy combined with diagnostic methods for CAF abundance, patient CAF expression levels can be detected using IHC and flow cytometry, thereby predicting chemotherapy efficacy.

Predicting Radiotherapy Efficacy: CAF intrinsic spatial heterogeneity and spatial distribution characteristics within TME are core biological elements determining and supporting precise radiotherapy efficacy prediction. Radiotherapy can induce CAFs to activate SASP phenotype, secrete large amounts of pro-tumorigenic factors like TGF-β, and mediate ECM remodeling, collectively enhancing tumor cell radioresistance [121]. Spatial Transcriptomics (ST) technology can analyze CAF spatial localization and molecular expression profiles, ultimately effectively reflecting heterogeneous functional characteristics and achieving precise radiotherapy efficacy prediction [122]. In head and neck cancer, primary tumor lesions exhibit high FAPI affinity, with mean SUV of 7.41±2.39; healthy head and neck tissues show only low background uptake, with mean SUV of 1.23±0.28. This difference can directly affect precise radiotherapy target area delineation, thereby impacting treatment efficacy [123]. Specific CAF-related genes (e.g., MMP9, FMOD) can serve as potential biomarkers for predicting radiotherapy respons [124]. Special interferon-type CAFs (ilCAFs) characterized by high IRF1 expression possess significant positive predictive value for tumor radiotherapy response. High IRF1 expression was significantly associated with improved OS in TCGA dataset, validated across various cancer types [125].

Predicting Targeted Therapy: vCAFs can secrete multiple alternative pro-angiogenic factors and mediate ECM remodeling, leading to tumor cell resistance to anti-angiogenic treatment [126]. Specific biomarkers can identify vCAF subpopulations and quantify infiltration abundance, thereby predicting patient response to anti-angiogenic targeted therapy [127]. In MCRC patients receiving bevacizumab, high IL-8 expression group showed significantly worse PFS and OS compared to low expression group; median PFS was 6.5 months and 12.6 months, respectively (HR=7.39, CI 2.76-19.83 P<0.0001), and median OS was 8.7 months and 28.8 months, respectively (HR=7.68, CI 2.59-22.77 P<0.001) [128]. IL-8 expression levels have potential to serve as prognostic biological marker for targeted therapy.

Predicting Immunotherapy Response: scRNA-seq technology can analyze CAF gene expression characteristics, thereby evaluating treatment sensitivity to immune checkpoint inhibitors and achieving precise immunotherapy efficacy prediction. Single-cell genomic data analysis revealed CRC prognosis and treatment response are closely related to different CAF subtypes. Cox proportional hazards analysis based on CAF marker genes showed low-CAF group individuals had better OS than high-risk group (HR=3.272, 95% CI 2.008-5.332, P<0.001), suggesting CAF gene risk scores have good CRC predictive value [129]. In advanced NSCLC patients, high FAP expression was significantly associated with reduced PD-1 monoclonal antibody immunotherapy efficacy, with data showing high FAP expression patients had lower objective response rate (16.4% vs 38.7%, P＜0.001) and worse DFS (HR=2.56, 95% CI 1.69-3.87, P＜0.001) [130]. By detecting CAF-specific biomarker expression levels, reliable evidence can be provided for precise immunotherapy response prediction.

5. Strategies for Targeting Cancer-Associated Fibroblasts to Sensitize Chemotherapy, Radiotherapy, Targeted Therapy, and Immunotherapy

CAFs are highly heterogeneous cell populations within the TME. They promote tumor cell growth and migration by secreting cytokines, promoting tumor angiogenesis, and remodeling ECM [131]. Due to CAF multifaceted functions, traditional cancer treatment strategies are often inhibited by them, leading to suboptimal treatment outcomes. However, as CAF intrinsic properties are continuously unveiled, researchers have designed strategies targeting CAFs to sensitize tumor treatment, aiming to enhance radiotherapy, chemotherapy, targeted therapy, and immunotherapy efficacy.

StrategyType	MainTargets	CombinationTherapyTypes	SensitizationMechanisms(forchemotherapy, radiotherapy, targetedtherapy, immunotherapy)	References
Direct targeting of CAFs	FAP	FAP inhibitors, FAP-CAR-T cells, FAP-ADC	1. Chemotherapy sensitization: Clear FAP+CAFs via targeted therapy, attenuate the pro-tumor effects of CAFs, and reverse chemoresistance.2. Radiotherapy sensitization: Precisely target and eliminate CAFs via FAPI-radiopharmaceuticals, inhibit SASP production, and interfere with pro-tumor signaling to enhance radiosensitivity.3. Targeted therapy sensitization: Break the physical and chemical barriers constructed by CAFs via FAP targeting, and promote the delivery of targeted drugs.4. Immunotherapy sensitization: Relieve the inhibitory effect of CAFs on T cells after FAP targeting, and induce specific anti-FAP immune responses via cancer vaccines.	[132,133,134,135]
Targeting CAF signaling pathways	TGF-β	Small-molecule TβRI inhibitors	1. Radiotherapy sensitization: Block TGF-尾 signal transduction, inhibit CAF activation and SASP induced by radiotherapy, and interrupt their DNA repair pathways in tumor cells.2. Targeted therapy sensitization: Inhibit CAF activation and ECM remodeling after pathway blockade, and improve the delivery of targeted drugs.3. Immunotherapy sensitization: Block TGF-β-mediated T cell exhaustion and Treg cell recruitment, restore the anti-tumor function of T cells, and enhance the therapeutic efficacy of immune checkpoint inhibitors.	[136,137,138]
	CXCR4/CXCL12 axis	CXCR4 antagonists	1. Radiotherapy sensitization: Block CXCR4 axis signal transduction, inhibit the interaction between CAFs and tumor cells, and maintain the radiosensitivity of tumor cells.2. Immunotherapy sensitization: Block the recruitment of immunosuppressive cells, and enhance the anti-tumor effect of T cells.	[139]
	IL-6	IL-6 neutralizing antibodies	1. Radiotherapy sensitization: Inhibit radiotherapy-induced SASP, and reduce the pro-tumor effects of inflammatory factors such as IL-6.2. Immunotherapy sensitization: Inhibit macrophage polarization and the recruitment of immunosuppressive cells.	[140]
CAF reprogramming	CAFs	Vitamin D analogs, Rho kinase inhibitors	Chemotherapy, radiotherapy, targeted therapy, immunotherapy: Redirect related drugs to CAFs, suppress the pro-tumor phenotype of CAFs, convert them into a quiescent state, and inhibit the pro-tumor effects of CAFs.	[141,142]
	CAF metabolic transporters (MCT4)	Metformin, SCD1	1. Chemotherapy and targeted therapy sensitization: Target the abnormal glycolysis and lipid metabolism of CAFs, inhibit their secretory and pro-tumor functions, and reverse tumor cell drug resistance.2. Immunotherapy sensitization: Alter the metabolic phenotype of CAFs, and inhibit lactate-driven T cell dysfunction.	[143]
Targeting ECM	Hyaluronic acid (HA)	Hyaluronidase	1. Chemotherapy and targeted therapy sensitization: Degrade ECM, destroy its dense physical and chemical barrier, reduce tumor interstitial pressure, and thereby promote drug penetration.2. Immunotherapy sensitization: Target ECM, relieve the physical exclusion of T cells by ECM effects, and promote T cell infiltration.	[144]
	Lysyl oxidase-like 2 (LOXL2)	LOXL2 antibodies		[145]

As evident from the table above, strategies targeting CAFs are complex and diverse. Current targeted intervention strategies have gradually moved from basic research to clinical trials. Researchers have conducted multiple clinical trials, primarily focusing on core mechanisms such as targeted elimination of CAFs and blocking CAF signaling pathways, to verify the feasibility of these strategies. Some current clinical studies on CAF-targeted therapies are presented in Table 3.

CancerType	Mechanism	CombinationTherapyRegimen	ClinicalTrials.govIdentifier	ClinicalTrials.govIdentifier	StartDate	Enrollment
Advanced esophageal cancer, head and neck cancer, cervical cancer	Target FAP-overexpressing cells using interleukin-2 variant (IL-2v), combined with anti-PD-L1 drugs for intravenous administration. Specifically eliminates FAP-high CAFs and enhances anti-tumor immune responses.	Simlukafusp Alfa in combination with Atezolizumab (MPDL3280A, an engineered anti-PD-L1 antibody)	NCT03386721	Discontinued	20180219	Actual: 256 patients
Solid tumors, breast cancer, head and neck cancer	Activate immunity via FAP-IL2v targeting. First validate safety with monotherapy, then combine with Trastuzumab or Cetuximab for synergistic tumor cell killing.	RO6874281 monotherapy; RO6874281 + Trastuzumab; RO6874281 + Cetuximab	NCT02627274	Completed enrollment	20151207	Actual: 134 patients
Breast cancer, bladder cancer, non-small cell lung cancer, pancreatic cancer	Simultaneously target CAFs and Nectin-4 using CAR-T cells. Co-express IL-7 and CCL19 or IL-12 to enhance CAR-T cell survival and targeting efficiency, thereby reversing immunosuppression.	Fourth-generation Nectin4/FAP-targeted CAR-T cells (expressing IL-7 and CCL19 or IL-12)	NCT03932565	Enrolling	20190213	Planned: 30 patients
Pancreatic ductal adenocarcinoma	Kill CAFs via OMTX705, an FAP-targeted antibody-drug conjugate (ADC). Combine with Pembrolizumab and Tislelizumab (BGB-A317) to improve drug penetration and relieve immunosuppression.	OMTX705 monotherapy; Pembrolizumab + OMTX705; Tislelizumab (BGB-A317) + OMTX705	NCT05547321	Enrolling	20221020	Planned: 150 patients
Non-small cell lung cancer, hepatocellular carcinoma	Combine Galunisertib with Nivolumab to block the TGF-尾 signaling pathway, reverse TGF-尾-mediated immunosuppression. Restore T cell activity in combination with anti-PD-1 monoclonal antibody to ultimately sensitize immunotherapy.	Galunisertib in combination with Nivolumab	NCT02423343	Completed enrollment	20150101	Actual: 41 patients

5.1. Sensitizing Chemotherapy by Targeting CAFs

During chemotherapy, CAFs induce chemoresistance through various mechanisms, obstructing the chemotherapy process. CAF-targeted therapy has emerged as highly promising strategy, significantly inhibiting CAF protective role on tumor cells through multiple pathways, thereby enhancing tumor sensitivity to chemotherapy. The core prerequisite for targeting CAFs is selecting appropriate targets, with CAF biomarkers being most ideal. This is because biomarkers are expressed at extremely low levels in other normal tissues and cells, reducing damage to other cells during targeting and improving targeted therapy precision. Current CAF targets include FAP, α-SMA, PDGFRβ [132].

FAP-targeted strategies include vaccines and immunotherapy, small molecule inhibitors, and CAR-T cell therapy [133]. FAP-CAR-T therapy has been designed and utilized in various solid tumors (e.g., lung cancer, pancreatic cancer) [134]. Current clinical trials indicate treatment leads to sharp reduction in FAP+CAFs in tumor stroma and degradation of dense collagen network in ECM, thereby enabling stromal remodeling. This remodeling improves chemotherapeutic drug distribution and quantity within tumor tissue, consequently sensitizing chemotherapy [133,134]. FAP can be used as target to deliver therapeutic agents directly to tumor stroma. FAP-targeted antibody-drug conjugates (ADCs) like OMTX705, upon reaching tumor stroma, clear CAFs, diminish CAF influence on tumor cells, and enhance chemical drug penetration. ADC and chemotherapy combination significantly improves chemotherapy efficacy [135]. FAP-targeted therapy can make FAP-enriched tumor cells more sensitive to chemotherapeutic drugs and induce tumor shrinkage [146]. FGFR inhibitors can suppress related FGFR signaling pathways, downregulate MARK/ERK signaling pathway in CAFs, thereby inhibiting CAF proliferation and activation, breaking physicochemical barriers, promoting drug infiltration, and ultimately enhancing chemo-sensitivity [147].

ECM remodeling and CAF phenotypic reprogramming can also sensitize chemotherapy. The primary mechanism involves targeting ECM to inhibit CAF activation and ECM regulation ability, thereby remodeling ECM and sensitizing chemotherapy. Specific strategies include using collagenase or hyaluronidase to degrade ECM, reducing interstitial pressure, improving drug delivery, and sensitizing chemotherapy [144]. Targeting CAF metabolic reprogramming, glycolysis inhibitors or related protein inhibitors (e.g., metformin, SCD1) can target CAF metabolic-related transporters (MCT4), inhibiting CAF-driven T cell functional suppression via lactate, alleviating TME, and sensitizing chemotherapy [143]. Vitamin D analogs, Rho protein kinase inhibitors, and other drugs can convert CAFs into quiescent state, inhibiting CAF-tumor cell interaction. By targeting CAFs with these drugs, promoting conversion to quiescent state, and inhibiting tumor cell chemoresistance, chemotherapy can be sensitized [142].

5.2. Sensitizing Radiotherapy by Targeting CAFs

During radiotherapy, CAFs can mediate radioresistance through various pathways. Irradiation induces CAFs to transform into SASP, secreting large amounts of pro-angiogenic factors, immune regulatory factors, ECM regulatory factors, and inflammatory cytokines driven by persistent DNA damage signals. These factors promote tumor cell survival and migration and interfere with DNA damage repair pathways, ultimately reducing radiotherapy efficacy.

Strategies include eliminating CAFs and their secreted cytokines, blocking signal transduction between CAFs and tumor cells. Specific recognition and clearance of CAFs can be achieved by directly targeting characteristic CAF biomarkers (primarily FAP). Related strategies include CAR-T cell therapy, ADCs, and small molecule drugs targeting FAP [134,135]. Researchers developed FAP-targeted radioligand therapy (FAP-RLT). The principle is linking small molecule inhibitors or peptides targeting FAP with radionuclides (e.g., 177-Lu) to form radioactive targeted drugs. These drugs accumulate in FAP-highly expressed tumor stroma, and in conjunction with radiation, precisely eliminate CAFs [148]. Several FAP-targeted radiopharmaceuticals including FAPI-46 and 3BP-3940 have entered early clinical trials [149].

Beyond directly eliminating CAFs, their major signaling pathways can also be targeted. The aim is blocking CAF-tumor cell interaction, inhibiting CAF-mediated radioresistance, and sensitizing radiotherapy. Targeting TGF-β signaling pathway: TGF-β receptor inhibitors have been identified in numerous clinical studies [136]. By employing small molecule TβRI inhibitors to competitively bind to relevant sites, signal transmission is severely hindered and entire pathway activation is suppressed [137]. Using inhibitors to target TGF-β pathway in conjunction with radiotherapy inhibits TGF-β signaling pathway, reduces CAF influence on tumor cells, thereby enhancing tumor cell radiosensitivity and ultimately improving radiotherapy efficacy [138]. For other relevant signaling pathways like HGF/cMet and IGF/IGF-1R signaling pathways, and CXCL12/CXCR4 chemokine axis, corresponding antibodies and inhibitory drugs can also be used. By targeting factors like CTGF, HGF, and CXCR4, their interaction with tumor cells can be blocked, ultimately maintaining tumor cell radiosensitivity [139].

For SASP and related cytokines produced by CAFs upon radiotherapy induction, targeted intervention can inhibit their generation, weakening SASP pro-tumor effect on tumor cells, thereby increasing radiosensitivity. For key inflammatory factors secreted by CAFs like IL-6, precise targeted blockade can be achieved using IL-6 neutralizing antibodies, inhibiting CAF paracrine function, reversing CAF-mediated tumor radioresistance, and ultimately enhancing tumor cell radiotherapy response [140].

5.3. Sensitizing Targeted Therapy by Targeting CAFs

During targeted therapy, CAFs induce targeted therapy resistance through mechanisms like secreting cytokines and remodeling ECM. Clinical strategies for sensitizing targeted therapy by targeting CAFs are gradually being developed, primarily focusing on targeting CAF-mediated resistance mechanisms.

Interventions targeting CAFs include:

Targeting CAF biomarkers (primarily FAP): Utilizing FAP-related small molecule inhibitors or ADCs, these reagents or antibodies precisely identify and eliminate CAFs, thereby reducing CAF impact on targeted therapy. CAR-T cell therapy can also enhance targeted drug infiltration, sensitizing targeted therapy [150].

Targeting major CAF signaling pathways: By targeting CAF activation pathways, CAF activation is inhibited, reducing influence on tumor cells. For example, by targeting TGF-β, CAF activation and ECM remodeling are suppressed, improving drug delivery and sensitizing targeted therapy [151].

Targeting CAF reprogramming: Inhibitors targeting CAF metabolic-related proteins can inhibit CAF metabolism, weakening CAF effect on tumor cells [143]; through novel targeted drugs (e.g., Vitamin D derivatives) targeting CAFs, inducing reprogramming into quiescent state, CAF role is diminished, enhancing tumor cell sensitivity to targeted therapy [152].

Beyond targeting CAFs, other TME components can be targeted:

Targeting PDGFR: PDGFR is tyrosine kinase receptor widely expressed in CAFs [105]. Its associated PDGFR/PDGF signaling pathway is involved in CAF activation and tumor cell vasculature formation and stabilization. TKIs can target PDGFR, inhibit tumor cell growth, improve drug delivery, and promote tumor cell sensitization to targeted drugs [153].

Targeting ECM: CAFs promote dense physicochemical barrier formation by ECM through various signaling pathways, impeding drug penetration and leading to tumor cell resistance to targeted drugs. By targeting enzymes or molecules involved in ECM synthesis, ECM remodeling can be inhibited and drug penetration improved. Lysyl oxidase like 2 (LOXL2) can promote tumor cell migration by remodeling ECM and participates in constructing dense physicochemical barrier within ECM. LOXL2 antibodies can be used for targeted intervention to reduce pro-cancer effects [141]. Additionally, ECM components can be targeted, for instance, by using hyaluronidase to target hyaluronic acid, thereby degrading ECM and ultimately sensitizing targeted therapy [145].

5.4. Sensitizing Immunotherapy by Targeting CAFs

CAFs are key factor mediating tumor cell immunosuppression. They primarily reduce immunotherapy efficacy by secreting immune-related inhibitory factors, inducing high expression of immune checkpoint molecule ligands (like PD-L1) on tumor cells, inhibiting T cell infiltration and function, and recruiting immune heterogeneous cells [154]. To improve immunotherapy, techniques targeting CAFs can be employed to inhibit CAFs, thereby reducing impact on tumor cells.

Targeted intervention strategies:

Directly targeting and eliminating CAFs: For example, by targeting specific CAF surface markers (like FAP) or using small molecule inhibitors, CAFs can be directly targeted and eliminated, thereby reducing influence and sensitizing immunotherapy [141].

Targeted intervention against CAF signaling pathways: Targeted signaling pathways include TGF-β, IL-6, and others. Through corresponding molecular inhibitors, these signaling molecules can be targeted to inhibit signal transduction, thereby reducing CAF impact on tumor cells and ultimately sensitizing immunotherapy [141]. Some studies found in breast cancer, knocking out signaling pathway adaptor protein Shcbp1 can significantly reduce CAF numbers. By targeting Shcbp1, the adaptor protein can be eliminated to decrease CAF numbers, thereby enhancing T cell infiltration and ultimately overcoming immunotherapy resistance [155].

Targeting CAF phenotype: Novel drugs (like Vitamin D derivatives) can target CAFs, inducing reprogramming into quiescent state, thereby weakening CAF role [152]. Some studies found NADPH oxidase 4 (NOX4) can convert CAFs into normal quiescent state. Pro-NOX4 enzyme-inducing drugs can be used for targeted intervention to induce CAF transformation, ultimately sensitizing immunotherapy [156].

CAF-targeted vaccines: Vaccines can induce the body to produce specific antibodies against CAFs, achieving active CAF clearance. For example, DNA vaccines (SynCon FAP) induce the body to produce anti-FAP antibodies and T cells. Dendritic cells transfected with FAP mRNA can be used for targeted vaccination to activate the body to produce specific cytotoxic T lymphocytes, clearing CAFs and inhibiting tumor growth [145].

Beyond directly intervening with CAFs, CAF-targeting strategies can involve targeted regulation of their secreted factors and ECM. This includes targeting CAF-secreted immunosuppressive factors and immunosuppressive ligands. CXCL12 inhibitors can target immune factors, blocking their mediated signaling pathways suppressing immune cells. Immune checkpoint ligand inhibitors can target checkpoint ligands (like PD-L1), reducing inhibitory effect on T cells, thereby sensitizing immunotherapy [81,157]. ECM can also be targeted. By using hyaluronidase to target hyaluronic acid, ECM is degraded, thereby enhancing T cell infiltration. In soft tissue sarcoma, FOXP1 can inhibit T cell infiltration, so by targeting FOXP1, T cell infiltration and immunotherapy sensitization can be enhanced [158].

6. Novel Strategies for Targeting Cancer-Associated Fibroblasts

6.1. Nanoengineering

Traditional CAF-targeting strategies are often limited by low drug delivery efficiency, insufficient target specificity, poor tumor tissue penetration, and ease with which single-target interventions are overcome by TME components [159]. Researchers have developed nanoengineering strategies. This strategy, owing to unique design flexibility and physicochemical properties, offers more comprehensive and precise therapeutic solution for cancer treatment [160].

Core therapeutic approaches:

Targeting CAF surface markers: Nanoparticles can be modified with antibodies or inhibitors corresponding to markers, enabling precise delivery. Functionalized H-protein nanocages corresponding to antibodies can achieve precise delivery and specific binding to CAFs with high FAP expression [161]. Nanoparticle carriers highly sensitive to TME can be designed to accelerate targeting process and enhance drug penetration. In pancreatic cancer, constructing dual-responsive lipid-albumin nanoparticles for FAP-α and NIR laser can enhance drug delivery and penetration [162].

Nanoengineering strategies for CAF reprogramming: Targeting CAF activation and pro-tumor signaling pathways (e.g., TGF-β), nanocarriers can be constructed to encapsulate corresponding signaling pathway inhibitors into nanoparticles for targeted delivery, inhibiting CAF functional expression and ultimately reversing CAF pro-tumor phenotype [163].Regarding CAF metabolic reprogramming, nanoparticles can deliver glycolysis inhibitors to reduce energy supply for CAFs and tumor cells, thereby inhibiting tumor growth [164].

Nanoengineering strategies for targeting ECM: Dense ECM and high interstitial fluid pressure contributed by CAFs severely restrict drug penetration [165]. Ultra-small nanoparticles can be designed to participate in targeted delivery of ECM-degrading enzymes (e.g., hyaluronidase). By degrading ECM, these nanoparticles reduce tumor interstitial pressure, improve targeted drug penetration, and enhance drug delivery efficiency within TME [166].

Synergistic combinations with nanoengineering include immunotherapy and chemotherapy. For immunotherapy, researchers developed nano-targeting strategy: mRNA-LNP. LNP encapsulating FAP-specific CAR mRNA are intravenously injected. These LNPs can be highly recognized by immune cells in vivo, and mRNA can be translated into CAR protein, enabling specific CAF killing. This method can reduce treatment cycle and improve targeting efficiency [167]. For immune checkpoint inhibitors, immunomodulators can be delivered via nanocarriers. After carrier delivery, these immunomodulators can transform TME into immune-activated state, effectively inhibiting CAF activation, reducing immunosuppressive factor secretion, and improving drug infiltration into tumor tissue, enhancing immune checkpoint inhibitor efficacy and ultimately boosting immune cell killing effect on tumor cells [168]. In chemotherapy combination strategies, utilizing nanocarriers to deliver chemotherapeutic drugs while simultaneously targeting CAFs can effectively reverse CAF-mediated chemotherapy resistance and improve tumor cell sensitivity to chemotherapy [169].Encapsulating nanoparticles with CAF membranes can effectively preserve their surface-specific markers, achieving homologous targeted delivery to CAFs. Simultaneously, this strategy can extend drug circulation time in vivo and enhance penetration ability into tumor tissue [170].

6.2. Novel Strategies

Strategies for targeting CAFs have gradually shifted from eliminating CAFs to precise regulation and reprogramming strategies. In specific circumstances, CAFs can even be “turned from foe to friend”[171].

Novel strategies include:

Multi-specific Antibody Engineering: The core principle is utilizing two or more antigen-binding sites on single molecule to bring different target cells into close proximity, thereby significantly enhancing biological effects. The most representative example is FAP-targeting T-cell engager (TCE), which can specifically bind to CAFs at one end and target T cell surface molecules at the other; when this engager simultaneously binds to both cell types, it can forcibly mediate CAF-T cell contact, directly initiating localized immune killing effect [171]. Multi-specific antibody design based on co-stimulatory molecules on immune cell surfaces is also employed, with common targets including 4-1BB and CD40[172]. FAP-4-1BB and FAP-CD40 bispecific antibodies can be constructed, with one end targeting FAP on CAF surface and the other end binding to co-stimulatory molecules of corresponding immune cells; when antibody binds to CAFs, co-stimulatory signals are locally activated, thereby efficiently recruiting and activating immune cells, achieving precise immunotherapy [173]. Additionally, bispecific antibodies can be designed as functional molecules directly inducing CAF apoptosis. For example, tetravalent FAP-DR5 bispecific antibodies can simultaneously efficiently bind to FAP on CAF surface and death receptor DR5, thereby activating apoptotic signaling pathway in CAFs, achieving precise CAF elimination [173].

Genetically Engineered Oncolytic Viruses: Oncolytic viruses are novel class of anti-cancer therapeutics that can kill tumor cells and activate anti-tumor immune responses [174]. However, under CAF influence, ECM often forms dense physicochemical barrier, severely hindering therapeutic virus spread and diffusion within TME [175]. To overcome this bottleneck, researchers are optimizing oncolytic viruses through genetic engineering to enhance targeted therapeutic effects. Viral capsid proteins can be engineered. By genetically modifying oncolytic adenovirus capsid protein, they can acquire specific recognition and targeting capabilities for CAFs [176].Thanks to oncolytic virus highly efficient replication characteristics, they can serve as ideal gene delivery vectors. After targeted modification of viral surface proteins, genes encoding bispecific antibodies can be delivered to tumor tissue; leveraging virus’s inherent potent replication ability, efficient expression and diffusion of antibodies can be achieved locally within tumor, thereby significantly enhancing anti-tumor efficacy [177].

Novel Reprogramming Strategies: Reprogramming CAFs to “turn foes into friends” is strategy aimed at fundamentally altering CAF biological characteristics, reversing pro-tumor phenotype to quiescent or tumor-suppressive state. Traditional targeting strategies mostly focus on CAF metabolic regulation, using small molecule inhibitors to block key metabolic pathways, inhibit glycolysis and lactate efflux, thereby cutting off energy supply and reducing CAF activity [171]. However, such methods can only achieve temporary inhibition and cannot fundamentally reshape CAF phenotype. To this end, researchers are exploring CAF phenotypic modification strategies based on genetic and epigenetic mechanisms [178]. Epigenetic regulators, such as DNA methyltransferase inhibitors and histone deacetylase inhibitors, can target CAFs, altering their phenotype and function [179]. Studies confirm vitamin D receptor agonists and retinoids can systematically remodel CAF gene expression profile, transforming them from activated pro-tumor state to quiescent tumor-suppressive state [171].

7. Summary and Outlook

Reviewing current research, significant progress has been made in the field of CAFs, and their central role in tumor development and treatment regulation has gained widespread consensus. Current research has established relatively systematic understanding of their intrinsic properties, diverse regulatory mechanisms, clinical significance, and targeted intervention strategies. Regarding intrinsic properties, CAF multiple origins have been clarified, primarily including normal fibroblast activation within TME, MSC differentiation, and EMT. Based on functional characteristics, CAFs can be categorized into nine main subtypes such as iCAFs, myCAFs, and apCAFs, with different subtypes exhibiting high heterogeneity in tumor progression. In therapeutic regulation, CAFs influence chemotherapy, radiotherapy, targeted therapy, and immunotherapy efficacy through various mechanisms including secreting inflammatory factors, forming dense physical barriers, mediating metabolic reprogramming, inducing senescence-associated phenotypes, activating drug resistance pathways, and remodeling immune microenvironment, thereby mediating tumor treatment resistance.

In clinical applications, CAFs demonstrate important diagnostic, prognostic, and therapeutic predictive value. Utilizing specific markers like FAP, combined with techniques such as IFC/IF, qPCR, flow cytometry, and scRNA-seq, precise qualitative and quantitative analysis and subtype identification of CAFs can be achieved. CAF abundance, subtype proportion, and spatial distribution characteristics can serve as prognostic indicators for various malignancies and be used to predict patient responses to different anti-tumor regimens, providing crucial references for precision diagnosis and treatment. In targeting strategies, CAF-centric sensitization therapy has become research hotspot. This primarily includes eliminating CAFs by targeting characteristic markers, blocking key signaling pathways like TGF-β to inhibit function, degrading ECM to improve drug delivery, and combining novel technologies such as nanoengineering, bispecific antibodies, and functional reprogramming to reverse CAF pro-tumor effects. These approaches offer new ideas and directions for overcoming tumor treatment resistance.

Despite existing research having elucidated CAF functions and mechanisms from multiple dimensions, this field still faces numerous unresolved issues and limitations in clinical application. Firstly, CAF specific origins and transformation pathways have not yet been fully confirmed, making it difficult to effectively implement prevention and inhibition strategies for transformed CAFs. Secondly, most clearance strategies centered on targeting CAF-specific markers have failed to achieve ideal results in clinical applications. The core reason lies in CAF high heterogeneity. This characteristic indicates targeting only a few markers cannot achieve comprehensive CAF clearance, which is also the central dilemma and challenge currently faced by CAF-targeted therapies. Therefore, future CAF-targeted therapy development needs to advance towards more refined direction, combining multidisciplinary treatment strategies. On one hand, targeted drugs capable of precisely identifying specific CAF subtypes within specific cancer types should be developed to ensure accuracy and comprehensiveness of targeted clearance, thereby guaranteeing therapeutic efficacy. On the other hand, further exploration is needed for drugs that can reprogram CAF phenotypes, allowing precise intervention tailored to different cancer types and CAF subtypes, achieving directional CAF phenotype regulation. Furthermore, personalized treatment should be promoted, developing individualized treatment strategies based on CAF subtype characteristics and tumor type to achieve better therapeutic outcomes. In summary, as one of the most critical TME components, CAF biological functions and regulatory mechanisms are gradually being revealed. In the future, continuous integration of multidisciplinary resources including basic research, drug development, and clinical application will be necessary to collaboratively tackle CAF-related challenges, thereby promoting further development of cancer treatment field.