
==== Front
Cell Biosci
Cell Biosci
Cell & Bioscience
2045-3701
BioMed Central London

39227992
1294
10.1186/s13578-024-01294-6
Review
Role of extracellular vesicle-associated proteins in the progression, diagnosis, and treatment of hepatocellular carcinoma
Liu Yao-Ge
Jiang Shi-Tao
Zhang Jun-Wei
Zheng Han
Zhang Lei
Zhao Hai-Tao
Sang Xin-Ting
Xu Yi-Yao xuyiyao@pumch.cn

http://orcid.org/0000-0003-1036-3369
Lu Xin luxin@pumch.cn

grid.506261.6 0000 0001 0706 7839 Department of Liver Surgery, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College (CAMS & PUMC), Beijing, China
3 9 2024
3 9 2024
2024
14 11317 5 2024
21 8 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by/4.0/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer, characterized by difficulties in early diagnosis, prone to distant metastasis, and high recurrence rates following surgery. Extracellular vesicles (EVs) are a class of cell-derived particles, including exosomes, characterized by a phospholipid bilayer. They serve as effective carriers for intercellular communication cargo, including proteins and nucleic acids, and are widely involved in tumor progression. They are being explored as potential tumor biomarkers and novel therapeutic avenues. We provide a brief overview of the biogenesis and characteristics of EVs to better understand their classification standards. The focus of this review is on the research progress of EV-associated proteins in the field of HCC. EV-associated proteins are involved in tumor growth and regulation in HCC, participate in intercellular communication within the tumor microenvironment (TME), and are implicated in events including angiogenesis and epithelial-mesenchymal transition (EMT) during tumor metastasis. In addition, EV-associated proteins show promising diagnostic efficacy for HCC. For the treatment of HCC, they also demonstrate significant potential including enhancing the efficacy of tumor vaccines, and as targeting cargo anchors. Facing current challenges, we propose the future directions of research in this field. Above all, research on EV-associated proteins offers the potential to enhance our comprehension of HCC and offer novel insights for developing new treatment strategies.

Keywords

Hepatocellular carcinoma (HCC)
Extracellular vesicles (EVs)
EV-associated proteins
Tumor microenvironment (TME)
Proteomics
Biomarkers
National High Level Hospital Clinical Research Funding2022-PUMCH-C-049 2022-PUMCH-A-237 issue-copyright-statement© Society of Chinese Bioscientists in America (SCBA) 2024
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pmcIntroduction

Primary liver cancer ranked as the sixth most frequently diagnosed cancer and was the third highest cause of cancer-related mortality worldwide in 2020 [1]. Hepatocellular carcinoma (HCC), as the most common form (comprising 75-85% of cases) of primary liver cancer, is characterized by insidious onset and a complex progression of biological processes. Due to the delayed manifestation of symptoms, HCC is diagnosed at advanced stages in over 50% of patients, which rendered the only potentially curative surgical approach unfeasible [2]. Despite achievements in the research of HCC, challenges including tumor regulation, early diagnosis strategies, and personalized precision medicine approaches still persist. Facing these challenges, extracellular vesicles (EVs) are extensively researched as promising hotspots based on their biological characteristics and cancer-specific cargo.

EVs are defined as particles surrounded by a lipid bilayer membrane that are discharged by various cells and cannot replicate independently [3]. According to the distinctions in size and specific origins of EVs, the nomenclature of EVs often encompasses various types. For example, small EVs are often described as < 200 nm in diameter while large EVs are often > 200 nm. Another common nomenclature, “exosome”, is a biogenesis-related term that specifically refers to vesicles originating from the endosomal system. Exosomes represent a subtype of small EVs with an intraluminal diameter < 200 nm. On account of the typically challenging experimental verification of the subcellular origin of exosomes, broad claims regarding research on exosomes may not be entirely accurate [4–7]. Currently, there are no recognized molecular markers of EVs or EV subtypes. The most widely tested tetraspanins CD9, CD63, and CD81 are often used to characterize exosomes, but they are not specific markers for exosomes [8]. As a consensus, the Minimal information for studies of extracellular vesicles (MISEV 2023) recommended extracellular vesicles as a standard nomenclature. Therefore, unless otherwise specified in the study, we strive to use “EVs” as a standard term in this review to summarize the relevant research as comprehensively as possible.

The tumor microenvironment (TME) plays a substantial role in the development of HCC, while HCC often presents an immunosuppressive TME. Stromal cells represented by macrophages and cancer-associated fibroblasts participate in the process of limiting immune cell infiltration [9], subsequently hindering the efficacy of immunotherapy. EVs serve as effective carriers containing a variety of substances including nucleic acids (miRNA, long noncoding RNA, and DNA), proteins, lipids, and glycoproteins [10], which provide a convenient approach for intercellular communication, and crosstalk between tumors and TME. The distinctive vesicular structures and targeted membrane signaling molecules of EVs enabled wide involvement in intercellular substance transfer and information exchange. Therefore, the properties of EVs have garnered great attention for exploring their role in biological functions, tumorigenesis, metastasis, angiogenesis, and drug resistance of HCC [11].

Moreover, EVs have exhibited promising capabilities in both the diagnosis and treatment of HCC. Alpha-fetoprotein (AFP), predominantly secreted by HCC cells, is recognized as a significant tumor biomarker for diagnosing HCC. Nonetheless, its sensitivity varies between 25% and 65% [12]. The exploration of novel biomarkers of HCC has never ceased. EVs have emerged as promising targets for HCC early diagnosis primarily based on their accessibility and specificity. Firstly, EVs are widely present in bodily fluids, including blood, urine, saliva, etc. [13]. This facilitates easy retrieval of EVs. On the other hand, the formation of EVs involves the process of inward budding from membrane and materials exchange with the endoplasmic reticulum and Golgi apparatus [14]. Consequently, EVs carry membrane and cytosolic proteins from the originating cells that accurately reflect the molecular features of the original tumor. These properties offer the potential for minimal tumor early diagnosis by using EVs as biomarkers. Furthermore, EVs as endogenous delivery carriers have the advantage of reduced immunogenicity, heightened biocompatibility, and increased permeability through cell membranes [15], they can serve as excellent drug delivery medium after undergoing bioengineering modifications. In terms of transport reliability, the protective biological membrane of EVs can guard their payload against degradation. By utilizing EVs as drug carriers, it is possible to enhance innate and tumor-specific immune responses [16, 17], promote apoptosis of HCC [18], and bolster the activity of targeted therapy [19].

In this review, we focused on the proteins from EVs in the field of HCC. As EV membranes contain a rich variety of protein components, as well as cargo with diverse protein constituents, proteomics studies on EVs can help with understanding the roles that proteins play in biological processes, including cell signaling, metabolism, and disease. By reviewing current research, we aim to gain insights into the tumor progression and TME of HCC from the perspective of EVs, summarize potential protein biomarkers and new therapeutic approaches, and furthermore discuss potential research directions in this field.

Biogenesis and characteristics of EVs

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].

Fig. 1 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 [23–25]. 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 [26–28]. 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).

Fig. 2 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 [40–42].

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.

The role EV-associated proteins play in HCC

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.

Fig. 3 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

Tumor progression and TME

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

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) [77]. Many proteins have been found to have pro-angiogenic functions. Clathrin light chain A (CLTA) is one subunit of the light chain of clathrin. It is found overexpressed in EVs of HCC and can enhance angiogenesis and break up the integrity of vascular endothelial barriers through the stabilization of basigin. The inhibitor of basigin can inhibit patient-derived xenografts (PDXs) tumor progression in mice [78]. DEAD-box helicase 55 (DDX55) is a member of the DEAD‐box RNA helicase family related to early recurrence of HCC after surgery. EVs containing DDX55 from HCC cells can enhance the proliferation and tube formation abilities of endothelial cells, thus stimulating angiogenesis and enhancing the malignancy of HCC cells [79]. Angiopoietin-2 (ANGPT2) plays a significant role in promoting tumor angiogenesis and inflammation. EVs containing ANGPT2 participate in the transportation from HCC cells to HUVECs, thereby enhancing angiogenesis notably [5]. One kind of EV-derived protein, von Willibrand factor (vWF) can upregulate the levels of VEGF-A and fibroblast growth factor 2 (FGF2) in endothelial cells to promote angiogenesis, and FGF2 can promote HCC cell growth in a positive feedback manner by activating the FGFR4/ERK1 signaling pathway [80]. Similar findings including EVs containing hypoxia-inducible factor (HIF) -1α can promote angiogenesis through the PI3K/Akt/mTOR pathway [81], and another EV-derived protein Rab13 can promote angiogenesis by upregulating VEGF in HCC cells [82], further demonstrated the positive effects of EV-associated proteins on angiogenesis.

Contrary to the above findings, researchers also revealed EVs containing proteins with anti-angiogenesis functions. C-Type Lectin Domain Family 3 Member B (CLEC3B) is a transmembrane Ca2+-binding protein expressed on EVs. High levels of CLEC3B can inhibit tumor migration and angiogenesis by targeting the VEGF pathway and inhibiting the EMT process. This suggests that VEGF-targeting therapy for HCC patients may benefit from CLEC3B-high EVs [83]. Another EV-derived protein secreted by HCC cells, general transcription factor II subunit H2 (GTF2H2), can inhibit the migration and permeability of HUVECs and thus suppress tumor angiogenesis [84], where the result needs further confirmation in animal models. Another study has demonstrated that HCC cells exposed to radiation can produce EVs rich in Maspin, which is regulated by histone deacetylase 5 (HDAC5). These EVs can inhibit angiogenesis, providing a new perspective for enhancing the sensitivity of radiotherapy [85]. Currently, multi-kinase inhibitors or anti-angiogenic drugs targeting the VEGF/VEGFR signaling pathway, such as sorafenib, lenvatinib, and bevacizumab, have been used in first-line and second-line treatments for HCC. However, due to the limited response rates of these drugs, exploring more effective therapies from the perspective of EVs holds promise.

Tumor metastasis

In the advanced stages of HCC, there is often a tendency for targeted organ metastasis, with lung metastasis being the most common, accounting for over 70% of HCC-related deaths [86]. This specific metastatic pattern relies on the formation of pre-metastatic niches, where the activation of EMT is considered a crucial mechanism for metastasis initiation. Research indicates that EVs play a widespread role in the process of HCC metastasis.

EMT is a biological process where epithelial cells lose their structural organization, including polarity and cell-to-cell junctions, and acquire a mesenchymal phenotype, boosting their migratory and invasive capabilities. The EMT process is typically accompanied by the increased expression of α-SMA and vimentin, and decreased expression of E-cadherin [87]. Studies suggested that EVs can promote the invasion and metastasis of HCC cells by inducing EMT. EVs from the highly metastatic MHCC97H cells can induce the low metastatic MHCC97L cells to undergo EMT through the MAPK/ERK pathway, and the levels of Rab27a from HCC cells influence the secretion levels of EVs [88]. Compared to complete EMT, HCC cells undergoing partial EMT retain both epithelial and mesenchymal characteristics to some extent. Early identification of the EMT process can be achieved through EV-associated proteins serving as biomarkers. Researchers revealed that enhanced secretion levels of post-translationally modified fibronectin 1 (FN1), collagen type II alpha 1 (COL2A1), and native fibrinogen gamma chain (FGG) in EVs can serve as biomarkers for chemo-resistance and partial EMT [89].

In addition to participating in the EMT process, proteins from EVs can also promote metastasis by boosting the stemness of tumor cells. Polymeric immunoglobulin receptor (pIgR) enriched in EVs from HCC cells can promote cancer stemness and aggressiveness by inducing the Akt/ β-catenin axis rather than activating the SMAD2/3 signaling and inducing EMT in HCC cells. In addition, the neutralizing of EV-pIgR with antibodies can provide an option for the treatment of HCC [90]. Another EVs-loaded protein S100A10 is found to enhance the stemness characteristics of HCC, promote pulmonary leakiness and EMT, and enhance HCC progression through epidermal growth factor receptor (EGFR) activation [91].

Based on inducing tumor stemness and EMT that promote tumor cell dissemination, EV-associated proteins play a crucial role in guiding “seed” to specific “soil”. The “seed and soil” hypothesis indicates that tumor cells (seeds) disseminate throughout the body but only grow in specific organ microenvironments (soil), and this underscores the importance of highlighting the significance of the pre-metastatic niche. Research suggests that EVs from HCC cells can promote lung metastasis formation by regulating circulating tumor cells (CTCs) proliferation and adhesion by releasing SMAD family member 3 (SMAD3) protein [92]. Actin-related protein 2/3 complex subunit 2 (ARPC2) is highly expressed in EVs of metastatic HCC cells, and ARPC2 inhibitor Pimozide can suppress the colonization of metastatic cells in the lung [93]. One EVs-loaded protein nidogen 1 (NID1) can enhance the permeability of pulmonary endothelial cells, promote angiogenesis, and increase the expression of tumor necrosis factor receptor 1 (TNFR1), to facilitate the formation of a pre-metastatic niche in the lung, thereby promoting lung metastasis of HCC [94]. Another research reveals that the levels of transmembrane serine protease 2 (TMPRSS2) in tumors are conversely correlated with the NID1 levels in EVs, indicating that inactivating TMPRSS2 can counteract the malignant characteristics of HCC [95]. For the bone metastasis of HCC, new evidence indicates that a transmembrane protein of EVs, VAMP-associated protein A (VAPA), can be recognized by osteoclast membranes and promote their activation, thereby fostering a fertile niche conducive to the growth of HCC cells [96].

Some other studies have explored the impact of EV-associated proteins on HCC metastasis from various perspectives, involving the complement system, reciprocal induction of cell transmigration, and drug-related events. In the complement system, complement Factor H (CFH), a kind of soluble protein abundant in EVs of metastatic HCC cell lines that can inhibit the alternative complement pathway, is capable of reducing complement-mediated cell lysis. By inhibiting the complement system to evade immune surveillance, the invasive ability of HCC cells can be enhanced [97]. In another proteomic quantitative analysis study involving 21 HCC patients and 15 healthy controls, a series of complement-related proteins were identified and validated to be upregulated in HCC patients, including complement C1Q subcomponent subunit B (C1QB), complement C1Q subcomponent subunit C (C1QC), C4B-binding protein alpha chain (C4BPA), and C4B-binding protein beta chain (C4BPB), further elucidating the role of the complement activation pathway in the tumorigenesis of HCC [98].

Caveolin-1 (CAV-1) as a structural protein of caveolae, is found highly-expressed in the EVs derived from metastatic HCC cell lines and can enhance the metastatic and invasive ability of immortalized hepatocytes [99, 100]. Carboxypeptidase E (CPE) is an exopeptidase upregulated in EVs from HCC cells. The proliferation and invasion ability of low-metastatic potential MHCC97L cells can notably enhance in the CPE-dependent pathway after incubation together with EVs derived from MHCC97H cells with high-metastatic potential [6]. In addition, EV-derived protein S100A4 has also been found to have similar intercellular crosstalk functions to promote the metastatic potential of MHCC97L cells [101].

There is also a drug-related event promoting tumor metastasis reported, where STA9090, as an inhibitor of HSP90, poses a risk of promoting HCC metastasis. Vacuolar protein sorting-associated protein 35 (VPS35) is found to be upregulated in the EVs from HCC cells when stimulated by STA9090 and can enhance tumor metastasis, primarily through the activation of the Bclaf1-VPS35-EVs axis [102].

The application of EV-associated proteins as diagnostic and therapeutic biomarkers for HCC

HCC diagnosis is based mainly on its clinical and imaging characteristics and is approved by guidelines. While similar imaging features, such as between HCC and intrahepatic cholangiocarcinoma (ICC), can result in misidentification and the implementation of inappropriate treatment strategies [103]. At present, there is limited consensus in clinical practice regarding the routine biopsy of newly detected liver tumors. Consequently, investigating non-invasive or minimally invasive liquid biopsy methods to identify dependable biomarkers holds promise for enhancing the diagnostic precision of HCC. Proteins within EVs serve as markers and can offer distinct advantages over traditional blood-based markers. Derived from parent cells and enclosed within protective lipid bilayers, EV proteins can provide increased stability and specificity for tumor diagnosis [10]. Notably, it should be noted that the sensitivity of minimally invasive liquid biopsy methods for detecting early-stage HCC may be constrained. This limitation could stem from the necessity for the tumor to advance to a vascularized stage before a substantial quantity of detectable EVs is generated [104].

Glypican-3 (GPC-3) is a heparin sulfate proteoglycan, typically located on the cell membrane of the fetal liver cells, but also expressed in malignant tumors, such as HCC and lung carcinoma [105]. Research indicates that GPC-3 is an optimal target for EV-based surveillance of HCC. GPC-3 is predominantly found within EVs, with only a minor presence of its soluble form in the serum. GPC-3 derived from EVs performs better than AFP in discriminating HCC patients from cirrhotic patients and healthy controls [106]. It can also be detected in patients with cirrhosis, and increased expression found in EVs is associated with impaired hepatocellular autophagy [107]. Additionally, as a membrane protein, GPC-3 can also be purified and analyzed through antibody selection using fluorescence nanoparticle tracking analysis (F-NTA) and is positively correlated with the total tumor size of HCC [108]. As a widely recognized EV membrane protein, in terms of treatment, GPC-3 single-chain scFv antibody can be utilized to target the induction of EVs loaded with IR780 and Lenvartinib, enhancing the efficacy of hyperthermia and chemotherapy [19].

EVs with specific protein expression profiles can be identified to recognize patients with HCC. Through fluorescence-activated cell scanning (FACS), AnnexinV+ EpCAM+ ASGPR1+ EVs are identified as potential biomarkers to distinguish between HCC patients and cirrhotic patients without detectable tumors, with an area under the curve (AUC) of 0.732 and exhibit a notable decrease by the seventh day following surgery [104]. Chen et al. also discovered HSP90α+ LC3B+ EVs as potential biomarkers to distinguish HCC patients from non-liver cancer controls (AUC = 0.960). The research further revealed a positive correlation between the levels of HSP90α+ LC3B+ EVs and PD-1high CD8+ exhausted T cells [68].

In recent years, researchers have further employed the use of tissue microarray methodology to aid in the quantitative analysis of EV subpopulations. An HCC EV ECG score, derived from the measurements of three distinct HCC EV subpopulations (EpCAM+ CD63+, CD147+ CD63+, and GPC3+ CD63+), was developed to detect early-stage HCC. In the validation cohort, the AUC of the ECG score for distinguishing early-stage HCC from cirrhosis reached 0.93 [109].

Through proteomic analysis of HCC cell lines followed by validation in clinical cohorts, one study identified two EV-enriched proteins, CCT8 and cofilin-1. These proteins demonstrate promise as serum biomarkers for diagnosing HCC, exhibiting respective AUC values of 0.698 and 0.677, notably surpassing that of AFP (AUC = 0.63). The combined utilization of all three biomarkers achieved the highest AUC (AUC = 0.84) [110].

The detection of tumor-associated (TA) autoantibodies holds promise for early tumor identification, and their secretion via EVs has been observed. Studies have identified several antigens recognized by TA autoantibodies, including ATIC, bromodomain-containing protein 2 (BRD2), and translation initiation factor 3 subunit A (EIF3A). These TA autoantibodies demonstrate effective discrimination between serum samples from HCC patients and healthy controls and are considered potential diagnostic biomarkers within EVs [111–113].

Other studies have identified several candidate protein biomarkers for HCC diagnosis or prognosis in EVs either within limited cohorts or solely validated using bioinformatics methods [114], including, haptoglobin (HP), transthyretin (TTR) [115], kinesin family member 2 C (KIF2C), targeting protein for xenopus kinesin-like protein 2 (TPX2) [116], LAPTM4B-35 [117], two isoforms MRP3A and MRP3B of ATP Binding Cassette Subfamily C Member 3 (ABCC3) [118], vWF together with other nine candidate proteins [119]. However, comprehensive explorations of their diagnostic efficacy remain unexplored.

In comparison to the blood proteome, the urine proteome is less complex, thus making it easier to detect changes in low-abundance proteins that may have potential significance. Currently, there is limited research focused on the proteomics of urinary EVs (uEVs). In an article published in 2023, Feng et al. reported the significant enrichment of three proteins, olfactomedin 4 (OLFM4), growth differentiation factor 15 (GDF15), and hepatocellular carcinoma-derived growth factor (HDGF), in uEVs from patients with HCC using an array-based amphiphilic supramolecular probe (ADSP)-modified NC membrane platform [120]. Another study explored the glycoproteomic profile of uEVs, focusing on the post-translational modification of proteins. The results revealed significantly elevated levels of galectin-3-binding protein (LG3BP), polymeric immunoglobulin receptor (pIgR), and kininogen-1 (KNG1) glycosylation in patients with HCC compared to the control group. Conversely, the level of apoptosis stimulating of p53 protein 2 (ASPP2) was found to be decreased [121]. However, further validation of the diagnostic efficacy of these studies is still needed.

EV-based proteomics has also been employed to predict treatment response. In a clinical trial involving 25 patients receiving selective internal radiation therapy (SIRT) plus sorafenib treatment and 20 patients receiving sorafenib alone for advanced HCC, high levels of EV-GPX3/ACTR3 and low levels of EV-ARHGAP1B were associated with greater efficacy of SIRT plus sorafenib treatment (AUC = 1) [122]. This encouraging result suggests that exploring EV-based biomarkers in a clinical trial setting is a promising avenue for future research.

Some other studies have revealed potential EV-associated protein markers, which await further exploration. One study indicated the RNA level of splicing factor 3b subunit 4 (SF3B4) derived from EVs can serve as a diagnostic biomarker for HCC, with the AUC surpassing that of AFP. However, further validation is essential to ascertain the protein expression levels of SF3B4 within EVs [123]. Another study has demonstrated the presence of tissue transglutaminase 2 (TGM2) protein, unique to HCC cell-derived EVs, suggesting its potential as a candidate diagnostic biomarker for HCC [124]. In order to display the existing research on EV-associated proteins in the field of HCC biomarkers, we listed Table 1 with key information to offer a concise and informative overview.

Table 1 The current status of research on EV-associated proteins as biomarkers for HCC

Biomarkers	Cohort number	AUC	Se/Spe (%)	PPV/NPV
(%)	Applications	Ref.	
AnnexinV+EpCAM+ASGPR1+	HCC: 86

Cirrhosis: 49

	0.732 (0.646–0.818)	81.40/41.94	72.92/58.97	Diagnosis of HCC in the background of cirrhosis	[104]	
EpCAM+ CD63+/

CD147+ CD63+/

GPC3+ CD63+

	HCC: 35

Cirrhosis: 37

(Validation cohort)

	0.93 (0.87–0.99)	91/81	82/91	Diagnosis of HCC in the background of cirrhosis	[109]	
CCT8	HCC: 132

Cirrhosis: 33

CHB: 25

Normal: 34

	0.698 (0.634–0.758)	46.21/93.48	58.93/91.05	Diagnosis of HCC	[110]	
cofilin-1	HCC: 132

Cirrhosis: 33

CHB: 25

Normal: 34

	0.677 (0.612–0.738)	40.91/94.57	91.53/52.73	Diagnosis of HCC	[110]	
CCT8 + cofilin-1 + AFP	HCC: 132

Cirrhosis: 33

CHB: 25

Normal: 34

	0.838 (0.783–0.884)	70.46/81.52	84.55/65.79	Diagnosis of HCC	[110]	
HSP90α+ LC3B+	HCC: 51

NMLD: 33

Normal: 30

	0.960 (NA)	86.00/96.67	NA	Diagnosis of HCC	[68]	
High GPX3/ACTR3

Low ARHGAP1B

	HCC: 45	1.0 (NA)	NA	NA	Predicting the efficacy of SIRT + sorafenib therapy	[122]	
Anti-ATIC	HCC: 144

Normal: 118

	0.875 (0.834–0.917)	70.83/90.63	NA	Diagnosis of HCC	[111]	
Anti-BRD2	HCC: 118

Cirrhosis: 32

Benign: 3

Normal: 91

	0.776 (0.714–0.839)	64.41/82.42	NA	Diagnosis of HCC	[112]	
Anti-EIF3A	HCC: 102

Normal: 85

	0.871 (0.822–0.922)	79.41/83.53	NA	Diagnosis of HCC	[113]	
GPC-3	HCC: 25

Cirrhosis: 25

Normal: 25

	1.0 (NA) (vs. normal)

0.95 (NA) (vs. cirrhosis)

	NA	NA	Diagnosis of HCC	[106]	
MRP3A/MRP3B	HCC: 14

Cirrhosis: 27

Normal: 15

	NA	NA	NA	Higher expression in EVs from HCC	[118]	
LAPTM4B-35	HCC: 43

Normal: 33

	NA	NA	NA	Higher expression in EVs from HCC	[117]	
HP	HCC: 15

Cirrhosis: 15

CHB: 15

	NA	NA	NA	Higher expression in EVs from HCC compared to cirrhosis and CHB	[115]	
TTR	HCC: 15

Cirrhosis: 15

CHB: 15

	NA	NA	NA	Lower expression in EVs from HCC compared to CHB	[115]	
vWF/ LGALS3BP/ TGFB1/ SERPINC1	HCC: 20

Normal: 10

	NA	NA	NA	Higher expression in EVs from HCC	[119]	
HPX/ HP/ HBA1/ FGA/

FGG/ FGB

	HCC: 20

Normal: 10

	NA	NA	NA	Lower expression in EVs from HCC	[119]	
COL1A2/ POSTN/ STAM/

COL6A1/ EXOC8

	HBV-related HCC cell lines

Normal liver cells

	NA	NA	NA	Higher expression in EVs from HBV-related HCC cells	[114]	
KIF2C/ TPX2	HBV-related HCC: 225

Normal: 220

	NA	NA	NA	Higher expression in EVs from HCC with poor OS and RFS	[116]	
TGM2	NA	NA	NA	NA	Unique in the EVs from HCC cells	[124]	
OLFM4/ GDF15/ HDGF	HCC: 18

Normal: 6

	NA	NA	NA	Higher expression in uEVs from HCC	[120]	
LG3BP/ pIgR/ KNG1	HCC: 21

Normal: 7

	NA	NA	NA	Higher expression in uEVs from HCC	[121]	
ASPP2	HCC: 21

Normal: 7

	NA	NA	NA	Lower expression in uEVs from HCC	[121]	
Se, sensitivity; Spe, specificity; PPV, positive predictive value; NPV negative predictive value; Ref., reference; CHB, chronic hepatitis B; HBV, hepatitis B virus; NMLD, non-malignant liver disease; OS, overall survival; RFS, relapse-free survival; NA, not available

The exploration of EV-associated proteins in HCC treatment

The current treatment methods for HCC include surgical treatment, chemotherapy, radiotherapy, targeted therapy, immunotherapy, and other approaches [125]. HCC exhibits high heterogeneity, with a high postoperative recurrence rate. Early-stage HCC frequently lacks symptomatic manifestations, leading to diagnosis at an advanced stage. Furthermore, patients with advanced HCC face a paucity of targeted pharmacological interventions tailored to their specific condition. Based on the specific protein expression profile of EVs and the ability to transport cargo, EVs demonstrate a remarkable level of selectivity. This selectivity is achieved through surface ligands that target specific receptors, as well as inherent cargo sorting mechanisms, thus enabling accurate delivery to target cells while minimizing off-target effects. Furthermore, EVs-mediated drug delivery is characterized by low toxicity, low immunogenicity, and high engineering versatility [126]. EVs differ from exogenous synthetic drug carriers in that the latter often exhibit strong immunogenicity and are prone to interact with drug proteins thus garnering increasing research attention [127].

LAMP2 (lysosomal associated membrane protein 2) B, as an abundantly expressed protein on the membrane of EVs, has been widely employed in the engineering of EVs. This enables the EVs to target tissues or organs carrying the corresponding receptors [128]. The tumor-targeted nano-delivery system, formed by the fusion of the SP94 peptide and the N-terminal RNA recognition motif (RRM) at both ends of the LAMP2 platform, can enhance sorafenib-induced ferroptosis in HCC [129]. We observed that to enhance the efficacy of ferroptosis and reduce liver toxicity, researchers have engineered EVs to achieve these aims. Researchers transfected CD47-expressing plasmids onto EVs to evade macrophage phagocytosis and displayed much lower toxicity. Additionally, they loaded the membrane with the ferroptosis inducer Erastin and internally loaded the photosensitizer RB through sonication. The combination of chemo-photodynamic therapy displayed significant efficacy and safety [130].

The clustered regularly interspaced short palindromic repeats (CRISPR)–associated nuclease protein 9 (Cas9)–based genome editing holds tremendous therapeutic potential but exhibits characteristics such as easy degradation in the bloodstream and lack of tissue-specific uptake, hampering its efficacy. Whereas EVs provide the most efficient delivery method for CRISPR-Cas9, particularly for the delivery of Cas9 ribonucleoprotein (RNP), serving as an excellent carrier. Research indicates that through electroporation, Cas9 RNP can be transported into EVs derived from HSCs and subsequently taken up specifically by liver cells. Notably, Cas9 RNP targeting lysine acetyltransferase 5 (KAT5) can suppress the growth of HCC, which suggests that therapeutics involving the editing of other HCC-related genes hold vast potential for treatment [131].

Recently, several EVs-loaded drugs have been developed to stimulate tumor-specific immune responses and can serve as tumor vaccines. Dendritic cells (DCs) are believed to enhance antigen presentation and improve immunogenicity upon stimulation by EVs derived from HCC. High-mobility group nucleosome-binding protein 1 (HMGN1) can enhance DC maturation and activation and consistently elicit Th1 immune responses. By conjugating HMGN1 with CP05, an anchoring protein on EVs, HMGN1 can be more efficiently delivered systemically into cells. Research evidence has demonstrated that this approach activates DCs, remodels the tumor microenvironment, activates memory T cells, and exhibits promising therapeutic prospects [132]. Furthermore, the research team proposed a universal immunotherapeutic approach that does not require the identification of tumor antigens. By engineering EVs derived from DCs simultaneously loaded with P47, AFP, and HMGN1, complete eradication of tumors in situ was achieved in mice with HCC [16]. One kind of histone deacetylase inhibitor, MS-275, has been shown to upregulate tumor-specific antigens, thereby enhancing both specific and nonspecific anti-tumor immune responses. EVs from HCC cells treated with MS-275 showed increased expression levels of HSP70 and major histocompatibility complex (MHC) class I polypeptide-related sequence B (MICB). Previous research has demonstrated that HSP70 from tumor EVs can selectively activate NK cell activity, which is also consistent with the enhanced NK cell activity observed in this study [133, 134]. Another inhibitor of DNA methyltransferase, 5-Aza-2’-deoxycytidine (5-Aza-CdR), has been shown to increase the production of EVs and immune-related protein components in hepatoma cells, including human leukocyte antigen-I (HLA-I) and NY-ESO-1 protein [135]. These findings suggest promising approaches for developing tumor vaccine strategies against HCC.

Some therapeutic prospects have been proposed to address challenges in chemotherapy, immunotherapy, and radiotherapy for HCC from the perspective of EV-associated proteins. Research indicates that the use of chemotherapy drugs, especially resistant anticancer drugs, can significantly increase the levels of HSP in EVs released by HCC cells. This further induces the biological activity of NK cells, suggesting that immunotherapy based on resistant anticancer drugs holds promising prospects [17]. Although HCC is not sensitive to radioiodine therapy with I131, it can still enhance the iodine uptake capacity of HCC cells through the transport of relevant proteins via EVs. Studies have shown that genetically engineered HCC cells expressing the sodium iodide symporter (NIS) protein can transfer NIS protein to other HCC cells in the form of EVs, exhibiting a high sensitivity to radiotherapy [136].

There are also several proteins with drug potential that deserve attention. The TRAIL protein, functioning as a transmembrane death receptor ligand, engages with cell surface receptors DR4 and DR5, prompting cellular apoptosis through Caspase-8-mediated signaling cascades. Evidence suggests that EV-delivered TRAIL effectively induces apoptosis in various cancer cell lines, including lung cancer and breast cancer. Moreover, several studies have identified a synergistic effect between TRAIL and sorafenib. This suggests that EV-mediated delivery of TRAIL may offer a promising therapeutic strategy to overcome drug resistance in HCC [18]. One recent study indicated that the Parkinson’s disease-related protein α-synuclein can be secreted in the form of EVs and can traverse the blood-brain barrier. It has been found to inhibit the proliferation and migration of HCC [137].

Based on the current research progress, it can be seen that studies on the application of EV-related proteins in HCC treatment are mostly at the cellular experiment stage. Some therapeutic potentials were confirmed in animal models; however, there is still a long way to go before clinical trials in humans can be conducted, such as the lack of standardized technologies for large-scale EVs production. In Table 2, we summarized the current research progress about the treatment of EV-associated proteins.

Table 2 The current progress of EV-associated proteins for the treatment of HCC

Protein	Origins	Location	Effects	Ref.	
LAMP2B	Naturally present in various types of EVs	Membrane	Serve as a kind of protein anchor	[128]	
SP94-LAPM2B-RRM complex	Transfection from plasmids into HEK-293T cells	Membrane	Enhance sorafenib-induced ferroptosis in HCC by silencing GPX4 and DHODH expression	[129]	
CD47	Transfection from plasmids into HEK-293T cells	Membrane	Ensure the EVs effectively escape the phagocytosis of mononuclear phagocyte system/

Induces ferroptosis in HCC with reduced toxicity when combined with Rose Bengal (a kind of photosensitizer) and Erastin (a kind of ferroptosis inducer)

	[130]	
Cas9 ribonucleoprotein	A kind of protein complexed with sgRNA which can be loaded into EVs isolated from LX-2 cells through electroporation	Cargo	Enable the efficient delivery of CRISPR-Cas9 system/

Cas9 RNP targeting lysine acetyltransferase 5 (KAT5) can suppress the growth of HCC

	[131]	
CP05	Naturally present in various types of EVs	Membrane	Serve as a kind of protein anchor	[16, 132]	
N1ND-CP05 complex	Incubated with EVs from Hepa1-6 cells	Membrane	Activate dendritic cells and activate memory T cells	[132]	
N1ND-CP05& AFP212- CP05& P47-CP05 complex	Incubated with EVs from DC2.4 cells	Membrane	Promote recruitment, accumulation and activation of dendritic cells/ Enhance cross-presentation of tumor neoantigens and T cell response	[16]	
MS-275	A kind of histone deacetylase inhibitor	NA	Increase the expression of HSP70 and MICB in EVs from HepG2 cells and augment the cytotoxicity of NK cells	[133]	
5-Aza-2’-deoxycytidine	A kind of DNA methyltransferase	NA	Increase the expression of HLA-I and NY-ESO-1 in EVs from HepG2 and Hep3B cells and stimulate anti-tumor-specific immune response	[135]	
HSP60&HSP70&HSP90	Naturally present in tumor-derived EVs	Cargo	Stimulate NK cell cytotoxicity and induce HSP-specific NK cell anti-tumor responses	[17]	
NIS	Transfection from plasmids into Huh7 cells	Cargo	Increase the I131 radioiodine uptake and DNA damage of HCC cells, show the potential to revert radioiodine-resistant cancers into radioiodine-sensitive cancers	[136]	
TRAIL	A kind of ligand to the death receptors, DR4 and DR5, which can be encapsulated into EVs	Cargo	Induce cellular apoptosis of tumor cells through the Caspase-8-mediated signaling cascades	[18]	
α-synuclein	A main component of

of Lewy bodies secreted by SH-SY5Y cells/

Transfection from plasmids into HEK-293T cells

	Cargo	Inhibited the cell viability,

migration, and invasion of HCC cells

	[137]	
Ref., reference; NA, not available

Conclusions and future prospects

This review introduces the biological characteristics and biogenesis of EVs, emphasizes the role of EV-associated proteins in the development of HCC, and summarizes current research on EV-associated proteins as tumor biomarkers and therapeutic targets for HCC. EVs, as important mediators of intercellular communication, play a significant role in the tumor progression of HCC. We have discussed the functions of EV-associated proteins from the perspectives of communication between HCC and components of the TME, and mechanisms of tumor metastasis including EMT and targeted metastasis. We outlined the advantages of EV-associated proteins as biomarkers for HCC and provided diagnostic data from existing studies, demonstrating their excellent diagnostic efficacy. From the perspective of therapeutic exploration, we have summarized that EV-associated proteins can serve as anchors for drug delivery, improving the accuracy of drug delivery. Additionally, they can directly exhibit anti-tumor effects, demonstrating promising therapeutic potential.

By reviewing existing research, we believe that the challenges identified in current studies can provide valuable insights for future research directions. Firstly, the protein composition of EVs is diverse and complex. In the same tumor process, EVs may contain proteins with opposite functions. For clinical applications, it is necessary to accurately isolate proteins in EVs that inhibit tumors and further consider methods for their effective delivery. Secondly, EV-associated proteins as tumor biomarkers for HCC hold significant clinical potential due to their stability and resistance to degradation. However, the high cost of EV extraction and the lack of standardized biomarkers to determine EV origin are challenges. Currently, most relevant research has not yet entered the clinical stage, and many potential protein markers lack diagnostic test data, highlighting the urgent need for further clinical validation in large cohorts. Thirdly, the exploration of therapeutic potential associated with EV-associated proteins warrants further investigation. In contrast to the extensive exploration of systemic treatment strategies for advanced HCC, such as targeted and immunotherapy combinations, there is limited research exploring the intersection of EV-associated proteins with current systemic treatment studies for advanced HCC.

Despite the challenges faced by current research, the undeniable potential of EV-associated proteins in the clinical application of HCC is evident. In conclusion, research on EV-associated proteins holds the promise to deepen our understanding of HCC and provide a unique perspective for the development of new treatment strategies.

Acknowledgements

For the completion of this study, I would like to express my deepest gratitude to Prof. Xu and Prof. Lu, who have given me the most valuable suggestions and guides.

Author contributions

XL and Y-YX conceived and designed the review. Y-GL wrote the manuscript, drew the figure, and participated in the design of the review. S-TJ collected relevant literature. All authors contributed to the article.

Funding

This article was partially sponsored by the National High Level Hospital Clinical Research Funding (2022-PUMCH-C-049 and 2022-PUMCH-A-237).

Data availability

Not applicable.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

This review has got consent for publication from all authors.

Competing interest

The authors declare that no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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References

1. Sung H Ferlay J Siegel RL Laversanne M Soerjomataram I Jemal A Bray F Global Cancer statistics 2020: GLOBOCAN estimates of incidence and Mortality Worldwide for 36 cancers in 185 countries CA Cancer J Clin 2021 71 209 49 10.3322/caac.21660 33538338
Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, Bray F. Global Cancer statistics 2020: GLOBOCAN estimates of incidence and Mortality Worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2021;71:209–49. 10.3322/caac.21660.33538338 10.3322/caac.21660
2. Yang C Zhang H Zhang L Zhu AX Bernards R Qin W Wang C Evolving therapeutic landscape of advanced hepatocellular carcinoma Nat Rev Gastroenterol Hepatol 2023 20 203 22 10.1038/s41575-022-00704-9 36369487
Yang C, Zhang H, Zhang L, Zhu AX, Bernards R, Qin W, Wang C. Evolving therapeutic landscape of advanced hepatocellular carcinoma. Nat Rev Gastroenterol Hepatol. 2023;20:203–22. 10.1038/s41575-022-00704-9.36369487 10.1038/s41575-022-00704-9
3. Welsh JA Goberdhan DCI O’Driscoll L Buzas EI Blenkiron C Bussolati B Cai H Minimal information for studies of extracellular vesicles (MISEV2023): from basic to advanced approaches J Extracell Vesicles 2024 13 e12404 10.1002/jev2.12404 38326288
Welsh JA, Goberdhan DCI, O’Driscoll L, Buzas EI, Blenkiron C, Bussolati B, Cai H, et al. Minimal information for studies of extracellular vesicles (MISEV2023): from basic to advanced approaches. J Extracell Vesicles. 2024;13:e12404. 10.1002/jev2.12404.38326288 10.1002/jev2.12404
4. Wang X Shen H Zhangyuan G Huang R Zhang W He Q Jin K 14-3-3zeta delivered by hepatocellular carcinoma-derived exosomes impaired anti-tumor function of tumor-infiltrating T lymphocytes Cell Death Dis 2018 9 159 10.1038/s41419-017-0180-7 29415983
Wang X, Shen H, Zhangyuan G, Huang R, Zhang W, He Q, Jin K, et al. 14-3-3zeta delivered by hepatocellular carcinoma-derived exosomes impaired anti-tumor function of tumor-infiltrating T lymphocytes. Cell Death Dis. 2018;9:159. 10.1038/s41419-017-0180-7.29415983 10.1038/s41419-017-0180-7
5. Xie JY Wei JX Lv LH Han QF Yang WB Li GL Wang PX Angiopoietin-2 induces angiogenesis via exosomes in human hepatocellular carcinoma Cell Commun Signal 2020 18 46 10.1186/s12964-020-00535-8 32183816
Xie JY, Wei JX, Lv LH, Han QF, Yang WB, Li GL, Wang PX, et al. Angiopoietin-2 induces angiogenesis via exosomes in human hepatocellular carcinoma. Cell Commun Signal. 2020;18:46. 10.1186/s12964-020-00535-8.32183816 10.1186/s12964-020-00535-8
6. Hareendran S, Albraidy B, Yang X, Liu A, Breggia A, Chen CC, Loh YP. Exosomal Carboxypeptidase E (CPE) and CPE-shRNA-Loaded exosomes regulate metastatic phenotype of Tumor cells. Int J Mol Sci. 2022;23. 10.3390/ijms23063113.
7. Liu Y, Tang Y, Jiang H, Zhang X, Chen X, Guo J, Jin C, et al. Exosome-related FTCD facilitates M1 Macrophage polarization and impacts the prognosis of Hepatocellular Carcinoma. Biomolecules. 2023;14. 10.3390/biom14010041.
8. Mathieu M Nevo N Jouve M Valenzuela JI Maurin M Verweij FJ Palmulli R Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking of CD63 and CD9 Nat Commun 2021 12 4389 10.1038/s41467-021-24384-2 34282141
Mathieu M, Nevo N, Jouve M, Valenzuela JI, Maurin M, Verweij FJ, Palmulli R, et al. Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking of CD63 and CD9. Nat Commun. 2021;12:4389. 10.1038/s41467-021-24384-2.34282141 10.1038/s41467-021-24384-2
9. Liu Y Xun Z Ma K Liang S Li X Zhou S Sun L Identification of a tumour immune barrier in the HCC microenvironment that determines the efficacy of immunotherapy J Hepatol 2023 78 770 82 10.1016/j.jhep.2023.01.011 36708811
Liu Y, Xun Z, Ma K, Liang S, Li X, Zhou S, Sun L, et al. Identification of a tumour immune barrier in the HCC microenvironment that determines the efficacy of immunotherapy. J Hepatol. 2023;78:770–82. 10.1016/j.jhep.2023.01.011.36708811 10.1016/j.jhep.2023.01.011
10. Zeng Y, Hu S, Luo Y, He K. Exosome cargos as biomarkers for diagnosis and prognosis of Hepatocellular Carcinoma. Pharmaceutics. 2023;15. 10.3390/pharmaceutics15092365.
11. Azmi AS Bao B Sarkar FH Exosomes in cancer development, metastasis, and drug resistance: a comprehensive review Cancer Metastasis Rev 2013 32 623 42 10.1007/s10555-013-9441-9 23709120
Azmi AS, Bao B, Sarkar FH. Exosomes in cancer development, metastasis, and drug resistance: a comprehensive review. Cancer Metastasis Rev. 2013;32:623–42. 10.1007/s10555-013-9441-9.23709120 10.1007/s10555-013-9441-9
12. Tateishi R Yoshida H Matsuyama Y Mine N Kondo Y Omata M Diagnostic accuracy of tumor markers for hepatocellular carcinoma: a systematic review Hepatol Int 2008 2 17 30 10.1007/s12072-007-9038-x 19669276
Tateishi R, Yoshida H, Matsuyama Y, Mine N, Kondo Y, Omata M. Diagnostic accuracy of tumor markers for hepatocellular carcinoma: a systematic review. Hepatol Int. 2008;2:17–30. 10.1007/s12072-007-9038-x.19669276 10.1007/s12072-007-9038-x
13. Thietart S Rautou PE Extracellular vesicles as biomarkers in liver diseases: a clinician’s point of view J Hepatol 2020 73 1507 25 10.1016/j.jhep.2020.07.014 32682050
Thietart S, Rautou PE. Extracellular vesicles as biomarkers in liver diseases: a clinician’s point of view. J Hepatol. 2020;73:1507–25. 10.1016/j.jhep.2020.07.014.32682050 10.1016/j.jhep.2020.07.014
14. Zhou X Jia Y Mao C Liu S Small extracellular vesicles: non-negligible vesicles in tumor progression, diagnosis, and therapy Cancer Lett 2024 580 216481 10.1016/j.canlet.2023.216481 37972701
Zhou X, Jia Y, Mao C, Liu S. Small extracellular vesicles: non-negligible vesicles in tumor progression, diagnosis, and therapy. Cancer Lett. 2024;580:216481. 10.1016/j.canlet.2023.216481.37972701 10.1016/j.canlet.2023.216481
15. Qiu X Li Z Han X Zhen L Luo C Liu M Yu K Tumor-derived nanovesicles promote lung distribution of the therapeutic nanovector through repression of Kupffer cell-mediated phagocytosis Theranostics 2019 9 2618 36 10.7150/thno.32363 31131057
Qiu X, Li Z, Han X, Zhen L, Luo C, Liu M, Yu K, et al. Tumor-derived nanovesicles promote lung distribution of the therapeutic nanovector through repression of Kupffer cell-mediated phagocytosis. Theranostics. 2019;9:2618–36. 10.7150/thno.32363.31131057 10.7150/thno.32363
16. Zuo B Zhang Y Zhao K Wu L Qi H Yang R Gao X Universal immunotherapeutic strategy for hepatocellular carcinoma with exosome vaccines that engage adaptive and innate immune responses J Hematol Oncol 2022 15 46 10.1186/s13045-022-01266-8 35488312
Zuo B, Zhang Y, Zhao K, Wu L, Qi H, Yang R, Gao X, et al. Universal immunotherapeutic strategy for hepatocellular carcinoma with exosome vaccines that engage adaptive and innate immune responses. J Hematol Oncol. 2022;15:46. 10.1186/s13045-022-01266-8.35488312 10.1186/s13045-022-01266-8
17. Lv LH Wan YL Lin Y Zhang W Yang M Li GL Lin HM Anticancer drugs cause release of exosomes with heat shock proteins from human hepatocellular carcinoma cells that elicit effective natural killer cell antitumor responses in vitro J Biol Chem 2012 287 15874 85 10.1074/jbc.M112.340588 22396543
Lv LH, Wan YL, Lin Y, Zhang W, Yang M, Li GL, Lin HM, et al. Anticancer drugs cause release of exosomes with heat shock proteins from human hepatocellular carcinoma cells that elicit effective natural killer cell antitumor responses in vitro. J Biol Chem. 2012;287:15874–85. 10.1074/jbc.M112.340588.22396543 10.1074/jbc.M112.340588
18. Thapa N, Chwae YJ, Yoo KH, Won TB, Kang D, Choi D, Kim J. Exosomal delivery of TRAIL and miR–335 for the treatment of hepatocellular carcinoma (review). Int J Mol Med. 2023;51. 10.3892/ijmm.2022.5206.
19. Huang S Xiong M Liu J Wang J Han X Chen Z Xie P Exosomes with IR780 and Lenvatinib loaded on GPC3 single-chain scFv antibodies for targeted hyperthermia and chemotherapy in hepatocellular carcinoma therapy Am J Cancer Res 2023 13 5368 81 38058824
Huang S, Xiong M, Liu J, Wang J, Han X, Chen Z, Xie P, et al. Exosomes with IR780 and Lenvatinib loaded on GPC3 single-chain scFv antibodies for targeted hyperthermia and chemotherapy in hepatocellular carcinoma therapy. Am J Cancer Res. 2023;13:5368–81.38058824
20. Cocucci E Meldolesi J Ectosomes and exosomes: shedding the confusion between extracellular vesicles Trends Cell Biol 2015 25 364 72 10.1016/j.tcb.2015.01.004 25683921
Cocucci E, Meldolesi J. Ectosomes and exosomes: shedding the confusion between extracellular vesicles. Trends Cell Biol. 2015;25:364–72. 10.1016/j.tcb.2015.01.004.25683921 10.1016/j.tcb.2015.01.004
21. Kalluri R, LeBleu VS. The biology, function, and biomedical applications of exosomes. Science. 2020;367. 10.1126/science.aau6977.
22. Hou PP Luo LJ Chen HZ Chen QT Bian XL Wu SF Zhou JX Ectosomal PKM2 promotes HCC by inducing macrophage differentiation and remodeling the Tumor Microenvironment Mol Cell 2020 78 1192 e12061110 10.1016/j.molcel.2020.05.004 32470318
Hou PP, Luo LJ, Chen HZ, Chen QT, Bian XL, Wu SF, Zhou JX, et al. Ectosomal PKM2 promotes HCC by inducing macrophage differentiation and remodeling the Tumor Microenvironment. Mol Cell. 2020;78:1192–e12061110. 10.1016/j.molcel.2020.05.004.32470318 10.1016/j.molcel.2020.05.004
23. Katzmann DJ Babst M Emr SD Ubiquitin-dependent sorting into the multivesicular body pathway requires the function of a conserved endosomal protein sorting complex ESCRT-I Cell 2001 106 145 55 10.1016/s0092-8674(01)00434-2 11511343
Katzmann DJ, Babst M, Emr SD. Ubiquitin-dependent sorting into the multivesicular body pathway requires the function of a conserved endosomal protein sorting complex. ESCRT-I Cell. 2001;106:145–55. 10.1016/s0092-8674(01)00434-2.11511343 10.1016/s0092-8674(01)00434-2
24. Babst M Katzmann DJ Snyder WB Wendland B Emr SD Endosome-associated complex, ESCRT-II, recruits transport machinery for protein sorting at the multivesicular body Dev Cell 2002 3 283 9 10.1016/s1534-5807(02)00219-8 12194858
Babst M, Katzmann DJ, Snyder WB, Wendland B, Emr SD. Endosome-associated complex, ESCRT-II, recruits transport machinery for protein sorting at the multivesicular body. Dev Cell. 2002;3:283–9. 10.1016/s1534-5807(02)00219-8.12194858 10.1016/s1534-5807(02)00219-8
25. Babst M Katzmann DJ Estepa-Sabal EJ Meerloo T Emr SD Escrt-III: an endosome-associated heterooligomeric protein complex required for mvb sorting Dev Cell 2002 3 271 82 10.1016/s1534-5807(02)00220-4 12194857
Babst M, Katzmann DJ, Estepa-Sabal EJ, Meerloo T, Emr SD. Escrt-III: an endosome-associated heterooligomeric protein complex required for mvb sorting. Dev Cell. 2002;3:271–82. 10.1016/s1534-5807(02)00220-4.12194857 10.1016/s1534-5807(02)00220-4
26. Babst M MVB vesicle formation: ESCRT-dependent, ESCRT-independent and everything in between Curr Opin Cell Biol 2011 23 452 7 10.1016/j.ceb.2011.04.008 21570275
Babst M. MVB vesicle formation: ESCRT-dependent, ESCRT-independent and everything in between. Curr Opin Cell Biol. 2011;23:452–7. 10.1016/j.ceb.2011.04.008.21570275 10.1016/j.ceb.2011.04.008
27. Wemmer M Azmi I West M Davies B Katzmann D Odorizzi G Bro1 binding to Snf7 regulates ESCRT-III membrane scission activity in yeast J Cell Biol 2011 192 295 306 10.1083/jcb.201007018 21263029
Wemmer M, Azmi I, West M, Davies B, Katzmann D, Odorizzi G. Bro1 binding to Snf7 regulates ESCRT-III membrane scission activity in yeast. J Cell Biol. 2011;192:295–306. 10.1083/jcb.201007018.21263029 10.1083/jcb.201007018
28. Henne WM Buchkovich NJ Zhao Y Emr SD The endosomal sorting complex ESCRT-II mediates the assembly and architecture of ESCRT-III helices Cell 2012 151 356 71 10.1016/j.cell.2012.08.039 23063125
Henne WM, Buchkovich NJ, Zhao Y, Emr SD. The endosomal sorting complex ESCRT-II mediates the assembly and architecture of ESCRT-III helices. Cell. 2012;151:356–71. 10.1016/j.cell.2012.08.039.23063125 10.1016/j.cell.2012.08.039
29. Raiborg C Stenmark H The ESCRT machinery in endosomal sorting of ubiquitylated membrane proteins Nature 2009 458 445 52 10.1038/nature07961 19325624
Raiborg C, Stenmark H. The ESCRT machinery in endosomal sorting of ubiquitylated membrane proteins. Nature. 2009;458:445–52. 10.1038/nature07961.19325624 10.1038/nature07961
30. Bache KG Brech A Mehlum A Stenmark H Hrs regulates multivesicular body formation via ESCRT recruitment to endosomes J Cell Biol 2003 162 435 42 10.1083/jcb.200302131 12900395
Bache KG, Brech A, Mehlum A, Stenmark H. Hrs regulates multivesicular body formation via ESCRT recruitment to endosomes. J Cell Biol. 2003;162:435–42. 10.1083/jcb.200302131.12900395 10.1083/jcb.200302131
31. Kostelansky MS Schluter C Tam YY Lee S Ghirlando R Beach B Conibear E Molecular architecture and functional model of the complete yeast ESCRT-I heterotetramer Cell 2007 129 485 98 10.1016/j.cell.2007.03.016 17442384
Kostelansky MS, Schluter C, Tam YY, Lee S, Ghirlando R, Beach B, Conibear E, et al. Molecular architecture and functional model of the complete yeast ESCRT-I heterotetramer. Cell. 2007;129:485–98. 10.1016/j.cell.2007.03.016.17442384 10.1016/j.cell.2007.03.016
32. Alam SL Langelier C Whitby FG Koirala S Robinson H Hill CP Sundquist WI Structural basis for ubiquitin recognition by the human ESCRT-II EAP45 GLUE domain Nat Struct Mol Biol 2006 13 1029 30 10.1038/nsmb1160 17057716
Alam SL, Langelier C, Whitby FG, Koirala S, Robinson H, Hill CP, Sundquist WI. Structural basis for ubiquitin recognition by the human ESCRT-II EAP45 GLUE domain. Nat Struct Mol Biol. 2006;13:1029–30. 10.1038/nsmb1160.17057716 10.1038/nsmb1160
33. Schoneberg J Lee IH Iwasa JH Hurley JH Reverse-topology membrane scission by the ESCRT proteins Nat Rev Mol Cell Biol 2017 18 5 17 10.1038/nrm.2016.121 27703243
Schoneberg J, Lee IH, Iwasa JH, Hurley JH. Reverse-topology membrane scission by the ESCRT proteins. Nat Rev Mol Cell Biol. 2017;18:5–17. 10.1038/nrm.2016.121.27703243 10.1038/nrm.2016.121
34. Amerik AY Nowak J Swaminathan S Hochstrasser M The Doa4 deubiquitinating enzyme is functionally linked to the vacuolar protein-sorting and endocytic pathways Mol Biol Cell 2000 11 3365 80 10.1091/mbc.11.10.3365 11029042
Amerik AY, Nowak J, Swaminathan S, Hochstrasser M. The Doa4 deubiquitinating enzyme is functionally linked to the vacuolar protein-sorting and endocytic pathways. Mol Biol Cell. 2000;11:3365–80. 10.1091/mbc.11.10.3365.11029042 10.1091/mbc.11.10.3365
35. Tarrason Risa G, Hurtig F, Bray S, Hafner AE, Harker-Kirschneck L, Faull P, Davis C, et al. The proteasome controls ESCRT-III-mediated cell division in an archaeon. Science. 2020;369. 10.1126/science.aaz2532.
36. Stuffers S Sem Wegner C Stenmark H Brech A Multivesicular endosome biogenesis in the absence of ESCRTs Traffic 2009 10 925 37 10.1111/j.1600-0854.2009.00920.x 19490536
Stuffers S, Sem Wegner C, Stenmark H, Brech A. Multivesicular endosome biogenesis in the absence of ESCRTs. Traffic. 2009;10:925–37. 10.1111/j.1600-0854.2009.00920.x.19490536 10.1111/j.1600-0854.2009.00920.x
37. Trajkovic K Hsu C Chiantia S Rajendran L Wenzel D Wieland F Schwille P Ceramide triggers budding of exosome vesicles into multivesicular endosomes Science 2008 319 1244 7 10.1126/science.1153124 18309083
Trajkovic K, Hsu C, Chiantia S, Rajendran L, Wenzel D, Wieland F, Schwille P, et al. Ceramide triggers budding of exosome vesicles into multivesicular endosomes. Science. 2008;319:1244–7. 10.1126/science.1153124.18309083 10.1126/science.1153124
38. Matsuo H Chevallier J Mayran N Le Blanc I Ferguson C Faure J Blanc NS Role of LBPA and Alix in multivesicular liposome formation and endosome organization Science 2004 303 531 4 10.1126/science.1092425 14739459
Matsuo H, Chevallier J, Mayran N, Le Blanc I, Ferguson C, Faure J, Blanc NS, et al. Role of LBPA and Alix in multivesicular liposome formation and endosome organization. Science. 2004;303:531–4. 10.1126/science.1092425.14739459 10.1126/science.1092425
39. van der Pol E Coumans FA Grootemaat AE Gardiner C Sargent IL Harrison P Sturk A Particle size distribution of exosomes and microvesicles determined by transmission electron microscopy, flow cytometry, nanoparticle tracking analysis, and resistive pulse sensing J Thromb Haemost 2014 12 1182 92 10.1111/jth.12602 24818656
van der Pol E, Coumans FA, Grootemaat AE, Gardiner C, Sargent IL, Harrison P, Sturk A, et al. Particle size distribution of exosomes and microvesicles determined by transmission electron microscopy, flow cytometry, nanoparticle tracking analysis, and resistive pulse sensing. J Thromb Haemost. 2014;12:1182–92. 10.1111/jth.12602.24818656 10.1111/jth.12602
40. Gardiner C Shaw M Hole P Smith J Tannetta D Redman CW Sargent IL Measurement of refractive index by nanoparticle tracking analysis reveals heterogeneity in extracellular vesicles J Extracell Vesicles 2014 3 25361 10.3402/jev.v3.25361 25425324
Gardiner C, Shaw M, Hole P, Smith J, Tannetta D, Redman CW, Sargent IL. Measurement of refractive index by nanoparticle tracking analysis reveals heterogeneity in extracellular vesicles. J Extracell Vesicles. 2014;3:25361. 10.3402/jev.v3.25361.25425324 10.3402/jev.v3.25361
41. Qi R Bai Y Li K Liu N Xu Y Dal E Wang Y Cancer-associated fibroblasts suppress ferroptosis and induce gemcitabine resistance in pancreatic cancer cells by secreting exosome-derived ACSL4-targeting miRNAs Drug Resist Updat 2023 68 100960 10.1016/j.drup.2023.100960 37003125
Qi R, Bai Y, Li K, Liu N, Xu Y, Dal E, Wang Y, et al. Cancer-associated fibroblasts suppress ferroptosis and induce gemcitabine resistance in pancreatic cancer cells by secreting exosome-derived ACSL4-targeting miRNAs. Drug Resist Updat. 2023;68:100960. 10.1016/j.drup.2023.100960.37003125 10.1016/j.drup.2023.100960
42. Wang Y Li Y Zhong J Li M Zhou Y Lin Q Zong S Tumor-derived Cav-1 promotes pre-metastatic niche formation and lung metastasis in breast cancer Theranostics 2023 13 1684 97 10.7150/thno.79250 37056561
Wang Y, Li Y, Zhong J, Li M, Zhou Y, Lin Q, Zong S, et al. Tumor-derived Cav-1 promotes pre-metastatic niche formation and lung metastasis in breast cancer. Theranostics. 2023;13:1684–97. 10.7150/thno.79250.37056561 10.7150/thno.79250
43. Vlassov AV Magdaleno S Setterquist R Conrad R Exosomes: current knowledge of their composition, biological functions, and diagnostic and therapeutic potentials Biochim Biophys Acta 2012 1820 940 8 10.1016/j.bbagen.2012.03.017 22503788
Vlassov AV, Magdaleno S, Setterquist R, Conrad R. Exosomes: current knowledge of their composition, biological functions, and diagnostic and therapeutic potentials. Biochim Biophys Acta. 2012;1820:940–8. 10.1016/j.bbagen.2012.03.017.22503788 10.1016/j.bbagen.2012.03.017
44. Hoshino A Kim HS Bojmar L Gyan KE Cioffi M Hernandez J Zambirinis CP Extracellular vesicle and particle biomarkers define multiple human cancers Cell 2020 182 1044 e10611018 10.1016/j.cell.2020.07.009 32795414
Hoshino A, Kim HS, Bojmar L, Gyan KE, Cioffi M, Hernandez J, Zambirinis CP, et al. Extracellular vesicle and particle biomarkers define multiple human cancers. Cell. 2020;182:1044–e10611018. 10.1016/j.cell.2020.07.009.32795414 10.1016/j.cell.2020.07.009
45. Li Y Yu S Li L Chen J Quan M Li Q Gao Y KLF4-mediated upregulation of CD9 and CD81 suppresses hepatocellular carcinoma development via JNK signaling Cell Death Dis 2020 11 299 10.1038/s41419-020-2479-z 32350244
Li Y, Yu S, Li L, Chen J, Quan M, Li Q, Gao Y. KLF4-mediated upregulation of CD9 and CD81 suppresses hepatocellular carcinoma development via JNK signaling. Cell Death Dis. 2020;11:299. 10.1038/s41419-020-2479-z.32350244 10.1038/s41419-020-2479-z
46. Wang L Chen X Wang L Wang S Li W Liu Y Zhang J Knockdown of ST6Gal-I expression in human hepatocellular carcinoma cells inhibits their exosome-mediated proliferation- and migration-promoting effects IUBMB Life 2021 73 1378 91 10.1002/iub.2562 34559939
Wang L, Chen X, Wang L, Wang S, Li W, Liu Y, Zhang J. Knockdown of ST6Gal-I expression in human hepatocellular carcinoma cells inhibits their exosome-mediated proliferation- and migration-promoting effects. IUBMB Life. 2021;73:1378–91. 10.1002/iub.2562.34559939 10.1002/iub.2562
47. Liu YG Jiang ST Zhang L Zheng H Zhang T Zhang JW Zhao HT Worldwide productivity and research trend of publications concerning tumor immune microenvironment (TIME): a bibliometric study Eur J Med Res 2023 28 229 10.1186/s40001-023-01195-3 37430294
Liu YG, Jiang ST, Zhang L, Zheng H, Zhang T, Zhang JW, Zhao HT, et al. Worldwide productivity and research trend of publications concerning tumor immune microenvironment (TIME): a bibliometric study. Eur J Med Res. 2023;28:229. 10.1186/s40001-023-01195-3.37430294 10.1186/s40001-023-01195-3
48. Huang LH, Rau CS, Liu YW, Lin HP, Wu YC, Tsai CW, Chien PC, et al. Cathelicidin Antimicrobial peptide acts as a tumor suppressor in Hepatocellular Carcinoma. Int J Mol Sci. 2023;24. 10.3390/ijms242115652.
49. Lin M Liao W Dong M Zhu R Xiao J Sun T Chen Z Exosomal neutral sphingomyelinase 1 suppresses hepatocellular carcinoma via decreasing the ratio of sphingomyelin/ceramide FEBS J 2018 285 3835 48 10.1111/febs.14635 30106227
Lin M, Liao W, Dong M, Zhu R, Xiao J, Sun T, Chen Z, et al. Exosomal neutral sphingomyelinase 1 suppresses hepatocellular carcinoma via decreasing the ratio of sphingomyelin/ceramide. FEBS J. 2018;285:3835–48. 10.1111/febs.14635.30106227 10.1111/febs.14635
50. Cheng Z Lei Z Yang P Si A Xiang D Tang X Guo G Exosome-transmitted p120-catenin suppresses hepatocellular carcinoma progression via STAT3 pathways Mol Carcinog 2019 58 1389 99 10.1002/mc.23022 30997702
Cheng Z, Lei Z, Yang P, Si A, Xiang D, Tang X, Guo G, et al. Exosome-transmitted p120-catenin suppresses hepatocellular carcinoma progression via STAT3 pathways. Mol Carcinog. 2019;58:1389–99. 10.1002/mc.23022.30997702 10.1002/mc.23022
51. Li L Zhao J Zhang Q Tao Y Shen C Li R Ma Z Cancer Cell-Derived Exosomes promote HCC Tumorigenesis through hedgehog pathway Front Oncol 2021 11 756205 10.3389/fonc.2021.756205 34692546
Li L, Zhao J, Zhang Q, Tao Y, Shen C, Li R, Ma Z, et al. Cancer Cell-Derived Exosomes promote HCC Tumorigenesis through hedgehog pathway. Front Oncol. 2021;11:756205. 10.3389/fonc.2021.756205.34692546 10.3389/fonc.2021.756205
52. Huang A Dong J Li S Wang C Ding H Li H Su X Exosomal transfer of vasorin expressed in hepatocellular carcinoma cells promotes migration of human umbilical vein endothelial cells Int J Biol Sci 2015 11 961 9 10.7150/ijbs.11943 26157350
Huang A, Dong J, Li S, Wang C, Ding H, Li H, Su X, et al. Exosomal transfer of vasorin expressed in hepatocellular carcinoma cells promotes migration of human umbilical vein endothelial cells. Int J Biol Sci. 2015;11:961–9. 10.7150/ijbs.11943.26157350 10.7150/ijbs.11943
53. Wan F Li H Huang S Sun J Li J Li Y Yang L Vasorin promotes proliferation and migration via STAT3 signaling and acts as a promising therapeutic target of hepatocellular carcinoma Cell Signal 2023 110 110809 10.1016/j.cellsig.2023.110809 37454705
Wan F, Li H, Huang S, Sun J, Li J, Li Y, Yang L, et al. Vasorin promotes proliferation and migration via STAT3 signaling and acts as a promising therapeutic target of hepatocellular carcinoma. Cell Signal. 2023;110:110809. 10.1016/j.cellsig.2023.110809.37454705 10.1016/j.cellsig.2023.110809
54. Jiang K Dong C Yin Z Li R Mao J Wang C Zhang J Exosome-derived ENO1 regulates integrin alpha6beta4 expression and promotes hepatocellular carcinoma growth and metastasis Cell Death Dis 2020 11 972 10.1038/s41419-020-03179-1 33184263
Jiang K, Dong C, Yin Z, Li R, Mao J, Wang C, Zhang J, et al. Exosome-derived ENO1 regulates integrin alpha6beta4 expression and promotes hepatocellular carcinoma growth and metastasis. Cell Death Dis. 2020;11:972. 10.1038/s41419-020-03179-1.33184263 10.1038/s41419-020-03179-1
55. Liu BHM Tey SK Mao X Ma APY Yeung CLS Wong SWK Ng TH TPI1-reduced extracellular vesicles mediated by Rab20 downregulation promotes aerobic glycolysis to drive hepatocarcinogenesis J Extracell Vesicles 2021 10 e12135 10.1002/jev2.12135 34401050
Liu BHM, Tey SK, Mao X, Ma APY, Yeung CLS, Wong SWK, Ng TH, et al. TPI1-reduced extracellular vesicles mediated by Rab20 downregulation promotes aerobic glycolysis to drive hepatocarcinogenesis. J Extracell Vesicles. 2021;10:e12135. 10.1002/jev2.12135.34401050 10.1002/jev2.12135
56. Li R Wang Y Zhang X Feng M Ma J Li J Yang X Exosome-mediated secretion of LOXL4 promotes hepatocellular carcinoma cell invasion and metastasis Mol Cancer 2019 18 18 10.1186/s12943-019-0948-8 30704479
Li R, Wang Y, Zhang X, Feng M, Ma J, Li J, Yang X, et al. Exosome-mediated secretion of LOXL4 promotes hepatocellular carcinoma cell invasion and metastasis. Mol Cancer. 2019;18:18. 10.1186/s12943-019-0948-8.30704479 10.1186/s12943-019-0948-8
57. Zhao L Pei R Ding Y Su Z Li D Zhu S Xu L LOXL4 shuttled by Tumor cells-derived Extracellular vesicles promotes Immune escape in Hepatocellular Carcinoma by activating the STAT1/PD-L1 Axis J Immunother 2024 47 64 76 10.1097/CJI.0000000000000496 38047403
Zhao L, Pei R, Ding Y, Su Z, Li D, Zhu S, Xu L, et al. LOXL4 shuttled by Tumor cells-derived Extracellular vesicles promotes Immune escape in Hepatocellular Carcinoma by activating the STAT1/PD-L1 Axis. J Immunother. 2024;47:64–76. 10.1097/CJI.0000000000000496.38047403 10.1097/CJI.0000000000000496
58. Tan HY Wang N Zhang C Chan YT Yuen MF Feng Y Lysyl Oxidase-Like 4 fosters an immunosuppressive Microenvironment during Hepatocarcinogenesis Hepatology 2021 73 2326 41 10.1002/hep.31600 33068461
Tan HY, Wang N, Zhang C, Chan YT, Yuen MF, Feng Y. Lysyl Oxidase-Like 4 fosters an immunosuppressive Microenvironment during Hepatocarcinogenesis. Hepatology. 2021;73:2326–41. 10.1002/hep.31600.33068461 10.1002/hep.31600
59. Chanmee T Ontong P Konno K Itano N Tumor-associated macrophages as major players in the tumor microenvironment Cancers (Basel) 2014 6 1670 90 10.3390/cancers6031670 25125485
Chanmee T, Ontong P, Konno K, Itano N. Tumor-associated macrophages as major players in the tumor microenvironment. Cancers (Basel). 2014;6:1670–90. 10.3390/cancers6031670.25125485 10.3390/cancers6031670
60. Li T Jiao J Ke H Ouyang W Wang L Pan J Li X Role of exosomes in the development of the immune microenvironment in hepatocellular carcinoma Front Immunol 2023 14 1200201 10.3389/fimmu.2023.1200201 37457718
Li T, Jiao J, Ke H, Ouyang W, Wang L, Pan J, Li X. Role of exosomes in the development of the immune microenvironment in hepatocellular carcinoma. Front Immunol. 2023;14:1200201. 10.3389/fimmu.2023.1200201.37457718 10.3389/fimmu.2023.1200201
61. Yang Q Liang Y Shi Y Shang J Huang X The ALKBH5/SOX4 axis promotes liver cancer stem cell properties via activating the SHH signaling pathway J Cancer Res Clin Oncol 2023 149 15499 510 10.1007/s00432-023-05309-6 37646828
Yang Q, Liang Y, Shi Y, Shang J, Huang X. The ALKBH5/SOX4 axis promotes liver cancer stem cell properties via activating the SHH signaling pathway. J Cancer Res Clin Oncol. 2023;149:15499–510. 10.1007/s00432-023-05309-6.37646828 10.1007/s00432-023-05309-6
62. Xie S Li X Yan J Yu H Chen S Chen K Knockdown of liver cancer cell-secreted exosomal PSMA5 controls macrophage polarization to restrain cancer progression by blocking JAK2/STAT3 signaling Immun Inflamm Dis 2024 12 e1146 10.1002/iid3.1146 38415977
Xie S, Li X, Yan J, Yu H, Chen S, Chen K. Knockdown of liver cancer cell-secreted exosomal PSMA5 controls macrophage polarization to restrain cancer progression by blocking JAK2/STAT3 signaling. Immun Inflamm Dis. 2024;12:e1146. 10.1002/iid3.1146.38415977 10.1002/iid3.1146
63. Kamerkar S Leng C Burenkova O Jang SC McCoy C Zhang K Dooley K Exosome-mediated genetic reprogramming of tumor-associated macrophages by exoASO-STAT6 leads to potent monotherapy antitumor activity Sci Adv 2022 8 eabj7002 10.1126/sciadv.abj7002 35179953
Kamerkar S, Leng C, Burenkova O, Jang SC, McCoy C, Zhang K, Dooley K, et al. Exosome-mediated genetic reprogramming of tumor-associated macrophages by exoASO-STAT6 leads to potent monotherapy antitumor activity. Sci Adv. 2022;8:eabj7002. 10.1126/sciadv.abj7002.35179953 10.1126/sciadv.abj7002
64. Kim IY Kim HY Song HW Park JO Choi YH Choi E Functional enhancement of exosomes derived from NK cells by IL-15 and IL-21 synergy against hepatocellular carcinoma cells: the cytotoxicity and apoptosis in vitro study Heliyon 2023 9 e16962 10.1016/j.heliyon.2023.e16962 37484408
Kim IY, Kim HY, Song HW, Park JO, Choi YH, Choi E. Functional enhancement of exosomes derived from NK cells by IL-15 and IL-21 synergy against hepatocellular carcinoma cells: the cytotoxicity and apoptosis in vitro study. Heliyon. 2023;9:e16962. 10.1016/j.heliyon.2023.e16962.37484408 10.1016/j.heliyon.2023.e16962
65. Kim HY Min HK Song HW Yoo A Lee S Kim KP Park JO Delivery of human natural killer cell-derived exosomes for liver cancer therapy: an in vivo study in subcutaneous and orthotopic animal models Drug Deliv 2022 29 2897 911 10.1080/10717544.2022.2118898 36068970
Kim HY, Min HK, Song HW, Yoo A, Lee S, Kim KP, Park JO, et al. Delivery of human natural killer cell-derived exosomes for liver cancer therapy: an in vivo study in subcutaneous and orthotopic animal models. Drug Deliv. 2022;29:2897–911. 10.1080/10717544.2022.2118898.36068970 10.1080/10717544.2022.2118898
66. Liu Z You Y Chen Q Li G Pan W Yang Q Dong J Extracellular vesicle-mediated communication between hepatocytes and natural killer cells promotes hepatocellular tumorigenesis Mol Ther 2022 30 606 20 10.1016/j.ymthe.2021.07.015 34601133
Liu Z, You Y, Chen Q, Li G, Pan W, Yang Q, Dong J, et al. Extracellular vesicle-mediated communication between hepatocytes and natural killer cells promotes hepatocellular tumorigenesis. Mol Ther. 2022;30:606–20. 10.1016/j.ymthe.2021.07.015.34601133 10.1016/j.ymthe.2021.07.015
67. Chen YQ Man ZS Zheng L Zhang Y Zhao CW Ma YT Zhou J Tumor cell-derived LC3B(+)extracellular vesicles mediate the crosstalk between tumor microenvironment and immunotherapy efficacy in hepatocellular carcinoma via the HSP90alpha-IL-6/IL-8 signaling axis Clin Immunol 2024 261 109925 10.1016/j.clim.2024.109925 38310993
Chen YQ, Man ZS, Zheng L, Zhang Y, Zhao CW, Ma YT, Zhou J, et al. Tumor cell-derived LC3B(+)extracellular vesicles mediate the crosstalk between tumor microenvironment and immunotherapy efficacy in hepatocellular carcinoma via the HSP90alpha-IL-6/IL-8 signaling axis. Clin Immunol. 2024;261:109925. 10.1016/j.clim.2024.109925.38310993 10.1016/j.clim.2024.109925
68. Chen YQ Zheng L Zhou J Wang P Wang L Zhang Y Man ZS Evaluation of plasma LC3B(+)extracellular vesicles as a potential novel diagnostic marker for hepatocellular carcinoma Int Immunopharmacol 2022 108 108760 10.1016/j.intimp.2022.108760 35398623
Chen YQ, Zheng L, Zhou J, Wang P, Wang L, Zhang Y, Man ZS, et al. Evaluation of plasma LC3B(+)extracellular vesicles as a potential novel diagnostic marker for hepatocellular carcinoma. Int Immunopharmacol. 2022;108:108760. 10.1016/j.intimp.2022.108760.35398623 10.1016/j.intimp.2022.108760
69. Ye L Zhang Q Cheng Y Chen X Wang G Shi M Zhang T Tumor-derived exosomal HMGB1 fosters hepatocellular carcinoma immune evasion by promoting TIM-1(+) regulatory B cell expansion J Immunother Cancer 2018 6 145 10.1186/s40425-018-0451-6 30526680
Ye L, Zhang Q, Cheng Y, Chen X, Wang G, Shi M, Zhang T, et al. Tumor-derived exosomal HMGB1 fosters hepatocellular carcinoma immune evasion by promoting TIM-1(+) regulatory B cell expansion. J Immunother Cancer. 2018;6:145. 10.1186/s40425-018-0451-6.30526680 10.1186/s40425-018-0451-6
70. Wang H Chen L Tumor microenviroment and hepatocellular carcinoma metastasis J Gastroenterol Hepatol 2013 28 Suppl 1 43 8 10.1111/jgh.12091 23855295
Wang H, Chen L. Tumor microenviroment and hepatocellular carcinoma metastasis. J Gastroenterol Hepatol. 2013;28(Suppl 1):43–8. 10.1111/jgh.12091.23855295 10.1111/jgh.12091
71. Chen QT Zhang ZY Huang QL Chen HZ Hong WB Lin T Zhao WX HK1 from hepatic stellate cell-derived extracellular vesicles promotes progression of hepatocellular carcinoma Nat Metab 2022 4 1306 21 10.1038/s42255-022-00642-5 36192599
Chen QT, Zhang ZY, Huang QL, Chen HZ, Hong WB, Lin T, Zhao WX, et al. HK1 from hepatic stellate cell-derived extracellular vesicles promotes progression of hepatocellular carcinoma. Nat Metab. 2022;4:1306–21. 10.1038/s42255-022-00642-5.36192599 10.1038/s42255-022-00642-5
72. Xia Y Zhen L Li H Wang S Chen S Wang C Yang X MIRLET7BHG promotes hepatocellular carcinoma progression by activating hepatic stellate cells through exosomal SMO to trigger hedgehog pathway Cell Death Dis 2021 12 326 10.1038/s41419-021-03494-1 33771969
Xia Y, Zhen L, Li H, Wang S, Chen S, Wang C, Yang X. MIRLET7BHG promotes hepatocellular carcinoma progression by activating hepatic stellate cells through exosomal SMO to trigger hedgehog pathway. Cell Death Dis. 2021;12:326. 10.1038/s41419-021-03494-1.33771969 10.1038/s41419-021-03494-1
73. Chen X Song E Turning foes to friends: targeting cancer-associated fibroblasts Nat Rev Drug Discov 2019 18 99 115 10.1038/s41573-018-0004-1 30470818
Chen X, Song E. Turning foes to friends: targeting cancer-associated fibroblasts. Nat Rev Drug Discov. 2019;18:99–115. 10.1038/s41573-018-0004-1.30470818 10.1038/s41573-018-0004-1
74. Qin W Wang L Tian H Wu X Xiao C Pan Y Fan M CAF-derived exosomes transmitted Gremlin-1 promotes cancer progression and decreases the sensitivity of hepatoma cells to sorafenib Mol Carcinog 2022 61 764 75 10.1002/mc.23416 35638711
Qin W, Wang L, Tian H, Wu X, Xiao C, Pan Y, Fan M, et al. CAF-derived exosomes transmitted Gremlin-1 promotes cancer progression and decreases the sensitivity of hepatoma cells to sorafenib. Mol Carcinog. 2022;61:764–75. 10.1002/mc.23416.35638711 10.1002/mc.23416
75. Petovari G, Toth G, Turiak L, Paloczi ALK, Sebestyen K, Pesti A. Dynamic interplay in Tumor ecosystems: communication between Hepatoma cells and fibroblasts. Int J Mol Sci. 2023;24. 10.3390/ijms241813996.
76. Morse MA Sun W Kim R He AR Abada PB Mynderse M Finn RS The role of Angiogenesis in Hepatocellular Carcinoma Clin Cancer Res 2019 25 912 20 10.1158/1078-0432.CCR-18-1254 30274981
Morse MA, Sun W, Kim R, He AR, Abada PB, Mynderse M, Finn RS. The role of Angiogenesis in Hepatocellular Carcinoma. Clin Cancer Res. 2019;25:912–20. 10.1158/1078-0432.CCR-18-1254.30274981 10.1158/1078-0432.CCR-18-1254
77. Yukawa H Suzuki K Aoki K Arimoto T Yasui T Kaji N Ishikawa T Imaging of angiogenesis of human umbilical vein endothelial cells by uptake of exosomes secreted from hepatocellular carcinoma cells Sci Rep 2018 8 6765 10.1038/s41598-018-24563-0 29713019
Yukawa H, Suzuki K, Aoki K, Arimoto T, Yasui T, Kaji N, Ishikawa T, et al. Imaging of angiogenesis of human umbilical vein endothelial cells by uptake of exosomes secreted from hepatocellular carcinoma cells. Sci Rep. 2018;8:6765. 10.1038/s41598-018-24563-0.29713019 10.1038/s41598-018-24563-0
78. Xu Y Yao Y Yu L Zhang X Mao X Tey SK Wong SWK Clathrin light chain A-enriched small extracellular vesicles remodel microvascular niche to induce hepatocellular carcinoma metastasis J Extracell Vesicles 2023 12 e12359 10.1002/jev2.12359 37606345
Xu Y, Yao Y, Yu L, Zhang X, Mao X, Tey SK, Wong SWK, et al. Clathrin light chain A-enriched small extracellular vesicles remodel microvascular niche to induce hepatocellular carcinoma metastasis. J Extracell Vesicles. 2023;12:e12359. 10.1002/jev2.12359.37606345 10.1002/jev2.12359
79. Yu B Zhou S Long D Ning Y Yao H Zhou E Wang Y DDX55 promotes hepatocellular carcinoma progression by interacting with BRD4 and participating in exosome-mediated cell-cell communication Cancer Sci 2022 113 3002 17 10.1111/cas.15393 35514200
Yu B, Zhou S, Long D, Ning Y, Yao H, Zhou E, Wang Y. DDX55 promotes hepatocellular carcinoma progression by interacting with BRD4 and participating in exosome-mediated cell-cell communication. Cancer Sci. 2022;113:3002–17. 10.1111/cas.15393.35514200 10.1111/cas.15393
80. Wong SWK Tey SK Mao X Fung HL Xiao ZJ Wong DKH Mak LY Small extracellular vesicle-derived vWF induces a positive Feedback Loop between Tumor and endothelial cells to promote angiogenesis and metastasis in Hepatocellular Carcinoma Adv Sci (Weinh) 2023 10 e2302677 10.1002/advs.202302677 37387563
Wong SWK, Tey SK, Mao X, Fung HL, Xiao ZJ, Wong DKH, Mak LY, et al. Small extracellular vesicle-derived vWF induces a positive Feedback Loop between Tumor and endothelial cells to promote angiogenesis and metastasis in Hepatocellular Carcinoma. Adv Sci (Weinh). 2023;10:e2302677. 10.1002/advs.202302677.37387563 10.1002/advs.202302677
81. Xu Q You R Yin G Gu J Hepatocellular carcinoma serum derived exosomal HIF-1alpha induces phosphoinositide-3 kinase/protein kinase B signaling to induce the angiogenesis and proliferation of HCC Transl Cancer Res 2019 8 1550 9 10.21037/tcr.2019.08.07 35116898
Xu Q, You R, Yin G, Gu J. Hepatocellular carcinoma serum derived exosomal HIF-1alpha induces phosphoinositide-3 kinase/protein kinase B signaling to induce the angiogenesis and proliferation of HCC. Transl Cancer Res. 2019;8:1550–9. 10.21037/tcr.2019.08.07.35116898 10.21037/tcr.2019.08.07
82. Huang XY Zhang JT Li F Li TT Shi XJ Huang J Huang XY Exosomal proteomics identifies RAB13 as a potential regulator of metastasis for HCC Hepatol Commun 2023 7 e0006 10.1097/HC9.0000000000000006 36633475
Huang XY, Zhang JT, Li F, Li TT, Shi XJ, Huang J, Huang XY, et al. Exosomal proteomics identifies RAB13 as a potential regulator of metastasis for HCC. Hepatol Commun. 2023;7:e0006. 10.1097/HC9.0000000000000006.36633475 10.1097/HC9.0000000000000006
83. Dai W Wang Y Yang T Wang J Wu W Gu J Downregulation of exosomal CLEC3B in hepatocellular carcinoma promotes metastasis and angiogenesis via AMPK and VEGF signals Cell Commun Signal 2019 17 113 10.1186/s12964-019-0423-6 31477130
Dai W, Wang Y, Yang T, Wang J, Wu W, Gu J. Downregulation of exosomal CLEC3B in hepatocellular carcinoma promotes metastasis and angiogenesis via AMPK and VEGF signals. Cell Commun Signal. 2019;17:113. 10.1186/s12964-019-0423-6.31477130 10.1186/s12964-019-0423-6
84. Li Z Li Y Ouyang Q Li X Huang J Exosome-derived GTF2H2 from Huh7 cells can inhibit endothelial cell viability, migration, tube formation, and permeability Tissue Cell 2022 79 101922 10.1016/j.tice.2022.101922 36116407
Li Z, Li Y, Ouyang Q, Li X, Huang J. Exosome-derived GTF2H2 from Huh7 cells can inhibit endothelial cell viability, migration, tube formation, and permeability. Tissue Cell. 2022;79:101922. 10.1016/j.tice.2022.101922.36116407 10.1016/j.tice.2022.101922
85. Lee SM Cho J Choi S Kim DH Ryu JW Kim I Woo DC HDAC5-mediated exosomal maspin and miR-151a-3p as biomarkers for enhancing radiation treatment sensitivity in hepatocellular carcinoma Biomater Res 2023 27 134 10.1186/s40824-023-00467-7 38102691
Lee SM, Cho J, Choi S, Kim DH, Ryu JW, Kim I, Woo DC, et al. HDAC5-mediated exosomal maspin and miR-151a-3p as biomarkers for enhancing radiation treatment sensitivity in hepatocellular carcinoma. Biomater Res. 2023;27:134. 10.1186/s40824-023-00467-7.38102691 10.1186/s40824-023-00467-7
86. Ge Y Mu W Ba Q Li J Jiang Y Xia Q Wang H Hepatocellular carcinoma-derived exosomes in organotropic metastasis, recurrence and early diagnosis application Cancer Lett 2020 477 41 8 10.1016/j.canlet.2020.02.003 32112905
Ge Y, Mu W, Ba Q, Li J, Jiang Y, Xia Q, Wang H. Hepatocellular carcinoma-derived exosomes in organotropic metastasis, recurrence and early diagnosis application. Cancer Lett. 2020;477:41–8. 10.1016/j.canlet.2020.02.003.32112905 10.1016/j.canlet.2020.02.003
87. Dan Hu Q Wang HL Liu J He T Tan RZ Zhang Q Su HW Btg2 promotes focal segmental glomerulosclerosis via Smad3-Dependent podocyte-mesenchymal transition Adv Sci (Weinh) 2023 10 e2304360 10.1002/advs.202304360 37749872
Dan Hu Q, Wang HL, Liu J, He T, Tan RZ, Zhang Q, Su HW, et al. Btg2 promotes focal segmental glomerulosclerosis via Smad3-Dependent podocyte-mesenchymal transition. Adv Sci (Weinh). 2023;10:e2304360. 10.1002/advs.202304360.37749872 10.1002/advs.202304360
88. Chen L Guo P He Y Chen Z Chen L Luo Y Qi L HCC-derived exosomes elicit HCC progression and recurrence by epithelial-mesenchymal transition through MAPK/ERK signalling pathway Cell Death Dis 2018 9 513 10.1038/s41419-018-0534-9 29725020
Chen L, Guo P, He Y, Chen Z, Chen L, Luo Y, Qi L, et al. HCC-derived exosomes elicit HCC progression and recurrence by epithelial-mesenchymal transition through MAPK/ERK signalling pathway. Cell Death Dis. 2018;9:513. 10.1038/s41419-018-0534-9.29725020 10.1038/s41419-018-0534-9
89. Karaosmanoglu O Banerjee S Sivas H Identification of biomarkers associated with partial epithelial to mesenchymal transition in the secretome of slug over-expressing hepatocellular carcinoma cells Cell Oncol (Dordr) 2018 41 439 53 10.1007/s13402-018-0384-6 29858962
Karaosmanoglu O, Banerjee S, Sivas H. Identification of biomarkers associated with partial epithelial to mesenchymal transition in the secretome of slug over-expressing hepatocellular carcinoma cells. Cell Oncol (Dordr). 2018;41:439–53. 10.1007/s13402-018-0384-6.29858962 10.1007/s13402-018-0384-6
90. Tey SK Wong SWK Chan JYT Mao X Ng TH Yeung CLS Leung Z Patient pIgR-enriched extracellular vesicles drive cancer stemness, tumorigenesis and metastasis in hepatocellular carcinoma J Hepatol 2022 76 883 95 10.1016/j.jhep.2021.12.005 34922977
Tey SK, Wong SWK, Chan JYT, Mao X, Ng TH, Yeung CLS, Leung Z, et al. Patient pIgR-enriched extracellular vesicles drive cancer stemness, tumorigenesis and metastasis in hepatocellular carcinoma. J Hepatol. 2022;76:883–95. 10.1016/j.jhep.2021.12.005.34922977 10.1016/j.jhep.2021.12.005
91. Wang X Huang H Sze KM Wang J Tian L Lu J Tsui YM S100A10 promotes HCC development and progression via transfer in extracellular vesicles and regulating their protein cargos Gut 2023 72 1370 84 10.1136/gutjnl-2022-327998 36631249
Wang X, Huang H, Sze KM, Wang J, Tian L, Lu J, Tsui YM, et al. S100A10 promotes HCC development and progression via transfer in extracellular vesicles and regulating their protein cargos. Gut. 2023;72:1370–84. 10.1136/gutjnl-2022-327998.36631249 10.1136/gutjnl-2022-327998
92. Fu Q Zhang Q Lou Y Yang J Nie G Chen Q Chen Y Primary tumor-derived exosomes facilitate metastasis by regulating adhesion of circulating tumor cells via SMAD3 in liver cancer Oncogene 2018 37 6105 18 10.1038/s41388-018-0391-0 29991801
Fu Q, Zhang Q, Lou Y, Yang J, Nie G, Chen Q, Chen Y, et al. Primary tumor-derived exosomes facilitate metastasis by regulating adhesion of circulating tumor cells via SMAD3 in liver cancer. Oncogene. 2018;37:6105–18. 10.1038/s41388-018-0391-0.29991801 10.1038/s41388-018-0391-0
93. Mei P Tey SK Wong SWK Ng TH Mao X Yeung CLS Xu Y Actin-related protein 2/3 complex subunit 2-enriched extracellular vesicles drive liver cancer metastasis Hepatol Int 2022 16 603 13 10.1007/s12072-022-10338-3 35556226
Mei P, Tey SK, Wong SWK, Ng TH, Mao X, Yeung CLS, Xu Y, et al. Actin-related protein 2/3 complex subunit 2-enriched extracellular vesicles drive liver cancer metastasis. Hepatol Int. 2022;16:603–13. 10.1007/s12072-022-10338-3.35556226 10.1007/s12072-022-10338-3
94. Mao X Tey SK Yeung CLS Kwong EML Fung YME Chung CYS Mak LY Nidogen 1-Enriched extracellular vesicles facilitate extrahepatic metastasis of Liver Cancer by activating pulmonary fibroblasts to Secrete Tumor Necrosis Factor Receptor 1 Adv Sci (Weinh) 2020 7 2002157 10.1002/advs.202002157 33173740
Mao X, Tey SK, Yeung CLS, Kwong EML, Fung YME, Chung CYS, Mak LY, et al. Nidogen 1-Enriched extracellular vesicles facilitate extrahepatic metastasis of Liver Cancer by activating pulmonary fibroblasts to Secrete Tumor Necrosis Factor Receptor 1. Adv Sci (Weinh). 2020;7:2002157. 10.1002/advs.202002157.33173740 10.1002/advs.202002157
95. Ma YB Qiao JW Hu X Transmembrane serine protease 2 cleaves nidogen 1 and inhibits extrahepatic liver cancer cell migration and invasion Exp Biol Med (Maywood) 2023 248 91 105 10.1177/15353702221134111 36408877
Ma YB, Qiao JW, Hu X. Transmembrane serine protease 2 cleaves nidogen 1 and inhibits extrahepatic liver cancer cell migration and invasion. Exp Biol Med (Maywood). 2023;248:91–105. 10.1177/15353702221134111.36408877 10.1177/15353702221134111
96. Zhang S Liao X Chen S Qian W Li M Xu Y Yang M Large oncosome-loaded VAPA promotes bone-tropic metastasis of Hepatocellular Carcinoma Via formation of Osteoclastic Pre-metastatic Niche Adv Sci (Weinh) 2022 9 e2201974 10.1002/advs.202201974 36169100
Zhang S, Liao X, Chen S, Qian W, Li M, Xu Y, Yang M, et al. Large oncosome-loaded VAPA promotes bone-tropic metastasis of Hepatocellular Carcinoma Via formation of Osteoclastic Pre-metastatic Niche. Adv Sci (Weinh). 2022;9:e2201974. 10.1002/advs.202201974.36169100 10.1002/advs.202201974
97. Mao X Zhou L Tey SK Ma APY Yeung CLS Ng TH Wong SWK Tumour extracellular vesicle-derived complement factor H promotes tumorigenesis and metastasis by inhibiting complement-dependent cytotoxicity of tumour cells J Extracell Vesicles 2020 10 e12031 10.1002/jev2.12031 33708358
Mao X, Zhou L, Tey SK, Ma APY, Yeung CLS, Ng TH, Wong SWK, et al. Tumour extracellular vesicle-derived complement factor H promotes tumorigenesis and metastasis by inhibiting complement-dependent cytotoxicity of tumour cells. J Extracell Vesicles. 2020;10:e12031. 10.1002/jev2.12031.33708358 10.1002/jev2.12031
98. Dong W Xia Z Chai Z Qiu Z Wang X Yang Z Wang J Proteomic analysis of small extracellular vesicles from the plasma of patients with hepatocellular carcinoma World J Surg Oncol 2022 20 387 10.1186/s12957-022-02849-y 36471393
Dong W, Xia Z, Chai Z, Qiu Z, Wang X, Yang Z, Wang J, et al. Proteomic analysis of small extracellular vesicles from the plasma of patients with hepatocellular carcinoma. World J Surg Oncol. 2022;20:387. 10.1186/s12957-022-02849-y.36471393 10.1186/s12957-022-02849-y
99. He M Qin H Poon TC Sze SC Ding X Co NN Ngai SM Hepatocellular carcinoma-derived exosomes promote motility of immortalized hepatocyte through transfer of oncogenic proteins and RNAs Carcinogenesis 2015 36 1008 18 10.1093/carcin/bgv081 26054723
He M, Qin H, Poon TC, Sze SC, Ding X, Co NN, Ngai SM, et al. Hepatocellular carcinoma-derived exosomes promote motility of immortalized hepatocyte through transfer of oncogenic proteins and RNAs. Carcinogenesis. 2015;36:1008–18. 10.1093/carcin/bgv081.26054723 10.1093/carcin/bgv081
100. Jiang X Wu S Hu C A narrative review of the role of exosomes and caveolin-1 in liver diseases and cancer Int Immunopharmacol 2023 120 110284 10.1016/j.intimp.2023.110284 37196562
Jiang X, Wu S, Hu C. A narrative review of the role of exosomes and caveolin-1 in liver diseases and cancer. Int Immunopharmacol. 2023;120:110284. 10.1016/j.intimp.2023.110284.37196562 10.1016/j.intimp.2023.110284
101. Sun H Wang C Hu B Gao X Zou T Luo Q Chen M Exosomal S100A4 derived from highly metastatic hepatocellular carcinoma cells promotes metastasis by activating STAT3 Signal Transduct Target Ther 2021 6 187 10.1038/s41392-021-00579-3 34035222
Sun H, Wang C, Hu B, Gao X, Zou T, Luo Q, Chen M, et al. Exosomal S100A4 derived from highly metastatic hepatocellular carcinoma cells promotes metastasis by activating STAT3. Signal Transduct Target Ther. 2021;6:187. 10.1038/s41392-021-00579-3.34035222 10.1038/s41392-021-00579-3
102. Tan W Zhang J Liu L Liang M Li J Deng Z Zheng Z Hsp90 inhibitor STA9090 induced VPS35 related extracellular vesicle release and metastasis in hepatocellular carcinoma Transl Oncol 2022 26 101502 10.1016/j.tranon.2022.101502 36137350
Tan W, Zhang J, Liu L, Liang M, Li J, Deng Z, Zheng Z, et al. Hsp90 inhibitor STA9090 induced VPS35 related extracellular vesicle release and metastasis in hepatocellular carcinoma. Transl Oncol. 2022;26:101502. 10.1016/j.tranon.2022.101502.36137350 10.1016/j.tranon.2022.101502
103. Yang X Yang C Zhang S Geng H Zhu AX Bernards R Qin W Precision treatment in advanced hepatocellular carcinoma Cancer Cell 2024 42 180 97 10.1016/j.ccell.2024.01.007 38350421
Yang X, Yang C, Zhang S, Geng H, Zhu AX, Bernards R, Qin W, et al. Precision treatment in advanced hepatocellular carcinoma. Cancer Cell. 2024;42:180–97. 10.1016/j.ccell.2024.01.007.38350421 10.1016/j.ccell.2024.01.007
104. Julich-Haertel H Urban SK Krawczyk M Willms A Jankowski K Patkowski W Kruk B Cancer-associated circulating large extracellular vesicles in cholangiocarcinoma and hepatocellular carcinoma J Hepatol 2017 67 282 92 10.1016/j.jhep.2017.02.024 28267620
Julich-Haertel H, Urban SK, Krawczyk M, Willms A, Jankowski K, Patkowski W, Kruk B, et al. Cancer-associated circulating large extracellular vesicles in cholangiocarcinoma and hepatocellular carcinoma. J Hepatol. 2017;67:282–92. 10.1016/j.jhep.2017.02.024.28267620 10.1016/j.jhep.2017.02.024
105. Mauro M, Ugo P, Walton Z, Ali S, Rastellini C, Cicalese L. Glypican-3 (GPC-3) structural analysis and Cargo in serum small extracellular vesicles of Hepatocellular Carcinoma patients. Int J Mol Sci. 2023;24. 10.3390/ijms241310922.
106. Aydin Y Koksal AR Thevenot P Chava S Heidari Z Lin D Sandow T Experimental validation of Novel Glypican 3 exosomes for the Detection of Hepatocellular Carcinoma in Liver cirrhosis J Hepatocell Carcinoma 2021 8 1579 96 10.2147/JHC.S327339 34917553
Aydin Y, Koksal AR, Thevenot P, Chava S, Heidari Z, Lin D, Sandow T, et al. Experimental validation of Novel Glypican 3 exosomes for the Detection of Hepatocellular Carcinoma in Liver cirrhosis. J Hepatocell Carcinoma. 2021;8:1579–96. 10.2147/JHC.S327339.34917553 10.2147/JHC.S327339
107. Koksal AR Thevenot P Aydin Y Nunez K Sandow T Widmer K Nayak L Impaired autophagy response in Hepatocellular Carcinomas enriches Glypican-3 in Exosomes, not in the Microvesicles J Hepatocell Carcinoma 2022 9 959 72 10.2147/JHC.S376210 36105695
Koksal AR, Thevenot P, Aydin Y, Nunez K, Sandow T, Widmer K, Nayak L, et al. Impaired autophagy response in Hepatocellular Carcinomas enriches Glypican-3 in Exosomes, not in the Microvesicles. J Hepatocell Carcinoma. 2022;9:959–72. 10.2147/JHC.S376210.36105695 10.2147/JHC.S376210
108. Koksal AR Ekmen N Aydin Y Nunez K Sandow T Delk M Moehlen M A single-step immunocapture assay to quantify HCC exosomes using the highly sensitive fluorescence nanoparticle-tracking analysis J Hepatocell Carcinoma 2023 10 1935 54 10.2147/JHC.S423043 37936599
Koksal AR, Ekmen N, Aydin Y, Nunez K, Sandow T, Delk M, Moehlen M, et al. A single-step immunocapture assay to quantify HCC exosomes using the highly sensitive fluorescence nanoparticle-tracking analysis. J Hepatocell Carcinoma. 2023;10:1935–54. 10.2147/JHC.S423043.37936599 10.2147/JHC.S423043
109. Sun N Zhang C Lee YT Tran BV Wang J Kim H Lee J HCC EV ECG score: an extracellular vesicle-based protein assay for detection of early-stage hepatocellular carcinoma Hepatology 2023 77 774 88 10.1002/hep.32692 35908246
Sun N, Zhang C, Lee YT, Tran BV, Wang J, Kim H, Lee J, et al. HCC EV ECG score: an extracellular vesicle-based protein assay for detection of early-stage hepatocellular carcinoma. Hepatology. 2023;77:774–88. 10.1002/hep.32692.35908246 10.1002/hep.32692
110. Cho HJ, Baek GO, Yoon MG, Ahn HR, Son JA, Kim SS, Cheong JY, et al. Overexpressed proteins in HCC Cell-Derived exosomes, CCT8, and Cofilin-1 are potential biomarkers for patients with HCC. Diagnostics (Basel). 2021;11. 10.3390/diagnostics11071221.
111. Heo CK, Hwang HM, Lim WH, Lee HJ, Yoo JS, Lim KJ, Cho EW. Cyclic peptide mimotopes for the detection of serum Anti-ATIC Autoantibody Biomarker in Hepato-Cellular Carcinoma. Int J Mol Sci. 2020;21. 10.3390/ijms21249718.
112. Heo CK, Lim WH, Park I, Choi YS, Lim KJ, Cho EW. Serum BRD2 autoantibody in hepatocellular carcinoma and its detection using mimotope peptide–conjugated BSA. Int J Oncol. 2022;61. 10.3892/ijo.2022.5448.
113. Heo CK Hwang HM Lee HJ Kwak SS Yoo JS Yu DY Lim KJ Serum anti-EIF3A autoantibody as a potential diagnostic marker for hepatocellular carcinoma Sci Rep 2019 9 11059 10.1038/s41598-019-47365-4 31363116
Heo CK, Hwang HM, Lee HJ, Kwak SS, Yoo JS, Yu DY, Lim KJ, et al. Serum anti-EIF3A autoantibody as a potential diagnostic marker for hepatocellular carcinoma. Sci Rep. 2019;9:11059. 10.1038/s41598-019-47365-4.31363116 10.1038/s41598-019-47365-4
114. Todorova VK, Byrum SD, Mackintosh SG, Jamshidi-Parsian A, Gies AJ, Washam CL, Jenkins SV, et al. Exosomal MicroRNA and protein profiles of Hepatitis B Virus-Related Hepatocellular Carcinoma cells. Int J Mol Sci. 2023;24. 10.3390/ijms241713098.
115. Huang H Zhang Q Zhang Y Sun X Liu C Wang Q Huang Y Identification of the level of Exosomal protein by parallel reaction Monitoring Technology in HCC patients Int J Gen Med 2022 15 7831 42 10.2147/IJGM.S384140 36267426
Huang H, Zhang Q, Zhang Y, Sun X, Liu C, Wang Q, Huang Y, et al. Identification of the level of Exosomal protein by parallel reaction Monitoring Technology in HCC patients. Int J Gen Med. 2022;15:7831–42. 10.2147/IJGM.S384140.36267426 10.2147/IJGM.S384140
116. Ji Y Yin Y Zhang W Integrated Bioinformatic Analysis Identifies Networks and promising biomarkers for Hepatitis B Virus-Related Hepatocellular Carcinoma Int J Genomics 2020 2020 2061024 10.1155/2020/2061024 32775402
Ji Y, Yin Y, Zhang W. Integrated Bioinformatic Analysis Identifies Networks and promising biomarkers for Hepatitis B Virus-Related Hepatocellular Carcinoma. Int J Genomics. 2020;2020:2061024. 10.1155/2020/2061024.32775402 10.1155/2020/2061024
117. Pang Y Zhang S Yang H Zhou RL [Serum LAPTM4B-35 protein as a novel diagnostic marker for hepatocellular carcinoma] Beijing Da Xue Xue Bao Yi Xue Ban 2021 53 710 5 10.19723/j.issn.1671-167X.2021.04.015 34393233
Pang Y, Zhang S, Yang H, Zhou RL. [Serum LAPTM4B-35 protein as a novel diagnostic marker for hepatocellular carcinoma]. Beijing Da Xue Xue Bao Yi Xue Ban. 2021;53:710–5. 10.19723/j.issn.1671-167X.2021.04.015.34393233 10.19723/j.issn.1671-167X.2021.04.015
118. Zertuche-Martinez C, Velazquez-Enriquez JM, Gonzalez-Garcia K, Baltierrez-Hoyos R, Carrasco-Torres G, Garcia-Roman R, Romero-Diaz RI et al. Identification of ABCC3 and its isoforms as potential biomarker in hepatocellular carcinoma. Toxicol Mech Methods 2023:1–10. 10.1080/15376516.2023.2294475
119. Zhao L Shi J Chang L Wang Y Liu S Li Y Zhang T Serum-derived exosomal proteins as potential candidate biomarkers for Hepatocellular Carcinoma ACS Omega 2021 6 827 35 10.1021/acsomega.0c05408 33458533
Zhao L, Shi J, Chang L, Wang Y, Liu S, Li Y, Zhang T, et al. Serum-derived exosomal proteins as potential candidate biomarkers for Hepatocellular Carcinoma. ACS Omega. 2021;6:827–35. 10.1021/acsomega.0c05408.33458533 10.1021/acsomega.0c05408
120. Feng X Jia S Ali MM Zhang G Li D Tao WA Hu L Proteomic Discovery and array-based validation of biomarkers from urinary exosome by Supramolecular Probe J Proteome Res 2023 22 2516 24 10.1021/acs.jproteome.3c00063 37126797
Feng X, Jia S, Ali MM, Zhang G, Li D, Tao WA, Hu L. Proteomic Discovery and array-based validation of biomarkers from urinary exosome by Supramolecular Probe. J Proteome Res. 2023;22:2516–24. 10.1021/acs.jproteome.3c00063.37126797 10.1021/acs.jproteome.3c00063
121. Li D, Jia S, Wang S, Hu L. Glycoproteomic analysis of urinary extracellular vesicles for biomarkers of Hepatocellular Carcinoma. Molecules. 2023;28. 10.3390/molecules28031293.
122. Shuen TWH Alunni-Fabbroni M Ocal E Malfertheiner P Wildgruber M Schinner R Pech M Extracellular vesicles may predict response to Radioembolization and Sorafenib Treatment in Advanced Hepatocellular Carcinoma: an exploratory analysis from the SORAMIC Trial Clin Cancer Res 2022 28 3890 901 10.1158/1078-0432.CCR-22-0569 35763041
Shuen TWH, Alunni-Fabbroni M, Ocal E, Malfertheiner P, Wildgruber M, Schinner R, Pech M, et al. Extracellular vesicles may predict response to Radioembolization and Sorafenib Treatment in Advanced Hepatocellular Carcinoma: an exploratory analysis from the SORAMIC Trial. Clin Cancer Res. 2022;28:3890–901. 10.1158/1078-0432.CCR-22-0569.35763041 10.1158/1078-0432.CCR-22-0569
123. Son JA Weon JH Baek GO Ahn HR Choi JY Yoon MG Cho HJ Circulating small extracellular vesicle-derived splicing factor 3b subunit 4 as a non-invasive diagnostic biomarker of early hepatocellular carcinoma J Exp Clin Cancer Res 2023 42 288 10.1186/s13046-023-02867-y 37899451
Son JA, Weon JH, Baek GO, Ahn HR, Choi JY, Yoon MG, Cho HJ, et al. Circulating small extracellular vesicle-derived splicing factor 3b subunit 4 as a non-invasive diagnostic biomarker of early hepatocellular carcinoma. J Exp Clin Cancer Res. 2023;42:288. 10.1186/s13046-023-02867-y.37899451 10.1186/s13046-023-02867-y
124. Zhu L Qu XH Sun YL Qian YM Zhao XH Novel method for extracting exosomes of hepatocellular carcinoma cells World J Gastroenterol 2014 20 6651 7 10.3748/wjg.v20.i21.6651 24914390
Zhu L, Qu XH, Sun YL, Qian YM, Zhao XH. Novel method for extracting exosomes of hepatocellular carcinoma cells. World J Gastroenterol. 2014;20:6651–7. 10.3748/wjg.v20.i21.6651.24914390 10.3748/wjg.v20.i21.6651
125. Vogel A Meyer T Sapisochin G Salem R Saborowski A Hepatocellular carcinoma Lancet 2022 400 1345 62 10.1016/S0140-6736(22)01200-4 36084663
Vogel A, Meyer T, Sapisochin G, Salem R, Saborowski A. Hepatocellular carcinoma. Lancet. 2022;400:1345–62. 10.1016/S0140-6736(22)01200-4.36084663 10.1016/S0140-6736(22)01200-4
126. Liang Y Duan L Lu J Xia J Engineering exosomes for targeted drug delivery Theranostics 2021 11 3183 95 10.7150/thno.52570 33537081
Liang Y, Duan L, Lu J, Xia J. Engineering exosomes for targeted drug delivery. Theranostics. 2021;11:3183–95. 10.7150/thno.52570.33537081 10.7150/thno.52570
127. Zhang X Han C Du B Nan D Zhang W He G Isolation and identification of adipose stem cell exosomes and the study of its potential as drug delivery carrier in Vitro Appl Biochem Biotechnol 2022 194 2594 603 10.1007/s12010-022-03835-6 35175564
Zhang X, Han C, Du B, Nan D, Zhang W, He G. Isolation and identification of adipose stem cell exosomes and the study of its potential as drug delivery carrier in Vitro. Appl Biochem Biotechnol. 2022;194:2594–603. 10.1007/s12010-022-03835-6.35175564 10.1007/s12010-022-03835-6
128. Qiao L Hu J Qiu X Wang C Peng J Zhang C Zhang M LAMP2A, LAMP2B and LAMP2C: similar structures, divergent roles Autophagy 2023 19 2837 52 10.1080/15548627.2023.2235196 37469132
Qiao L, Hu J, Qiu X, Wang C, Peng J, Zhang C, Zhang M, et al. LAMP2A, LAMP2B and LAMP2C: similar structures, divergent roles. Autophagy. 2023;19:2837–52. 10.1080/15548627.2023.2235196.37469132 10.1080/15548627.2023.2235196
129. Li X Yu Q Zhao R Guo X Liu C Zhang K Zhang W Designer exosomes for targeted delivery of a Novel Therapeutic Cargo to enhance sorafenib-mediated ferroptosis in Hepatocellular Carcinoma Front Oncol 2022 12 898156 10.3389/fonc.2022.898156 35814401
Li X, Yu Q, Zhao R, Guo X, Liu C, Zhang K, Zhang W, et al. Designer exosomes for targeted delivery of a Novel Therapeutic Cargo to enhance sorafenib-mediated ferroptosis in Hepatocellular Carcinoma. Front Oncol. 2022;12:898156. 10.3389/fonc.2022.898156.35814401 10.3389/fonc.2022.898156
130. Du J Wan Z Wang C Lu F Wei M Wang D Hao Q Designer exosomes for targeted and efficient ferroptosis induction in cancer via chemo-photodynamic therapy Theranostics 2021 11 8185 96 10.7150/thno.59121 34373736
Du J, Wan Z, Wang C, Lu F, Wei M, Wang D, Hao Q. Designer exosomes for targeted and efficient ferroptosis induction in cancer via chemo-photodynamic therapy. Theranostics. 2021;11:8185–96. 10.7150/thno.59121.34373736 10.7150/thno.59121
131. Wan T Zhong J Pan Q Zhou T Ping Y Liu X Exosome-mediated delivery of Cas9 ribonucleoprotein complexes for tissue-specific gene therapy of liver diseases Sci Adv 2022 8 eabp9435 10.1126/sciadv.abp9435 36103526
Wan T, Zhong J, Pan Q, Zhou T, Ping Y, Liu X. Exosome-mediated delivery of Cas9 ribonucleoprotein complexes for tissue-specific gene therapy of liver diseases. Sci Adv. 2022;8:eabp9435. 10.1126/sciadv.abp9435.36103526 10.1126/sciadv.abp9435
132. Zuo B Qi H Lu Z Chen L Sun B Yang R Zhang Y Alarmin-painted exosomes elicit persistent antitumor immunity in large established tumors in mice Nat Commun 2020 11 1790 10.1038/s41467-020-15569-2 32286296
Zuo B, Qi H, Lu Z, Chen L, Sun B, Yang R, Zhang Y, et al. Alarmin-painted exosomes elicit persistent antitumor immunity in large established tumors in mice. Nat Commun. 2020;11:1790. 10.1038/s41467-020-15569-2.32286296 10.1038/s41467-020-15569-2
133. Xiao W Dong W Zhang C Saren G Geng P Zhao H Li Q Effects of the epigenetic drug MS-275 on the release and function of exosome-related immune molecules in hepatocellular carcinoma cells Eur J Med Res 2013 18 61 10.1186/2047-783X-18-61 24359553
Xiao W, Dong W, Zhang C, Saren G, Geng P, Zhao H, Li Q, et al. Effects of the epigenetic drug MS-275 on the release and function of exosome-related immune molecules in hepatocellular carcinoma cells. Eur J Med Res. 2013;18:61. 10.1186/2047-783X-18-61.24359553 10.1186/2047-783X-18-61
134. Gastpar R Gehrmann M Bausero MA Asea A Gross C Schroeder JA Multhoff G Heat shock protein 70 surface-positive tumor exosomes stimulate migratory and cytolytic activity of natural killer cells Cancer Res 2005 65 5238 47 10.1158/0008-5472.CAN-04-3804 15958569
Gastpar R, Gehrmann M, Bausero MA, Asea A, Gross C, Schroeder JA, Multhoff G. Heat shock protein 70 surface-positive tumor exosomes stimulate migratory and cytolytic activity of natural killer cells. Cancer Res. 2005;65:5238–47. 10.1158/0008-5472.CAN-04-3804.15958569 10.1158/0008-5472.CAN-04-3804
135. Xiao WH Sanren GW Zhu JH Li QW Kang HR Wang RL Song LP Effect of 5-aza-2’-deoxycytidine on immune-associated proteins in exosomes from hepatoma World J Gastroenterol 2010 16 2371 7 10.3748/wjg.v16.i19.2371 20480522
Xiao WH, Sanren GW, Zhu JH, Li QW, Kang HR, Wang RL, Song LP, et al. Effect of 5-aza-2’-deoxycytidine on immune-associated proteins in exosomes from hepatoma. World J Gastroenterol. 2010;16:2371–7. 10.3748/wjg.v16.i19.2371.20480522 10.3748/wjg.v16.i19.2371
136. Son SH Gangadaran P Ahn BC A novel strategy of transferring NIS protein to cells using extracellular vesicles leads to increase in iodine uptake and cytotoxicity Int J Nanomed 2019 14 1779 87 10.2147/IJN.S189738
Son SH, Gangadaran P, Ahn BC. A novel strategy of transferring NIS protein to cells using extracellular vesicles leads to increase in iodine uptake and cytotoxicity. Int J Nanomed. 2019;14:1779–87. 10.2147/IJN.S189738.10.2147/IJN.S189738
137. Hou TZ Yang HM Cheng YZ Gu L Zhang JN Zhang H The Parkinson’s disease-associated protein alpha-synuclein inhibits hepatoma by exosome delivery Mol Carcinog 2023 62 1163 75 10.1002/mc.23553 37144864
Hou TZ, Yang HM, Cheng YZ, Gu L, Zhang JN, Zhang H. The Parkinson’s disease-associated protein alpha-synuclein inhibits hepatoma by exosome delivery. Mol Carcinog. 2023;62:1163–75. 10.1002/mc.23553.37144864 10.1002/mc.23553
