Hepatocellular carcinoma (HCC) is the sixth most frequently diagnosed cancer worldwide and ranks as the third leading cause of cancer deaths, characterized by its high malignancy rate and poor prognosis [1]. Although significant advances have been made in the diagnosis and treatment of HCC, effective therapies for advanced stages of HCC remain scarce [2], [3]. In the emerging era of cancer immunotherapy, adoptive cell therapy (ACT) has attracted considerable attention for demonstrating clinical efficacy in an increasing number of clinical trials. [4], [5] Dendritic cell (DC)-based immunotherapy is regarded as a promising approach to cancer immunotherapy [6], [7]. Nevertheless, it still faces numerous challenges, such as securing sources of antigens, achieving broad-spectrum effects, and the effective mobilization of antigen-specific T cells [8], [9], [10], [11]. These hurdles have driven efforts to identify workable solutions.

It has been widely recognized that tumor cells express tumor-specific or –associated antigens exhibiting potential immunogenicity [12], [13]. Exosomes, derived from multivesicular bodies, are a class of double-layer membrane structures with a diameter ranging from 30 to 150 nm and are released to extracellular environments by many cell types following multivesicular body fusion with the plasma membrane [14], [15], [16]. Extracellular exosomes are found to contain many bioactive materials, including protein, DNA and RNA [17], [18], and exosomes have become a major player in cell-cell communications [19]. Tumor-derived exosome (Tex), which is considered an important mechanism of anti-tumor immune responses [20], [21], [22], has been used as a novel tumor antigen-derived cell-free cancer vaccine [23], [24]. Previous studies have shown that stimulation of dendritic cells (DCs) with Tex, or cytokines- or stress protein genes-modified Tex may elicit protective anti-tumor responses [25], [26], [27], and tumor antigens targeting the exosome membrane surface may increase the immunogenicity of Tex [28]. However, there is increasing evidence demonstrating that Tex may facilitate cancer immune escape [29], [30]. It is reported that Tex may negatively mediate the functions of T cells and natural killer (NK) cells, and suppress DC differentiation [31], [32], [33]. These findings demonstrate that Tex has immunoinhibitory activities and helps tumors escape from immunosurveillance [34]. Acquisition of more high-immunogenic exosome-associated antigens and reducing the negative effect of Tex on DCs have been paid much attention during DCs-based immunotherapy [35].

Small GTPase Rab27a is a member of the Rab family [36], and plays a critical role in exosome secretion [37]. Rab protein has been reported to be involved in various stages of vesicle trafficking and the fusion of multiple vesicle transport intermediates [38]. In addition, Rab27 gene-modified tumor cell is found to further promote the secretion of more vesicles by tumor cells, and the acquisition of more exosome antigens may elicit a stronger anti-tumor activity, which is of great importance in DC-based immunotherapy [39].

IL-12 is a type I cytokine, originally discovered in the early 1990s [40], [41], that is naturally produced as an interleukin by dendritic cells and macrophages in response to antigenic stimulation. It is known for its potent activation of CD8+ T and natural killer (NK) cells and its role in increasing interferon-gamma (IFN-γ) production [42].IL-12 is a heterodimer consisting of a light chain p35 or IL-12α and a heavy chain p40 or IL-12β. The IL-12 receptor (IL-12R) is also made up of two distinct subunits, IL- 12R-β1 and IL-12R-β2, which can bind IL-12 with low affinity, and are part of the cytokine receptor superfamily, which is composed or induced to be expressed in several types of immune cells, including NK cells, T-lymphocytes and B-lymphocytes [43], [44], [45]. IL-12 production is induced by the interaction of CD40 on antigen-presenting cells (APCs) with CD40 ligand (CD40L) expressed on activated T cells, and IL-12 induces the production of IFN-γ through the activation of IL-12Rs in T cells and NK cells [46].

In this study, we generated exosomes derived from human hepatocellular carcinoma cells overexpressing Rab27a and pulsed them onto DCs to investigate their impact on DC biological activity. Our preliminary findings confirm that tumor-derived exosomes suppress the secretion of IL-12 by DCs. Consequently, we utilized Tex as a novel antigen source to load onto DCs, in combination with IL-12, to stimulate T cell proliferation and HCC-directed cytotoxicity. Accumulated evidence supports the establishment of a specific and broad-spectrum immunotherapy approach.