The Tumor Microenvironment: Interactions Between Cancer Cells and Their Surroundings

1. Introduction

Since the stronghold of the idea that a tumor is merely an accumulation of malignant cells, the concept of the tumor microenvironment (TME) has developed into one of the most significant paradigms in contemporary cancer biology [1]. Tumors are not just comprised of cancer cells, but they’re also myriad adjacent non-malignant components, including immune cells, fibroblasts, endothelial cells, the extracellular matrix (ECM), signaling molecules and various biochemical factors affecting tumor behavior; thus, tumors are best described as complex and interactive ecosystems in which endless interplay amongst each constituent translates to respective roles in the proliferation, dissemination and response to therapy of cancer [2].

The TME can thus be defined as a heterogeneous and dynamic milieu in which cancer cells live within a mass of tissue and immune components that together represent an active cellular niche supporting or restricting tumor development depending on contextual cues [3]. This notion is not at odds with Paget's classical "seed and soil" hypothesis, where it has been proposed that the presence of permissive and supportive (the "soil") microenvironments is required for soluble (the "seed") tumor cells to survive, grow and metastasize, Within this framework, TME does not serve as a passive aggregator but participates in defining many facets of tumoral biology via biochemical signaling, mechanical communications or metabolic regulation [4].

At the cellular level, the TME is composed of various cell types and undergoes dynamic reciprocal interactions with cancer cells. Immune cells such as T lymphocytes may be anti-tumor or pro-tumor depending on their activation status and signals received. Tumor-associated macrophages (TAMs) are also highly relevant to remodeling the extracellular matrix, assisting in invasion, and secretion of growth factors that promote tumor proliferation [5].

Endothelial cells assist in angiogenesis, which is the formation of aberrant blood vessels supplying nutrients and oxygen to expand a growing tumour mass [4]. In addition to its cellular constituents, non-cellular components, including cytokines, chemokines, growth factors, and extracellular matrix proteins, establish a complex biochemical and structural milieu shaping cellular behaviors within the tumor niche [6].

The Cooperativity of TME signature feature of the tumor microenvironment is its dynamic, bidirectional nature; cancer cells are in constant communication through direct cell contact, paracrine signaling and the secretion of bioactive extracellular vesicles such as exosomes with surrounding cells and matrix components. Such interactions trigger activation multiple pathways that affect critical neoplastic processes including proliferation, invasion, metastasis, immunoediting, and microenvironment adaptation [7]. As an example, tumor-mediated immune evasion occurs because tumor cells skew responses toward immunosuppressive populations or export inhibitory cytokines to escape immune detection while stromal cells essentially support specific properties of tissues by modifying physical and biochemical characteristics leading to dissemination capabilities [8].

TME also differs extensively from healthy tissues through unique physiological conditions, including low oxygen levels due to poor and irregular blood supply, acidic pH arising from elevated glycolytic metabolism (the Warburg effect) [9], and competition between tumor cells and immune cells for nutrients. These types of conditions exert selective pressure in cancer cell clones toward increasing aggressiveness and hinder the functions of anti-tumor immune cells. Chronic inflammation represents another major characteristic feature of the TME It triggers genomic instability, further promotes tumorigenesis, and potentiates pro-carcinogenic environments [10].

This review covers a range of issues on the TME components and their significance for facilitating cancer progression. This considers the complex relationship between the cancer, stromal, immune cells, and extracellular matrix components that collectively impact tumor proliferation, metastasis, escape from immunosurveillance, and therapy resistance. Also, highlight new strategies for cancer therapy aimed at manipulating the TME.

2. Stromal Cellular Components of the TME

In general, TME consists of many malignant and non-malignant cells (immune and stromal cells), which are in crosstalk and exchange signals with each other via signaling molecules, cytokines, and others. They are closely related to boosting and supporting all stages of tumor development, progression, and metastasis, as well as affecting treatment response. Also, it is involved in mechanisms that feed the tumor and protect it from normal defenses, for example, angiogenesis, immune escape, and cancer cell stemness.

2.1. Mesenchymal Stem/Stromal Cells (MSCs)

MSCs are tissue-resident mesenchymal cells that are widely distributed in the body and are multipotent cells capable of differentiating into various lineages, including fibroblasts and endothelial cells [11]. Generally, MSCs have different functions in providing structural and functional support for organs and forming the extracellular matrix (ECM) [12]. Recently, they found that MSCs have an immune-modulatory function by expressing immune regulators and cytokine-signaling molecules and interacting with immune cells [12]. They also play a role in wound healing and inflammatory hemostasis by preventing excess inflammation, which is achieved by transforming into reactive stroma [13,14].

However, during tumorigenesis, tumor and non-tumor cells can transition MSC into tumor-associated stromal cells (TASCs). TASCs secrete many pro-tumorigenic substances, including IL-6, IL-8, stromal-derived factor-1 alpha, vascular endothelial growth factor, tenascin-C, and matrix metalloproteinases, which recruit additional tumor and pro-tumorigenic cells to the developing microenvironment [15,16]. Moreover, TASCs accumulate at the primary site to support tumor growth and recruit immune cells [17]. This pathological stroma can play a promotive or suppressive role in cancer development through various mechanisms [18].

In harsh conditions for the tumor, it secretes many molecules such as growth factors, angiogenesis cytokines, and MPP, which in turn boost blood and lymphatic vessels to nutrient supply and metastasis. Recently, many studies have been conducted to reveal the facts about these complex communications between stromal cells and tumor components.

It is referred to as a cancer-supportive agent that promotes the establishment of immune-shielded niches or the formation of new tumors through shifting into cancer-associated fibroblasts (CAFs) and cancer stem cells [17,19]. Conversely, because MSCs have a distinct ability to home to tumor sites after intravenous injection, they can be utilized as a vector to carry therapeutic agents selectively to cancerous regions [19,20]. An in vivo study showed that MSCs loaded with radiosensitive bismuth selenide (Bi₂Se₃) NPs enhanced accumulation in the tumor site by 20 times [21].

TASCs enhance cancer proliferation and Epithelial to Mesenchymal Transition (EMT) transformation, which is associated with invasion risk through upregulation of EMT-related genes and downregulation of E-cadherin [22,23].

Stromal cells have been shown to modulate effects on immune cells, such as macrophages, by different mechanisms to improve a favorable immunosuppression environment. It plays a role in macrophages switching from a pro-inflammatory M1 to an anti-inflammatory tumor-promoting M2 phenotype by lowering pro-inflammatory molecules (TNF-α, IP10, RANTES, MIP-1α) and increasing anti-inflammatory molecules (TGF-β, IL-10) [24]. It also enhances M2 recruitment via increase its migration mediators including (MCP-1, SDF-1, Chi3L1, M-CSF1, IL-6, IL-8) [24]. Moreover, it was found that macrophage phagocytosis suppression in colorectal cancer is mediated, in part, through an increase in expression of PD-L1 and CD47 on both human and murine stromal cells [21].

2.2. Endothelial Cells (EC)

Angiogenesis is essential for tumor proliferation and metastasis. ECs serve as the lining of the luminal side of blood and lymphatic vessels and are continuously in contact with blood and its circulating biomolecules, thereby serving as sensors of and responding to hemodynamic, paracrine, and endocrinal stimuli [25]. Tumor-associated endothelial cells (TECs) are mainly derived from tumor vasculature, and they may be formed by trans-differentiation from tumor stem cells, endothelial progenitor cells, or fusion between transformed cells and normal stem cells [26]. Due to the tumors’ vascular aberrant structure and function, characterized by irregular diameter and loss of tight intracellular junctions between ECs, this results in low intraluminal shear stress, which hinders the transport of antitumor agents and affects the quality and quantity of immune cell infiltration.

It also plays a critical role as a gatekeeper regulating adaptive immune cells' recruitment via chemokines and adhesion molecules, and then prompts its dysfunction, thus enhancing immune surveillance escape [27]. Moreover, TEC protein abnormalities promote hypoxia and acidosis that reinforce immune evasion by recruiting immunosuppressive cells and preventing effector T-cell infiltration through suppression of key integrins and selectins, which serve as a guide for leukocytes [27].

ECs can be transformed into mesenchymal cells via endothelial-to-mesenchymal transition (EndMT) to promote tumor angiogenesis and cancer invasion by enhancing tumor cell motility [28]. This results in further compromise in the above tumor vascular irregularities while facilitating tumor cell intravasation [29]. During the EndoMT process, ECs lose their endothelial identity, downregulating endothelial-specific markers such as VE-cadherin, CD31, Tie-1, Tie-2, and vWF, while concomitantly upregulating mesenchymal-related markers, including SMA, SM22α, N-cadherin, vimentin, COL1/III, and FSP-1 [30]. Mesenchymal cells also contribute significantly to the pool of CAFs, which promote tumor metastasis through secreting cytokines such as TGF-β1 [31].

Across various tumors, ECs may exhibit different upregulated markers, which give them new phenotypes to further modulate tumor growth. Recent EC subtypes are listed in Table (1).

Table 1. Endothelial cell subtypes and mechanisms that promote cancer growth and metastasis.

Cancer type	EC Subtype	Mechanism Of Enhancing Metastasis	Reference
Gastric cancer (GC)	COL1A1+ ECs play a critical role in tumor progression and metastasis.	Undergo EndoMT by upregulation of CEBPB and crosstalk with malignant cells via the ANGPTL4-SDC4 axis	[28]
Gallbladder cancer (GBC)	CD34+CD90+ ECs (SAEndo2) exhibit EndoMT properties	Activation of TGF-β signaling, showing EndoMT properties	[32]
Colorectal cancer	Nrdp1lowECs, which is closely correlated with a higher metastatic probability	Cancer cell-derived VEGF triggers Nrdp1 degradation in tumor ECs and increases Fam20C secretion, disrupting vascular basement integrity	[33]
breast cancer	Plasma EC VWF: Ag is inversely correlated with cancer metastasis and patient survival.	Breast cancer-induced VWF release from ECs by secretion of VEGF-A. Promotes tumor adhesion and trans endothelial migration,	[34]
Non-small cell lung cancer	miR-186low induces angiogenesis.	Whose gene is PKCα, downregulated by hypoxia via activation of HIF1a	[35]

Table 1. Endothelial cell subtypes and mechanisms that promote cancer growth and metastasis.

Cancer type	EC Subtype	Mechanism Of Enhancing Metastasis	Reference
Gastric cancer (GC)	COL1A1+ ECs play a critical role in tumor progression and metastasis.	Undergo EndoMT by upregulation of CEBPB and crosstalk with malignant cells via the ANGPTL4-SDC4 axis	[28]
Gallbladder cancer (GBC)	CD34+CD90+ ECs (SAEndo2) exhibit EndoMT properties	Activation of TGF-β signaling, showing EndoMT properties	[32]
Colorectal cancer	Nrdp1lowECs, which is closely correlated with a higher metastatic probability	Cancer cell-derived VEGF triggers Nrdp1 degradation in tumor ECs and increases Fam20C secretion, disrupting vascular basement integrity	[33]
breast cancer	Plasma EC VWF: Ag is inversely correlated with cancer metastasis and patient survival.	Breast cancer-induced VWF release from ECs by secretion of VEGF-A. Promotes tumor adhesion and trans endothelial migration,	[34]
Non-small cell lung cancer	miR-186low induces angiogenesis.	Whose gene is PKCα, downregulated by hypoxia via activation of HIF1a	[35]

2.3. Cancer-Associated Fibroblasts (CAFs)

CAFs serve as primary constituents of the ECM of connective tissues and have a tissue reparative role in injury conditions [36]. They also have a secretory role by enhancing angiogenesis and coordinating immune responses through releasing VEGF-A, chemokines, and cytokines, respectively [36]. CAFs originate from MSCs and tumor-associated endothelial cells [37].

CAFs are widely known for their significant plasticity; CAFs can respond to local autocrine and paracrine signaling from the microenvironment, which could influence CAF functions within a tumour. This explains the substantial subpopulation of CAFs and their dual function as tumor-promoting and tumor-restraining [38]. CAFs play a critical role in ECM shaping, crosstalk with immune cells to enhance immunosuppression status, and promoting cancer cell proliferation, angiogenesis, and drug resistance [37,38].

It was found that direct CAF-tumor interactions act as a tumor-promoting mechanism, mediated by myCAF and iCAF, while the opposite effects are seen in type I collagen-expressing myCAF and myCAF216-CM [39,40].

MyCAFs and iCAFs are frequently observed across various cancers and differ in spatial distribution, where myCAFs are positioned adjacent to tumor cells, while iCAFs are distributed distantly[41]. The emergence of many intermediate groups that share biomarkers of the main groups, such as newly reported αSMA-highIL-6high CAFs, which contribute to chemoresistance and share i-and my-CAFs subtypes and they can transform from one subtype to another under the influence of signals from the surrounding environment [42,43]. This makes it important to carefully reclassify CAFs.

myCAFs or iCAFs can be distinguished from each other based on the expression of the following markers: FAP, ACTA2, HOPX, MYH11 and POSTN, and iCAFs: IL6, CXCL12 and PDGFA, CCL11, and CXCL14 [40,44,45,46]. CXCL12 (C-X-C motif ligand 12) is a chemotactic chemokine, a marker of ICAFs.

It was shown that iCAFs associate with polarization of M2 macrophages and inactivation of CD8+T cells and induce their apoptosis via Galectin-1 (LGALS1), TGFB1, NFATC2, and macrophage migration inhibitory factor (MIF)-CXCR4 interaction[45]. CAFs compromise the activation and recruitment of T lymphocytes in cancerous regions by secreting cytokines and chemokines like TGF-β and CXCL12. [38] CXCL12 induces transformation of T cells into Tregs, which enhance immunosuppression, promoting the generation of proangiogenic macrophages and dysfunctional dendritic cells (DCs) [38]. In addition, CAFs induce Siglec-10 on macrophages and NK cells, which can attenuate macrophage phagocytosis and NK cell cytotoxicity, thereby enhancing immune escape [37]. Fibroblast activation protein (FAP), which is highly expressed by CAFs in pancreatic cancer, acts by secreting TSLP, a cytokine induce tumor infiltration of Th2 cells and is associated with poor survival [47].

CAFs are associated with a pro-angiogenic effect. CAFs produce angiogenesis factors, such as VEGFA, PDGFC, FGF2, CXCL12, osteopontin, and CSF3, to stimulate and accelerate tumor-associated blood vessel formation by recruiting myeloid cells, vascular endothelial cells, and monocytes [38]. CAFs also induce angiogenesis by secreting CHI3L1-induced IL-8 and CTGF [38,48]. Moreover, another mechanism of neovascularization CAFs by it induce activation of ECs by STAT3 activation via IL-6 and CCL2 secretion; in part, PDPN + CAF is involved [49,50]. CAFs can also facilitate vascular mimicry (VM) formation via the Notch2-Jagged1 pathway [51].

3. Extracellular Matrix and Tumor Architecture

The ECM is a complex network of proteins and glycans that provides structural support to tissues and regulates cellular processes such as proliferation, differentiation, and migration[52]. ECM can be classified into two groups: the interstitial matrix and the basement membrane. Basement membranes are thin layers of ECM that form the supporting structure under epithelial and endothelial cells. The basement membrane has a distinctive composition containing several types of collagens, especially type IV, laminins, and proteoglycans [53]. The interstitial matrix, which is primarily produced by stromal cells, fills in the interstitial space between cells, and it contains the interstitial fluid and connective tissue matrix, including collagen, elastin, and glycosaminoglycans [54].

Cancer development and progression require extensive reorganization of the extracellular matrix. Altering the balance of the ECM signal is sufficient to induce a variety of cancers to develop and progress [53].

In the early stages of tumor development, stromal cells produce large amounts of ECM components to protect normal tissue. This leads to increased tissue stiffness due to the accumulation of collagen and hyaluronic acid[55]. With chronic damage, excessive ECM production results in fibrosis, and this heightened ECM deposition is strongly associated with tumor initiation and progression [56].

The ECM is composed of a diverse array of molecules, including proteoglycans, glycosaminoglycans, collagen, elastin, fibronectin, laminin, and matrix metalloproteinases (MMPs) [57].

The ECM also consists of glycoproteins, which have an important role in the migration of tumors. A glycoprotein consists of α, β, and γ chains that come together to form trimeric proteins. Laminins are one type of glycoprotein, and they are found within the basal lamina and contribute to differentiation, adhesion, migration, as well as play a crucial role in wound healing [58].

Numerous studies have demonstrated that dysregulation of laminin (LM) expression and distribution, including alterations in specific laminin chains, is closely linked to tumor progression and cancer development across a broad spectrum of human cancers. For example, in breast cancer, there is a downregulation in LM-111 that will cause tumor proliferation, but in pancreatic cancer, there is upregulation in LM-332 that will encourage tumor migration and metastasis [58].

Fibronectin (FN) is a major ECM protein involved in a wide range of physiological processes, including cell migration. FN has several domains. It exists as a dimer, with each monomeric FN unit consisting of repeating type I, type II, and type III domains, and is involved in the interactions between the ECM and cells. Fibronectin forms a fibrillar network and is key to cell differentiation, adhesion, and migration [59].

Phosphorylation of FN modulates its biological activity and influences cellular behavior. In vitro studies have demonstrated that phosphorylated FN promotes fibroblast adhesion, spreading, and metabolic activity, while also enhancing cellular traction forces and mechanical strain, thereby strengthening cell–ECM interactions. [59]

Fibronectin can bind to collagens, laminins, and other proteins, allowing cells to adhere to the ECM and migrate. The binding of cancer cells to ECM proteins, including fibronectin, can protect cells from drug-induced apoptosis compared to cells attached to plastic [56].

3.1. Cell Adhesion Molecules

Tumor invasion and metastasis are the primary cause of the fatal outcome of cancer diseases. A feature of metastatic cells is the considerable flexibility in their adhesive interactions with other cells or components of the extracellular matrix. [60]

The main adhesion molecules are integrins, cadherins, and selectins. These adhesion molecules enable cancer cells to bind more effectively to the ECM [61]. Some of these adhesion molecules are for cell-cell adhesion (Cadherin, Selectin), and others for cell-ECM adhesion (Integrins).

3.1.1. Cadherins

Cadherin–catenin complexes are integral components of adherens junctions that are essential for mediating cell–cell adhesion and maintaining tissue homeostasis. There are three major types of cadherin: E-cadherin, N-cadherin, and P-cadherin [62]. Dysregulation of these complexes contributes to cancer development by altering cell-intrinsic oncogenic signaling pathways as well as modulating the tumor microenvironment [63].

All cadherins are transmembrane proteins composed of three main domains:

• an extracellular cadherin (EC) domain that mediates homophilic cadherin–cadherin interactions;
• a single-pass transmembrane domain; and
• a cytoplasmic domain that links the cell surface to the cytoskeleton [64].

The cytoplasmic domain of E-cadherin binds to the members of the catenin protein family, β-catenin, and p120[64]. So, the frequent loss of p120 expression promotes the degradation of E-cadherin in lung cancer. Consequently, β-catenin expression is upregulated in association with reduced E-cadherin levels, leading to activation of downstream signaling pathways involved in tumorigenesis[65].

In many types of cancers, the loss of E-cadherin is mediated by the activation of transcription factors such as SNAIL, SLUG, TWIST1, ZEB1, and ZEB2. These factors bind to E-box sequences within the CDH1 gene promoter, thereby repressing its transcription and reducing E-cadherin expression[65]Loss of E-cadherin is the primary cause of tumor invasion[66].

N-cadherin is anchored to the intercellular actin cytoskeleton by interaction with the β-, α-, and γ-catenin complex; its expression is low in normal cells [64]. However, it has been reported that overexpressed N-cadherin is associated with cell migration and angiogenesis [67].

3.1.2. Integrins

Integrin-mediated adhesion to the ECM consists of 18 α and 8 β subunits that assemble into 24 different heterodimeric receptors. These receptors display ligand-specific binding properties and are regulated at both transcriptional and translational levels in a tissue-specific manner, thereby controlling cell–extracellular matrix interactions[68]It is essential for normal tissue development and homeostasis, but its regulation is often disrupted in cancer, contributing to tumor progression and metastasis [69]. The phosphorylation of the integrin β chain is important for the function of adhesion of integrins with the ECM [70].

Integrins transmit signals that regulate cell survival by pathways like PI3K/AKT, the dysregulated of these pathway make dysfunction of integrins and can enable cancer cells to resist apoptosis, helping abnormal cells survive [71].

The role of integrin in tumor migration is that β4 integrin interacts with several receptor tyrosine kinases (TKRs), including EGFR, HER2, MET, and RON, sustaining their signaling activity. Upon ligand binding, these receptors activate SRC-family kinases (SFKs), which phosphorylate the β4 integrin cytoplasmic tail and TKR-associated proteins. These phosphorylation events create an amplified signaling platform that enhances downstream TKR pathways, leading to hemidesmosome disassembly and promoting cancer cell migration and invasion [69].

3.1.3. Selectins

Selectins are cell adhesion proteins it binds the cells to the ECM. Selectins represent the first group of cell adhesion molecules composed of three different glycoproteins, expressed either at the surface of endothelial cells (E-Selectin), leukocytes (L-Selectin), or platelets (P-Selectin), it plays a distinct role in inflammation, hematopoiesis, and tumor biology [72].

P-selectin (SELP) is rapidly expressed on activated endothelial cells and platelets, where it mediates leukocyte recruitment and cell adhesion through its primary ligand, PSGL-1. Binding of SELP to PSGL-1 activates intracellular signalling pathways, including Src and ERK kinases, regulating cellular functions and cytokine production. Additional SELP ligands, such as CD44 and CD24, are involved in EMT, cancer stem cell biology, and tumor metastasis [73].

3.2. Matrix Remodelling

3.2.1. Matrix Metalloproteases (MMPs)

MMPs represent an important group of proteases involved in the breakdown of ECM constituents, thereby contributing to ECM remodelling and reconstruction.[74]. MMPs constitute a family of 23 zinc-dependent endopeptidases involved in a wide range of physiological and biological functions. The activity and expression of MMPs are tightly controlled by multiple regulatory factors, including cytokines, hormones, and growth factors. [57]. Each of these MMPs is synthesized by different cell types; for example, MMP-1, synthesized by macrophages, fibroblasts, and dendritic cells, contributes to cell survival and may facilitate tumor development.[75].

These proteins, including MPPs, are involved in the degradation of the extracellular matrix and whose functions are modulated by tissue metalloproteinase inhibitors called TIMPs. Maintenance of tissue homeostasis depends on a finely regulated balance between MMPs and tissue inhibitors of metalloproteinases (TIMPs). Any imbalance favoring MMP activity can promote ECM remodeling and degradation, leading to the progression of inflammation, tissue damage, and disease development [75].

Research on MMP-2, MMP-7, MMP-9, MMP-13, and MMP-14 has received considerable attention since they play several roles in cancer. Each MMP is overexpressed in different types of cancer; for example, in breast cancer, MMPs -1,-9,-15,-23 are overexpressed, and in liver cancer, MMPs -3,-9,-12,-14 are overexpressed [74]. Cancer cells stimulate the surrounding cells to increase the production of MMPs by stimulating the secretion of interferon, interleukins, growth factors, and especially the extracellular MMPs [57].

Gelatinase A (MMP-2) and Gelatinase B (MMP-9) are key members of the MPP family that play crucial roles in ECM remodeling by degrading type IV collagen, a major component of the basement membrane. MMP-2, a 72-kDa enzyme, is additionally capable of degrading type I, II, and III collagens[74].

Gelatinase B (MMP-9) has a molecular weight of 92 kDa, it is found in very small amounts in normal tissues, and is secreted by dendritic, hematopoietic, macrophage, neutrophil, fibroblast, and lymphocyte cells[57]. Polymorphisms in the promoter region of the MMP-9 gene may modulate transcription and protein synthesis, thereby altering extracellular matrix degradation. Consequently, these genetic variations may contribute to increased susceptibility to the development and progression of disease.[75]. Gelatinase B has a critical role in Inflammation, metastasis, and degradation of collagens (types I, IV, V, XI, and XIV), gelatin, elastin, vitronectin, and laminin[74]. Interestingly, melatonin has been reported to inhibit the MMP-2 and MMP-9 signaling pathways in breast cancer cells [76].

MMP-14, also known as membrane-type 1 matrix metalloproteinase (MT1-MMP), is expressed by a variety of cell types, including fibroblasts, macrophages, endothelial cells, and hematopoietic cells. Cancer has a role in the degradation of collagens (Types I, II, and III), Gelatin, Fibronectin so promoting the angiogenesis [74].

3.2.2. Collagen Deposition

Collagen is the most abundant structural protein in the extracellular matrix (ECM) and plays a central role in the dynamic interactions between tumor cells and their surrounding microenvironment. Beyond providing mechanical support, collagens actively regulate various cellular processes, including cell adhesion, migration, proliferation, differentiation, and survival. Alterations in collagen deposition, organization, and remodeling contribute significantly to tumor progression, invasion, metastasis, and therapeutic resistance [77].

Collagens are a family of 28 extracellular matrix proteins containing α chains, classified into fibrillar and non-fibrillar types based on their structure [77].

Fibrillar collagens represent approximately 90% of the total collagen content and include collagen types I, II, III, V, XI, XXIV, and XXVII. They provide structural integrity and tensile strength to tissues and are the principal components of the interstitial extracellular matrix.[78]

The increased stiffness of the tumor extracellular matrix can arise from several biomechanical mechanisms. These include an imbalance between extracellular matrix deposition and degradation, dynamic interactions between tumor cells and surrounding stromal cells, and, most importantly, alterations in collagen cross-linking. Together, these processes contribute to extracellular matrix remodeling and the development of a mechanically rigid tumor microenvironment that promotes cancer progression [79]. Increased collagen accumulation and remodeling enhance extracellular matrix stiffness, creating a microenvironment that favors tumor growth and survival. Through interactions with integrins and other collagen-binding receptors, cancer cells receive signaling cues that promote proliferation, invasion, migration, metabolic alterations, therapeutic resistance, and immune escape, thereby facilitating cancer progression and metastasis[80].

Discoidin domain receptors (DDRs), a family of receptor tyrosine kinases consisting of DDR1 and DDR2. Different collagen types preferentially bind to specific DDRs [80].Activation of DDRs by collagen regulates several cellular processes involved in tumor progression. For instance, activation of the collagen IV–DDR1 signaling axis stimulates the AKT pathway and promotes the expression of matrix metalloproteinase-9 (MMP-9), enhancing the migration and adhesion of leukemia cells within the bone marrow microenvironment [81].

Monomeric collagen has been demonstrated to inhibit p21 expression in tumor cells, disrupting normal cell cycle control and facilitating increased cellular proliferation[80].

Under normal conditions, p53 regulates collagen production and promotes the formation of anti-angiogenic factors that suppress tumor growth, whereas p53 mutations disrupt this control, leading to abnormal collagen remodeling, enhanced angiogenesis, and tumor progression [81].

Increased collagen density promotes integrin activation, which subsequently triggers the PI3K/Akt signaling pathway. Activation of this pathway upregulates key metabolic mediators, such as GLUT1 and PKM2, thereby enhancing glucose uptake and aerobic glycolysis. These metabolic alterations contribute to the Warburg effect and support cancer cell proliferation and survival [82].

4. Immune Evasion in Cancer

4.1. Antigen-Loss Variants

Antigen-loss variants are subsets of cells, typically cancerous cells or viruses, that have either completely lost or greatly diminished expression of a particular target antigen [83]. This process is a key mechanism in immune evasion, where the immune system’s selective pressure, whether spontaneously or brought on by treatment, allows cells that the immune system is no longer able to recognize to survive and proliferate under the selective pressure of the immune system [84,85]. In the context of current immunotherapy, specifically Chimeric Antigen Receptor (CAR) T-cell therapy, antigen loss variants are a significant factor in treatment resistance and clinical relapse [86].

4.1.1. Mechanisms of Antigen Loss in Cancer

The disappearance of target antigens in cancerous cells is a complicated, multifaceted phenomenon that can take place at the genomic, transcriptomic, or protein levels. These processes enable tumor cells to evade targeted treatments while still retaining their cancer-causing capabilities [87].

• Genomic and Transcriptomic Alterations:

In cases of B-cell cancers treated with CAR-T cells targeting CD19, relapses often occur due to genomic losses caused by deletions or mutations [88]. Alternative splicing, on the other hand, has become a highly complex mechanism. For example, tumor cells may generate a CD19 variant that omits exon 2, which has the epitope addressed by the majority of CAR-T designs [89]. Although the cell continues to express CD19, the “target” is essentially absent [89].

• Trogocytosis and internalization

Antigen loss is not necessarily permanent or genomic. Trogocytosis, sometimes referred to as “shaving,” happens when CAR-T cells bind with tumor cells and physically remove the target antigen in combination with the pores of the tumor cell membranes [90]. In addition to lowering the tumor’s antigen density, this can result in “fratricide”, a situation in which CAR-T cells unintentionally target one another after acquiring the tumor antigen on their own surface [91].

• Lineage Plasticity and Switching

Specifically in leukemias with KMT2A (Lysine Methyltransferase 2A), formerly known as MLL (Mixed-Lineage Leukemia ) rearrangements, lineage switching may result from lymphoid-targeted therapy’s selective pressure [92,93]. In this process, B-cell markers such as CD19 and CD22 are lost as a result of malignant cells’ trans differentiation from a lymphoid to a myeloid phenotype [94].

4.2. Immune Suppression Mechanisms

4.2.1. Immune-Checkpoint Mediated Suppression

Our immune system incorporates multiple regulatory mechanisms to maintain immune homeostasis and prevent severe immune responses that can result in tissue damage or autoimmunity. As part of these mechanisms, immune checkpoints are inhibitory pathways that control T-cell activation and effector responses to retain immune homeostasis and self-tolerance under physiological conditions [95].

The major immune checkpoints include: programmed cell death protein (PD-1), Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), Lymphocyte-activation gene 3 (LAG-3), and B7-Homolog 3 (B7-H3). PD-1 is a checkpoint that arises in naive T-cells 2-6 hours after the activation of the T-cell receptor, which contributes to T-cell suppression across priming, differentiation, and memory stages. PD-1 has two ligands: PD-L1 and PD-L2; the first one is regularly expressed on vascular endothelium, epithelial, and lymphoid cells, allowing for maintaining appropriate immune responses, while the second ligand is largely expressed on immune cells like antigen-presenting cells (APCs) [96]. In normal conditions, PD-1 is an inhibitory immune checkpoint that helps in preventing excessive activation of T-cells and safeguarding tissues from damage caused by the immune response [97].

4.2.2. Regulatory T-Cells and Immune Tolerance

Regulatory T cells (Tregs) are specialized T-cell subtypes that maintain immune homeostasis and self-tolerance by limiting excessive immune responses. In oncology, their presence in the TME promotes immune evasion, enabling tumor cells to escape host surveillance. The suppressive function of Tregs is regulated by the forkhead box protein P3 (FoxP3) transcription factor, which operates as the key regulator of their formation and stability [98].

Tregs are widely classified into three groups: induced Tregs (iTregs), peripheral Tregs (pTregs), and thymus-derived Tregs (tTregs). Each of these aids in immunological tolerance by diverse molecular and cellular targets [99]. In the context of cancer, effector Tregs (eTregs) are highly activated subsets that are notably abundant in the TME. eTregs exhibit enhanced proliferation and potent suppressive capacity, marked by elevated expression of activation markers such as CD44 and immune checkpoints such as CTLA4 and TIGIT [100].

Primary experimental research has explained various ways by which Tregs inhibit anti-tumor immunity. A fundamental pathway, CTLA-4-driven trans endocytosis and Trogocytosis of costimulatory ligands. Ovcinnikovs et al. (2019) showed that utilizing in vivo models outperforms ordinary T-cells in collecting CD80 and CD86 from migrating dendritic cells (DCs) through trans endocytosis, thereby reducing CD28-mediated stimulation [101]. Further research by Tekguc et al.2021 revealed that CTLA-4 expressed by Tregs helps in the formation of stable immune synapses with antigen-presenting cells, allowing for the extraction of CD80/CD86 via Trogocytosis. Notably, this reduction of CD80 on DCs increases the amount of PD-L1 on the DC surface, which inhibits PD-1+ effector T-cells [102].

Another critical mechanism is the deprivation of interleukin- 2 (IL-2) from the local environment. Chinen et al. (2016) employed genetic gain and loss of function strategies to demonstrate that the high-affinity IL-2 receptor (CD25) on Tregs behaves as a “sink” sequestering IL-2 and restricting the activation of CD8+ T-cells. This sequestration is associated with IL-2R-dependent STAT 5 signaling, which is essential for maintaining Treg-suppressive activity independently of TCR signaling [103]. In addition to that, the adenosine pathway acts as a significant metabolic barrier. While human Tregs often lack sufficient CD73 to completely convert ATP into adenosine, Schneider et al. 2021 found that activated CD8+ T-cells emit extracellular vesicles containing CD73. These vesicles work in conjunction with Treg-expressed CD39 to break down extracellular ATP into immunosuppressive adenosine, which subsequently elevates the levels of intracellular cAMP in effector cells, thereby reducing their proliferation [104].

4.2.3. Myeloid-Derived Suppressor Cells (MDSCs)

MDSCs are a heterogeneous population of immature myeloid cells that exhibit potent immunosuppressive activity. These cells undergo expansion during chronic inflammation and malignancy, exerting a pivotal function in immune evasion [105]. MDSCs are categorized into two subsets: monocytic and polymorphonuclear. Both subsets are involved in immune suppression with distinct mechanisms and varying prevalence in different tumor types [106]. In cancer patients, polymorphonuclear type constitutes the majority of the MDSC population.

4.3. Mechanisms of Immune Suppression by MDSCs

4.3.1. Arginine Depletion

The prominent mechanism involves the depletion of L-arginine, an amino acid necessary for T-cell proliferation and function. MDSCs accomplish this by upregulating arginase -1, an enzyme responsible for metabolizing L-arginine into urea and L-ornithin crating an arginine-poor environment that disrupts T-cell activation and proliferation [107]

4.3.2. Reactive Oxygen Species (ROS) and Nitric Oxide Production

MDSCs suppress antitumor activity through the production of reactive oxygen species and nitric oxide. ROS generated by NADPH oxidase 2 and nitric oxide synthesized by inducible nitric oxide synthase induce oxidative stress and impair T-cell function. In addition to that, the interaction of nitric oxide with superoxide anions forms pyroxinitrite (PNT), a highly reactive molecule that affects the signaling of the T-cell receptor and decreases T-cell migration. Polymorphonuclear MDSCs mediate suppression mainly through ROS and PNT production, while monocytic MDSCs rely mainly on inducible nitric oxide synthase-derived nitric oxide [108].

5. Tumor-Derived Factors and Exosomes

5.1. Overview of Tumor-Derived Factors

Tumor progression has shifted away from a purely genetic model governing the autonomous changes of malignant cells, Recent evidence shows that cancer cells engage in active regulation of their niche [109], continuously releasing biologically active molecules termed collectively as tumor-derived factors, They modify stroma, immune populations, endothelium and extracellular matrix composition to promote a favourable context of tumor growth, dissemination and resistance to therapy [110]. Tumor-derived mediators are a heterogeneous panel of soluble and vesicle-associated material including cytokines, chemokines, growth factor, proteolytic enzymes, metabolites, and extracellular vesicles such as exosomes [111].

Tumor cells can act in an autocrine manner, where they stimulate themselves and have a paracrine effect by modifying neighboring cells [112]. These signalling pathways are coopted by cancer cells to drive angiogenesis, immune evasion, metabolic adaptation, and metastatic spread.

Tumor-derived factors play many roles, perhaps the most prominent of which is converting normal tissue into a supportive tumor microenvironment [113]. A major example of molecules that promote neovascularization includes vascular endothelial growth factor (VEGF), which enables the tumor to receive oxygen and nutrients as it increases in mass [114]. Perhaps low-grade chronic physical inflammation is maintained by inflammatory cytokines such as IL6[115], while TGF-β induces Epithelial-to-Mesenchymal Transition (EMT), fibrosis, and immune suppression [116]; MMPs secreted from stromal cells can characterize and simultaneously degrade extracellular matrix barriers and promote local invasion [117]. In addition to local effects, tumor-derived factors may exert systemic actions across the host, such as cachexia, systemic immune suppression, altered hematopoiesis and pre-metastatic niche formation [118]. This suggests that tumors are instead more accurately described as dynamic pathophysiological organs, which can modulate biology at distant sites rather than the masses of cancerous cells [119].

Exosomes have attracted special attention as mediators derived from tumor cells, as they can transfer proteins and lipids, DNA fragments, messenger RNA (mRNA), and microRNA (miRNA) between cells [111]. Such nanovesicles broaden the communication distance of tumor cells and are indispensable for angiogenesis, immune modulation, metastasis as well as resistance to chemotherapy and immunotherapy [120]. Explaining these molecular networks is now a part of modern oncology, as effective cancer treatment requires not just attacking the tumor cells themselves, but also disrupting what they do to counter and shape their microenvironment [121]. Additionally, metabolic byproducts like lactate, produced via aerobic glycolysis (the Warburg effect) [122], exacerbate the acidification of the TME, further inhibiting immune cell activity and promoting tumour viability [123].

Tumor-derived exosomes can promote immunosuppression through the delivery of inhibitory cytokines such as TGF-β and IL-10, leading to reduced T-cell activation and proliferation. Conversely, exosomes can also contribute to immunostimulant by facilitating antigen presentation to immune cells, including T and B lymphocytes, thereby enhancing adaptive immune responses under certain conditions [124].

5.2. Soluble Tumor-Derived Mediators

Tumor-derived soluble mediators are biologically active molecules secreted by malignant cells into the TME, in which they mediate communication between cancer cells and surrounding stromal, vascular, and immune components [125]. Such mediators consist of cytokines, chemokines, growth elements, proteolytic enzymes, and metabolic items [126]. There are many therapeutic targets among those mediators because soluble tumor-derived mediators are core regulators of cancer biology [127].

5.2.1. Cytokines

Cytokines are small soluble proteins that serve as critical mediators of inflammation, immunity, proliferation, differentiation and cell survival within the tumor microenvironment [128]. Cytokine signaling can frequently be dysregulated in malignancy, contributing to chronic inflammation and driving tumor progression [129]. Some pro-inflammatory cytokines, such as interleukin-1 (IL-1), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α) and interleukin-8 (IL-8) and several signaling pathways, including NF-kB [128,130], the Janus kinase-signal transducer signal activating transcription factors pathway, collectively referred to as JAK/STAT3 pathway, and MAPK, have been activated[131]. These pathways contribute to proliferation, evasion of apoptosis, angiogenesis, and metastatic potential [132]. As those, immunosuppressive cytokines such as transforming growth factor-β (TGF-β) or interleukin (IL)-10 render inhibition of cytotoxic T lymphocytes and natural killer cells and the recruitment of regulatory T cells and TAMs [128]. This immunosuppressive microenvironment, in turn, inhibits immune-remediated elimination [133].

5.2.2. Chemokines

Chemokines are a class of cytokine-like proteins, specifically tuned for the purpose of directing cell migration via chemotaxis [134]. In contrast, in cancer, chemokine signaling is frequently commandeered to support cancer growth by enhancing angiogenesis and immune evasion as well as facilitating metastasis [135]. Major cancer-related chemokine axes and their cognate chemokine receptors: CXCL12/CXCR4, CCL2/CCR2 and CCL5/CCR5 [136]. These pathways promote tumor cell invasion, recruitment of stroma, and infiltration with immune-suppressive populations (myeloid-derived suppressor cells and regulatory T cells)[137]. CXCR4-positive tumor cells have frequently been shown to be responsible for metastases to organs, such as bone marrow, lung, and liver [134].

5.2.3. Growth Factors

Growth factors are soluble proteins that guide proliferation in continuous form, survival, migration, and differentiation [138]. These become commonly overexpressed by tumor or stromal cells in cancer and result in sustained activation of pro-cancer signaling pathways [129]. Such as epidermal growth factor (EGF), transforming growth factors − alpha and beta, insulin-like growth factor (IGF), vascular endothelial growth factor/vascular permeability factor (VEGF/VPF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF) , Such molecules activate PI3K/AKT, MAPK and mTOR pathways, which are involved in stimulating cell-cycle progression and inhibiting apoptosis [139,140]. VEGF is a key regulator in angiogenesis, whereas TGF-β also promotes epithelial–mesenchymal transition and metastatic dissemination [141].

5.2.3. Proteolytic Enzymes

These molecules have the ability to degrade components of the ECM, which under normal physiologic conditions are typically associated with biological processes like inflammation, tissue remodeling, or wound healing; but when over-activated in cancer these factors become dysregulated conferring invasion and metastasis [142]. One of the most studied families is the subclass of MMPs, especially MMP-2 and MMP-9 that are important for collagen, laminin, and fibronectin degradation [143]. If these adhesive junctions are disrupted, tumor cells may proliferate into adjacent tissues and enter the blood or lymphatic system [144]. An equally famous proteolytic pathway activated from proenzyme is the urokinase plasminogen activator (uPA) system that activates plasmin for the degradation of matrix [145]. The data on the high expression of uPA and the fact that it predicts more aggressive behavior for tumors, as well as poor prognosis [146].

5.3. Role of Tumor-Derived Factors in Tumor Progression

One of the most important functions regarding cancer progression by tumor-derived factors is communication and manipulation of the microenvironment surrounding malignant cells [147]. Such factors include cytokines, chemokines, growth factors, proteolytic enzymes, extracellular vesicles, and metabolic products secreted by tumor cells [148]. They act not in isolation but instead as an interconnected signaling network that drives proliferation, angiogenesis, invasion, metastasis, the evasion of immunity and therapy resistance [129].

5.3.1. Tumor Cell Proliferation

Tumor-derived factors play crucial roles in tumor-cell proliferation, and these growth-promoting ligands can be epidermal growth factor (EGF), transforming growth factor-alpha (TGF-α), insulin-like growth factors (IGFs), and hepatocyte growth factor (HGF), which are secreted by the cancer cells to activate receptor tyrosine kinases and downstream pathways PI3K/AKT, MAPK and mTOR, enabling the cells to re-enter the next phase of the cell cycle along with protecting them from programmed death as well [132].

5.3.2. Angiogenesis

When tumors become larger, demand for oxygen exceeds supply, leading to hypoxia [149]. Consequently, pro-angiogenic mediators (VEGF, fibroblast growth factor [FGF], platelet-derived growth factor [PDGF], and angiopoietins) are produced by tumor cells [150]. These molecules induce proliferation, migration and new blood vessel formation in endothelial cells (Lee et al., 2025). Vessels have a dual role, which sustains the necessary oxygen and nutrients for the tumor-associated vasculature [151]. Yet these vessels are usually aberrant, tortuous, and leaky [152]. This altered architecture leads to inadequate perfusion, regional hypoxia, increased interstitial hypertension, and lower delivery of the drug, which in turn aids cancer progression [153].

5.3.3. Invasion and Metastasis

Tumor-derived factors play an essential role in invasion and metastatic dissemination. Proteolytic enzymes, such as MMPs, cathepsins, or urokinase plasminogen activator (uPA), degrade the barriers of basement membranes and extracellular matrix, allowing malignant cells to infiltrate surrounding tissues [154]. Simultaneously, chemokines and growth factors evoke the EMT process, where tumor cells lose adhesion molecules, especially E-cadherin (acting as a tumor suppressor), thus gaining migratory phenotypes and invasive properties [155].

5.3.4. Immune Suppression

The tumor-derived factors exert potent immunosuppressive activities, resulting in immune evasion by malignant cells [156]. Through various cytokines (e.g., transforming growth factor-beta [TGF-β], interleukin-10 [IL-10] and colony-stimulating factors) [128]. Immune responses are inhibited from activated cytotoxic T lymphocytes and natural killer cells and instead drive a strong regulatory, anti-tumor cell response with recruited or expanded as well as infiltrating types of Tregs, MDSCs and Tumor-associated macrophages [156]. Tumor-related alterations in innate immunity pathways allow tumor evasion by inhibition of effector function and recruitment of non-tumoral immune populations [157]. Lactate and adenosine are metabolic byproducts that are present within the tumor microenvironment that suppress immune-cell activation and inhibit cytokine secretion [156].

5.3.5. Metabolic Reprogramming and Acidic Microenvironment

Metabolic reprogramming is one of the hallmarks of cancer-related processes, where malignant cells adjust normal metabolic pathways to meet the increased demands of rapid proliferation and environmental stress protection for tumor progression requirements [158]. In contrast to normal differentiated cells, which depend mostly on mitochondrial oxidative phosphorylation for ATP production, most tumor cells undergo a preference towards glucose glycolysis and other anabolic pathways even in cytoplasmic oxygenated conditions [159]. One important consequence of this changed metabolism is the creation of an acidic tumor microenvironment. Excessive glycolysis results in lactate and proton overproduction. In addition, low perfusion of tumors coupled with aberrant vasculature impedes the clearance of acid metabolites [144]. From a biological point of view, acidic microenvironment is essential for tumor cells. It leads to the degradation of the ECM, enhances invasion and angiogenesis, induces immune-cell suppression, and selects for aggressive phenotypes that can prosper under difficult conditions [160]. Accordingly, Tumor Energetics metabolic reprogramming directly relates to microenvironmental remodeling and disease progression [161]. Manipulation of tumor metabolism is an important therapeutic strategy in modern oncology since it modulates treatment response, and hence targeting glycolysis, lactate transport, mitochondrial adaptation, and pH regulation have all been exploited for this purpose [156].

5.4. Exosomes: Definition and Biological Characteristics

Exosomes are nanoscale (30–150 nm in diameter) membrane-derived small extracellular vesicles secreted by most cell types [162]. These, however, are a consequence of the endosomal pathway and, after fusion of multivesicular bodies with the plasma membrane, are secreted into the extracellular space [163]. Exosomes have been identified as key mediators of intercellular communication, carrying biologically active cargo from donor cells to recipient cells and modulating cellular behavior and signaling pathways [164]. Tumor-derived EVs are of an extreme importance in cancer biology, as they have been shown to be involved in angiogenesis, immunosuppression, remodeling of stromal tissues, invasion and metastasis as well as chemo resistance/immune resistance [165]. Extracellular vesicles (EVs) are released by all types of cells and can be detected in biological fluids [166]. therefore, they have potential as noninvasive biomarkers for cancer diagnosis and prognosis via a liquid biopsy strategies [167]. Their endosomal origin, nanoscale size range, selective molecular composition, and relative stability in circulation distinguish exosomes from all other extracellular vesicles [168]. Due to their role as the molecular face of parent cells, exosomes hold great potential for studying tumor biology and disease status [169].

5.4.1. Exosomes as Mediators of Intercellular Communication

Exosomes are one of the major factors involved in inter-cellular communication in TME [164]. Exosomes allow tumor cells to affect neighboring stromal, immune, and endothelial cells and distant tissues by virtue of their ability to transport proteins, lipids, mRNA (messenger RNA), miRNAs (microRNA), DNA fragments, and signalling molecules [170]. While soluble factors can diffuse freely, exosomes transfer a secure and concentrated molecular cargo from the originating cell to recipient cells through membrane fusion, receptor-mediated uptake, or endocytosis [163,171]. Such a communication network enables the tumor to orchestrate associated biological processes such as remodelling of the extracellular matrix, angiogenesis, immune evasion, metabolic adaptation, and metastatic niche formation in a spatiotemporal fashion [111].

5.4.2. Role of Exosomes in Angiogenesis

Movements of proangiogenic signals from tumor cells to endothelial cells and stroma are mimicked by exosomes, which serve as the most common carriers in tumor angiogenesis [172]. As an example, revealed tumor exosomes may harbour vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), fibroblast growth factors (FGF) and matrix metalloproteinase, as well as angiogenesis-related miRNAs like miR-210 and miR-221 [173,174]. These cargo molecules, taken up by endothelial cells, act as stimulators of biological processes including proliferation, migration, tube formation, and vascular permeability [175].

5.4.3. Exosomes in Metastasis and Pre-Metastatic Niche Formation

Prior to circulating tumor cells arriving at distant organ sites, the primary tumor communicates with those organs using exosomes, which are crucial for the dissemination of metastasis [176]. This process contributes to the establishment of a pre-metastatic niche [177]. They also carry proteins, lipids, enzymes, cytokines, and nucleic acids involved in the genetic engineering of surrounding stroma cells, immune system cells, endothelial cells, or extracellular matrix components in secondary organs [111]. Once in these tissues, exosomes facilitate vascular permeability, inflammation, fibroblast activation and remodelling of the extracellular matrix and recruitment of bone marrow-derived cells [178,179]. This reprogramming creates a permissive milieu, facilitating the metastatic tumor cells' survival and implantation.

5.4.4. Exosomes in Immune Modulation

Most of the tumor-derived exosomes are found to be immunosuppressive. These include programmed death-ligand 1 (PD-L1), TGF-β, and IL-10 [180], and many suppressive miRNAs, which prevent the cytotoxic T lymphocytes and natural killer cells from eradicating them [181]. Such vesicles suppress the proliferation, cytokine production, and killing activity of immune cells [182]. The same tumor exosomes also induce regulatory immunostimulatory effects by enhancing antigen presentation and activating anti-tumor immune responses [183]. All these mechanisms together allow for tumor cells to escape immune surveillance and gain resistance to immunotherapy [184]. Most tumor-derived exosomes are suppressive, but some exosomes can also elicit pro-immune effects, although in a context-dependent manner [182]. Exosomes derived from activated dendritic cells, T cells, or NK cells are being studied as therapeutic vaccines since they can promote adaptive and innate anti-tumor immunity [185]. Engineered exosomes that deliver immune stimulants or tumor antigens are also being developed as a new type of immunotherapy [186].

Therefore, exosomes are a double-edged weapon in terms of immune evasion and cancer immunity [187].

5.4.5. Clinical Applications of Tumor-Derived Exosomes

Tumor-derived exosomes have attracted considerable attention in clinical oncology for their abundance and stability in body fluids and their ability to reflect the molecular phenotype of their parental tumors [188]. Minimally invasive sampling propelled their use as a tool in diagnosis, prognosis, and treatment monitoring, allowing for precision-guided therapy [189]. Liquid biopsy tools, symptom discovery and precision therapy/ drug delivery systems are currently being explored for use with exosomes [190]. Exosomes, however, are well adapted for this purpose because they protect nucleic acids and proteins from degradation, allowing reliable molecular analysis[191]. Exosomes are well characterized as a natural carrier of tumor-specific proteins, RNAs, lipids and DNA fragments; thus can be potentially used as diagnostic biomarkers [192]. Exosomes can be used as a drug delivery vehicle due to the following reasons: they are biocompatible, exhibiting immune tolerance and can also transport molecules across biological barriers[193]. These preliminary data of these systems indicate their potential for targeted delivery to brain tumors, metastatic lesions and therapy-resistant cancers [194]. Therefore, personalized exosomes will be among the most clinically oriented applications of extracellular vesicle biology in precision oncology [195].

6. Therapeutic Targeting of the Tumor Microenvironment

Designing the rational therapeutic target should be built on studying the heterogeneous assembly of the TME constituents and how they influence the tumor's survival and growth. The therapeutic strategies that target the TME are including Anti-angiogenesis / Vascular Normalization, ECM Remodeling, and Anti-inflammatory Strategies. Moreover, Combinatorial therapies incorporating these approaches have great potential in overcoming resistance in therapy and creating a platform for personalized treatment regimens for cancer.

6.1. Inhibition of Angiogenesis and Vascular Normalization

Recent publications have demonstrated the efficacy of anti-angiogenic agents in reducing the vascular density of tumors and slowing down the process of tumor growth. However, it’s important to note that a dysfunctional vascular structure can hinder drug delivery and cell infiltration. [196], and this emphasizes the need to study the concept of vascular normalization [197,198]. It involves modification of abnormal, leaky, and dysfunctional vessels, along with restoration of pericyte coverage and endothelial cell junctions. This collectively will ensure improved perfusion and oxygenation [199] Vascular normalization also facilitates drug delivery and prevents the development of hypoxia-driven resistance mechanisms [200,201]. Thereby, inhibition of immune cells such as TAMs and regulatory T cells along with angiogenic pathways could amplify the inhibition of new blood vessel formation, which will result in normalizing vessels [202].

Other molecular targets apart from VEGF that have been examined are the angiopoietin/Tie2 signaling pathway, PDGF signaling pathway, and FGF signaling pathway; they play key roles in regulating blood vessel maturation and stabilization [203]. However, Studies have reported that Inhibitors targeting these pathways have demonstrated efficacy in preclinical and clinical studies and are frequently combined with VEGF inhibition to optimize vascular normalization and tumor regression [204]. Another potential target for antiangiogenic therapy involves regulating endothelial cell metabolism, such as glycolysis and fatty acid oxidation. [205]. Recent examples revealed that proper vascularization enhances immune cells' ability to enter tumors and function, thereby enhancing the effectiveness of immune checkpoint blockers [206]. In Table (2), a focused overview of anti-angiogenic agents and their mechanisms is outlined.

Table 2. Anti-Angiogenic Agents and Vascular Normalization Strategies.

Agent	Target	Drug Class	Their Strategy	Approval Status / Clinical Example	References
Bevacizumab	VEGF-A	Monoclonal antibody	Blocks VEGF-A ligand to inhibit VEGF signaling, reducing new blood vessel formation and tumor vascular density	FDA-approved; used in colorectal, lung, and renal cancers	[207]
Ramucirumab	VEGFR-2	Monoclonal antibody	Targets the VEGFR-2 receptor to prevent VEGF binding, inhibiting angiogenesis and decreasing tumor vascular permeability	FDA-approved; used in gastric and lung cancers	[208]
Axitinib	VEGFR-1, VEGFR-2, VEGFR-3	Tyrosine kinase inhibitor	Inhibits multiple VEGF receptors’ tyrosine kinase activity to block endothelial proliferation and new vessel formation	FDA-approved; used in renal cell carcinoma	[209]
Imatinib	PDGF receptor	Tyrosine kinase inhibitor	Inhibits PDGF receptor signaling to impair pericyte recruitment and vessel stabilization, contributing to vascular normalization	FDA-approved; used in gastrointestinal stromal tumors (GIST)	[210]
Nintedanib	PDGF, VEGF, FGF receptors	Tyrosine kinase inhibitor	Blocks multiple growth factor receptors to inhibit vessel maturation and stromal support, enhancing vascular normalization	FDA-approved; used in idiopathic pulmonary fibrosis and lung cancer	[211]
Ponatinib	FGFR	Tyrosine kinase inhibitor	Targets FGFR signaling to disrupt angiogenesis and stromal interactions, complementing VEGF pathway inhibition	Clinical trials: FGFR pathway targeting	[213]

Table 2. Anti-Angiogenic Agents and Vascular Normalization Strategies.

Agent	Target	Drug Class	Their Strategy	Approval Status / Clinical Example	References
Bevacizumab	VEGF-A	Monoclonal antibody	Blocks VEGF-A ligand to inhibit VEGF signaling, reducing new blood vessel formation and tumor vascular density	FDA-approved; used in colorectal, lung, and renal cancers	[207]
Ramucirumab	VEGFR-2	Monoclonal antibody	Targets the VEGFR-2 receptor to prevent VEGF binding, inhibiting angiogenesis and decreasing tumor vascular permeability	FDA-approved; used in gastric and lung cancers	[208]
Axitinib	VEGFR-1, VEGFR-2, VEGFR-3	Tyrosine kinase inhibitor	Inhibits multiple VEGF receptors’ tyrosine kinase activity to block endothelial proliferation and new vessel formation	FDA-approved; used in renal cell carcinoma	[209]
Imatinib	PDGF receptor	Tyrosine kinase inhibitor	Inhibits PDGF receptor signaling to impair pericyte recruitment and vessel stabilization, contributing to vascular normalization	FDA-approved; used in gastrointestinal stromal tumors (GIST)	[210]
Nintedanib	PDGF, VEGF, FGF receptors	Tyrosine kinase inhibitor	Blocks multiple growth factor receptors to inhibit vessel maturation and stromal support, enhancing vascular normalization	FDA-approved; used in idiopathic pulmonary fibrosis and lung cancer	[211]
Ponatinib	FGFR	Tyrosine kinase inhibitor	Targets FGFR signaling to disrupt angiogenesis and stromal interactions, complementing VEGF pathway inhibition	Clinical trials: FGFR pathway targeting	[213]

6.2. Remodeling the Extracellular Matrix for Therapeutic Benefit

Therapeutic approaches that focus on targeting enzymes such as matrix metalloproteinases (MMPs) and lipoxygenases (LOXs), which are responsible for regulating ECM degradation and crosslinking, respectively [212], can restore ECM integrity and normalize the TME [213], thereby diminishing and slowing down the disease progression. Another Example is that the selective inhibition of LOX-mediated collagen crosslinking can optimize the efficacy of chemotherapeutic agents by improving the vascular perfusion and drug delivery, and that's through reducing the ECM stiffness and mitigating the metastatic potential [213]. Altogether, the rational therapeutic remodeling of the ECM should be aimed at reducing its barriers to enhance immune cell trafficking and activation. Furthermore, stimulating the ECM with growth factors such as the TGF-β family, especially TGF-β1, enhances collagen production and deposition, which is essential for tissue repair. [214]. Enzymatic breakdown of ECM components such as collagen and hyaluronic acid is being researched to decrease the tumor stiffness and interstitial pressure, thereby enhancing the efficacy of chemotherapies. Moreover, another example is PEGylated hyaluronidase (PEGPH20), which works on degrading hyaluronic acid to reduce its amount in the ECM, therefore lowering the intratumoral pressure and facilitating the drug delivery. These enzymatic approaches help overcome the physical barriers imposed by the ECM, allowing for more effective chemotherapy by increasing drug access to cancer cells [215,216].

6.3. Targeting Hypoxia and Metabolic Adaptations in the TME

Targeting the hypoxic and metabolic adaptations within the TME is an essential therapeutic approach that must be considered owing to the significant impact of these responses on tumor development and resistance to existing therapies [217].

Antagonists of glycolysis enzymes like HK2 and LDHA, as well as compounds targeting mitochondria, have shown effectiveness in preclinical studies, reversing the metabolic phenotype due to hypoxia and enhancing the sensitivity of tumors to chemotherapies and radiation therapy [218,219].

Recent evidence reported that under hypoxia, pancreatic cancer cells depend on micropinocytosis to acquire aspartate, which is a critical metabolite for nucleotide synthesis. This process is driven by HIF1A-dependent upregulation of carbonic anhydrase 9 (CA9) to boost the bicarbonate availability and stimulate micropinocytosis, thereby blocking micropinocytosis, or CA9 can impair hypoxic tumor cell growth [220].

On the other hand, Normalization of tumor vasculature through enhanced oxygenation or inhibition of the HIF pathway plays a crucial role in addressing the effects of hypoxia on the TME, which will allow the reprogramming of immune cells for increased effectiveness of cancer immunotherapies, such as immune checkpoint inhibitors [221,222].

6.4. Therapeutic Strategies Against Tumor-Promoting Inflammation

Targeting pro-inflammatory cytokines such as interleukin-6 (IL-6) and tumor necrosis factor–alpha (TNF-α), which are fundamental factors in maintaining the milieu conductive to tumor survival and proliferation. [223]. Another approach is through therapeutic agents that can neutralize or block these cytokines, or their receptors, to reduce tumor-induced inflammation and promote an effective anti-tumor immunity response [224,225]. Another example, Immune checkpoint inhibitors (ICIs) represent a revolutionary advancement in cancer therapy, in which the function of exhausted T cells is revitalized; nevertheless, these therapeutics' efficiency can be compromised by the immunosuppressing microenvironment created by inflammation. Thus, a combination approach using ICIs and agents targeting inflammatory mechanisms shows promise in overcoming resistance to such therapeutics [226,227,228].

However, in terms of targeting molecular pathways underlying chronic inflammation, the targeting of various transcription factors, e.g., NF-κB and STAT3, represents a targeted approach to disrupt the inflammatory signaling networks within tumors. Small molecule inhibitors and biologics that target these pathways can reduce the production of inflammatory cytokines and chemokines, thereby mitigating the pro-tumorigenic effects of chronic inflammation [229]. An overview of the main therapies that act on inflammation mechanisms to fight cancer is described in Table (3).

Table 3. Therapeutic Strategies Targeting Tumor-Promoting Inflammation in the TME.

Therapeutic Target	Immunological Rationale	Strategy	Representative Agent(s)	Clinical Stage	References
TAMs	TAMs promote immunosuppression and tumor growth via pro-inflammatory mediator secretion and T cell suppression	Depletion, recruitment blockade, reprogramming to tumoricidal phenotype	CSF1R inhibitors (e.g., Pexidartinib), CD40 agonists, CCR2 antagonists	Phase I/II	[221,230]
MDSCs	MDSCs suppress cytotoxic lymphocytes and maintain immunosuppressive TME	Elimination, recruitment blockade, functional inhibition	PDE5 inhibitors, ARG1 inhibitors, CXCR2 antagonists	Early clinical	[230,231]
Pro-inflammatory Cytokines (IL-6, TNF-α)	Key drivers of tumor-supportive inflammatory milieu; sustain immunosuppression and tumor proliferation	Neutralization/blockade of cytokines or receptors	Tocilizumab (anti-IL-6R), Infliximab (anti-TNF-α)	Approved/Phase III	[233]
Transcription Factors (NF-κB, STAT3)	Central regulators of inflammatory signaling networks promoting tumor progression	Small molecule inhibitors, biologics	Bortezomib (NF-κB pathway), STAT3 inhibitors (e.g., Napabucasin)	Early clinical	[229,232]
ICIs	Restore exhausted T cell function suppressed by inflammatory TME	PD-1/PD-L1 and CTLA-4 blockade	Nivolumab, Pembrolizumab, Ipilimumab	Approved	[228,233]
Metabolic Reprogramming of Immune Cells	Modulate immune cell metabolism to shift from immunosuppressive/pro-inflammatory to antitumoral state	Target glycolysis, oxidative phosphorylation pathways	Metformin, 2-DG (glycolysis inhibitor)	Preclinical/early clinical	[234]
Combination Therapies (ICIs + Anti-inflammatory Agents)	Synergistically overcome immune suppression and resistance by targeting multiple inflammatory pathways	Combination regimens	ICIs + corticosteroids or cytokine blockers	Early clinical	[235]

Table 3. Therapeutic Strategies Targeting Tumor-Promoting Inflammation in the TME.

Therapeutic Target	Immunological Rationale	Strategy	Representative Agent(s)	Clinical Stage	References
TAMs	TAMs promote immunosuppression and tumor growth via pro-inflammatory mediator secretion and T cell suppression	Depletion, recruitment blockade, reprogramming to tumoricidal phenotype	CSF1R inhibitors (e.g., Pexidartinib), CD40 agonists, CCR2 antagonists	Phase I/II	[221,230]
MDSCs	MDSCs suppress cytotoxic lymphocytes and maintain immunosuppressive TME	Elimination, recruitment blockade, functional inhibition	PDE5 inhibitors, ARG1 inhibitors, CXCR2 antagonists	Early clinical	[230,231]
Pro-inflammatory Cytokines (IL-6, TNF-α)	Key drivers of tumor-supportive inflammatory milieu; sustain immunosuppression and tumor proliferation	Neutralization/blockade of cytokines or receptors	Tocilizumab (anti-IL-6R), Infliximab (anti-TNF-α)	Approved/Phase III	[233]
Transcription Factors (NF-κB, STAT3)	Central regulators of inflammatory signaling networks promoting tumor progression	Small molecule inhibitors, biologics	Bortezomib (NF-κB pathway), STAT3 inhibitors (e.g., Napabucasin)	Early clinical	[229,232]
ICIs	Restore exhausted T cell function suppressed by inflammatory TME	PD-1/PD-L1 and CTLA-4 blockade	Nivolumab, Pembrolizumab, Ipilimumab	Approved	[228,233]
Metabolic Reprogramming of Immune Cells	Modulate immune cell metabolism to shift from immunosuppressive/pro-inflammatory to antitumoral state	Target glycolysis, oxidative phosphorylation pathways	Metformin, 2-DG (glycolysis inhibitor)	Preclinical/early clinical	[234]
Combination Therapies (ICIs + Anti-inflammatory Agents)	Synergistically overcome immune suppression and resistance by targeting multiple inflammatory pathways	Combination regimens	ICIs + corticosteroids or cytokine blockers	Early clinical	[235]

6.5. Challenges and Future Directions in Therapeutic Targeting of the TME

Approaches involving depletion or redirection of the immunosuppressive cells have shown some promise, but they have been hindered by concerns regarding off-target effects and systemic immune imbalance due to their potential impact on healthy tissues [236]. and the differential spatial distribution of immune cell subsets, which varies between tumors, poses an obstacle to the delivery and efficacy of the therapeutic interventions [237]. Additionally, targeting various components of ECM and/or MMPs showed only limited clinical success, often due to the existence of compensation mechanisms and possible impact on tissue integrity in healthy organs [238]. Recent evidence has established that development in new methods based on the latest achievements in nanotechnology and biomaterial science can provide an opportunity for further improving the efficacy of targeted treatments. Although it still needs extensive clinical validation [239].

While anti-angiogenic therapy has already become an integral part of oncological practice, resistance mechanisms such as vessel co-option and recruitment of pro-angiogenic immune cells make its efficiency low [240]. Moreover, modern technological developments like single-cell RNA sequencing and spatial transcriptomics have contributed significantly to providing deeper insight into TME complexity, including heterogeneity of cells and their interactions within the environment, leading to the discovery of new targets and mechanisms [241]. However, difficulties in applying this information clinically due to problems related to data integration, identification of biomarkers, and patient selection are obstacles that hamper further progress in this field [242].

One important aspect regarding the future development of this field is associated with multi-modal therapeutic strategies targeting various aspects of TMEs. Such an approach based on combining immune-checkpoint inhibitors and drugs affecting stroma, metabolism, and angiogenesis might prove to be particularly effective and reduce the probability of resistance [243]. Nonetheless, due to the complexity of such combinations, profound insight into TMEs becomes a prerequisite for the successful implementation of such treatment strategies.

7. Conclusions

In conclusion, therapeutic targeting of the TME has many complexities, starting from the heterogeneity of cells within it, immune networks involved in suppressing the immune system, the presence of an ECM barrier, and the plasticity of its vasculature. Altogether, underline the need for an integrated approach to therapy that utilizes novel technologies and molecular knowledge to boost its efficacy and overcome its hurdles