Currently, the classification of EV subtypes is challenged by experimental detection and isolation methods, where EVs mainly refer to two main categories, exosomes and ectosomes [20]. Ectosomes are vesicles secreted directly from the plasma membrane by direct outward budding, with a larger diameter ranging from 50 to 1000 nm compared to exosomes [21]. Although research on ectosomes of HCC is scarce, there are still studies demonstrating the role of ectosomal proteins such as PKM2 in reshaping the tumor microenvironment and its potential role as a biomarker of HCC [22]. In comparison, exosomes have attracted more attention in the research field due to their formation through a unique intracellular regulatory mechanism, which makes the exploration of their compositions and functions more appealing. Current research predominantly focuses on small EVs, often referred to as “exosomes.” Therefore, for a better understanding of the characteristics of exosomes, it is essential to provide a detailed explanation of their biogenesis process.

The generation of exosomes initially begins with inward budding from the plasma membrane, and in this process, endosomes are generated. During the maturation of endosomes, they undergo material exchange with the endoplasmic reticulum and the Golgi apparatus [14]. The endosomal membrane invagination of mature endosomes forms multiple intraluminal vesicles (ILVs), as precursors of exosomes, which further develop into multivesicular bodies (MVBs). Ultimately, ILVs can be discharged as exosomes through exocytosis, after the membrane of MVBs merges with the plasma membrane, or are degraded by lysosomes or autophagosomes (Fig. 1). In summary, exosome formation involves two steps of membrane invagination, resulting in the structure of large vesicles known as MVBs containing smaller ILVs. These smaller vesicles, upon secretion from the cell, are referred to as exosomes, typically with diameters ranging from 40 to 160 nm [21].

The secretion process and structure of exomes. Endosomes are generated through cellular invagination and further invaginate to exchange components with the intracellular membrane system, forming intraluminal vesicles (ILVs), which further develop into multivesicular bodies (MVBs). Endosomes can either be degraded within the cell or release ILVs through exocytosis to become exosomes. Exosomes have a lipid bilayer structure, with several characteristic membrane and cargo components labeled in the figure. 1. ESCRT-dependent pathway; 2. ESCRT-independent pathway

The sorting machinery of ILVs can rely on both the ESCRT (Endosomal sorting complexes required for transport)-dependent pathway and the ESCRT-independent pathway. The proposal of ESCRT were based on studies of a series of vacuolar protein sorting (VPS) mutants in budding yeast [23,24,25]. The endosomal sorting complexes are a kind of cytosolic protein complexes, which are currently recognized as four subtypes: ESCRT-0, ESCRT-I, ESCRT-II, and ESCRT-III. The endosomal sorting complexes, together with other accessory proteins including Vps4 and Bro1, participate in the process of endosomal sorting of ubiquitinated cargo proteins [26,27,28]. Typically, the ubiquitylation of proteins in the form of lysine-63-linked polyubiquitin chains functions as sorting signals for endocytosis [29]. The ubiquitinated proteins are sorted into ILVs through the sequential action of the ESCRT complexes, while researchers noticed that ESCRT-0, -I, and -II all possess subunits that could bind to ubiquitin and interact directly with ubiquitylated cargos, which provided supporting evidence for the ESCRT-independent pathway [24, 25].

In the process of a typical ESCRT-dependent pathway, the recognition of ubiquitylated proteins is initially mediated by ESCRT-0, which consists of the subunits Hrs and STAM (known as Vps27 and Hse1 in yeast). ESCRT-0 participates in recruiting ESCRT-I, and the recruitment of ESCRT-I to endosomal membranes will be impeded without ESCRT-0 [30]. Crystallographic study on yeast has confirmed the structure of ESCRT-I with four subunits: Vps23, Vps28, Vps37, and Mvb12. The endpiece of ESCRT-I which contains the ESCRT-0-binding domain contributes to the recruitment of ESCRT-I to the membrane. The characteristic structure of ESCRT-I is the long stalk domain which is essential for the correct disposition of cargo [31]. ESCRT-II is composed of four subunits: one Vps22, one Vps36, and two Vps25. It interacts with the subunit of ESCRT-I Vps28 through the GLUE (GRAM-like ubiquitin-binding in Eap45) domain of Vps36 [32], and binds with ESCRT-III through the subunit Vps25. ESCRT-III is composed of multiple small and highly charged subunits and is mainly recruited by ESCRT-II. ESCRT-III can assemble into filamentous oligomers which can further transform into helical tubes or conical funnels to enable the attachment and entrance of cargo to the invaginations of membranes. The inverse budding into MVBs is mediated by ESCRT-III and Vps4 through the process of plasma-membrane abscission. Currently, the mechanism of the scission process remains unclear, one model explains this process as the strong binding between the helix ESCRT-III complex and the lipid membrane causes the vesicle neck to contract, leading to vesicle fission [33]. Before the sorting of proteins is finished, deubiquitylating enzymes are recruited by ESCRT-III to maintain the recycling of ubiquitin [34], and Vps4 as a kind of ATPase participates in dissociating ESCRT-III for the recycling of ESCRTs [35] (Fig. 2a-c).

The ESCRT-dependent pathway (a-c) and ESCRT-independent pathway (d,e) in protein sorting. (a) The recognition and sorting of ubiquitinated proteins mediated by ESCRT complexes. This figure highlights the way ESCRT complexes interact and function together. (b) This figure illustrates how cargo proteins are sorted into ILVs, and the transforming of ESCRT-III to a helical filament structure that aids in membrane invagination and the entry of proteins. (c) The endosomal membrane further invaginates during the ESCRT-III helical process, and the Vps4 protein participates in the disassembly and recycling of the ESCRT-III complex. (d, e) In the ESCRT-independent pathway discovered in mammalian cells, ILV formation and protein transport can still occur after the silencing of key subunits of ESCRT proteins. This process primarily relies on the interactions between membrane lipids (such as ceramide and LBPA) and intracellular proteins (such as Alix)

The ESCRT-dependent pathway has been thoroughly researched in yeast, while in mammalian cells, ILVs can still form in the absence of ESCRT components [36] (Fig. 2d and e). This kind of ESCRT-independent pathway can be driven by the presence of lipid molecules along with essential proteins. Ceramide is the product of sphingomyelin hydrolysis by sphingomyelinases. In mouse oligodendroglial cells, researchers revealed that the release of exosomes decreased by inhibiting sphingomyelinases. Furthermore, sphingomyelinases can increase the budding of small vesicles from the giant unilamellar vesicles model [37]. Lysobisphosphatidic acid (LBPA), a kind of lipid molecule that is abundant in the endosomal membrane, can induce the creation of membrane invaginations in acidic liposomes by interaction with Alix [38]. Currently, researchers are able to analyze the complete lipid and protein components of the membrane, but functional studies of specialized cellular membrane regions remain a challenge. Evidence suggests that although ESCRTs can ensure the efficiency and accuracy of protein sorting, they are not necessarily required for the formation of ILVs, and their specific functional region within this context is not indispensable [26].

To characterize exosomes, it is essential to demonstrate from the perspectives of structural identification and qualitative detection of biomarkers. The observation of exosomes requires microscopes with sufficiently high resolution. Transmission electron microscopy (TEM), with a resolution that can reach 0.1 ~ 0.2 nm, enables the visualization of exosomes featuring distinct lipid bilayers, alongside a unique cup-shaped structure [39]. In recent years, nanoparticle tracking analysis (NTA) has been increasingly utilized for exosome detection. NTA tracks and analyzes observed particles, ultimately providing analysis results of particle size distribution and particle concentration. It is possible to statistically analyze the size and quantity of exosomes, as well as perform preliminary quality assessment [40,41,42].

In addition to morphology detection, exosomes possess unique protein biomarkers that offer characteristics for their identification. The most commonly detected proteins from exosomes are the tetraspanin family (mainly refers to CD9, CD63, CD81, and CD82), which can be demonstrated in various studies focusing on HCC. Other commonly detected marker proteins include membrane transport proteins (Rab GTPases and Annexins), heat shock proteins (HSPA8 and HSP90), Alix, and TSG101 [43] (Fig. 1). In one pan-cancer analysis of EVs from 426 human samples, CD9, HSPA8, Alix, and HSP90 were identified as the most prominent markers. In addition, ACTB, MSN, and RAP1B can serve as novel pan-EV markers [44].

Functionally, research has revealed that the overexpression of CD9 and CD81 can inhibit HCC cell proliferation through the Krüppel-like factor 4 (KLF4)-CD9/CD81-Jun N-terminal kinase (JNK) signaling pathway. Changes in the levels of CD9 and CD81 do not impact the expression of exosomal CD63, Alix, and TSG101 [45]. Another exosomal marker CD63 was discovered as a kind of sialoglycoprotein, and the glycosylation of CD63 mediated by silencing α2,6-sialyltransferase I (ST6Gal-I) can alleviate the effects of HCC-derived EVs in promoting tumor progress, mainly through blocking the Akt/Glycogen synthase kinase (GSK)-3β or JNK1/2 pathways [46]. Although EVs still possess many other characteristic markers, including lipids and glycoproteins, researchers are increasingly focused on functionally significant molecules and proteins, including cargo contained within them.

EVs carry a variety of proteins that participate in the regulation of HCC. During the progression of HCC, EVs produced by tumor cells and stromal cells in the TME serve as communication tools, capable of altering the proliferation, metabolism, phenotype, and function of recipient cells. As HCC progresses, tumor cells exhibit a tendency for metastasis, often targeting specific sites, which involves processes including angiogenesis, reacquisition of tumor stemness, and epithelial-mesenchymal transition (EMT). The study of EV-associated proteins holds significant importance in the understanding of the tumor and microenvironment characteristics of HCC, tumor metastasis mechanisms, and their potential prospects in HCC treatment. In Fig. 3, we provide an overview of the functions of different EV-associated proteins in the context of HCC development and progression.

The role EV-associated proteins play in HCC. The interactions between HCC cells and TME components, including TAM, NK cell, CD8+ T cell, TIM-1+ Breg cell, HSC cell, CAF cell, and cancer stem cell, and essential processes in the development of HCC including angiogenesis, EMT, metastasis, and the formation of the pre-metastatic niche based on EV-associated proteins are summarized. EVs with different colors represent different cellular origins

The progression of HCC not only depends on its biological characteristics but is also closely associated with its surrounding tumor microenvironment. The tumor microenvironment refers to the complex environment composed of tumor cells and the surrounding stromal cells and matrix components [47]. In this intricate environment, studies on EV-associated proteins mainly involve HCC cells, intrinsic immune cells, adaptive immune cells, hepatic stellate cells (HSCs), and cancer-associated fibroblasts (CAFs) among others.

Researchers have observed that EVs derived from HCC cells harbor a variety of proteins, some of which can inhibit tumor growth while others can promote tumor progression. Proteins with tumor growth inhibitory functions act through multiple pathways. Through cell cycle analysis, researchers have observed that elevated levels of cathelicidin antimicrobial peptide (CAMP) are associated with reduced cell proliferation and a significant delay in the G1-S transition. This phenomenon was observed to be diminished in the circulating EVs of HCC patients [48]. Neutral sphingomyelinase 1 (NSMase1) within EVs secreted from HCC cells, which can convert sphingomyelin to ceramide, is able to inhibit cell growth and induce apoptosis of HCC cells via reducing the ratio of sphingomyelin/ceramide [49]. Another EV-derived protein, p120-catenin, secreted from HCC cells can suppress the growth and progression of HCC cells by inhibiting the signal transducer and activator of transcription (STAT) pathway [50]. Besides inhibiting tumor growth, emerging evidence suggests proteins contained in HCC-associated EVs also possess functions that promote tumor growth. A kind of hedgehog protein in EVs, sonic hedgehog (SHH), is revealed to promote HCC progression through the SHH pathway and facilitate the formation of cancer stem cells (CSCs) [51]. Another transmembrane glycoprotein expressed in EVs from HCC cells, Vasorin (VASN), is reported with pro-angiogenic functions and can promote tumor cell proliferation and migration through activation of the STAT3 signaling pathway [52, 53].

Tumor cells exhibit a preference for glycolysis in the TME, which is a metabolic pathway to maintain an elevated growth rate. Even when oxygen is abundant, tumor cells prefer aerobic glycolysis to mitochondrial oxidative phosphorylation. This process is known as the Warburg effect. For HCC, Alpha-enolase (ENO1) is an essential enzyme for glycolysis that contributes to the lactic acid production in tumor cells. EV-derived ENO1 can upregulate integrin α6β4 expression and activate the focal adhesion kinase (FAK)/Src/p38 pathway to promote tumor growth and metastasis of HCC cells [54]. Another protein from EVs, triose-phosphate isomerase 1 (TPI1), as a kind of homodimer glycolytic enzyme that participates in the glycolysis, is found to decrease the aerobic glycolysis in the recipient HCC cells. The decreased levels of EV-TPI1 enhance aerobic glycolysis-driven tumorigenesis. Furthermore, the level of TPI1 in EVs is positively correlated with the level of Rab27 in HCC cells, which is often downregulated in HCC [55].

In the TME of HCC, EVs serve as mediators for intercellular communication. These EVs can originate from HCC cells themselves or from surrounding stromal and immune cells. Investigating the functions of proteins within these EVs provides crucial insights into the developmental patterns of the TME. Among numerous proteins, lysyl oxidase-like 4 (LOXL4) has garnered considerable research attention. LOXL4 is a member of the lysyl oxidase family, exhibiting multiple tumorigenic effects. It can spread among HCC cells via EVs, facilitating tumor metastasis through the FAK/Src pathway and promoting angiogenesis [56]. In addition, LOXL4 shuttled by EVs can induce the expression of programmed death ligand 1 (PD-L1) on macrophages and immunosuppression by activating the STAT1/PD-L1 pathway, thus promoting an immunosuppressive microenvironment and inducing the immune escape of HCC [57, 58].

For the innate immunity of HCC, tumor-associated macrophages (TAMs) play a crucial role in immune evasion and are a focal point of investigation. TAMs are one of the most common stromal cells in the TME of HCC which derive primarily from circulating monocytes. Research reveals that HCC cells can promote monocyte-to-macrophage differentiation through the pyruvate kinase M2 isoform (PKM2)-dependent manner by ectosomes. The reshaped monocytes/macrophages can further secrete cytokines to promote the proliferation of HCC cells [22]. Typically, macrophages can be divided into two subtypes: the classical M1 and the alternative M2 macrophages. In the early stage of tumors, TAMs mainly exhibit the M1 phenotype to inhibit angiogenesis and promote immunity. As the tumor progresses, the tumor microenvironment typically induces polarization from the M1 phenotype towards the M2 phenotype. M2 macrophages possess a limited antigen-presenting capacity and can promote angiogenesis, enhance tumor cell invasion, and inhibit T-cell immune responses by releasing immunosuppressive factors IL-10 and TGF-β [59, 60]. Some studies have revealed the mechanisms that EV-associated proteins contribute to TAM polarization. AlkB homolog H5 (ALKBH5) is a kind of N6-methyladenosine (m6A) demethylase. Elevated levels of ALKBH5 can promote HCC cell stemness and are associated with poor prognosis, mainly through activating the SOX4/SHH signaling axis. EVs originating from HCC cells have the potential to transfer ALKBH5 to THP-1 cells (a kind of human monocytic cell), which is related to macrophage M2 polarization [61]. EVs containing proteasome subunit alpha 5 (PSMA5) possess similar functions in promoting M2 polarization of macrophages, mainly by activating Janus Kinase 2 (JAK2)/ STAT3 pathway [62]. Conversely, EVs from HCC cells containing formimidoyltransferase-cyclodeaminase (FTCD) have the ability to promote macrophage polarization towards M1 and suppress the proliferation of HCC cells [7]. To further suppress the M2 phenotype conversion, researchers have engineered EVs by conjugating antisense oligonucleotides (ASOs) to the prostaglandin F2 receptor negative regulator (PTGFRN) on the EV membrane. This approach effectively silences the expression of STAT6, a critical transcription factor involved in M2 polarization, reshaping the tumor microenvironment of HCC. Consequently, it promotes the polarization of M1-type TAMs and inhibits HCC growth [63].

Natural killer (NK) cells can produce EVs containing cytotoxic proteins to kill tumor cells. Research indicates that stimulating NK cells with IL-15 and IL-21 can lead to the production of EVs containing perforin and granzyme B, enhancing cytotoxicity and apoptosis of HCC cells [64]. Another research illustrates that NK cell-derived EVs can exert potent anti-tumor effects by inhibiting serine/threonine kinase pathway-associated cell proliferation and enhancing caspase activation pathway-associated apoptosis [65]. Correspondingly, HCC cells can also influence the function of NK cells through EVs. One characteristic of HCC cells is the suppression of gluconeogenic function. This leads to the secretion of pyruvate kinase (PKLR)-attenuated EVs, which can inhibit the function of NK cells, thereby promoting the tumorigenic process [66].

In the TME of HCC, tumor-infiltrating T lymphocytes (TILs) play a pivotal role in adaptive immunity. CD8+ CTLs specifically inhibit tumor growth by killing tumor cells through cytotoxicity. Mounting evidence suggests that EVs produced by HCC predominantly exert inhibitory effects on the function of CD8+ CTLs. High expression of 14-3-3 protein zeta in both HCC cells and CD8+ TILs can facilitate the proliferation, EMT of HCC cells and CD8+ TILs exhaustion. It is suggested that 14-3-3 protein zeta may be transferred from HCC cells to CD8+ TILs, potentially via EVs, contributing to these effects [4]. The B isoform of microtubule-associated protein 1 light chain 3 (LC3B) functions as an EV marker. Evidence suggests that LC3B+ EVs hinder the immune response by inducing inflammation. These EVs stimulate leukocytes to secrete IL-6 and IL-8 by transporting HSP90α. IL-6 and IL-8 contribute to the suppression of CD8+ T cell function, thus impacting the effectiveness of immunotherapy. While blocking HSP90α from LC3B+ EVs has the potential to improve the effectiveness of anti-PD-1 treatment. This provides a new perspective for enhancing the response rate of immunotherapy for HCC [67, 68].

Breg cells as a subset of B cells, contribute to immune modulation and suppress immune responses in HCC. T cell Ig and mucin domain (TIM)-1+ Breg cells as a subgroup of Breg cells, can impair the functions of CD8+ T cells and accelerate HCC progression by producing abundant IL-10. EVs containing high mobility group box 1 (HMGB1) released by HCC cells can enhance the accumulation of TIM-1+ Breg cells via the toll-like receptor (TLR) 2/4 and mitogen-activated protein kinase (MAPK) pathway [69]. This suggests that EVs derived from HCC primarily promote the proliferation of Breg cells and accelerate tumor progression.

HSCs are resident mesenchymal cells that can be activated in response to liver injury and participate in the formation of liver fibrosis. In addition, HSCs are a significant contributor to CAFs [70]. Both HSCs and CAFs primarily serve to promote tumor progression. In the TME of hepatic fibrosis, hexokinase 1 (HK1) secreted from HSCs via large EVs can be captured by HCC cells and can promote tumor glycolysis and progression. In addition, the small molecule PDNPA disrupts Akt-mediated degradation of Nur77, resulting in reduced release of HK1. This finding holds promise for inhibiting HCC progression [71]. On the other hand, HCC cells can actively produce EVs to activate HSCs, ultimately promoting tumor development. EVs derived from HCC cells transmit SMO, a key signal transducer in the Hedgehog pathway, to HSCs, leading to the proliferation, invasion, migration, and EMT of HSCs, and further accelerating HCC development in a positive feedback manner [72]. CAFs promote tumorigenic features by remodeling the extracellular matrix to facilitate tumor proliferation, metastasis, angiogenesis, and drug resistance [73]. EVs secreted from CAFs containing Gremlin-1 can induce EMT of hepatoma cells and induce resistance to sorafenib, possibly through activation of the Wnt/β-catenin pathway [74]. Additional research has also revealed tumor-specific communication between HCC cells and CAFs. EVs isolated from HCC cells can stimulate the phospho-extracellular regulated protein kinases (pERK)1/2 signaling and upregulate mitogen-activated protein kinase (MAPK) and Wnt in fibroblast cells, while EVs from fibroblast cells can notably increase the levels of SPOCK1 (also known as testican-1), a proteoglycan recognized as oncogenic, in HCC cells [75].

Angiogenesis is a characteristic feature of tumor growth, and hypoxia is often considered to strongly stimulate tumor angiogenesis. At the molecular level, upregulated expression of pro-angiogenic factors such as vascular endothelial growth factor (VEGF) and platelet-derived growth factors (PDGF) in the TME can promote angiogenesis by activating the phosphatidylinositol-3 kinase (PI3K)/Akt/mTOR pathway [76]. Proteins from EVs have been found to widely participate in this process.

The imaging of angiogenesis has confirmed that the number of EVs secreted from HCC cells could affect the lumen formation of human umbilical vein endothelial cells (HUVECs) [