Exosome-based immunotherapy in hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC) is a significant global health concern and ranks as the third leading cause of cancer-associated mortality. Systemic therapy faces the emergence of resistance, which hinders the clinical benefits. Recent evidence suggests that exosomes, measuring between 30 and 150 nm in size, which impact the antitumor immune responses, making them a promising candidate for cancer immunotherapy. Owing to their unique physical and chemical characteristics, exosomes can be tailored and engineered for a range of therapeutic objectives. In the present review, we outline the immunomodulatory functions of exosomes in the tumor microenvironment (TME) of HCC, aiming to decipher the underlying mechanisms of exosomes in remodeling suppressive TME. Moreover, we provide detailed and intuitive resource for leveraging the potential of exosomes in immunotherapy, presenting valuable strategies to improve and optimize HCC treatment. Despite the huge therapeutic potential of exosomes, significant challenges persist, including the need for standardization in exosome production, optimization of cargo loading techniques, and the assurance of safety and effectiveness in clinical applications. Addressing these challenges may pave the way for exosome-based immunotherapy for HCC patients.

Introduction

Liver cancer represents a significant global health challenge due to its widespread prevalence in numerous countries. It is among the leading causes of cancer-related mortality globally and ranks as the fifth most common malignancy [1]. Hepatocellular carcinoma (HCC) is the most prevalent form of primary liver cancer, accounting for approximately 90% of liver cancer cases [2]. HCC is characterized by its aggressive behavior, high rates of recurrence, and limited treatment options, which results in most patients being diagnosed at advanced stages. This condition contributes to a poor prognosis, with an overall 5-year survival rate of approximately 24% [3, 4]. Systemic therapy, primarily centered on sorafenib over the past decade, has become the standard regimen for HCC. Following a series of late-stage clinical trials, several treatment options have been introduced for both first-line and second-line therapies for advanced HCC patients, including lenvatinib, regorafenib, cabozantinib, and ramucirumab [5]. However, these therapies offer only marginal survival benefits and frequently result in significant toxicities [6, 7]. Furthermore, the increasing prevalence of drug resistance contributes to the limited efficacy [8]. Consequently, there is an urgent demand for novel treatment strategies to improve outcomes for HCC patients.

Cancer immunotherapy can strengthen the antitumor immune responses to effectively elicit tumor cells [9]. Unlike traditional therapies, immunotherapy is based on a deep understanding of tumor immune evasion mechanisms, whereby cancer cells evade immune recognition and destruction through diverse pathways, enabling their survival and proliferation [10]. The utilization of immune checkpoint inhibitor (ICI) therapy that targets the programmed cell death-1 (PD-1)/programmed cell death ligand 1 (PD-L1) and cytotoxic T-lymphocyte antigen 4 (CTLA-4) pathways signifies a remarkable advancement in the treatment of advanced HCC patients [11]. For example, the first-line treatment combining atezolizumab and bevacizumab for HCC patients with advanced stages is linked to an objective response rate (ORR) of 30% [12]. The combination strategy sets the standard of care for evaluating novel therapeutic regimens. Nonetheless, the use of immunotherapy has limitations in effectiveness and is vulnerable to immune evasion and immunotherapeutic resistance induced by intrinsic factors within the tumor microenvironment (TME) [13]. Therefore, there is a significant demand for developing strategies to enhance the effectiveness of immunotherapy in HCC.

TME encompasses the intricate ecosystem of tumor cells and their adjacent stromal cells, which plays essential role in mediating immune response [14]. In HCC, TME is consist of various immune cells, including tumor-associated macrophages (TAMs), lymphocytes, neutrophils, natural killer (NK) cells, cancer-associated fibroblasts (CAFs), and dendritic cells (DCs) [15]. Additionally, it also includes various non-cellular components such as extracellular matrix (ECM), chemokines, cytokines, and multiple signaling factors [15]. The persistence of HCC progression—such as tumor growth, metastasis, and metabolic reprogramming—is influenced to varying extents by factors within the TME [16, 17]. Moreover, TME contributes to immunosuppressive characteristics of HCC, largely facilitating immune evasion by releasing immunosuppressive factors and activating immunosuppressive pathway, which create opportunities for therapeutic strategies aimed at targeting specific elements or signaling pathways associated with the TME [17]. Additionally, the immune cell components within the TME are regarded as significant predictors of HCC patient prognosis [18]. This highlights an essential foundation for further research into immunotherapy targeting HCC, suggesting that modulating the TME may enhance therapeutic efficacy.

Exosomes, typically measuring around 30 to 150 nm, are vital for paracrine signaling and serve a key function in facilitating intercellular communication [19]. Exosomes are released by almost all types of eukaryotic cells, including immune cells and tumor cells, which can influence both local and distant TME [20]. Recent investigations have increasingly recognized the role of exosomes as essential constituents of the HCC TME in shaping this microenvironment [21, 22]. Moreover, exosomes have unique physical and chemical properties that enable their modification and design for a variety of therapeutic purposes [23]. Understanding how exosomes contribute to TME remodeling may enhance our grasp of the molecular mechanisms underlying HCC immunotherapy. In this review, we comprehensively elaborate on the mechanisms of exosomes in the immunomodulatory of HCC TME and outline the advancements in the therapeutic applications of exosomes in HCC. The potential of these engineered exosomes to serve as an innovative tool in the fight against cancer drives continued research and progress in HCC immunotherapy.

Biogenesis and functions of exosomes

Extracellular vesicles (EVs) are produced by various cells during normal physiological processes as well as under pathophysiological conditions [24]. EVs can be primarily classified into two types: ectosomes and exosomes [24]. Exosomes are approximately 30–150 nm in size, which play a vital role in paracrine signaling and are essential for facilitating intercellular communication [25]. The biogenesis of exosomes begins with sequential invagination of the plasma membrane, leading to the formation of early endosomes by the contributions of the trans-Golgi network and endoplasmic reticulum [26] (Fig. 1). Furthermore, early endosomes develop into late-sorting endosomes (LSEs), ultimately leading to the formation of multivesicular bodies (MVBs) [27]. They arise from the inward invagination of the endosomal limiting membrane and include multiple intraluminal vesicles (ILVs) [27]. The release of ILVs as exosomes is enabled by the fusion of these MVBs with the plasma membrane, which transport a variety of cellular components, including DNA, messenger RNA (mRNA), non-coding RNAs (ncRNAs), lipids, metabolites, and various proteins [28]. While certain protein or RNA cargoes are sorted into exosomes, the mechanisms by which cells selectively sort these molecules into exosomes remain inadequately understood. Liu et al. reported that Y-box protein 1 (YBX1) was essential for the sorting of miR-223 into cellular exosomes [29]. Mechanistically, YBX1condensated selectively recruited miR-223 in vitro and within exosomes secreted by cultured cells. These findings revealed that local enrichment of cytosolic RNA-binding proteins and their corresponding RNAs, driven by phase separation, enabled their effective targeting and packaging into vesicles that bud from multivesicular bodies [30]. Additionally, the generation of ILVs is classified into two main mechanisms: endosomal sorting complex required for transport (ESCRT)-dependent and ESCRT-independent pathways [31]. The ESCRT machinery comprises ESCRT-0, -I, -II, -III, and vacuolar protein sorting 4–vesicle trafficking 1 (VPS4–VTA1), along with several accessory proteins, including the homodimer of ALG-2-interacting protein X (ALIX) [32, 33].

Fig. 1.

The biogenesis process of exosomes. The biosynthesis of exosomes begins with the inward budding of the cell membrane, forming early endosomes. These endosomes then undergo further inward budding, resulting in the generation of multivesicular bodies (MVBs), during which various miRNAs, proteins, and other selected molecules are incorporated. MVBs can either fuse with the cell membrane, releasing extracellular DNA, or merge with lysosomes, where their biological contents are degraded. Moreover, exosome production involves both ESCRT-dependent pathways

Exosomes influence the biological activities of recipient cells by transferring various cargoes as they move through the extracellular environment [26]. Exosomes display intricate structures and functional roles in mediating cellular communications. Exosomes can initiate downstream signaling cascades and induce epigenetic modifications through the release of proteins, mRNAs, and various ncRNAs [34–36]. Additionally, exosomes can facilitate membrane fusion by utilizing adhesion molecules, leading to specially targeting cells for activating intracellular signals [37]. These functions enable that exosomes play essential roles in the context of cancers, cardiovascular diseases, pathogenic infections, and neurological disorders [38, 39]. Notably, exosomes present unique benefits as natural, endogenous drug delivery systems, owing to their low immunogenicity, high stability in circulation, effective transport of drugs to target cells, and improved retention [40].

Regulatory roles of exosomes in immune responses and cancer immunotherapy

Exosomes can modulate immune responses by presenting antigens, delivering immunoregulatory molecules, or directly interacting with immune cells [41, 42]. Mounting evidence has indicated that tumor-derived exosomes (TEXs) can facilitate immunosuppression by transmitting inhibitory signals. The engagement of PD-L1 on tumors with PD-1 on T cells has been recognized for a long time as a mechanism for escaping immune surveillance [43]. Exosomal PD-L1 is essential for cancer cells to escape immune detection [44]. When TEXs that express PD-L1 enter the lymphatic system from the bloodstream, they can inhibit T cell functions and prevent immune cells from identifying and eliminating tumor cells [45, 46]. Metastatic tumors released exosomes, which displayed PD-L1 on their surface. Interferon-γ (IFN-γ) stimulation further increased PD-L1 levels, thereby inhibiting CD8⁺ T cell function and promoting tumor progression. [44]. In the experimental glioblastoma models, exosomal PD-L1 secreted by TEXs suppressed the proliferation of both CD4⁺ and CD8 + T cells and reduced the expression of CD69 and CD25, which function as essential markers of T cell activation [47]. Additionally, exosomal PD-L1 significantly inhibited the phosphorylation of ERK and suppressed the NF-κB pathway, as well as inhibited IL-2 secretion stimulated by phytohemagglutinin (PHA), thereby effectively suppressing the cytotoxic function of T cells [48]. After phosphorylation mediated by ERK, hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) interacted with PD-L1 and facilitated its selective incorporation into exosomes, which subsequently inhibited the migration of CD8⁺ T cells toward the tumor [49]. Exosomes indirectly influenced tumor immunity via upregulating PD-L1 expression in secondary types of immune cell. These TEXs-derived exosomes exploited the toll-like receptor 7 (TLR7) signaling pathway in macrophages to enhance PD-L1 expression [50]. TEXs induced TLR2 and NF-κB pathway to activate glycolysis, leading to polarization of macrophages toward an immunosuppressive phenotype marked by heightened PD-L1 expression [51]. Exosomal microRNA (miRNA)-372-5p derived from colorectal cancer cells can be phagocytosed by macrophages, regulating PD-L1 expression in both cancer and macrophage cells by targeting the PTEN/AKT/NF-κB pathway [52]. TEXs stimulated the chemotaxis of neutrophils, and this prolonged interaction led to a transition toward a pro-tumor phenotype, as well as elevated expression of CD73 and PD-L1, resulting in an immunosuppressive secretome [53]. Gaining insight into the crosstalk mediated by TEX between tumors and PD-L1 provided valuable information for immunomodulation strategies aimed at enhancing cancer therapies.

Exosomes can also facilitate an immunosuppressive effect through reprogramming energy metabolism of T cells. For example, exosomal IL-8 was found to overstimulate PPARα and downregulated glycolysis enzymes including glucose transporter 1 (GLUT1) and hexokinase2 (HK2), while promoting fatty acid catabolism. This metabolic shift ultimately diminished the ability of CD8⁺ T cells to proliferate [54]. Exosomal ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1) decreased extracellular ATP levels, while also enhancing the conversion of ATP to adenosine, thus promoting the adenosine-A2AR signaling pathway. This ATP-adenosine metabolism reprogramming significantly promoted the inhibition of CD8.⁺ T cellular function [55]. Additionally, lung cancer cells incorporatedCD39 into exosomes and transferred them into interacting T cells, driving metabolic adaption and promoting mal-differentiation of CD4 + T cells, leading to suppressive antitumor immune responses [56].

Additionally, exosomes have been shown to be involved in TME regulation by interacting with various immune cells. Exosomal circular RNA (circRNA)_0013936 derived from bladder cancer cells markedly targeted miR-320a/JAK2 and miR-301b/CREB1 signals in MDSCs, thus promoting suppressive immunity [57]. Notably, TEXs contribute to the decline in immune function of cancer patients by delivering EGFR to host macrophages, which inhibits type I interferon production [58]. Myofibroblastic cancer-associated fibroblasts (myCAFs) derived exosomes inculpated PWAR6, which facilitated immune evasion by impairing NK cell functions via diminishing glutamine availability [59]. It has been shown that small interfering RNA (siRNA)-loaded-exosomes can induce macrophages to polarize into an M1 phenotype, thereby enhancing the antitumor effects of macrophages [60]. All of these mechanisms collectively result in immune suppression remain to be explored further.

Moreover, several specific signaling pathways and mechanisms can be utilized to distinguish between immunosuppressive and immunostimulatory cargoes within exosomes. TGF-β pathway is generally associated with immunosuppressive properties and may enhance the sorting of certain inhibitory cargoes, including specific immunosuppressive cytokines [61]. Additionally, NF-κB pathway is related to immune stimulation, activation of this pathway can lead to the expression of pro-inflammatory cytokines, such as IL-6 and TNF-α, which may be encapsulated within exosomes to evoke an immune response [62]. Besides, IL-10 and other anti-inflammatory factor are often enriched in immunosuppressive exosomes [63], and their transport mechanisms may involve interactions with specific receptors, which await further investigations.

Regulatory roles of exosomes in HCC immune responses

The liver encompasses robust immune characteristics that collectively establish a powerful immune network to combat the significant immunogenicity and microbial environment associated with HCC [16]. The TME comprises various cell types, cytokines, and additional components. In this setting, cytotoxic immune responses are notably weakened, as demonstrated by the accumulation of immunosuppressive cell types and molecules that promote tumor growth [64]. Understanding the mechanisms by which these immune cells facilitate tumor immune evasion is critically important for informing immunotherapy strategies [65]. The TME of HCC possesses distinct characteristics, with both cellular and environmental factors playing essential roles in the initiation and progression of the disease [66]. Exsomes, as the essential communicational mediators, play a critical role in remodeling TME, thus providing immunotherapeutic targets in HCC (Fig. 2 and Table 1).

Fig. 2.

The regulatory role of exosomes within the TME of HCC

Table 1.

Exosomes mediate immune responses in HCC

Cargo	Mechanism	Effect	Tumor progression	References
PD-L1	Golgi membrane protein 1 facilitated the transport of exosomal PD-L1 into TAMs	Suppressed functions of CD8+ T cells	Enhanced	[66]
miR-200b-3p	Downregulated ZEB1 expression and upregulated IL-4, leading to activated STAT pathway	Induced M2 macrophage polarization	Enhanced	[67]
miR-21-5p	Targeted RhoB	Induced M2 macrophage polarization	Enhanced	[68]
PCED1B-AS1	Enhanced PD-L1 expression tumor cells by sponging hsa-miR-194-5p	Inhibited functions of T cells	Enhanced	[69]
miR-146a-5p	SALL4 regulated expression of miR-146a-5p, leading to a decrease in the expression of PD-1 and CTLA-4 on T cells	Reversed T cell exhaustion and delayed M2 TAMs polarization	Inhibited	[70]
circGSE1	circGSE1 regulated the miR-324-5p/TGFBR1/Smad3 pathway	Promoted the expansion of Tregs	Enhanced	[71]
LOXL4	Enhanced PD‐L1 through IFN–mediated STAT pathways	Inhibited functions of T cells	Enhanced	[72]
miR-1290	miR-1290 inactivated AKT2 while simultaneously increasing the expression PD-L1	Suppressed functions of CD8+ T cells	Enhanced	[74]
HMMR-AS1	HIF-1α promoted the secretion of exosomal HMMR-AS1 that sequestered miR-147a and indirectly targeted ARID3A	Induced M2 macrophages polarization	Enhanced	[76]
miR-23a-3p	ER stress prompted the release of exosomal miR-23a-3p, which suppressed PTEN expression and upregulated PD-L1 in TAMs by activating the PI3K/AKT pathway	Suppressed functions of CD8+ T cells	Enhanced	[79, 80]
lncMMPA	Exosomal lncMMPA sponged miR-548 and upregulated ALDH1A3	Induced M2 macrophage polarization	Enhanced	[81]
NEAT1	Exosomal NEAT1 targeted KLF5/galectin-3 axis	CD8+ T cell exhaustion	Enhanced	[82]
circHIF1A	Exosomal circHIF1A enhanced the expression of PD-L1	CD8+ T cell exhaustion	Enhanced	[83]
ZNF250	Exosomal ZNF250 activated the expression of PD-L1 in HCC cells	CD8+ T cell exhaustion	Enhanced	[84]

TEXs play a crucial role as immune modulators within the TME. Evidence indicates that these exosomes facilitated the interactions between tumor cells and various types of immune cell to promote immune evasion and tumor progression [67]. T cells that infiltrate human cancers significantly influence disease progression and impact the likelihood of clinical responses to cancer immunotherapies [68]. The primary distinction among T cell subsets is between CD4⁺ and CD8⁺ T cells [68]. CD8⁺ T cells, upon recognizing antigens presented by MHC class I molecules via their T cell receptors (TCRs), release cytotoxic substances like perforin and granzymes, which are vital for effective tumor immunity [69]. In contrast, CD4 + T cells primarily regulate other immune cells’ activities through cytokine secretion. This process is essential for the immune system’s responses to tumor development [69, 70]. Golgi membrane protein 1 (GOLM1) was found to facilitate the suppression of CD8 + T cells by enhancing the stabilization of PD-L1 and mediating the transport of PD-L1 into TAMs through an exosome-dependent mechanism, which providing a promising strategy for HCC immunotherapy [71].

TEXs and exosomal miR-200b-3p were internalized by macrophages, which subsequently induced M2 macrophages polarization through the downregulation of zinc finger E-box-binding homeobox protein 1 (ZEB1) and the upregulation of interleukin-4 (IL-4) [72]. Furthermore, the JAK/ signal transducer and activator of transcription (STAT) signaling pathway was activated in M2 TAMs, leading to elevated expression of PIM1 and vascular endothelial growth factor (VEGF)α, contributing to the acceleration of HCC progression, establishing a feedback loop between tumor cells and TAMs [72]. Exosomal miR-21-5p facilitated the polarization of M2-like TAMs through targeting RhoB [73]. TEXs secreted PCED1B-AS1, which enhanced PD-L1 expression in recipient tumor cells while simultaneously inhibiting functions of T cells by sponging hsa-miR-194-5p, leading to induced suppression of immune responses [74]. Sal-like protein-4 (SALL4) has the ability to interact with miR-146a-5p, directly regulating its expression in TEXs. Inhibition of the SALL4/miR-146a-5p interaction in HCC led to a decrease in the expression of PD-1 and CTLA-4 on T cells, reversing T cell exhaustion and delaying M2 TAMs polarization, thus slowing tumor progression in HCC mouse models [75]. Furthermore, Huang et al. exploited that TEXs contained circGSE1 that facilitated HCC progression by promoting the expansion of regulatory T cells (Tregs) through the regulation of the miR-324-5p/TGFBR1/Smad3 signaling pathway [76]. TEXs secreted Lysyl Oxidase-Like 4 (LOXL4) that could be localized in hepatic macrophages through the internalization of exosomes. Label-free proteomics analysis indicated that the immunosuppressive effects of LOXL4 primarily depended on the activation of PD‐L1 through IFN–mediated STAT pathways [77].

Hypoxia is a crucial factor in the tumor formation of HCC [78]. Hypoxic tumor cells could secret exosomes that promoted the polarization of M2 macrophages, the pro-tumoral phenotype of TAMs in the TME. Infiltered M2 TAMs significantly promoted apoptosis of CD8⁺ T cells, leading to the suppressive TME in HCC [79]. Further experimental evidence revealed that TEXs from hypoxic HCC cells were packaged within miR-1290 that inactivated AKT2 while simultaneously increasing the expression PD-L1, which offered a promising immunotherapeutic target for HCC therapy [79]. Hypoxia-inducible factor-1α (HIF-1α) is a nuclear protein that regulates the transcription of numerous target genes, including those associated with hypoxic adaptation, angiogenesis, and tumor development [80]. HIF-1α promoted the secretion of exosomal long non-coding RNAs (lncRNAs) HMMR-AS1 that sequestered miR-147a and indirectly targeted AT-rich interaction domain 3A(ARID3A), resulting in enhanced macrophage polarization toward M2 TAMs and fostered HCC progression [81]. These findings revealed hypoxia plays an essential role in exosome secretion and mediating immune responses and tumor progression in the context of HCC.

In HCC, various oncogenic, transcriptional, and metabolic irregularities contribute to establish hostile microenvironments that disrupt endoplasmic reticulum (ER) homeostasis, inducing a state of chronic ER stress, which has been shown to reprogram the functions of immune cells within TME [82, 83]. ER stressed tumor cells secreted exosomes that activated STAT3 pathway, resulting in remodeling macrophage function by upregulating expression of IL-6, IL-10, and TNF-α [84]. ER stress prompted tumor cells to release exosomes that were enriched with miR-23a-3p, which targeted M2-type TAMs. Upon internalization by M2 TAMs, miR-23a-3p suppressed PTEN expression, which in turn led to the upregulation of PD-L1 in TAMs by activating the PI3K/AKT pathway [85].

Researchers are exploring the immunomodulatory role of TAM-derived exosomes in the TME of HCC. TAMs enhanced aerobic glycolysis and cell proliferation in HCC through the extracellular transmission of a myeloid-derived long non-coding RNA, known as M2 macrophage polarization-associated lncRNA (lncMMPA) [86]. Mechanistically, exosomal lncMMPA facilitated the polarization of M2 TAMs and functioned as a ceRNA for miR-548, thus elevating expression of ALDH1A3 [86]. Treatment with M2-like TAM-derived exosomes enhanced the expression of exosomal lncRNA NEAT1, which targeted KLF5/galectin-3 axis, thereby leading to the exhaustion of CD8⁺ T cells and further accelerating immune escape [87]. Additionally, exosomal circHIF1A derived from hypoxia-induced CAFs enhanced the immune evasion in HCC cells through the upregulation of PD-L1 in a HuR-dependent manner [88]. In line with this, CAF-derived exosomal zinc finger protein 250 (ZNF250) also transcriptionally activated the expression of PD-L1 in HCC cells, thus promoting tumor growth [89]. These findings underscore the essential of CAFs-derived exosomes in mediating the immune evasion of HCC cells.

Engineering exosomes in HCC immunotherapy

Exosomes as drug delivery tools in HCC immunotherapy

Immunotherapy is emerging as one of the most prevalent therapeutic strategies for HCC patients [90]. However, the suppressive TME of metastatic HCC limits its responsiveness to immunotherapeutic treatments [90]. Consequently, it is critically important to enhance immunotherapeutic approaches to improve their effectiveness in HCC. In recent years, engineered exosomes have gained attention as “natural nanoparticles” for drug delivery vehicles attributed to their low cytotoxicity, potential to enhance drug bioavailability, and precise targeting specificity [91]. Recent research has demonstrated that exosomes possess the capability to traverse physical barriers, indicating that exosomes have significant potential as novel drug delivery systems [92] (Fig. 3).

Fig. 3.

Exosome-based immunotherapy in HCC

M2 TAMs play an essential role in immune evasion within the TME of HCC, while reprogram TAMs hol immense potential for immunotherapy [93]. STAT6 serves as a crucial regulator of the transcriptional program associated with M2 TAMs in HCC [94, 95]. Therefore, reducing STAT6 levels in TAMs may provide an effective strategy for reprogramming these macrophages. Kamerkar et al. outlined an innovative exosome-based method for reprogramming M2-like TAMs into the M1 phenotype through the selective delivery of a specific antisense oligonucleotide (ASO) targeting STAT6 in these macrophages [96]. They developed engineered exosomes, known as exoASO-STAT6, exhibited optimal biodistribution and STAT6-silencing efficacy, which exerted significantly antitumor effects on HCC models. Mechanistic studies have revealed that treatment with exoASO-STAT6 increased markers of M1 macrophages, decreased the infiltrating of M2 TAMs, and activated the CD8⁺ T cell-dependent immune responses, leading to complete tumor remission in the majority of treated subjects along with minimal toxicity [96]. These findings indicated that exoASO-STAT6 was a pioneering therapy designed to selectively target TAMs while causing minimal systemic toxicity.

The application of exosome-like nanocarriers represents a significant progress in the delivery of tumor-targeted [97, 98]. Chen et al. presented an innovative nanomedicine, MPDA/ICG-coloaded exosome-like nanoparticles (MPDA/ICG@M1NVs), designed for synergistic tumor-targeted photo-immunotherapy in the context of HCC. These exosome-like nanocarriers combined M1-like macrophages with MPDA/ICG, which functioned as photosensitizers and photothermal agents. Following intravenous administration, MPDA/ICG@M1NVs enhanced the polarization of TAMs toward M1 phenotype, effectively reversing the immunosuppressive TME and boosting the antitumor effects, demonstrating improved therapeutic effects of photo-immunotherapy [99]. Furthermore, exosomes were used as carriers to collaborate with pegylated iron oxide nanoparticles loaded with chlorin e6 (PIONs@E6) to enhancing macrophage polarization toward M1 phenotype, thus improving antitumor immune responses in HCC [100]. Additionally, hypoxic exosomes were subjected to continuous stimulation using low-intensity ultrasound (LICUS), which significantly enhanced exosome uptake and increased substantial internal localization. Furthermore, LICUS-treated H-Exos were loaded with hydrophobic curcumin (H-Exo-Cur) exhibited significantly antitumor effects in HCC [101], which presented a potential safe and effective strategy for the clinical implementation of exosome-based immunotherapy in HCC.

Role of exosomes in tumor vaccines

Tumor vaccines are classified as a form of tumor immunotherapy, operating on the fundamental principle of stimulating the patient’s autoimmune response by injecting tumor-specific antigens, enabling immune cells to target and destroy cancer cells [102, 103]. Exosomes obtained from α-fetoprotein (AFP)-expressing dendritic cells (DEXAFP) reversed the immunosuppressive TME and functioned as critical nanovaccines for HCC immunotherapy [104]. Additionally, Zou et al. engineered DEXs by coupling exosomes with P47-P and AFP212-A2 peptides and a domain of the high mobility group nucleosome-binding protein 1 (N1ND-N), thus creating a nanovaccine referred to as DEXP&A2&N. This nanovaccine remarkedly promoted the accumulation and activity of DCs, resulting in enhanced cross-presentation of tumor neoantigens and the activation of novel T cell responses [105]. Furthermore, microwave ablation combined with DEX vaccines significantly suppressed tumor growth by improving the TME, which was evidenced by increased number of CD8 + T cells and a reduction in Treg cell numbers in HCC [106]. This strategy provides a versatile framework for personalized immunotherapy in HCC, eliminating the necessity of identifying specific tumor antigens.

Role of exosomes in immune checkpoint blockade therapeutic resistance

Recent advancements in understanding the TME have paved the way for developing and applying new immunotherapy strategies [107]. Although immunotherapy has shown impressive durable response rates in cancer patients, many still do not respond due to therapeutic resistance [108]. Exosomes serve as mediators of cell-to-cell communication and potentially impact immunotherapy by secreting various biological molecules [108]. Exosomes exert critical impacts on modifying the TME [109]. Research indicates that tumor cells can express PD-L1, and the PD-L1 found in exosomes can also inhibit T cell activation, allowing cancer cells to evade antitumor immunity [110]. Moreover, anti-PD-L1 antibodies appear to be ineffective at completely suppressing exosomal PD-L1 [110]. Exosomal PD-L1 derived from tumors suppressed T cell activation in draining lymph nodes. Systemically delivered exosomal PD-L1 was shown to enhance the growth of tumors incapable of producing their own PD-L1. Moreover, exposure to tumor cells deficient in exosomal PD-L1 inhibited the growth of wild-type tumor cells at a distant site [111]. Exosomal PD-L1 serves as a potential therapeutic target that could address resistance to existing antibody-based treatments [111]. Furthermore, immune cells have been shown to infiltrate the TME. Exosomes carried tumor-associated antigens that interfered with antitumor immunotherapy, which indicated that exosomes served as mediators of immune cells’ response to anticancer therapies, facilitating interactions between the TME and HCC cells, which resulted in the transfer of resistance [112]. For example, Hu et al. revealed that TEXs wrapped circCCAR1, which functioned as a molecular sponge for miR-127-5p and targeted WTAP. Mechanistically, exosomal circCCAR1 was absorbed by CD8⁺ T cells, causing T cell dysfunction by stabilizing the PD-1 protein, thus contributing to resistance against anti-PD-1 therapy in the setting of HCC [113]. Moreover, exosomal circUHRF1 derived from HCC cells suppressed the secretion of IFN-γ and TNF-α from NK cells. Furthermore, circUHRF1 hindered NK cell activity by enhancing T cell immunoglobulin domain and mucin domain-3 (TIM-3) expression through the degradation of miR-449c-5p, which contributed to resistance against anti-PD1 immunotherapy [114]. Exosomal circTMEM181 suppressed miR-488-3p and heightened CD39 expression in TAMs, which, in combination with CD73 in HCC cells, synergistically triggered the eATP-adenosine pathway, resulting in elevated adenosine production. This ultimately impaired the function of CD8⁺ T cells and promoted therapeutic resistance to anti-PD-1 antibodies [115]. Exosomal PD-L1 has been identified as a biomarker for disease status and the clinical efficacy of immunotherapy. During anti-PD-1 treatment, the observed increase in exosomal PD-L1 levels is believed to be stimulated by IFN-γ produced by reactivated CD8 + T cells, indicating that changes in tumor-derived exosomal PD-L1 represent an adaptive response of tumor cells to T cell regeneration [116]. Research has shown that in patients with metastatic tumors undergoing immunotherapy, those with an increased levels of exosomal PD-L1 are more likely to respond positively to treatment [44].

It is noteworthy that ongoing research indicates that exosomes derived from mesenchymal stem cells (MSCs) play an essential role in immune regulation [117]. Glioma-associated mesenchymal stem cells (GA-MSCs) were found to secret exosomal miR-21 that targeted SP1/DNMT1 axis, leading to amplified the immunosuppressive signals of myeloid-derived suppressor cells (MDSCs). Moreover, modified DEXs loaded with miR-21 inhibitors were able to target GA-MSCs, leading to a reduction in the immunosuppressive functions of MDSCs and enhanced the efficacy of anti-PD-1 antibodies [118]. Gao et al. revealed that exosomal miR-124 derived from bone marrow MSCs targeted moncarboxylate transporter 1 (MCT1), leading to reduced lactate uptake and ultimately diminishing the immune-suppressive capabilities of Treg cells, thus functioning as a promising strategy for treating immunotherapy resistant cancer patients [119]. In the context of HCC, miR-199-modified exosomes from adipose tissue-derived MSCs enhanced the sensitivity of cancer cells to anticancer drugs by inhibiting the mTOR signaling pathway [120]. Given the essential role of MSCs-derived exosomes in remodeling the TME, these exosomes may exert regulatory impacts on HCC immunotherapy, which await further investigations in preclinical studies.

Challenges in the application of exosome-based cancer treatment

Exosomes, as effective key mediators of cellular communication, serve as valuable biomarkers for early disease diagnosis and monitoring [121]. Their biocompatibility and protective lipid bilayer structure safeguard genetic material from degradation and minimize immunogenicity, allowing them to actively participate in drug delivery, targeted therapy, and immune regulation [39]. Furthermore, their small size and unique membrane composition enable them to traverse the blood–brain barrier, offering new insights into disease treatment [122]. Despite the numerous advantages of exosomes, accurately understanding their roles in drug therapy and delivery applications still presents several challenges. Here, we outline the challenges in the application of exosome-based cancer treatment. Firstly, exosome biogenesis is regulated by multiple mechanisms. However, the precise ways in which these distinct mechanisms cooperate in a finely tuned manner remain unclear. Additionally, the processes by which most cargo is accurately sorted into exosomes are still not well understood. Recent studies have also identified several subpopulations of MVBs [123]; are there other subfamilies of MVBs and what are their mechanisms of generation? Notably, it is crucial to recognize that the mechanisms of exosome biogenesis can vary significantly based on the cargo, cell type, and environmental context; hence, caution should be exercised when drawing conclusions about specific mechanisms observed in any given study [124, 125]. Secondly, current standards for distinguishing exosomes are their intracellular origin. However, recent evidence suggests that multiple subpopulations may exist within these exosome groups. This heterogeneity may play various roles in complex biological processes, adding a layer of complexity to the study of exosome biology and function [126]. The limitations of current isolation and characterization techniques remain a significant barrier to effectively addressing the issue of exosome subpopulations. Nonetheless, the scientific community is actively pursuing the development of new and improved technologies to tackle this challenge and enhance the understanding of exosome biology and function [127]. Furthermore, exosomes play an essential role in tumorigenesis, and drug resistance in the HCC, which hold immense potential for HCC therapy [128]. However, concerns regarding the safety of exosome-based therapy have persisted, often considered their “Achilles’ heel” in clinical applications [129]. Although exosomes have demonstrated promising therapeutic delivery potential; however, off-target accumulation of exosomes remains a critical concern, as it may compromise treatment efficacy and safety [130]. Such pharmacological agents often exhibit polypharmacological properties, characterized by concurrent inhibition of multiple kinase pathways or unintended off-target interactions, and induce pleiotropic biological effects [131]. In order to address this challenge, surface functionalization approaches—such as synthetic material coatings or overexpression of specific membrane proteins—have been explored to improve vesicle specificity, paving the way for personalized therapeutic platforms [132].

It is crucial to investigate the impact of specific exosomes to further understand their underlying mechanisms. Consequently, identifying the most suitable cell sources for producing bioengineered exosomes could enhance treatment for cancer therapy while minimizing adverse effects [133]. Developing methods to deactivate or eliminate undesirable and harmful exosomal components may emerge as a significant novel engineering strategy. Additionally, the application of an in vivo monitoring platform is also essential for monitoring drug distribution, refining dosage regimens, and confirming therapeutic safety [129, 133]. Moreover, blocking the M2 polarization of macrophages induced by HCC-derived exosomes represents a highly promising immunotherapeutic strategy; yet, its success relies on the following critical factors: highly specific target screening (e.g., membrane proteins or miRNAs unique to HCC-derived exosomes); development of efficient delivery systems (e.g., macrophage-targeted nanocarriers); and optimization of combination therapies (e.g., combined use of immune checkpoint inhibitors, chemotherapy, or TAM depletion therapies). Future research should employ single-cell sequencing and spatial transcriptomics to unravel the spatiotemporal dynamics of exosome-macrophage interactions, thereby providing a foundation for precise therapeutic interventions [134]. The main technical challenge lies in producing sufficient quantities of exosomes. To achieve an effective dose response, at least 10 µg of exosomes are needed [135]. However, the yield of exosomes isolated from biological fluids can be both limited in quantity and variable in composition. Additionally, exosomes isolated from these fluids are often highly heterogeneous, impure, and show low yields. Physical, chemical, and biological stresses can stimulate the production of exosomes; however, potential contamination with apoptotic bodies necessitates a thorough evaluation of the contents, therapeutic efficacy, and safety of these exosomes [136]. Moreover, the high cost of advanced production techniques can increase treatment expenses, limiting accessibility for patients [137].

Additionally, the gut microbiota plays a pivotal role in modulating hepatic immune responses, which critically influences the efficacy of immunotherapy in HCC [138]. Mechanistically, microbiota-mediated bile acid metabolism has been shown to upregulate the abundance of CXCR6⁺ natural killer T cells within the liver, a process integral to suppressing HCC progression. This immunomodulatory effect is driven by CXCL16, a chemokine secreted by liver sinusoidal endothelial cells. The localized accumulation of these immune cells correlates with enhanced tumor-suppressive activity, including inhibition of neoplastic proliferation and induction of immunogenic cell death [139]. Given the pivotal role of gut microbiota in orchestrating immune regulation, exosomes secreted by commensal bacteria may serve as critical mediators to modulate the therapeutic outcomes of HCC immunotherapy. However, the mechanistic contributions of gut microbiota-derived exosomes to HCC immunotherapy remain unexplored in current literature. Systematic investigations integrating multi-omics approaches (metagenomics, proteomics, and spatial transcriptomics) and preclinical models are urgently warranted to elucidate their immunomodulatory mechanisms, therapeutic synergies with ICIs, and translational potential for clinical biomarker discovery.

Conclusions

Given that HCC is among the deadliest cancers globally and that traditional treatments often fail due to resistance, it is vital to explore innovative strategies for managing this disease. The use of exosomes in HCC therapy offers a promising and novel therapeutic approach due to their inherent advantages. In this review, we categorize the research on the role of exosomes in HCC immunotherapy. Exosomes exert crucial impacts on the immune regulation within the TME, thereby serving as potential immunotherapeutic targets in HCC. Additionally, we discuss the application of exosomes in HCC treatment, including the delivery of immunotherapeutic agents, cancer antigens used as nanovaccines, and regulators of immunotherapy resistance.

Exosomes are increasingly recognized as essential tools in cancer immunotherapy, garnering substantial interest over the past few decades for their effectiveness in delivering various chemical and natural therapies. However, exosome-based therapies for HCC face notable challenges, such as limited targeting efficiency that affects accurate tumor navigation, as well as technical difficulties related to drug loading and controlled release, which are essential for successful treatment. Concerns regarding immunogenicity and safety complicate clinical applications, highlighting the need for extensive research and strategies to mitigate these issues. Researchers are exploring innovative delivery systems to surmount biological barriers and improve the distribution of exosomes.

These collaborative efforts hold immense potential for advancing the field and unlock the complete therapeutic potential of exosomes in HCC treatment. Clinical trials are crucial for evaluating the safety and effectiveness of exosome-based therapies in cancer treatment. The resulting data will support the development of personalized treatment protocols and the optimization of therapeutic strategies tailored to individual patient responses.

Author contributions

H.L was contributed conceptualization, data curation, investigation, methodology, software, writing—original draft, and writing—review and editing. GW.W was involved in project administration, resources, supervision, validation, and writing—review and editing. ZY.L was performed methodology and writing—review and editing. XT.Z was done data curation and formal analysis. WD.Z did writing—review and editing and investigation. X.Z was carried out data curation, investigation, methodology, and writing—review and editing. F.L and J.G was responsible for conceptualization, data curation, investigation, methodology, and writing—review and editing.

Funding

The study was funded by the Medical Health Science and Technology Project of HangZhou Health Commission [grant number: A20251404].

Data availability

No datasets were generated or analyzed during the current study.

Declarations

Conflict of interest

The authors declare no competing interests.

Ethical approval and consent to participate

Not applicable.