The Role of Tumor Microenvironment in Triple-Negative Breast Cancer and Its Therapeutic Targeting

Academic Editor: Alexander E. Kalyuzhny Received: 2 August 2025 Revised: 25 August 2025 Accepted: 29 August 2025 Published: 30 August 2025 Citation: Vuleti´c, A.; Mirjaˇci´c Martinovi´c, K.; Juriši´c, V. The Role of Tumor Microenvironment in Triple-Negative Breast Cancer and Its Therapeutic Targeting. Cells 2025,14, 1353. https://doi.org/10.3390/ cells14171353 Copyright: © 2025 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/ licenses/by/4.0/). Review The Role of Tumor Microenvironment in Triple-Negative Breast Cancer and Its Therapeutic Targeting Ana Vuleti´c 1, Katarina Mirjaˇci´c Martinovi´c 1and Vladimir Juriši´c 2,* 1Department of Experimental Oncology, Institute of Oncology and Radiology of Serbia, Pasterova 14, 11000 Belgrade, Serbia 2Faculty of Medical Sciences, University of Kragujevac, P.O. Box 124, 34000 Kragujevac, Serbia *Correspondence: [email protected] Abstract Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer characterized by rapid proliferation and infiltration of immune cells into tumor microenvironment (TME). The treatment of TNBC still remains challenging due to the lack of expression of effective molecular targets pertaining to the tumor cell itself. In TNBC standard of care, therapies such as chemotherapy, together with recently introduced immunotherapy with checkpoint inhibitors, often do not result in durable clinical response. Therefore, better understanding of complex interactions between tumor cells, immune cells, and stromal cells mediated by multiple cytokines, chemokines, enzymes, and metabolites in TME is crucial for understanding the mechanisms that underlie tumor cell immune evasion strategies. The aim of this review is to give comprehensive overview of immune cell network and their interactions with cells in TME and possibilities for therapeutic targeting of TME in TNBC. We discuss cancer-associated fibroblasts (CAFs) as an important recently characterized player in TNBC with respect to their role in interactions with immune cells and their impact on tumor invasion. Based on the recently accumulated knowledge, therapies targeting immune suppressive mechanisms and CAF-related tumor-promoting mechanisms in TME hold great potential for clinical evaluation in TNBC. Keywords: triple negative breast cancer; tumor microenvironment; cancer associated fibroblasts 1. Introduction Tumor microenvironment (TME) or tumor stroma is a complex biological network in which malignant cells coexist with a variety of nonmalignant cells, including immune cells, tumor-associated fibroblasts (CAFs), mesenchymal stem cells (MSCs), tumor-associated adipocytes, endothelial cells, and pericytes in glycoprotein-rich extracellular matrix (ECM). The crosstalk between malignant cells and TME contributes to epithelial-to-mesenchymal transition (EMT), acquisition of invasive phenotype, increased motility of tumor cells, and subsequent formation of secondary tumor deposits [1]. Breast cancer is one of the most prevailing malignancies [ 2 ]. Triple-negative breast cancer (TNBC) is characterized by the lack of expression of estrogen receptor α (ERα ), progesterone receptor (PR), and the absence of expression/amplification of human epidermal growth factor receptor 2 (HER2). TNBC exhibits a higher proliferation rate and higher incidence of metastases compared to other breast cancer types [ 3 ]. The heterogeneous molecular characteristics of TNBC reduce the options for targeted therapies, making Cells 2025,14, 1353 https://doi.org/10.3390/cells14171353 Cells 2025,14, 1353 2 of 31 this tumor particularly aggressive and resulting in poor prognosis [ 4 ]. Accordingly, systemic chemotherapy based on anthracyclines and taxanes remains the primary treatment option [5] . In the past decade, based on studies involving genomic analyses, TNBC was classified into distinct molecular subtypes that were further refined according to the immunomodulatory gene expression profile attributed to the tumor infiltrating lymphocytes (TILs) and stromal cells [ 6 , 7 ]. The classification proposed subtypes displaying distinct clinical and pathological characteristics with disease prognosis differing significantly between the subtypes, and each subtype showing a specific pattern of gene expression [ 8 , 9 ]. However, the classification of TNBC due to the complexity and heterogeneity of the tumor, remained without the true consensus. Nevertheless, the four subtypes identified based on gene expression profiles named basal-like immune activated (BLIA), basal-like immune suppressed (BLIS), luminal androgen receptor (LAR), and mesenchymal (MES) proposed by Burstein et al. are the most thoroughly validated [ 8 , 10 ]. In one study TNBC was classified according to genomic immune profile in TME [11]. Recently, immunotherapy based on the inhibition of immune checkpoint (IC) molecules, most notably of the programmed cell death receptor (PD)-1 and cytotoxic T lymphocyte antigen (CTLA)-4, has shown potential to enhance immune response against tumor cells and thereby modulate TME [ 12 , 13 ]. In this sense, PD-/PD-L1 blockade with Food and Drug Administration (FDA)-approved monoclonal antibodies such as anti-PD-1 (pembrolizumab, nivolimab, cemiplimab, camrelizumab), anti-PD-L1 (atezolizumab, durvalumab, avelumab), and CTLA-4 blockade with ipilimumab, were introduced in clinical trials for breast cancer in TNBC and have shown therapeutic potential [ 14 ]. However, monotherapy with IC inhibitors resulted in limited clinical benefit as tumor cells are prone to immune escape and drug resistance due to highly dynamic interplay between the cells in TME. In the context of available therapeutic options including chemotherapy, radiotherapy and immunotherapy, there is a constant need for better understanding of cellular and molecular components of TME and their interactions that could be targeted in order to overcome therapeutic limitations and improve treatment outcome. 2. Immune Cells in TME Immune cells in TME include immunoreactive cells such as CD8 cytotoxic T lymphocytes (CTL)s, CD4 helper T cell, B lymphocytes, natural killer (NK) cells, dendritic cells (DC)s, M1 macrophages, and suppressive immune cells such as myeloid-derived suppressor cells (MDSC)s, tumor-associated macrophages (TAM)s, regulatory T cells (Treg)s, etc. [ 15 ] (Figure 1). TILs in TME encompass stromal TILs, which are positioned in the tumor margins without direct contact with cancer cells and intratumoral TILs, defined as lymphocytes in direct cell-to-cell contact with tumor cells with no intervening stroma. Intratumoral TILs make the smaller proportion of TILs as endothelial cells and stromal tissue aggravate their infiltration into the tumor [16]. The abundance and the composition of TILs regarding the presence of distinct lymphocyte populations has been evaluated in multiple studies in the context of breast cancer disease prognosis and response to chemotherapy [ 16 – 19 ]. TNBC is characterized by a greater abundance of TILs compared to other molecular subtypes of breast cancer [ 20 ]. Moreover, in TNBC and HER2+ breast tumors, higher proportions of TILs have been associated with better response to chemotherapy. The same study indicated the presence of TILs as a favorable prognostic marker in early-stage TNBC [ 21 ]. Furthermore, the deficiency of stromal TILs and low number of CD8+ T cells were found to independently predict mortality in TNBC while the prognostic value of TILs and their CD8+ subset varied with respect to the cancer compartment [ 22 ]. In patients with TNBC distant metastases were shown to exhibit scarce presence of TILs compared to the primary tumor. The same study reported Cells 2025,14, 1353 3 of 31 the longer median overall survival (OS) in the cohort of patients with metastatic lesions containing TILs>10% compared to patients with TILs<10% [ 23 ]. In distant metastases of TNBC, the presence of TILs identified patients with enhanced response to immunotherapy, suggesting the importance of immune activation for improving survival outcomes [24]. Figure 1. Immunosuppressive and immunoreactive cells in tumor microenvironment (TME). Cells in TME: M2 macrophages, cancer-associated fibroblasts (CAF), N2 subset of neutrophils, regulatory T cells (Treg), myeloid-derived suppressor cells (MDSC), M1 macrophages, natural killer (NK) cells, cytotoxic T lymphocytes (CTL), CD4 helper T cells, and B lymphocytes. CD8+ T cells play an initial role in antitumor immune defense. The naïve T cells undergo priming by tumor-derived antigens presented by antigen-presenting cells (APCs) in regional lymph nodes, and migrate to TME via blood stream or lymphatics, where they differentiate into cytotoxic T cells (CTLs). CTLs directly kill tumor cells by producing cytotoxic enzymes perforin and granzymes. CD4+ T cells play an immunoregulatory role. Based on the cytokines they generate, CD4+ T cells are divided into Th1, Th2, Th17, and regulatory T (Treg) cell subsets, each of which has a unique function in immune responses [ 25 ]. Th1 T cells are the primary source of the inflammatory cytokines interferon (IFN)- ꝩ , interleukin (IL)-2, and tumor necrosis factor (TNF), which induce maturation of APCs, more effective presentation of tumor antigens, and maturation of CD8+ cells to fully functional CTLs. Conversely, Th2 cells secrete IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13, which are responsible for activation of humoral immunity, eosinophil activation, inhibition of macrophage functions and suppression of antitumor immune responses [ 26 ]. Th17 cytokines IL-17, IL-21, and IL-22 are reported to facilitate cancer progression by promoting inflammation and angiogenesis. The elevated level of these cytokines in peripheral blood has been related to progression of breast cancer [27]. Cells 2025,14, 1353 4 of 31 B cells in TME of breast cancer have been less studied in primary tumors compared to axillary lymph nodes. In antitumor immune response, B cells have a primary role in presenting antigens to CD8 T cells and secreting antibodies that induce tumor cell destruction via antibody-dependent cellular cytotoxicity (ADCC), as well as producing cytokines that further support antigen presentation by professional APCs. In invasive breast cancer, B cells in TIL population are associated with higher grade tumors and lymph node positivity, indicating the role of B cells in tumor aggressiveness [ 28 ]. There are opposing results regarding the association of B cells in TME with disease prognosis in TNBC. While several studies report the association of infiltrating B cell with larger tumor size, high histological grade, lymphovascular invasion, and lymph node metastases in early breast cancer [ 29 ], there are also data showing association of high densities of infiltrating B cells in primary tumors with a better response to neoadjuvant chemotherapy [ 30 ]. According to some research data, interactions between TNBC cells and B cells increase gene expression for a number of inflammatory cytokines, most notably IL-1 β , which results in chronic inflammation [31] . IL-1 β by activating nuclear factorκ B (NF κ B), promotes angiogenesis and EMT. Furthermore, NF κ B signaling was associated with large tumor size, high histological grade, and negativity for ER and PR expression in invasive breast cancer [ 32 ]. Newly defined subtype of B cells, the regulatory B cells (Bregs), have a role in immune tolerance, similar to regulatory T cells. In this sense, CD25+ and IL10+ B cells were recently related to the induction of Tregs in TIL aggregates and the development of metastasis in breast cancer [33]. NK cells are the subpopulation of innate lymphoid cells (ILCs) that have a unique ability to directly recognize malignantly transformed cells via cognate NK cell receptors and kill tumor cells by releasing cytotoxic enzymes perforin and granzymes. Aside from cytotoxic activity, which is performed without major histocompatibility complex (MHC) class I restriction, NK cells produce multiple cytokines—IFNꝩ , TNF, IL-10, IL-13—and granulocyte macrophage colony-stimulating factor (GM-CSF) that regulate immune responses [ 34 ]. NK cell antitumor activity is tightly regulated by the balance of signals transmitted by activating and inhibitory receptors bound to their ligands on tumor cells. NK cells are able to distinguish normal from transformed cells by killer cell immunoglobulin-like receptors (KIRs) that inhibit NK cell cytotoxic activity by binding to MHC class I molecules [ 35 , 36 ]. In tumor tissues, NK cells are mostly present during early tumorigenesis, but they become scarce in advanced stages of tumor development due to increasing immunosuppression in TME [37]. In this sense, several tumor evasion mechanisms overcoming NK cell-mediated tumor control have been identified, including the impaired NK cell recruitment to the tumor bed, shedding of ligands for NK cell activating receptors, and the induction of the inhibitory ligands on tumor cells [ 38 , 39 ]. The suppressive effect of TME on NK cytotoxic activity of NK cells can be mediated by cytokines such as TGFβ and IL-10, L-kynurenine, a product of tryptophan degradation, and prostglandin E2 (PGE2), secreted by tumor cells, suppressive immune cells, and CAFs [ 40 ]. These soluble factors in TME downregulate the expression of NK cell-activating receptors, including NK group 2D (NKG2D), NKp30, and DNAM1, as well as their signaling pathways [ 41 – 43 ]. Moreover, persistent stimulation of NKG2D receptor by its tumor cell ligands, as well as soluble ligands generated by proteolytic shedding mediated by matrix metalloproteinases (MMPs) produced by tumor cells or CAFs, may also lead to functional exhaustion of NK cells [42,44]. However, IFN-γ released during acute early inflammation in the context of antitumor immune response induces the resistance of tumor cells to NK cell-mediated lysis by upregulating classical and non-classical MHC class I molecules [45]. Cells 2025,14, 1353 5 of 31 Cell-to-cell contacts in TME significantly affect NK cell functions. In this sense, ligand– receptor interaction between CAFs and NK cells was reported to inhibit NK cell cytotoxicity toward cancer cells, downregulate the expression of activating NK cell receptors, and thereby promote cancer cell escape from NK cell surveillance, according to a study that used a mouse model of breast cancer. Moreover, the tumor samples obtained from patients with TNBC showed enrichment of NK cells in CAF-rich regions, accompanied with upregulated binding of NK cell receptors to ligands on CAFs that altogether correlated with poor disease outcome [46]. Tumor infiltrating NK cells appear to have lower antitumor activity compared to circulating NK cells [ 47 , 48 ]. Moreover, in early stages of tumor development, NK cells exhibit antitumor activity, while in the later cancer stages they show impaired cytotoxic capacities and become senescent cells, as shown in the murine model of TNBC [ 49 ]. In one study in mice, a unique subcluster of Socs3highCD11b-CD27of immature NK cells found specifically in TNBC samples showed reduced expression of cytotoxic granzyme signature and the ability to activate cancer stem cells through Wnt signaling. Furthermore, the same study reported increased numbers in functionally less mature CD56bright NK cell subset in tumor samples, which correlated with poor OS in patients with TNBC [ 50 ]. A recent study identified the distinctive IL-10+secreting immunosuppressive NK cell population in TILs with a strong relation to poor survival prognosis in TNBC patients treated with neoadjuvant IC blockade immunotherapy [51]. 2.1. Immune Checkpoint Receptors IC receptors are upregulated as a consequence of immune responses, with their primary physiological role being the prevention of excessive immune reactions. IC receptor PD-1 and its ligand PD-L are important negative regulators of immune activity. During tumorigenesis, oncogenic pathways, genetic, and epigenetic factors intrinsic to tumor cells upregulate the expression of PD-L1 (B7H1) and PD-L2 (PD-L2) on tumor cells. In this sense, microRNA-200/ZEB1 axis, aberrant activation of Wnt signaling, loss of phosphatase and tensin homolog (PTEN), activation of phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mechanistic target of rapamycin (mTOR) signaling pathway, and MUC1C/Myc/NF-K β axis were shown to upregulate the expression of PD-L1 in TNBC [ 52 – 55 ]. Inflammatory cytokines in TME, such as IFNꝩ , IL-1 β , IL-6, and TNF, further favorize cancer immune escape by augmenting PD-L1/PD-L2 expression on tumor cells including TNBC [ 55 , 56 ]. Aside from tumor cells, multiple cells in TME, including immune cells (DCs, macrophages, Tregs) and CAFs, express PD-L1 which further contributes to suppressing antitumor immunity [ 57 – 59 ]. In this sense, in TME and tumor-draining lymph nodes, PD-L1 is often upregulated by IFNꝩ on APCs, leading to the inhibition of T cell activation [60]. The process of EMT is associated with augmentation of PD-L1 expression in multiple tumors. EMT-inducing transcription factors such as ZEB1 have been shown to bind to gene promoter region of PD-L1 gene [ 61 ]. By blocking the glycogen synthase kinase (GSK)3 β -mediated phosphorylation, ubiquitination, and degradation of the EMT-inducing transcription factor Snail, PD-L1 has been shown to enhance EMT in TNBC cells, hence increasing the likelihood of metastasis [62]. Aside from PD-1 and CTLA-4, chronic inflammation in TME can result in upregulated expression of several other IC receptors contributing to immunosuppression and resistance of tumor cells to the antitumor activity of lymphocytes infiltrating the TME. In this sense, T cell immunoglobulin and mucin domain-containing protein 3 (TIM3), lymphocyte activation gene-3 (LAG-3), T cell immunoreceptor with immunoglobulin and ITIM domain (TIGIT), and V-domain Ig-containing suppressor of T cell activation (VISTA) are upregulated on T and NK cells [ 13 ]. Therefore, therapeutic targeting of multiple IC molecules Cells 2025,14, 1353 6 of 31 on immune cells represents a challenge and a potential option for treatment of TNBC [reviewed in 10]. 2.2. Suppressive Immune Cells in TME 2.2.1. Regulatory T Cells Regulatory T cells represent the functionally distinctive subpopulation of CD4+ T cells that express forkhead box P3 (FoxP3) transcription factor and high-affinity IL-2 receptor α chain (CD25), depleting IL-2 from the TME and thereby disturbing T cell proliferation and activation. Tregs play the key role in immunologic tolerance primarily by inhibiting the proliferation, cytotoxic activity, and cytokine production of T cells. Tregs suppress immune reactions by multiple mechanisms such as the production of immunosuppressive cytokines and other soluble factors, including TGFβ , IL-10, IL-35, and PGE2. Additionally, Tregs express IC molecules that altogether create an immunosuppressive environment by impairing antitumor activity of T and NK cells [ 63 , 64 ]. Breast cancer and stromal cells in TME cells secrete chemokines CCL2, CCL5 (RANTES), CCL20, CCL22, and CXCL12 which, by binding to the corresponding receptors, induce Treg homing to tumor tissue. It has been reported that by releasing CCR5-associated chemokines, mainly CCL3 (MIP-1 α ), CCL4 (MIP-1 β ), and CCL5, which are strong attractants for lymphocytes and macrophages, Tregs facilitate metastatic invasion of TNBC to lymph nodes and bone marrow [64–66]. Tregs constitutively and highly express IC receptor CTLA-4 and are able to downregulate the expression of costimulatory molecules CD80/CD86 on APCs thereby hindering antitumor activity of T cells [ 67 ]. Inflammatory conditions in TME such as type I IFN signature, PD-L1, and expression of indoleamine 2,3-dioxygenase (IDO), which characterize immunomodulatory subtype of TNBC, contribute to acquisition of a strong immunesuppressive functional Treg phenotype [ 8 , 9 ]. Furthermore, it was reported that PD-1 in contact with PD-L1 could convert naive CD4+ T cells to Tregs through the downregulation of Akt, mTOR, and ERK2 and simultaneous upregulation of PTEN signaling pathways [ 68 ]. Although the presence of lymphocyte infiltrates seems to reflect favorable host antitumor immune responses, suggesting that immune activation is important for improving survival outcomes, the composition of lymphocyte infiltrate varies across molecular subtypes of breast cancer [ 69 ]. In this sense, the increased presence of Foxp3+ Treg subsets was reported in TNBC and HER-2-positive tumors compared to luminal subtypes [ 70 ]. Moreover, the same study indicated the association of greater Treg to CD8+ T cell ratios with higher grade tumors and higher Ki-67 expression. Changes in Treg infiltration were reported during the course of metastatic progression, indicating the relevance of Treg infiltration for unfavorable disease prognosis in breast cancer [ 71 ]. Furthermore, the presence of FOXP3+ lymphocytes in tumor tissues, along with TNM stage, has been identified as an independent prognostic factor for OS in TNBC [ 72 ]. Aside from their role in promoting metastatic invasion, high infiltration rate of Tregs in patients with TNBC has been associated with inhibited immune activation pathways and resistance to anti-PD1 immunotherapy [66]. 2.2.2. Tumor-Associated Macrophages Tumor-associated macrophages (TAMs) may include macrophages of M1 antitumor phenotype that are present mostly in early phases of immune response to tumor and immunosuppressive M2 subset which is more abundant in TME of the growing tumor. M2 macrophages enhance immune tolerance, angiogenesis, tumor growth, and invasion. This subset of macrophages produces numerous anti-inflammatory factors (TGFβ , IL-4, IL-13, IL-10, IL-1RA), lower levels of inflammatory cytokines (IL-6, IL-12, IL-23, TNFα ), and considerable amounts of growth factors (EGF, vascular endothelial growth factor Cells 2025,14, 1353 7 of 31 (VEGF)) [ 73 ]. Furthermore, by lowering the levels of arginine through the conversion of L-arginine to ornithine through arginase (Arg) activity, TAMs suppress T cell antitumor function [74]. The ornithine is further translated into polyamines (putrescine, spermidine, and spermine), which are linked to more immunosuppressive TME as they promote the growth and function of MDSCs, macrophages and Tregs, as well as collagen production in the process of tissue repair and survival of tumor cells [ 75 , 76 ]. Moreover, the expression level of Arg-1, which is one of the molecular markers of M2 macrophages, was elevated in peripheral blood, lymph nodes and tumor tissue of breast cancer patients [ 77 ]. Furthermore, TAMs produce multiple enzymes that participate in metastatic tumor invasion, such as MMPs, serine proteases, and cathepsins, and decompose collagen and other components of the ECM, thereby helping the migration of tumor and stromal cells [ 73 ]. There are also data indicating the role of M2 macrophages in EMT, as CCL2 produced by cancer and myeloid cells was shown to attract M2 macrophages and induce Wnt-1 upregulation. Wnt-1 in turn downregulates E-cadherin junctions in HER2+ early breast cancer [ 78 ]. Accordingly, in TNBC, the presence of M2 TAMs in TME was correlated with stromal fibroblast infiltration, EMT, and poor patient survival [79]. 2.2.3. MDSC MDSCs are immature myeloid cells that arise after prolonged exposure of monocytes or neutrophils to inflammatory conditions in TME. Various tumor-derived factors induce differentiation of MDSCs in vitro , including PGE2, IL-6, IL-10, IL-1 β , TGFβ , stem cell factor (SCF), and proangiogenic VEGF [ 80 ]. By expressing IC molecules and secreting immunosuppressive cytokines, most notably TGFβ , IL-1 β , and IL-10, MDSCs suppress the activity of T and NK cells [ 81 , 82 ]. MDSCs also block T cell activation by sequestering cystine and limiting the availability of cysteine in TME [ 83 ]. Additionally, MDSCs contribute to tumor-induced immune suppression by blocking the production of IL-12 by TAMs and DCs that induces polarization of macrophages toward a tumor-promoting M2 phenotype. Furthermore, M2 macrophages play a role in the development and proliferation of FoxP3+ Treg cells [84]. Increased infiltration of MDSCs was reported in TNBC compared to other molecular subtypes of breast cancer [ 85 ]. The same study reported that the number of MDSCs population in patient with basal-like TNBC correlated with the level of transcription factor ∆ Np63, which promotes tumor growth, progression, and metastasis in humans and in the mouse model of TNBC. The mechanism involved in MDSC recruitment was the ∆ Np63dependent activation of chemokines CXCL2 and CCL22. Furthermore, it has been revealed that MDSCs recruited into the TME of TNBC release the enzymes chitinase 3-like and MMP9, which are implicated in metastatic invasion and the promotion of cancer stem cell function [85,86]. 2.2.4. Tumor-Associated Neutrophils Tumor mediated signals such as TGFβ promote the differentiation of a protumorigenic N2 subtype of neutrophils. Tumor-associated neutrophils (TANs) produce high levels of TNF, inducible nitric oxide synthase (iNOS), NO, and H 2 O 2 , damaging DNA and inducing genetic instability. Therefore, N2 neutrophils represent active players in inflammation-related tumorigenesis and tumor development. Furthermore, this population of neutrophils supports tumor invasion, angiogenesis, and metastasis by secreting VEGFA and MMP9. Besides the secretion of soluble factors, TANs have been shown to impair the antitumor immune response by expressing immunosuppressive molecules, such as PD-L1. TANs are present in most TNBC tumors, indicating their potential to impact breast cancer prognosis [87]. Cells 2025,14, 1353 8 of 31 3. Cancer-Associated Fibroblasts Fibroblasts are mesenchymal cells with diverse physiological functions such as ECM synthesis and remodeling, tissue repair, mesenchymal lineage maintenance, secretion of signaling molecules, and immune regulation. Cancer-associated fibroblasts (CAFs) play a complex role in the TME because of their ability to influence many aspects of tumorigenesis, including the proliferation of malignant cells, motility, angiogenesis, metastatic invasion, inflammation, and metabolic reprogramming [ 88 ]. In breast cancer, up to 80% of normal fibroblasts in breast tissue acquire CAF phenotype during tumor progression [89,90]. Aside from “normal” fibroblasts, diverse cells of mesenchymal origin, such as adipocytes and mesenchymal stem cells, can give rise to CAF differentiation when exposed to the soluble factors in TME due to intrinsic intercellular plasticity. In this sense, bone marrow mesenchymal stem cells can be transformed into CAFs when exposed to conditioned medium in which tumor cells were cultivated [ 91 ]. Similarly, adipocytes and pericytes, as cells of mesenchymal origin, were reported to differentiate into fibroblasts when exposed to conditioned media from tumor cells [ 92 , 93 ]. Furthermore, in the proximity of the developing tumor and through the process of EMT triggered by soluble factor in TME, epithelial and endothelial cells can downregulate epithelial and endothelial markers, respectively, and acquire fibroblast-like characteristics [88]. Morphologically, CAFs are spindle-shaped mesenchymal cells characterized by overexpression of α -smooth muscle actin ( α -SMA) that regulates cytoskeleton rearrangements contributing to cellular mobility. In breast cancer, the expression of α -SMA was reported to correlate with lymph node metastases and poor prognosis [ 89 ]. CAFs also express cytoskeletal protein vimentin, which is also a marker of EMT [94]. In tumor surroundings, growth factors, cytokines, various signaling molecules, metabolites released by cancer and immune cells, mechanical stress, alterations in histone acetylation, and DNA damage, all trigger differentiation of CAFs [ 95 ]. TGFβ plays a leading role in the generation of CAFs by activating canonical (SMAD-dependent) and noncanonical (non-SMAD) pathways [ 96 , 97 ]. In addition, TGF β -1 secreted by activated fibroblasts (TGFβ RII) creates a positive feedback loop by binding to the type 2 TGFβ receptor, further increasing fibroblast activation. Aside from TGFβ , osteopontin (OPN), IL-1 β and IL-6, secreted by cancer or immune cells, induce the conversion of stromal fibroblasts to CAFs by activating downstream TGFβ /Smads, NF-kB signaling, and signal transducer and activator of transcription (STAT)3 that are pivotal for modulating expression of genes linked to the CAF lineage [ 98 ]. Furthermore, metabolic reprograming towards aerobic glycolysis induced by lysophosphatidic acid (LPA), TGFβ 1 or platelet-derived growth factor (PDGF) produced by tumor cells, activate hypoxia-inducible factor (HIF)-1 α pathway [ 96 ] contributing to the differentiation of fibroblasts into CAFs. The central role in the establishment and maintenance of CAFs is associated with function of Yes-associated protein (YAP)1 and its paralog, transcriptional coactivator with a PDZ-binding motif (TAZ). In CAFs and tumor cells, the lack of YAP1 phosphorylation results in its translocation to the nucleus, its subsequent binding to TEAD transcription factors, and the activation of mitogenic pathways [ 99 ]. YAP regulates the expression of several cytoskeletal proteins. The activation of YAP occurs as a consequence of the ECM remodeling and stiffening induced by factors such as LPA and TGFβ . Src-family kinase function is required for YAP activity and expression of myosin light chain (MYL) 9, matrix stiffening, and many pro-tumorigenic properties of fibroblasts [ 100 ]. Moreover, cancer-derived exosomes induce fibroblast conversion to CAFs by shuttling not only microRNAs (miRNAs) and long noncoding RNA Gm26809, but also growth factors such as TGFβ 1 [ 101 ]. Furthermore, PDGF drives pericyte differentiation into fibroblasts [90,92]. Cells 2025,14, 1353 9 of 31 Aside from previously mentioned cytoskeletal markers, CAFs were also found to express fibroblast activation protein (FAP), CD29 (integrin β 1), PDGF receptors α and β (PDGFR α and PDGFR β ), CD90 (THY-1), podoplanin (PDPN), fibroblast-specific protein 1 (FSP-1, S100A4), and caveolin 1. However, these markers are neither uniquely expressed nor necessarily coexpressed in CAFs [102]. The FAP serine protease is exclusively present in the TME and absent in healthy tissues. Its function is to enzymatically remodel the ECM, thereby enabling cellular migration. In HER2+ and TNBC, this tumor-promoting protein is enriched in CAFs expressing PD-L1/2 which, by binding to PD-1, have a potential to inhibit T cell activity [ 102 ]. Based on a recent study comprising integrated analysis of single-cell RNA sequencing data, clinical specimens, in vivo , and in vitro experiments, FAP+ CAFs have been identified as the predominant stromal population associated with poor clinical outcomes and immunosuppressive features in breast cancer patients. FAP+ subset of CAFs by secreting high levels of fibronectin 1 which binds to integrin α 5 β 1 on macrophages was found to triggers macrophage polarization toward immunosuppressive M2-like phenotype by activating focal adhesion kinase (FAK)-AKT-STAT3 signaling [103]. The CD90 glycoprotein plays a major role in cell migration, adhesion, and fibrosis by regulating interactions between cells or between cells and the ECM. High expression of CD90 in breast cancer CAFs was associated with increased cell transformation and unfavorable disease prognosis, particularly in the basal-like TNBC subtypes [104]. The high expression of integrin α 11/PDGFR β on stromal cells was associated with high tumor grades and unfavorable clinical outcome in breast cancer patients. Proinvasive function of integrin α 11 relies on its ability to interact with PDGFR β that promotes downstream JNK activation, leading to the production of pro-invasive ECM protein tenascin C [105]. FSP-1, the small calcium-binding integral membrane protein, is a serine protease often expressed in CAFs in primary breast cancer and in matching lymph node metastasis as well as in macrophages, other immune cells, and cancer cells. FSP-1+ CAFs produce the ECM component tenascin C, cytokines, MMPs, and VEGF-A, thereby facilitating cell motility, angiogenesis and metastatic invasion [ 106 ]. Moreover, the expression of FSP-1 was associated with unfavorable prognosis and may be considered a prognostic indicator of metastatic disease in early breast cancer [107,108]. Pro-tumorigenic CAFs in breast cancer often express CD10, a Zn-dependent matrix metalloproteinase which was predominantly related to ER-negative invasive breast cancer, whereas CD10CAFs were associated with luminal type invasive breast cancer [ 109 ]. GPR77, the non-G protein-coupled complement receptor, when expressed in combination with CD10, defines distinct CAF subpopulation that was related to chemotherapy resistance and poor prognosis in patients with lung and breast cancer. CD10+GPR77+ CAFs supply the TME with IL-8 and IL-6, which through the activation of NFκ B signaling via p65 phosphorylation and acetylation, protect cancer stem cells from chemotherapy-induced cell death [110–112]. However, the low expression of some markers, such as caveolin 1 scaffold protein, in CAFs is related to a more advanced tumor stage, early cancer recurrence, lymph node metastasis, and poor disease prognosis in breast cancer [113]. 3.1. Subpopulations of Fibroblasts in TNBC CAFs are a heterogeneous population with respect to their embryonic and spatial origin and functional characteristics. Several attempts have been made to classify CAFs in breast cancer according to the expression of molecular markers and functional properties. In this sense, single-cell RNA sequencing and histological characterization performed on Cells 2025,14, 1353 16 of 31 Most notably, anti-TGFβ therapy has been used to improve the effect of IC inhibitors. In this sense, clinical phase Ib study (NCT02423343) investigating the combination of anti-PD-1 therapy (nivolumab) with novel TGFβ RI kinase inhibitor galunisertib monohydrate (LY2157299) in advanced solid tumors has been completed and showed good tolerability [167]. In the past decade, bifunctional antibodies targeting IC and TGFβ signaling have been developed. In this sense, bifunctional antibody SHR-1701 directed against extracellular domain of TGFβ RII and PD-L1 is currently being investigated in a phase I study (NCT03710265) in subjects with metastatic or locally advanced solid tumors to assess the safety and tolerability of different dose levels [168]. Furthermore, bintrafusp alfa, another bifunctional antibody targeting TGFβ RII and PD-L1 in phase Ib/II clinical (NCT03579472) study in patients with metastatic TNBC, was investigated in combination with eribulin mesylate. The study showed that the agent was generally well tolerated and showed promising antitumor activity [169]. HCW9218, a bifunctional antibody combining simultaneous TGFβ -neutralizing and immune-stimulating properties, was designed to target extracellular domains of human TGFβ RII and the IL-15 receptor α . HCW9218 is presently being investigated in a phase I first in-human clinical trial (NCT05322408) to determine the maximum tolerated dose in advanced or metastatic solid tumors. Furthermore, the study in mice treated with HCW9218 in combination with anti-PD-1 therapy demonstrated modulation of immune landscape in tumor-draining lymph nodes and enhanced T cell antitumor activity [170]. 5.1.1. Targeting IL-6 Targeting IL-6/JAK/STAT-3 pathway is another therapeutic approach directed against the TME, as IL-6 regulates many tumor-promoting functions. According to previous studies, TNBC cells secrete autocrine IL-6 and are less responsive to paracrine IL-6 signaling. The exposure to IL-6 leads to the chronic induction of STAT3 phosphorylation, which promotes further growth and invasion of these tumor cells [ 171 ]. Monoclonal antibody against IL-6 receptor, tocilizumab (TCZ), inhibited in vivo growth of MDA-MB-231 tumor cells and reduced the formation of metastasis in mice. Furthermore, phase I and II clinical trials demonstrated the efficacy of monoclonal antibodies against IL-6 and its receptor, either as single agents or in combination with other chemotherapeutic agents, radiation, and targeted therapies in various types of cancer [ 172 ]. In recent years the clinical use of TCZ was mainly restricted to mitigation of immune-related adverse events in patients treated with IC inhibitors and showed promising results in phase II clinical study (NCT04375228) [173]. The inhibition of the STAT3 signaling represents another experimental therapeutic strategy. In this sense, OPB-51602, the small-molecule inhibitor of STAT3 phosphorylation was evaluated in phase I (NCT01423903) multiple dose escalation clinical trial to determine safety and tolerability in subjects with advanced cancers for whom there is no standard treatment available. Furthermore, STAT3 inhibitor VVD-130850 is currently being investigated in a phase I clinical study (NCT06188208), both as a single agent and in combination with IC inhibition to evaluate its safety, tolerability, and preliminary antitumor activity in participants with advanced solid and hematological tumors [174]. Additionally, the polykinase inhibitor IMX-110 targeting STAT3 in combination with anti-PD1 agent tislelizumab is currently being investigated a phase I/IIa in dose escalation/dose expansion clinical study (NCT05840835) designed to assess its safety, tolerability, pharmacokinetics and antitumor activity in patients with advanced solid tumors [125]. The next-generation antisense oligonucleotide inhibitors of STAT3, AZD9150, and AZD5069, are currently being evaluated in combination with MEDI4736 (durvalumab), in a phase I clinical study (NCT02499328) in advanced solid malignancies to compare the Cells 2025,14, 1353 17 of 31 effects of STAT3 inhibition monotherapy and its combination with anti-PD-L1 therapy. A similar phase I clinical study (NCT03394144) conducted on Japanese adult patients with advanced solid malignancies showed good tolerability for monotherapy and combination of this agent with durvalumab [ 175 ]. However, comparing the number of STAT3 inhibitors developed, only a small fraction is currently in clinical trials, perhaps due to the severe toxicities of the most of them [176]. 5.1.2. Targeting IL-8 Due to its role as a chemokine that promotes EMT of tumor cells, the recruitment of MDSCs to tumor site, subsequent immune escape, and its frequent overexpression in malignancies, including TNBC, IL-8 has been evaluated as a therapeutic target. Preclinical data showed therapeutic potential for clinical-stage monoclonal antibody that neutralizes IL-8 (HuMax-IL8) as it was shown to revert EMT in claudin-low TNBC models. The same study showed a significant decrease in the recruitment of MDSCs to tumor sites, an effect substantiated in treatment combination with docetaxel in TNBC [ 171 , 177 ]. Anti-IL-8 monoclonal antibody HuMax-IL8 (BMS-986253) was evaluated in phase Ib clinical study (NCT02536469) and showed good safety and tolerability, while the ongoing studies are presently evaluating the combination of IL-8 blockade and IC inhibitors in melanoma [ 178 ]. 5.1.3. CXCR4 Antagonist A potent selective CXCR4 antagonist, the bilixafortide (B)- POL6326 is a synthetic cyclic peptide. POL6326 showed encouraging safety/efficacy data in phase I clinical trial (NCT01837095) in combination with eribulin, the synthetic analog of a natural product that inhibits microtubule dynamics and induces apoptosis of cancer cells, in second line treatment options of patients with relapsed TNBC and hormone refractory ER-positive metastatic breast cancer [ 179 ]. The preliminary activity of the combination showed promising results in patients with HER-negative metastatic breast cancer that led to phase III clinical trial (NCT03786094) in locally recurrent or metastatic breast cancer. 5.1.4. IDO1 Inhibition IDO1 allows tumor escape through kynurenine production which stimulates Treg activity while suppressing the proliferation of effector T cells. Preclinical findings have revealed that IC inhibitors, while removing molecular brakes on cytotoxic immune cells, also stimulate the production of IDO1 in TME, thereby activating a negative feedback loop in immune responses. Based on this, pharmacological IDO1 inhibitors are currently being tested in clinical studies (Table 2), mostly in combination with IC inhibitors [ 180 ]. In this sense, phase I clinical study (NCT02658890) evaluating BMS-986205 (Linrodostat) IDO-1 inhibitor, in combination with nivolumab alone, and both nivolumab and ipilimumab (anti-CTLA-4) (NCT02658890), showed favorable safety and efficacy in heavily pretreated patients with advanced solid tumors [181]. Furthermore, two clinical studies investigated the possibility of using BMS-986205 IDO1 inhibitor in combination with LAG-3 inhibitor relatlimab, CTLA-4 inhibitor ipilimumab, and PD-1 inhibitors in advanced and metastatic solid tumors (NCT03335540; NCT03459222), concluding that this therapeutic approach should be further investigated [182]. Epacadostat represents another IDO1 inhibitor that competes with Trp for binding to the catalytic site of the enzyme. Recently, epacadostat was evaluated in addition to chemotherapy and pembrolizumab in phase I/II clinical study (NCT03085914) and showed acceptable safety profile and antitumor activity across multiple types of advanced or metastatic solid tumors [ 183 ]. However, a phase Ia/1b clinical study (NCT03343613), investigating an anti-IDO1 agent LY3381916 administered alone or in combination with Cells 2025,14, 1353 18 of 31 PD-L1 inhibitor LY3300054 in advanced solid tumors including TNBC, was terminated without a conclusion [184]. Table 2. Summary of clinical trials targeting IDO1 in TNBC. Agent Therapy Design Group of Patients Clinical Trial Number and Phase Status BMS-986205 (Linrodostat) BMS-986205+ nivolumab BMS-986205 + nivolumab + ipilimumab BMS-986205 + ipilimumab Advanced malignant tumors NCT02658890 Phase I/IIa Completed BMS-986205 BMS-986205+ nivolumab/relatlimab/ipilimumab Advanced malignant tumors NCT03335540 (ADVISE) Phase I Completed BMS-986205 BMS-986205 + relatlimab (anti-LAG-3) + nivolumab Advanced malignant tumors NCT03459222 Phase I/II Completed Epacadostat Epacadostat + pembrolizumab + chemotherapy Advanced or metastatic solid tumors NCT03085914 (ECHO207/KEYNOTE-723) Phase I/II Completed LY3381916 LY3381916 + antiPD-L1 (LY3300054) Solid tumors NCT03343613 Phase I Terminated E7046 Monotherapy Selected advanced malignancies NCT02540291 Phase I Terminated HTL0039732 Monotherapy HTL0039732 + atezolizumab Advanced solid tumors NCT05944237 Phase Ib/IIa Recruiting Buparlisib (AN0025) Buparlisib (AN2025) + AN0025 AN2025 + AN0025 + atezolizumab Advanced solid tumors NCT04975958 Phase Ia Completed eganelisib (IPI-549) Monotherapy PI-549 + nivolumab Advanced solid tumors NCT02637531 Phase I/Ib Unknown status IPI-549 IPI-549+ tecentriq (atezolizumab) + nab-paclitaxel Front-line TNBC NCT03961698 MARIO-3 Phase II Active, not recruiting Triple-negative breast cancer (TNBC). 5.1.5. Targeting PGE2 PGE2, another potential therapeutic target in TME, mediates its activity mainly through its G protein-coupled receptors EP2 and EP4 [ 185 ]. PGE2 suppresses the immune response by inhibiting the function of T cells, NK cells, and M1 macrophages, thus promoting tissue regeneration and inducing the differentiation of Tregs. In vivo studies have shown that the administration of EP4 antagonists restored the cytotoxic activity of NK cells in the context of progressive tumor growth [ 186 ]. Preclinical studies in breast cancer have shown NK cell-dependent antimetastatic activity of two PGE2 inhibitors: frondosideA, the antagonist of EP4/EP2 receptors derived from the sea cucumber, and RQ-15986, the newly synthetized antagonist of the EP4 receptor [ 186 , 187 ]. Further studies led to the development of several antagonists for the EP4 receptor for clinical use. In this sense, the first in-human phase I study of E7046, a highly selective small-molecule antagonist, showed manageable tolerability in patients with advanced malignancies, immunomodulatory effects, the best clinical response of stable disease, and recommended the dose for phase II clinical investigation (Table 3) [ 188 ]. Phase I/IIa clinical trial (NCT05944237) is currently investigating the best dose of HTL0039732, a potent small-molecule antagonist of EP4, for its sole application in metastatic solid tumors and its joint use with atezolizumab [ 189 ]. Furthermore, a novel EP4 antagonist AN0025 was investigated in combination with panclass I PI3K inhibitor buparlisib (AN2025) and atezolizumab in a phase Ia clinical study (NCT04975958) in patients with advanced solid tumors [190]. Cells 2025,14, 1353 19 of 31 Table 3. Summary of clinical trials targeting PGE2 in TNBC. Agent Therapy Design Group of Patients Clinical Trial Number and Phase Status E7046 Monotherapy Advanced malignancies NCT02540291 Phase I Terminated HTL0039732 Monotherapy HTL003973+ atezolizumab Advanced solid tumors NCT05944237 Phase I/IIa Recruiting AN0025 AN0025 + AN2025 (pan-class I PI3K inhibitor) AN0025 +atezolizumab Advanced malignant tumors NCT04975958 Phase Ia Completed Triple-negative breast cancer (TNBC). 5.1.6. PI3KγInhibition In preclinical studies, a selective inhibitor of PI3Kγ , eganelisib (IPI-549), showed potent ability to reshape the TME by reducing myeloid cell recruitment to tumors and reprogramming TAMs from immune-suppressive to immune-activating phenotype. This effect enhanced the activity of anti-PD-1/PD-L1 therapy [ 191 ]. Two complementary clinical studies have investigated eganelisib in combination with IC inhibitors. In this sense, eganelisib alone, and in its combination with nivolumab, was evaluated for safety and tolerability in a phase I/Ib clinical study (NCT02637531) in patients with advanced solid tumors. Simultaneously, the phase II MARIO-3 clinical study (NCT03961698) investigated the inhibition of PI3Kꝩ , in combination with tecentriq (atezolizumab) and abraxane (nabpaclitaxel) in front-line TNBC (Table 4). The MARIO-3 study revealed elevated gene signatures of TAM reprogramming and immune activation in patients with TNBC with longer progression-free survival, regardless of the baseline PD-L1 status [192]. Table 4. Summary of clinical trials targeting PI3K-γin TNBC. Agent Therapy Design Group of Patients Clinical Trial Number and Phase Status Eganelisib (IPI-549) Monotherapy Advanced solid tumors NCT02637531 Phase I/Ib Terminated IPI-549 IPI-549 + nabpaclitaxel + atzolizumab Front-line triple TNBC NCT03961698 Phase II Unknown status Triple-negative breast cancer (TNBC). 5.2. CAF-Directed Therapies Based on recently defined roles of CAFs in immune suppression and resistance to therapy, direct targeting of CAFs and their surface markers was established as annovel and attractive target for anticancer therapies in advanced breast cancer (Table 5) [102,110]. RO7300490 represents a bifunctional antibody (Table 3) with potential immunostimulatory and antineoplastic activity. RO7300490 is a CD40 agonist that also targets the inducible tumor stromal antigen FAPα protease, which is broadly expressed in CAFs in various solid tumors. RO7300490 displays its immunostimulatory effect by activating CD40 cell surface stimulatory TNF family receptor expressed on immune cells. CD40 engagement induces the proliferation and activation of B lymphocytes, differentiation of immunostimulatory macrophages, and activation of DCs to secrete inflammatory cytokines. This promotes the proliferation and activation of CTLs to kill tumor cells. A phase I clinical study (NCT04857138) evaluating safety, pharmacokinetics, and antitumor activity of RO7300490, as a single agent or in combination with atezolizumab, in participants with advanced and/or metastatic solid tumors, showed a favorable safety profile, and supported further clinical investigations of this agent in combination with other anticancer therapies [193]. Cells 2025,14, 1353 20 of 31 Table 5. Summary of clinical trials targeting cancer-associated fibroblasts in TNBC. Activity/Mechanism Agent Therapy Design Group of Patients Clinical Trial Status Targeting FAPα FAPαtargeted CD40 agonist antibody RO7300490 Monotherapy RO7300490 + atezolizumab Advanced and/or metastatic solid tumors NCT04857138 Phase I Completed Targeting FGFR signaling Pan FGFR 1-4 inhibitor Erdafitinib Monotherapy Advanced solid tumors NCT04083976 RAGNAR Phase II Completed Erdafitinib + Fulvestrant+ Palbociclib ER+/HER2-/FGFRamplified BC NCT03238196 Phase I Active, not recruiting Targeting Hedgehog signaling Smoothened receptor (SMO) antagonist Sonidegib (LDE225) Sonidegib + docitaxel Advanced TNBC NCT02027376 EDALINE Phase Ib Completed Inhibitor of SMO Vismodegib (GDC-0449) Vismodegib + standard neoadjuvant chemotherapy TNBC NCT02694224 Phase II Unknown status Reverting CAFs into quiescent state Vitamin D receptor agonist 19-nor-1,25dihydroxyvitamin D2 19-nor-1,25dihydroxyvitamin D2 + standard Neoadjuvant Chemotherapy Metastatic Breast Cancer NCT00637897 Phase I Completed RARβ agonist All trans retinoic acid (ATRA) ATRA + non-steroidal aromatase inhibitor anastrozole Hormonal receptor+/HER2early BC NCT04113863 Phase II Recruiting Targeting ECM Inhibition of hyaluronidase activity PEGPH20 PEGylated Recombinant Human hylaronidaze PEGPH20 + eribulin mesylate HER2-, high-hyaluronan metastatic BC NCT02753595 Phase Ib/II Terminated Inhibition of colagen and hyaluron production Losartan (angiotensin receptor blocker) Losartan + camrelizumab +doxorubicin TNBC treated with no more than 1 Prior line of chemotherapy NCT05097248 Phase II Unknown status (TNBC), estrogen receptor (ER), estrogen receptor (ER), breast cancer (BC), human epidermal growth factor receptor2 (HER2), fibroblast growth factor receptor (FGFR), retinoic acid receptor (RAR). Preclinical studies have consistently implicated FGFR signaling in breast cancer progression, while clinical evidence failed to support these findings. This may indicate that the clinical significance of FGFR should be analyzed in the context of the stroma, as the activation of resident fibroblasts can be a crucial factor for CAF generation and tumor progression [ 194 ]. Erdafitinib, a tyrosine kinase inhibitor of FGFR, was investigated in a phase Ib clinical study (NCT03238196) in combination with fulvestrant and palbociclib in ER+/HER2-/FGFR-amplified metastatic breast cancer. The study suggested some antitumor efficacy of this combination. Further investigations of erdafitinib in phase II RAGNAR (NCT04083976) clinical trial showed clinical benefit in tumor-agnostic setting in patients with advanced solid malignancies with susceptible FGFR alterations who have exhausted other treatment options. The results of this trial support the continued development of pharmacological inhibitors of FGFR for patients with advanced solid tumors [195]. The hedgehog signaling pathway is activated in the stroma of TNBC in response to hedgehog ligand secreted by cancer epithelial cells. Accordingly, a therapeutic approach that combines targeting cancer epithelial cells with chemotherapy and CAFs with hedgehog pathway inhibitor emerged as a potential approach to improve TNBC treatment outcome [ 196 ]. Recently, it was shown that the hedgehog signaling inhibitor sonidegib to downregulates the expression of CSC markers and increases the sensitivity of TNBC to paclitaxel in phase I clinical study (NCT02027376), thus improving patient survival and reducing metastasis in advanced TNBC [ 197 ]. Another inhibitor of hedgehog signaling, vismodegib (GDC-0449), was added to standard neoadjuvant chemotherapy in phase II clinical study (NCT02694224) due to its ability to inhibit breast CSC self-renewal and mammosphere formation [198]. The status of this clinical trial remains unknown. Cells 2025,14, 1353 21 of 31 5.2.1. Reverting CAFs into Quiescent State Therapies reverting CAFs to a quiescent state, such as targeting vitamin D receptor that represents a transcriptional suppressor of activated CAFs, have been shown to contribute to enhanced delivery of anticancer drug in animal models. In this sense, a phase I clinical trial (NCT00637897) investigated the effect of paricalcitol (19-nor-1,25-dihydroxyvitamin D2) in assisting chemotherapeutic drugs to more efficiently kill tumor cells. The study showed safety and feasibility in women with metastatic breast cancer receiving taxanes or ixabepilone [199]. All trans retinoic acid (ATRA) activates retinoic acid receptor (RAR) β and introduces CAFs into quiescent state by downregulating actin myosin contractility, thereby reducing CAF activities related to ECM remodeling and cell migration [ 200 ]. An Italian single-center randomized phase II clinical study (NCT04113863) is presently investigating preoperative activity of ATRA in HR+/HER2early breast cancer in combination with non-steroidal aromatase inhibitor anastrozole. 5.2.2. Targeting ECM As desmoplasia and increased interstitial pressure exert compression on tumor vasculature impeding the effective distribution of chemotherapeutic agents into tumor tissue, the inhibition of ECM production by CAFs represents another CAF-targeted therapeutic strategy. For example, enzymatic activity of hyaluronidase can be used to modify tumor architecture for a more effective delivery of chemotherapeutics. In this sense, hyaluronidase PEGPH20 in combination with eribulin versus eribulin alone showed in phase II clinical trial (NCT02753595) improved antitumor effects in subjects with HER2-negative breast cancer [201] . Furthermore, angiotensin receptor blocker losartan, also known for inhibiting the production of collagen and hyaluronan and suppressing TGFβ -mediated fibrotic signaling, represents another agent targeting the ECM that showed some efficacy in the preclinical model of TNBC [ 202 ]. Losartan was included in the treatment protocol in a phase II clinical trial (NCT05097248) evaluating PD-1 inhibitor camrelizumab and liposomal doxorubicin in patients with advanced or locally advanced TNBC, who were previously treated with no more than one line of chemotherapy. 6. Conclusions The accumulating evidence shows the importance of complex crosstalk between cancer cells, immune cells, fibroblasts, endothelial cells, molecular stromal components, and extracellular matrix components for tumor progression and response to therapy. The importance of TME for response to therapy is very pronounced in TNBC due to the lack of adequate molecular therapeutic targets confined to tumor cells and the absence of effective treatment options except for chemotherapy. Although multiple clinical trials have recently implied the benefits of introducing immunotherapy into the treatment of TNBC, the response rates remained low due to activation of additional suppressive mechanisms in TME during the course of therapy that undermine the therapeutic benefit. In this sense therapeutic targeting of the TME and its factors, including cytokines, enzymes, metabolites, chemokines, and signaling pathways in TME, represents potential complementary therapeutic strategy in TNBC. Furthermore, CAFs emerge as relevant targets due to their multiple roles in tumor initiation and progression. Moreover, CAFs confer cancer cell resistance to therapy, as they orchestrate immunosuppressive TME by interacting with immune cells and play a crucial role in the formation of ECM that serves as a physical barrier for therapeutic drugs and immune cells from reaching the tumor. However, based on the recent data obtained from clinical investigations, the most of currently available agents targeting cytokines, cytokine receptors, prostaglandin receptors, Cells 2025,14, 1353 22 of 31 IDO-1 activity and CAF-related targeting agents have reached phase I/II clinical trials. The leading number of developed pharmacological agents and clinical trials investigating them was related to blockade of TGFβ signaling and IDO inhibition. For efficient clinical application, further development of therapeutics with high specificity for their target in TME is needed, as well as experimental evaluations of strategies that simultaneously target several components in the TME. Furthermore, better understanding of the interactions between cells and molecular components in the TME is crucial for discovering novel druggable targets related to molecular and immunological aspects of TNBC. Author Contributions: Conceptualization, A.V. and V.J.; writing—original draft preparation, A.V., K.M.M. and V.J.; writing—review and editing, A.V., K.M.M. and V.J.; supervision, V.J.; funding acquisition, V.J. All authors have read and agreed to the published version of the manuscript. Funding: Ministry of Science, Technological Development and Innovation of the Republic of Serbia (Agreements No. 451-03-136/2025-03/200111 and 451-03-136/2025-03/200043). Institutional Review Board Statement: Not applicable. 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