1.1 Biogenesis and Signaling Pathways of Tumor-Derived Exosomes

Tumor-derived exosomes (TDEs) have gained significant attention in cancer research due to their immunoregulatory roles and involvement in tumor pathophysiology. The biogenesis of exosomes involves four main stages: cargo sorting, endocytosis, multivesicular body (MVB) formation, and exosome release. This process begins with the recruitment of various molecules—proteins, nucleic acids, and lipids—into early endosomes. Cargo sorting is regulated by distinct mechanisms; for example, proteins often require monoubiquitination, while miRNAs are guided into exosomes by binding to heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) [29-32].

Early endosomes form through plasma membrane invagination and undergo maturation into late endosomes, which involves changes in membrane composition, such as the conversion of sphingomyelin to ceramides and the replacement of Rab5 with Rab11, facilitating endosomal trafficking [33-36]. During this maturation, inward budding of the endosomal membrane creates dynamic MVBs. These MVBs can either fuse with lysosomes, leading to cargo degradation, or with the plasma membrane, releasing their intraluminal vesicles (ILVs) as exosomes into the extracellular space [37-40] as shown in Figure 1.

Exosome synthesis and release involve complex pathways, including Endosomal Sorting Complexes Required for Transport (ESCRT)-dependent and ESCRT-independent mechanisms. Released exosomes deliver their cargo—such as DNA, microRNA, and proteins—to recipient cells through mechanisms like direct fusion with the plasma membrane, surface protein binding, or endocytosis [41]. By utilizing these mechanisms, TDEs significantly contribute to intercellular communication, supporting tumor growth, proliferation, angiogenesis, and metastasis. They deliver immunosuppressive molecules to stromal and immune cells, dampening immune responses against cancer [42].

Exosomes derived from tumors express immunosuppressive proteins such as programmed death-ligand 1 (PD-L1), which inhibits T-cell activation [43, 44]. TDEs also mediate immune suppression through the FAS/FASL pathway, promoting T-cell apoptosis and signaling via exosomal TGF-β1 [45]. The ESCRT-associated protein ALIX regulates PD-1 expression on cell membranes; its downregulation increases PD-1 expression in breast cancer cells, enhancing immune evasion [46]. Cancer-associated fibroblast (CAF)-derived exosomes overexpress miR-92, which upregulates PD-L1 in breast cancer cells, linking reduced cell division and increased T-cell apoptosis [47]. TDE signaling further suppresses natural killer (NK) cells by inhibiting interleukin-2 and transporting heat shock protein-72 to regulate myeloid-derived suppressor cells (MDSCs) via the Signal transducer and activator of transcription 3 (STAT3) pathway. Additionally, TDEs transfer epidermal growth factor receptor (EGFR) to adjacent macrophages, contributing to immune suppression [46]. Exosomes from MSCs influence tumor-associated macrophages (TAMs), converting them into immunosuppressive type-2 macrophages that enhance arginase-1 activity and interleukin-10 secretion, promoting malignancy and immune suppression at the tumor site [48]. Exosomes deriving from MSCs have a large amount of semaphorins, TGF-β and C1Q which incentivize the expression of PD-L1 in macrophages [49]. The epithelial–mesenchymal transition (EMT) also stimulates exosome biogenesis and release in tumor cells. CAFs secrete TGF-β1 through the TGF-β1-SMAD pathway, facilitating EMT and supporting exosome-mediated signaling [50-52]. Exosomes from these fibroblasts can activate the Wnt pathway in breast cancer cells, promoting metastasis [53].

Exosome-mediated delivery of miRNA-rich cargo activates key pathways such as Ras, PI3K-Akt, and MAPK, driving cancer proliferation [15]. For example, exosomes containing miR-374a-5p, miR-200b-3p, and miR-21-5p activate the Wnt/β-catenin and PI3K/Akt pathways in HCC, enhancing tumor aggressiveness [15]. miR-17-5p, another exosomal miRNA, promotes proliferation by inhibiting MAPK9 and suppressing the G1/S cell cycle checkpoint [54]. Downregulation of critical mRNAs, like GADD45A, further destabilizes genomic integrity, contributing to aggressive cancer phenotypes [15]. Furthermore, genes associated with cell proliferation and collagen production were found to be upregulated, whereas those related to immune functions, such as CLEC1B and CLEC4G, were downregulated [16].

1.2 Tumor Microenvironment

The TME is a dynamic and intricate ecosystem that evolves continuously to support cancer development. It consists of diverse cellular and non-cellular components that play pivotal roles in all stages of carcinogenesis. The TME typically comprises immune cells, stromal cells, blood vessels, and the ECM, though its composition varies by tumor type [55, 56]. The interaction between cancer cells and the surrounding environment fosters tumor survival, local invasion, and metastatic spread from the early stages of tumor growth, illustrated in Figure 2 [55].

1.2.2 Stromal Cells and the ECM

Stromal cells, such as CAFs, adipocytes, and endothelial cells, play critical roles in tumor growth, angiogenesis, and metastasis. CAFs are particularly influential in restricting immune cell access to the tumor and secreting factors that support cancer cell survival [60]. Adipocytes in adipocyte-rich environments, such as breast cancer, release metabolic substrates, and cytokines that promote cancer cell proliferation and migration [61, 62].

In liver cancer, hepatic stellate cells (HSCs) transform into myofibroblasts in response to tissue injury, contributing to the fibrotic and inflammatory environment that supports HCC development [63, 64]. Endothelial cells (ECs) in the vascular endothelium are essential for tumor angiogenesis, which restores oxygen and nutrient supply to growing tumors that have outgrown passive diffusion [65]. This angiogenic process is often driven by hypoxia and is critical to the survival of tumors larger than 1–2 mm³ [55, 65].

The ECM, a three-dimensional network of macromolecules such as collagen, fibronectin, elastin, laminin, and proteoglycans, provides structural support to the tumor and plays a crucial role in facilitating metastasis [66]. ECM consists of collagen, fibronectin, elastin, laminin, glycoproteins, and proteoglycans [67]. In solid tumors, the ECM forms extensive deposits, making up as much as 60% of the tumor's mass [55, 68].

In the TME, cells interact through various signaling mechanisms that promote carcinogenesis. These interactions can be direct, such as autocrine signaling where a cell binds to its own secreted messenger (Figure 3a) [69], or juxtacrine signaling where membrane-bound ligands on one cell interact with receptors on an adjacent cell (Figure 3b) [70]. Alternatively, indirect communication occurs locally through paracrine –such as VEGF-driven angiogenesis (Figure 3c) [71, 72], or synaptic signaling, particularly in neural cancers such as brain cancer [73] (Figure 3d). Additionally, cells communicate over longer distances through endocrine signaling mechanisms, including the transfer of biological information via exosomes [74-76], or by releasing signaling molecules and mediators such as cytokines, chemokines, and growth factors (Figure 3e) [75]. Consequently, these mechanisms modulate immune responses, reprogram stromal cells, and remodel the ECM, driving tumor proliferation, invasion, metastasis, and angiogenesis [9, 72, 77]. As tumors grow, hypoxia and metabolic waste accumulation create a stressful microenvironment that triggers angiogenesis to restore oxygen and nutrient levels [65]. Hypoxia-induced factors, such as hypoxia-inducible factor 1-alpha (HIF-1α), stimulate the secretion of pro-angiogenic factors like VEGF, promoting the formation of new blood vessels. This newly formed vasculature is often abnormal, contributing to inefficient blood flow and exacerbating the hypoxic and acidic conditions within the TME, further driving tumor progression [65].

3.1 Promoting Cell Proliferation and Resisting Cell Death

EVs play a central role in cancer progression by inducing the transformation of normal cells into cancerous ones and supporting the phenotypic transformation of tumor cells. This transformation promotes tumor growth by stimulating proliferative signaling through pathways such as MAP/ERK, PI3K/AKT, and WNT. Sustained autocrine or paracrine signaling mediated by cancer EVs provides tumor cells with a proliferative advantage [100]. For example, EVs from GBM cells have been shown to enhance tumor growth by transferring oncogenic proteins to neighboring glioma cells, activating growth-promoting pathways. Studies involving GBM cells injected into mice demonstrated that EVs containing specific proteins enhanced tumor growth compared to EVs lacking those proteins [98]. Additionally, EVs from aggressive cancer cells, such as MDA-MB231 breast cancer cells, have been shown to transfer malignancy traits like anchorage-independent growth and survival under nutrient-limiting conditions to normal cells. EVs also play a critical role in resisting apoptosis. GBM EVs, for example, have been found to carry splicing factors that induce the expression of oncogenic gene isoforms in recipient cells, promoting cell survival. Additionally, microRNAs (miRNAs) in EVs, such as miR-1246 in breast cancer EVs and miR-205 in cholangiocarcinoma EVs, have been implicated in promoting cell proliferation and suppressing apoptosis [100].

3.2 Evading Immune Responses

One of the hallmark abilities of cancer cells is their capacity to evade immune detection and destruction. TDEs contribute to this immune evasion by releasing immunosuppressive EVs that interact with immune cells in the TME [98, 100]. Many of these EVs contain Fas Ligand (FasL), which induces apoptosis in activated anti-tumor immune cells, such as T-cells, and reduces the activity of NK cells [98].

Studies have shown that TDEs can carry immune-modulating proteins such as PD-L1, which binds to PD-1 receptors on cytotoxic T-cells, blocking their activation and proliferation. For example, GBM-derived EVs expressing PD-L1 can directly inhibit T-cell function [101]. Additionally, EVs from breast cancer cells have been shown to suppress T-cell activation by delivering TGF-β, which modulates T-cell behavior through the TGF-β/Smad signaling pathway.

TDEs also influence the polarization of immune cells, such as macrophages. For example, EVs released by HCC cells have been shown to contain lncRNA TUC339, which regulates macrophage activation and induces M2 macrophage polarization. M2-like macrophages are known to support tumor growth by promoting immune suppression and tissue remodeling [100]. Furthermore, TDEs can drive the expansion of regulatory T cells (Tregs) and the differentiation of MDSCs, both of which suppress effector T-cell activation and contribute to immune suppression in the TME [100, 102].

3.3 Reprogramming Energy Metabolism

Cancer cells rely on altered energy metabolism to support rapid growth and proliferation, and EVs play a role in this metabolic reprogramming. TDEs can increase the production of metabolic acids, contributing to the acidification of the TME, which enhances the secretion and uptake of EVs [98]. For instance, EVs from adriamycin-resistant breast cancer cells contain high levels of glutathione S-transferase P1 (GSTP1), a metabolic enzyme that helps detoxify harmful chemicals and reprogram glucose metabolism in recipient cells [100]. Moreover, EVs from breast cancer cells can transfer miR-122 to non-tumor cells, promoting cancer progression by altering glucose metabolism. Another example involves miRNA-105, which is found in tumor-derived EVs from breast cancer cells and enhances glutamine and glucose metabolism in CAFs, providing energy to tumor cells. In nutrient-scarce environments, CAFs expel metabolic waste products such as lactate, which are then taken up by cancer cells to fuel oxidative phosphorylation, creating a metabolic symbiosis that supports tumor growth. Cancer cells stimulate the expression of HIF1a to support this relationship, creating “pseudo-hypoxia” conditions that then promote the release of exosomes in a calcium-dependent manner via MCT1, supporting glycolysis and facilitating tumor growth and survival [103].

3.4 Angiogenesis Formation

Angiogenesis, the formation of new blood vessels, is a critical process in tumor development, allowing tumors to grow beyond a few millimeters by ensuring a supply of oxygen and nutrients. EVs play a significant role in this process by delivering angiogenic factors to endothelial cells in the TME [104]. Studies have demonstrated that hypoxic tumor cells release exosomes that contribute to angiogenesis by carrying pro-angiogenic factors like tissue factor VIIa, which activates the ERK1/2 pathway and upregulates heparin-binding EGF-like growth factor [96]. Additionally, TDEs can contain Delta-like 4 (Dll4), a molecule that promotes angiogenesis by suppressing the Notch signaling pathway, which normally inhibits the development of new blood vessels. In HCC, exosomes carrying miR-210 have been shown to promote angiogenesis by inhibiting negative regulators such as SMAD4 and STAT6 [105].

Exosomal miRNAs also regulate cancer invasion and metastasis by altering gene expression in endothelial and immune cells, creating a premetastatic environment. For example, exosomal miR-200 has been shown to modify gene expression patterns, enhancing the metastatic potential of poorly metastatic cancer cells [96].

4.1 Brain Tumor

Exosomes play a pivotal role in brain tumors, especially GBM, where they promote cell proliferation and inhibit apoptosis. Proteomic analyses of GBM exosomes have revealed over 1000 proteins, including pro-angiogenic factors like interleukin-6 (IL-6), IL-8, and angiogenin, which contribute to hypoxia in brain endothelial cells and tumor malignancy [23, 107]. miR-21 and miR-451, found in GBM exosomes, are highly expressed in the cerebrospinal fluid (CSF) and plasma of GBM patients, making them useful biomarkers for diagnosing and monitoring tumor progression. miR-21 inhibits caspase activity, promoting cell survival, while miR-451 regulates proliferation and migration by suppressing the AMPK signaling pathway, which adapts to metabolic stress [106-109].

In neuroblastoma (NB), exosomes carry proteins involved in key biological processes like cell differentiation and proliferation. Exosomal miR-17-5p, secreted by MYCN-amplified NB cells, enhances the proliferation and migration of non-MYCN-amplified cells, highlighting the malignant role of exosomes in tumor progression [110, 111]. Additionally, exosomal miR-375 has been implicated in bone marrow metastasis in NB, as it promotes the differentiation of MSCs into osteogenic cells, creating a microenvironment conducive to tumor growth [112]. Targeting miR-375 could potentially reduce metastasis, making it a promising biomarker and therapeutic target for NB patients [112, 113].

4.2 Liver Tumor

In HCC, exosomal miRNAs and circular RNAs (circRNAs) play crucial roles in promoting tumor proliferation and immune evasion. miR-21 is carried by HCC-derived exosomes [114, 115]. It Transforms HSCs into CAFs by downregulating PTEN and activating the PDK1/AKT pathway. This leads to increased secretion of pro-angiogenic factors such as VEGF and TGF-β, supporting angiogenesis and tumor invasion [25, 116-118]. miR-665, another exosomal miRNA, promotes cellular proliferation through the MAPK/ERK pathway, enhancing metastatic potential [119]. Conversely, the downregulation of miR-100b and miR-125 in HCC exosomes activates the IGF2/AKT/mTOR pathway, essential for maintaining the stemness of HCC cells [120].

CircRNAs within HCC-derived exosomes also facilitate immune evasion by inducing T-cell exhaustion and promoting regulatory T cell (Treg) expansion. For instance, cirCCAR1, found in exosomes, inhibits CD8+ T-cell activity by upregulating PD-1 expression, leading to reduced cytotoxicity against HCC cells [19]. Additionally, circTMEM45A in HCC exosomes sponges miR-665, promoting IGF2 expression and cell proliferation by accelerating the G1/S cell cycle transition, contributing to tumor growth [121, 122].

Exosomes also modify the TME through EMT pathways. Circ_0004277 from HCC exosomes activates the EMT pathway by stimulating mesenchymal markers like N-cadherin, which facilitates HCC cell migration and the progression of normal cells into malignant ones [26]. Similarly, circ_0061395 enhances tumor proliferation by sponging miR-877-5p, leading to increased expression of the oncogene PIK3R3 [123, 124]. Circ_002136 derived from HCC cells Huh7 and HA22T cells increased proliferation, migration, and invasiveness of HCC cells through sponging of miR-19a-3p and downregulating RAB1A, which are members of Rab proteins needed for the trafficking of amino acids from the Golgi apparatus and endoplasmic reticulum, along with upregulation of the mTOR signaling pathway for cellular growth and migration [125].

4.3 Breast Cancer

Breast cancer-derived exosomes play a critical role in tumor progression by influencing stromal cells in the TME. Exosomal miR-135b-5p promotes the transition of MSCs into CAFs by inhibiting TXNIP, while miR-146a in exosomes converts normal fibroblasts into CAFs, which enhances metastasis in MCF-7 cells [126, 127]. Additionally, exosomal Survivin from breast cancer cells transforms fibroblasts into CAFs by upregulating superoxide dismutase 1 (SOD1), contributing to tumor growth [27, 128].

Exosomal miRNAs, including miR-21, miR-19b-3p, and miR-5100, also regulate the EMT, promoting metastasis [10, 129]. miR-1910-3p, encapsulated in breast cancer exosomes, enhances metastasis by activating the Wnt/β-catenin and NF-κB signaling pathways, while downregulating tumor suppressors like myotubularin-related protein 3 [28].

Long non-coding RNAs (lncRNAs) in exosomes also contribute to breast cancer progression [130, 131]. For example, lncRNA BCRT1, overexpressed in breast cancer exosomes, is associated with poor prognosis and promotes macrophage polarization to a tumor-supportive phenotype [132]. Additionally, Lnc GS1-600G8.5, carried by exosomes, facilitates brain metastasis by disrupting the BBB, while reduced expression of lncXIST has been linked to increased brain metastasis via EMT and c-Met signaling pathways [133].

5.1 Brain Cancer

The BBB poses a significant challenge for the effective treatment of brain cancers such as GBM. Exosomes have emerged as potential nanocarriers for anticancer therapies due to their ability to cross the BBB. Their stability, low immunogenicity, and high loading capacity make them ideal candidates for delivering drugs or therapeutic molecules directly to the tumor site.

One promising approach uses dendritic cell-derived exosomes, which have demonstrated immune-related antitumor activity in GBM mouse models [136]. Additionally, exosomes from MSCs have shown potential in delivering therapeutic miRNAs to glioma cells. For example, miR-199a delivered via MSC-derived exosomes inhibits glioma cell proliferation and migration by downregulating AGAP2 expression, leading to increased apoptosis in glioma cells [137].

Another promising strategy involves the use of exosomal miR-151a, which has been linked to overcoming temozolomide (TMZ) resistance in GBM. Chemo-resistant GBM cells have lower levels of miR-151a in their exosomes, contributing to resistance. Restoring miR-151a in exosomes could reverse this resistance, making it a potential therapeutic target for refractory GBM [138-140].

Exosomes also offer potential for drug delivery. Studies in zebrafish and mouse models have shown that exosomes derived from glioma and brain endothelial cells can effectively deliver chemotherapeutic agents such as paclitaxel (PTX) and doxorubicin (DXR) across the BBB, reducing tumor progression and overcoming chemoresistance [24, 141]. Additionally, exosomes containing microRNA-29a-3p have shown promise in suppressing angiogenesis and migration in resistant gliomas, offering a novel anti-angiogenic therapeutic approach [8].

5.2 Liver Cancer

Hepatocellular carcinoma is commonly treated with surgical resection, ablation, or targeted drug therapies. While conventional therapies such as sorafenib and regorafenib are widely used, exosome-based therapies are emerging as novel strategies for enhancing treatment outcomes [142]. Dendritic cell-derived exosomes (DEX), for instance, have shown promise in activating T cells and inducing antitumor responses in HCC by upregulating IFN-γ and IL-2 while downregulating inhibitory cytokines like TGF-β and IL-10 [143, 144].

NK cell-derived exosomes also show potential in inducing apoptosis in HCC cells through perforin and granzyme B pathways, leading to the activation of caspases and promoting cell death [145, 146]. Moreover, engineered exosomes carrying miR-654-5p have been shown to induce ferroptosis in HCC cells by inhibiting HSPB1, enhancing the efficacy of sorafenib, a drug that is often hindered by resistance due to HSPB1 overexpression [147]. Combination therapies using exosomes have demonstrated enhanced therapeutic effects. For instance, dendritic cell-derived exosomes combined with microwave ablation resulted in reduced tumor size by decreasing Tregs and increasing CD8+ T cells, a more effective outcome than either treatment alone [148, 149]. The combination of exosomes derived from dendritic cells, along with P47 (peptide targeting the HCC cells), HMGN1 (recruiting the DC), and AFP212-A2 (exosomal anchors), on injection, suppressed tumor growth and increased the immune responses through immune-stimulatory CD8+ effector T-cell recruitment and IFN-g along with a decrease in immunosuppressive factors such as TGF-b and IL-10 [150]. This approach highlights the potential for exosome-based combination therapies in improving immune responses and reducing tumor progression in HCC.

5.3 Breast Cancer

Breast cancer-derived exosomes (TDEs) have garnered significant interest for their potential in diagnosis and therapy. Engineered exosomes, enriched with miRNAs, have been explored as therapeutic alternatives. For example, exosomal miR-134 has been shown to inhibit the invasion and migration of breast cancer cells while increasing their sensitivity to anti-HSP90 inhibitors like PU-H71 [151]. Another miRNA, miR-503, inhibits breast cancer cell invasion by targeting CCND3 and CCND2, both of which are involved in cell cycle regulation, as shown in Figure 4 [152, 153].

Exosomes also play a role in modulating the TME. Studies have shown that exosomes carrying miR-135b-5p can induce the transition of MSCs into CAFs, promoting tumor growth and metastasis [104]. Additionally, exosomal long non-coding RNA (lncRNA) BCRT1, overexpressed in breast cancer cells, promotes macrophage polarization and enhances tumor progression [154-156]. The potential of exosomes in breast cancer treatment is evident in their ability to carry therapeutic molecules and modulate the TME. However, further research is needed to develop reliable protocols for isolating, characterizing, and utilizing exosomes in clinical applications.