The production of exosomes is not strictly programmed but is influenced by a multitude of factors, such as cellular state and external stimuli, leading to variations in the composition and biological functions of exosomes derived from different sources.

Exosome biogenesis initiates with endocytosis at the cell membrane surface, forming early endosomes that undergo cargo loading and transform into multivesicular bodies (MVBs) containing intraluminal vesicles (ILVs). The biosynthesis of MVBs is critical for exosome formation. Subsequently, plasma membrane budding generates cup-shaped structures that further develop into early sorting endosomes (ESEs). The trans-Golgi network and endoplasmic reticulum contribute to ESE formation. ESEs mature into late sorting endosomes, followed by invagination of the internal limiting membrane, resulting in the formation of multiple ILVs within MVBs. Consequently, MVBs encapsulate these ILVs, which will eventually become exosomes. Finally, MVBs either fuse with the plasma membrane to release ILVs as exosomes into the extracellular space or fuse with lysosomes or autophagosomes for degradation and metabolism ( Figure 1 ) (12, 13).

Under non-hypoxic conditions, multiple mechanisms for MVB generation have been proposed. These include the ESCRT-dependent pathway, specific lipid molecules, and tetraspanin family proteins (14). In the classical ESCRT-dependent pathway, ubiquitinated receptor proteins in endosomal membranes are recognized and recruited, followed by vesicle budding and deubiquitination to form the final cargo proteins. The invagination of the vesicles leads to their contraction and separation from the membrane, ultimately producing MVBs (15–17). The mechanism by which hypoxia mediates MVB production remains unclear; however, it is well established that hypoxia activates multiple signaling pathways that stimulate accelerated exosome secretion. These pathways include hypoxia-inducible factor (HIF), phosphoinositide 3-kinase (PI3K)-Akt, Wnt/β-catenin, mitogen-activated protein kinase (MAPK), Rab GTPases, nuclear factor-κB (NF-κB), and NADPH oxidase (NOX). All these signaling pathways are thought to be involved in exosome synthesis (18–20). Wang et al. found that HIF expressed by hypoxic cells triggered the expression and transcription of the gene encoding the small GTPase RAB22A, leading to enhanced shedding of microvesicles (MVs) (21). Additionally, hypoxia has been shown to induce the expression of Rho-associated protein kinase (ROCK) (22), which plays a role in MV biogenesis in various tumor cell types (23). These studies reveal that hypoxia affects exosome synthesis and release by modulating the expression of signaling pathways, which will be discussed in detail later.

Under normal physiological conditions, ligands on the cell membrane of recipient cells interact with surface molecules on exosomes to facilitate specific exosome uptake. For instance, tetraspanins CD9 and CD81 specifically enhance exosome internalization in certain cell types (24). Research has demonstrated that while exosome uptake is not strictly cell type-specific, the efficiency of uptake varies among different cell types. Peritoneal exudate cells exhibit the highest uptake of exosomes derived from pancreatic cancer, whereas granulocytes show the lowest uptake (25). It can be inferred that there exists a specific targeting transfer function between recipient and donor cells, which is activated only when the ligand-receptor interaction is precise (26). In breast cancer cells, glycoproteins enriched with high mannose or NeuAcα2,3/6 structures are secreted, leading to preferential targeting of certain cell types (27). Conversely, glioblastoma-derived exosomes, which are enriched in phosphatidylethanolamine, selectively target glioblastoma, fibrosarcoma, and breast cancer cells (28). Exosomes derived from lymph node stromal cells expressing Tspan8-α4 complexes are more readily taken up by endothelial and pancreatic cells compared to their parental lymph node stromal cells (29).

Under normal physiological conditions, the cell membrane of recipient cells interacts with surface molecules on exosomes to facilitate exosome uptake. For example, Tetraspanins CD9 and CD81 specifically promote the internalization of exosomes by certain cell types (24). Studies have demonstrated that exosome uptake is not cell type-specific; however, there are variations in uptake efficiency among different cell types. Peritoneal exudative cells exhibit the highest uptake of exosomes derived from pancreatic adenocarcinoma; granulocytes display the lowest uptake (25). We can assume that there is a specific targeting transfer function between recipient cells and donor cells, but only when the ligand and receptor are correctly combined (26). Therefore, the specificity of exosomes relies on the interaction between recipient cell ligands and molecular complexes to which exosomes adhere. Breast cancer cells secrete glycoproteins rich in high mannose or NeuAcα2,3/6 constructs, specifically targeting certain cell types (27). In contrast, glioblastoma-derived exosomes rich in phosphatidyleglycolamine preferentially target glioblastoma, fibrosarcoma, and breast cancer cells (28). Lymph node stromal cell-derived exosomes expressing Tspan8-α4 complex are more likely to be taken up by endothelial cells and pancreatic cells than they are to be taken up by parental lymph node stromal cells (29).

On this basis, the addition of hypoxia factors significantly impacts the transport of specific proteins to the cell surface. Yoon et al. observed that hypoxia promotes the translocation of vesicles containing α6 integrin from the perinuclear region to the plasma membrane, thereby increasing the surface expression of integrin α6β4 via a Rab11-dependent pathway (30). This finding suggests that hypoxia may influence exosome uptake by modulating integrin α6β4. Menard et al. reported that the uptake of major lipoproteins, including high-density lipoproteins (HDL), low-density lipoproteins (LDL), and very low-density lipoproteins (VLDL), is enhanced under hypoxic conditions, with the initial increased uptake likely mediated by heparan sulfate proteoglycans (HSPGs) (31). Given that extracellular vesicles (EVs) directly regulate HSPG raft endocytosis, EV internalization is augmented under hypoxic conditions (32). These observations collectively support the hypothesis that hypoxia enhances the uptake of exosomes by cancer cells, thereby improving the efficiency of tumor exosome uptake by tumor parent cells.

In this process, several researchers have observed that exosomes produced under hypoxic conditions exhibit reduced size, facilitating faster internalization compared to those generated under normoxic conditions. This characteristic enables them to more readily cross physiological barriers, reach other cells, and effectively transport to metastatic sites via the bloodstream (11, 33). Ye et al. noted that the bending modulus of small extracellular vesicles (sEVs) decreases with increasing malignancy, while stiffness and osmolality increase with malignancy but decrease with sEV size (34). These observations suggest an inverse correlation between exosome size and tumor malignancy; however, the underlying mechanisms require further investigation.

Exosomes transport a diverse array of cargos, including DNA, mRNA, miRNA, proteins, lipids, metabolites, and other molecules. Under hypoxic conditions, exosomes selectively enrich tumor-promoting factors, resulting in higher concentrations of these components compared to non-hypoxic conditions. This enrichment facilitates tumor progression and development while also presenting potential targets for tumor diagnosis and therapy. In summary, hypoxia not only modifies exosomal cargo but also increases exosome heterogeneity.

Under hypoxic conditions, the imbalance between reactive oxygen species (ROS) and antioxidants predisposes cells to oxidative damage, disrupting REDOX signaling and homeostasis as well as causing molecular damage. This phenomenon is commonly referred to as oxidative stress (67, 68). Oxidative stress caused by hypoxia can disrupt the intracellular REDOX balance, leading to ROS accumulation and the production of other oxidative by-products, and these oxidators can regulate various intracellular signaling pathways and regulatory mechanisms, thereby affecting exosome biogenesis.

As one of the key responses initiated by tumors, REDOX pathway has been found to directly affect the release of exosomes through post-translational modification of exosome surface proteins (69). In a REDOX dependence mercaptan modification to explore, discover protein thiol REDOX modification may be directly regulate EV release in response to a change in the cell oxidation-reduction environment, act as regulate secrete body release switch (70). Hedlund et al. showed that oxidative stress exacerbated exosome secretion by cancer cells, including leukemia/lymphoma T and B cell lines (71). Atienzar‐Aroca et al. discovered that oxidative stress in retinal pigment epithelial cells enhanced exosome release and promoted endothelial cell angiogenesis (72). In an experiment in which Pt nanoparticles enhanced the release of exosomes from human lung epithelial adenocarcinoma cells, oxidative stress was also found to promote the secretion of exosomes from cancer cells (73). These observations suggest that hypoxia can elevate intracellular oxidative stress levels and impact exosome secretion.

And oxidative stress also affects cargo in exosomes, The redoxin sensitive signal path PI3K/Akt/eNOS regulate secrete body release Angiopoietin 2 (Ang2), and the Ang2 is an important participant of tumor vascular remodeling (74, 75). Maria Eldh et al. also found that exosomes released from mouse mast cells in response to oxidative stress differed in mRNA content (76).

It is worth noting that studies now generally believe that the effects of exosomes and oxidative stress are reciprocal, and while oxidative stress stimulates exosome secretion, exosomes from tumor sources also exacerbate the toxicity of oxidative stress, thus further supporting malignant tumor growth. For instance, exosomes derived from glioblastoma cells augment oxidative stress-induced toxicity by reducing total antioxidant capacity and glutathione (GSH) levels (77). Pancreatic cancer cells overexpressing Vanin-1 (VNN1) secrete cysteamine and exosomes, inducing oxidative stress and thereby exacerbating the dysfunction of paraneoplastic islets (78).

During cell transformation, the lactate metabolic pathway undergoes reprogramming, causing tumor cells to redirect their original glucose metabolism from efficient oxidative phosphorylation to the less efficient glycolytic pathway, known as the “Warburg effect” (79). Hypoxia enhances the Warburg effect, leading to intracellular accumulation of lactate and bicarbonate and a consequent decrease in pH within the TME. Ban et al. discovered that acidic pH can enhance exosome stability in vitro, improving exosome isolation yield (80). Parolini et al. showed that acidic conditions promote exosome release and melanoma cell metastasis (81). Logozz et al. demonstrated that an acidic pH increases exosome release from human cancer cells independent of the original cancer type, and buffering the acidic TME reduces exosome release (82). Hypoxia activates pyruvate kinase isoform PKM2 to stimulate glycolysis and lactate production, shaping an acidic microenvironment (83, 84). PKM2 also enhances exosome secretion (85).

Hypoxia can induce genetic and proteomic alterations in cancer cells, exerting selective pressure that promotes the emergence of a more aggressive phenotype. Notably, hypoxic conditions predominantly occur at the distal end of the invasion front, typically at the tumor periphery (39). Consequently, exosomes serve as critical signaling mediators facilitating intercellular communication between peripheral and central tumor regions. Many studies have shown that exosomes facilitate tumor cell communication within hypoxic microenvironments in various cancers, such as lung (86), prostate (39), breast (21), colorectal (87), and oral (55) cancers.

Exosomes also play a role in inhibiting apoptosis, regulating tumor cell proliferation, and inducing tumorigenesis. In glioma cells, exosomes enriched with linc-CCAT2 are internalized by human umbilical vein endothelial cells (HUVEC), resulting in reduced HUVEC apoptosis under hypoxia through Bcl-2 upregulation and downregulation of Bax and caspase-3 (88). In HCC cells, hypoxia induces increased exosome production, promoting miR-1273f expression in normoxic cells and enhancing malignant phenotype partially by targeting LHX6 downregulation and facilitating the proliferation and malignant transformation of normoxic tumor cells (89). Maja et al. observed that the induction of latent membrane protein 1 (LMP1), the major oncoprotein produced by Epstein-Barr virus found in nasopharyngeal carcinoma (NPC), significantly elevated HIF1-α levels within exosomes; these exosomes retained transcriptional activity upon uptake by recipient cells (90).

Angiogenesis is a crucial process in tumor growth and metastasis, and tumors might undergo an “angiogenic switch” to activate their vascular network when reaching a size limit of typically 1–2 mm³ (91). Tumors must trigger the angiogenic switch and expand their vascular network to surpass this restricted size and sustain indefinite proliferatio.

Angiogenesis is an inevitable outcome of the complex interplay between pro-angiogenic and anti-angiogenic factors. Under normal conditions, a delicate equilibrium exists between these opposing forces. However, this balance is disrupted during the early stages of tumor progression due to hypoxia, inflammation, and other precipitating factors. Hypoxia directly triggers the activation of numerous pro-angiogenic factors, such as VEGF and its receptors FLT-1 and FLK-1, via the HIF pathway. Additionally, plasminogen activator inhibitor-1 (PAI-1), angiopoietins (ANG-1 and ANG-2), and platelet-derived growth factor-B (PDGF-B) are also directly involved in tumor angiogenesis (92, 93).

In hypoxia-driven pro-angiogenic tumor responses, exosomes function as potent mediators facilitating hypoxia-dependent intercellular communication between malignant cells and vascular cells, such as endothelial cells and pericytes. miR-21-5p is significantly upregulated in the exosomes derived from hypoxia in thyroid papillary carcinoma BCPAP cells, which directly targets and inhibits TGFBI and COL4A1, enhancing angiogenesis in HUVEC (94). Renal cancer cells exposed to hypoxic stimulation secrete increased CA9-containing exosomes that bind to endothelial cells, promoting angiogenesis (95). The impact of exosomes on blood vessels extends beyond promoting angiogenesis. Lung cancer cell-derived exosome miR-23a under hypoxia inhibits the tight junction protein ZO-1, leading to increased vascular permeability and facilitating transendothelial migration during cancer progression (96).

During metastasis, a specific subset of circulating tumor cells, known as metastasis-initiating cells (MICs), plays a crucial role. These MICs reprogram distant cells by secreting exosomes, thereby transforming the distant microenvironment into one that is more conducive to tumor survival and growth, which in turn promotes tumor metastasis to distant sites (90). Consequently, exosomes function to optimize the microenvironment and establish pre-metastatic niches (PMNs) prior to the arrival of circulating tumor cells at these distant locations (97). In a mouse model of ductal adenocarcinoma (PDAC), PDAC releases exosomes highly expressing macrophage migration inhibitory factor (MIF), which are engulfed by Kupfer cells and transported from the bloodstream to the liver. This process induces the release of transforming growth factor β (TGFβ), promoting fibronectin production by hepatic stellate cells (hStCs). Fibronectin deposition further facilitates bone marrow-derived macrophages’ and neutrophils’ arrest within the liver, leading to PMN formation. Plasma exosomes isolated from patients with progressive PDAC exhibit significantly higher levels of MIF than those exhibited by exosomes isolated from patients without disease progression (98).

Studies have demonstrated that exosomes exhibit a propensity to target specific organs and tissues. This targeting facilitates the rapid establishment of pre-metastatic niches (PMNs) in these sites, thereby enhancing the metastatic potential of orthotopic tumors. Moreover, under hypoxic conditions, this tropism is further intensified by increased exosome secretion. In a hypoxic environment, colorectal cancer (CRC) cells can stimulate the release of exosomes rich in miR-135a-5p. Upon phagocytosis by KC cells, miR-135a-5p activates the large tumor inhibitory kinase 2-Yes-associated protein-matrix metalloproteinase 7 axis, leading to PMN formation and promoting liver metastasis in CRC (99). Lysyl oxidase-like 2 (LOXL2) is more abundant in hypoxic exosomes than it is in normoxic ones in head and neck squamous cell carcinoma cells. These LOXL2-rich exosomes are assimilated by distant fibroblasts and activate FAK/Src signaling in recipient fibroblasts, enhancing fibronectin production and recruiting myeloid-derived suppressor cells for PMN formation (100). Furthermore, studies on hypoxic exosomes (HypoExos) derived from prostate cancer (PCa) cells revealed elevated levels of metalloproteinases MMP2 and MMP9, extracellular matrix proteins such as fibronectin and collagen, and an increased number of CD11b+ cells at selective sites conducive for PMN formation (101). Therefore, the selection of target organs for tumor metastasis is not random; rather, it is facilitated by exosomes, which establish a fertile TME in these organs while delivering essential biomolecules.

Hypoxia induces the expression of diverse immunosuppressive molecules, including death receptor ligands (e.g., FasL or TRAIL), checkpoint receptor ligands (e.g., PD-L1), inhibitory cytokines (e.g., IL-10 and TGF-β1), prostaglandin E2 (PGE2), VEGF, and others, to suppress immune responses (102, 103). Exosomes carry immunosuppressive factors that can interfere with immune cell function and transfer them to host immune cells to block innate and adaptive immunity (104). PD-L1 and TGF-β carried by exosomes can interact with T cells, inhibit their proliferation, and suppress the immune response (105, 106). Under hypoxic conditions, miR-24-3p levels in nasopharyngeal carcinoma cell-derived exosomes increase. miR-24-3p inhibits T cell proliferation and Th1/Th17 differentiation while inducing regulatory T cell expansion (107).

Macrophages play a crucial role in tumor progression. Exosomes derived from hypoxic lung cancer cells can enhance the enrichment of miR-103a, augment the expression of M2-type cytokines and pro-angiogenic factors, and induce a phenotypic shift in macrophages towards pro-tumor activity (108). In hypoxic ovarian cancer (109) and pancreatic cancer (110), exosomes mediate the polarization of M2-type macrophages into immunosuppressive M2-like macrophages expressing PD-L1 and IL-10, inhibiting the proliferation of CD4+ and CD8+ T cells in vitro; in vivo, this process promotes tumor growth through PD-L1 (111). Natural killer group 2 member D (NKG2D), an activating receptor expressed on natural killer (NK) cells and CD56+ and CD8+T cells, plays a pivotal role in innate immunity. Hypoxia can transfer TGF-β1 to NK cells leading to reduced cell surface expression of activating receptor NKG2D, impairing NK cell function. More exosomes containing miR-23a are released, which target CD107a expression in NK cells, attenuating their cytotoxicity and resulting in ineffective eradication of cancer cells by NK cells (112).