Systemic presence and tumor-growth promoting effect of ovarian carcinoma released exosomes

Exosomes are membrane vesicles that are released from many different cell types. Tumor derived-exosomes play a role in immune suppression. We hypothesized that in ovarian carcinoma patients exosomes initially produced at the local abdominal site may become systemic. We examined paired samples of ascites and blood from ovarian carcinoma patients for the presence of exosomes. We also studied the requirements for exosomal uptake by immune cells, the role of phosphatidyl-serine (PS) as uptake signal and the effect of exosome application on tumor growth. We used exosomes from ovarian carcinoma cell lines, malignant ascites and sera from ovarian carcinoma patients isolated by ultracentrifugation. PS-displayed by exosomes was detected by Anexin-V-FITC staining of latex beads adsorbed exosomes. For uptake experiments, labeled exosomes were exposed to cells in the presence or absence of cold Annexin-V as competitor. Uptake was examined by fluorescent microscopy and cytofluorographic analysis. Effects of exosomes on tumor growth were studied using SKOV3ip ovarian carcinoma cells in CD1 nu/nu mice. We found that malignant ascites-derived exosomes cargo tumor progression related proteins such as L1CAM, CD24, ADAM10, and EMMPRIN. We observed that exosomes become systemic via the blood stream. Uptake of ovarian carcinoma exosomes by NK cells was found to require PS at the exosomal surface but the presence of PS was not sufficient. Application of malignant ascites-derived exosomes to tumor bearing mice resulted in augmented tumor growth. Exosomes from the serum of tumor patients could be isolated from only one ml of blood and this analysis could serve for diagnostic purposes. We propose that tumor-derived exosomes could play a role in tumor progression.     

MHC independent anti-tumor immune responses induced by Hsp70-enriched exosomes generate tumor regression in murine models

The ideal cancer vaccine should work regardless of MHC types but currently the barrier generated by MHC specificity hampers the development of human cancer vaccines, requesting to identify strong immunogenic molecules that can induce anti-cancer immune responses without being affected by MHC polymorphism. Tumor-derived exosomes are small membrane vesicles containing tumor antigens as well as other immunologically important molecules such as MHC molecules and heat shock proteins (HSPs). Because of their potential immunogenicity, the plausible utility of tumor-derived exosomes as an MHC independent cancer vaccine was proposed. Here, we investigated whether Hsp70-enriched tumor exosomes can induce stronger immunogenicity as compared to normal tumor-derived exosomes in autologous as well as allogeneic murine models in vitro and in vivo. Western blotting showed that the exosomes of heat-treated tumor cells (HS Exo) contained higher amounts of Hsp70 than the exosomes of untreated cells (CNTL Exo). In both MHC type-identical and -irrelevant antigen-presenting cell models in vitro, HS Exo triggered the increased expressions of MHC class II molecules. Crucially, HS Exo performed greater therapeutic capability in regressing pre-established MHC type-identical and -irrelevant tumors than CNTL Exo in vivo. The analyses of anti-tumor function in allogeneic mouse model demonstrated that HS Exo elicited Th1-polarized immune responses defined by the increased productions of IgG2a and IFN-γ. In summary, the Hsp70-enriched exosomes extracted from heat-treated tumors induced strong Th1 immune responses, resulting in eliminating cancer cells in allogeneic hosts in vivo. These results indicate that HS Exo is a potent MHC independent cell-free cancer therapeutic agent that can be developed for clinical trials.     

外体及其在肿瘤生物治疗中的应用

外体是活细胞释放到胞外的囊泡,含脂质双层膜,是细胞与外界信息传递的载体。肿瘤细胞来源的外体含肿瘤相关抗原,经树突状细胞呈递,可激发肿瘤特异性细胞毒作用和交叉抗肿瘤作用。树突状细胞释放的外体表面有大量的抗原呈递分子,而且性质稳定,易保存,可快速批量生产。因此,外体作为非细胞瘤苗在恶性肿瘤生物治疗方面意义重大。     

Exosomes and anti-tumour immunotherapy

Exosomes are 60 to 90 nm membrane vesicles originating from late endosomes and secreted from most hematopoietic and epithelial cells in vitro. B cell derived-exosome antigenicity was first reported in 1996 in MHC class II restricted CD4+ T lymphocytes. In 1998, we reported that dendritic cell derived-exosomes are immunogenic in mice leading to tumor rejection. These findings have renewed the interest in exosomes. The current challenge consists in understanding the mechanisms and the physiological relevance of exosomes that could contribute to the design of the optimal exosome based-vaccination. Here, we will focus on the biological features pertaining to dendritic cell- and tumor cell derived-exosomes and will discuss their potential clinical implementation.     

EXPRESSION OF IMMUNE－RELATED MOLECULES IN GLIOBLASTOMA MULTIFORM CELLS

Objective: To investigate the expression of immune-related molecules in glioblastoma multiform(GBM) cells.Methods: The expression of major histocompatibility complex (MHC), β2-microglobulin, Fas, CD80 and CD86 molecules on the surface of GBM cells were evaluated by flow cytometry. The expression of TAP-l, TAP-2 and Tapasin in the GBM cells were evaluated by RT-PCR method. Results: MHC class I, 62 microglobulin, TAP-l,TAP-2 and tapasin were expressed in most GBM cell lines. Except U87, there was no MHC class II molecule expression on any of the other GBM cell lines. Fas was expressed on all the GBM cell lines examined.Conclusion: The mechanism by which GBM escapes immune surveillance may involve down regulation of expression of MHC class I molecules and MHC class Ⅱ molecules. MHC class I positive GBM may be the suitable target of immunotherapy.     

T cell recognition of HLA‐A2 restricted tumor antigens is impaired by the oncogene HER2

The HER2 oncogene is frequently over‐expressed in human cancers and a promising target for immune therapy. Previous studies have shown that over‐expression of mouse or rat HER2 leads to markedly reduced levels of major histocompatibility complex (MHC) class I and molecules of the antigen processing and presentation machinery (APM), thus resulting in a phenotype promoting tumor escape from the immune system. Our study focuses on analyzing the effect of HER2 on MHC class I antigen presentation and sensitivity to tumor‐antigen specific cytotoxic T lymphocytes (CTLs) in HLA‐A2.1⁺ melanoma cell lines. We demonstrate significant inverse correlations both between the expression of HER2 and total MHC class I surface expression as well as between HER2 and HLA‐A2. A significant reduction of HLA‐A2 levels was found when melanoma and carcinoma cell lines were transfected with a human HER2 gene. A signaling‐competent HER2 molecule was crucial for the observed HLA‐A2 down‐regulation, as transfectants expressing high levels of HER2 mutated in the tyrosine signaling domain did not show altered HLA‐A2 expression. Importantly, the human melanoma cell line EST049 demonstrated reduced HER2 and melanoma antigen‐specific recognition by CTLs upon HER2 transfection. In addition, high expression of HER2 prevented both IFN‐γ mediated HLA‐A2 up‐regulation and improved recognition by HLA‐A2‐restricted CTLs in treated cells. Moreover, key APM molecules were down‐regulated by HER2. These findings implicate that HER2 over‐expressing tumors may be more prone to escape from HLA‐A2 restricted CTLs suggesting that immunotherapy approaches inducing an integrated humoral, cellular and innate immune response would be most effective.     

Exosomes from CIITA-Transfected CT26 Cells Enhance AntitumorEffects

Aim: To study anti-tumor effects of exosomes from class II transactivator (CIITA) gene transfected CT26cells. Methods: In this study, we established an MHC class II molecule-expressing murine colon cancer cellline (CT26-CIITA) by transduction of the CIITA gene. Immune effects in vitro and tumor protective results invivo were tested and monitored. Results: Exosomes from CT26-CIITA cells were found to contain a high levelof MHC class II protein. When loaded on dendritic cells (DCs), exosomes from CT26-CIITA cells significantlyincreased expression of MHC class II molecules, CD86 and CD80, as compared to exosomes from CT26 cells.In vitro assays using co-culture of immunized splenocytes and exosome-loaded DCs demonstrated that CIITAExoenhanced splenocyte proliferation and IFN-γ production of CD4+T cells, while inhibiting IL-10 secretion.In addition, compared to exosomes from CT26 cells, CT26-CIITA-derived exosomes induced higher TNF-α andIL-12 mRNA levels. A mouse tumour preventive model showed that CT26-CIITA derived exosomes significantlyinhibited tumour growth in a dose-dependent manner and significantly prolonged the survival time of tumourbearingmice. Conclusion: Our findings indicate that CT26-CIITA-released exosomes are more efficient to induceanti-tumour immune responses, suggesting a potential role of MHC class II-containing tumour exosomes ascancer vaccine candidates.     

Prodrug suicide gene therapy for cancer targeted intracellular by mesenchymal stem cell exosomes

The natural behavior of mesenchymal stem cells (MSCs) and their exosomes in targeting tumors is a promising approach for curative therapy. Human tumor tropic mesenchymal stem cells (MSCs) isolated from various tissues and MSCs engineered to express the yeast cytosine deaminase::uracil phosphoribosyl transferase suicide fusion gene (yCD::UPRT-MSCs) released exosomes in conditional medium (CM). Exosomes from all tissue specific yCD::UPRT-MSCs contained mRNA of the suicide gene in the exosome's cargo. When the CM was applied to tumor cells, the exosomes were internalized by recipient tumor cells and in the presence of the prodrug 5-fluorocytosine (5-FC) effectively triggered dose-dependent tumor cell death by endocytosed exosomes via an intracellular conversion of the prodrug 5-FC to 5-fluorouracil. Exosomes were found to be responsible for the tumor inhibitory activity. The presence of microRNAs in exosomes produced from naive MSCs and from suicide gene transduced MSCs did not differ significantly. MicroRNAs from yCD::UPRT-MSCs were not associated with therapeutic effect. MSC suicide gene exosomes represent a new class of tumor cell targeting drug acting intracellular with curative potential.     

Renal cell carcinoma-infiltrating natural killer cells express differential repertoires of activating and inhibitory receptors and are inhibited by specific HLA class I allotypes

Among tumor-infiltrating lymphocytes (TILs) directly isolated from renal cell carcinomas (RCCs), we found substantial numbers of natural killer (NK) cells in most tumor tissues. They could be identified reliably in situ with an antibody directed against the activating receptor (AR) NKp46 that is exclusively expressed by all NK cells. NK-enriched TILs (NK-TILs) showed cytotoxicity against major histocompatibility complex (MHC) class I-negative cell lines. The ability to detect lysis of target cells was dependent on the percentage of NK cells within the TILs, and cytotoxicity was only observed after overnight activation with low-dose interleukin-2 (IL-2). Infiltrating NK cells were found to express various inhibitory receptors (IRs); among these the CD94/NKG2A receptor complex was overrepresented compared to the autologous peripheral blood mononuclear cell (PBMC) population. Other IRs were underrepresented, indicating that NK subpopulations vary in their tumor-infiltrating capacity. IRs expressed by NK-TILs are functional since receptor engagement with MHC class I ligands presented by human leukocyte antigen (HLA)-transfected target cell lines was able to inhibit NK-mediated cytotoxicity. NK-TILs were also able to lyse autologous or allogeneic tumor cell lines in vitro. This activity correlated with low HLA class I surface expression since lysis could be inhibited by interferon (IFN)-gamma-expressing RCC transductants that displayed a higher surface density of HLA class I molecules. Therefore, NK cells infiltrating tumor tissues have an inherent ability to recognize transformed cells, but they require cytokine activation and are sensitive to inhibition by IR ligands.     

Retaliation against tumor cells showing aberrant HLA expression using lymphokine activated killer-derived T cells.

Lymphokine activated killer (LAK) cells represent mixtures of natural killer (NK) and non-MHC-restricted CTLs that have the capacity to lyse a variety of tumor cells and MHC class I-negative target cells. Although it is clear that NK cells are negatively regulated by interactions with MHC class Ia or class Ib molecules, the regulation of LAK-derived T cells has not been clarified to date. In the studies presented here, we demonstrate that IFN-gamma treatment of tumor cells can induce their resistance to LAK-derived T cells in a manner similar to that seen for NK cells. The IFN-gamma-mediated suppression of LAK activity correlates with increased MHC class I expression by the tumor cells, and the inhibition of LAK-mediated cytotoxicity can be reversed in the presence of class I-specific antibody. Furthermore, the expression of MHC class Ia or class Ib molecules in class I-negative cell lines can reduce their susceptibility to LAK-mediated cytotoxicity. This principle of negative regulation by MHC class I molecules applies to LAK-derived T cells generated from peripheral blood lymphocytes of renal cell carcinoma patients and healthy, control donors. Although LAK-derived T cells can be inhibited in their lytic activity through interactions with MHC class Ia and class Ib molecules, they do not express the known inhibitory receptors specific for these ligands that are found on NK cells. Apparently, LAK-derived T cells are negatively regulated by as yet undefined inhibitory receptors.     

Frequent lack of translation of antigen presentation-associated molecules MHC class I, CD1a and Beta(2)-microglobulin in Reed-Sternberg cells

Epstein-Barr virus (EBV) is present in Reed-Sternberg (RS) cells of a substantial proportion of Hodgkin's lymphoma cases. Most EBV-positive cases are also MHC class I-positive, whereas the majority of EBV-negative cases lack detectable levels of MHC class I expression. Application of the SAGE technique has led to the identification of tags corresponding to MHC class I and beta(2)-microglobulin genes in the EBV- and MHC class I-negative L428 Hodgkin's cell line. Further expression studies indicated that single RS cells that do not express HLA class I also lack beta(2)-microglobulins but frequently contain mRNA coding for these proteins. Another tag was identified corresponding to CD1a, a thymocyte and Langerhans cell antigen structurally related to the MHC class I genes. CD1a expression studies revealed mRNA in all cell lines and in several of the single cells, whereas immunostaining showed a cytoplasmic signal in only 2 of the 4 cell lines and in none of the Hodgkin's lymphoma tissue samples. In conclusion, RS cells frequently lack MHC class I, beta(2)-microglobulin and CD1a protein expression but contain mRNA coding for these proteins in some of the RS cells, suggesting a common mechanism affecting the translation of these antigen presentation-associated molecules.     

Role of exosomes in the immune microenvironment of ovarian cancer

Exosomes are excretory vesicles that can deliver a variety of bioactive cargo molecules to the extracellular environment. Accumulating evidence demonstrates exosome participation in intercellular communication, immune response, inflammatory response and they even play an essential role in affecting the tumor immune microenvironment. The role of exosomes in the immune microenvironment of ovarian cancer is mainly divided into suppression and stimulation. On one hand exosomes can stimulate the innate and adaptive immune systems by activating dendritic cells (DCs), natural killer cells and T cells, allowing these immune cells exert an antitumorigenic effect. On the other hand, ovarian cancer-derived exosomes initiate cross-talk with immunosuppressive effector cells, which subsequently cause immune evasion; one of the hallmarks of cancer. Exosomes induce the polarization of macrophages in M2 phenotype and induce apoptosis of lymphocytes and DCs. Exosomes further activate additional immunosuppressive effector cells (myeloid-derived suppressor cells and regulatory T cells) that induce fibroblasts to differentiate into cancer-associated fibroblasts. Exosomes also induce the tumorigenicity of mesenchymal stem cells to exert additional immune suppression. Furthermore, besides mediating the intercellular communication, exosomes carry microRNAs (miRNAs), proteins and lipids to the tumor microenvironment, which collectively promotes ovarian cancer cells to proliferate, invade and tumors to metastasize. Studying proteins, lipids and miRNAs carried by exosomes could potentially be used as an early diagnostic marker of ovarian cancer for designing treatment strategies.     

Recent advances on the role of tumor exosomes in immunosuppression and disease progression

Exosomes are endosomal-derived nanovesicles released by most cells types, including tumor cells, and principally involved in intercellular communication in physiology and disease. Tumor exosomes are gaining increasing interest in medicine and oncology as efficient tools for the delivery of defined signals. Representing the acellular replicas of tumor cells, they contain a great variety of bioactive molecules, such as proteins, RNA, miRNA and DNA. Their great ability to recirculate in body fluids and their structure allow them to transport their cargo to distant targets. Major studies have shown that tumor exosomes convey information not only between tumor cells but also to other cell types, including different immune cell components. There is increasing evidence that these nanovesicles may contribute to cancer progression by influencing different immune cell types, likely blunting specific T cell immunity and skewing innate immune cells toward a pro-tumorigenic phenotype. Because of this function and the additional property to deliver molecular signals modulating neoangiogenesis and stroma remodeling, tumor exosomes are believed to play a role in tumor progression by favoring metastatic niche onset. This review outlines the recent knowledge on immune suppressive mechanisms mediated by tumor exosomes. We will discuss our view on the role of these nanovesicular structures in cancer progression and how their presence could interfere with cancer therapy.     

exosomes在胃癌中作用机制的研究进展

exosomes是一种大小为30～100 nm的囊泡状小体，能被多种细胞分泌，存在于各种体液中。exosomes作为一种全新的细胞间信息传递系统，广泛参与肿瘤的发生、发展。胃癌来源的exosomes通过运输miRNA、细胞因子等关键物质，影响靶细胞内PI3K／Akt、MAPK／ERK等信号转导通路，参与胃癌细胞增殖、侵袭、转移、免疫逃逸等过程。现就近年来exosomes在胃癌中作用机制的研究进行综述。     

Favorable outcome in clinically stage II melanoma patients is associated with the presence of activated tumor infiltrating T-lymphocytes and preserved MHC class I antigen expression

In this study we investigated whether the presence of specific populations of tumor infiltrating lymphocytes (TILs) in diagnostic primary melanoma biopsies are related to outcome in clinically stage II melanoma patients. Moreover, we investigated whether the presence of TILs correlates with expression of MHC class I antigen and MHC class II antigen on tumor cells and/or tumor infiltrating antigen presenting cells. Diagnostic primary melanoma samples of 15 patients with an unfavorable outcome were compared with 20 patients with favorable outcome. Patients were matched for age, gender and Breslow thickness. Biopsies were examined for the presence of granzyme B+, CD8+, CD4+ and CD56+ TILs and for expression of MHC class I antigen and MHC class II antigen on tumor and/or tumor infiltrating cells. A favorable clinical outcome was strongly associated with the presence of GrB+ and CD4+ TILs, with expression of MHC class I antigen on tumor cells and with expression of MHC class II antigen on intratumoral antigen presenting cells. These data strongly support the notion that in melanoma patients the cellular immune response is a major factor in preventing melanoma cell dissemination.     

Immunization with MHC class I-negative but not -positive HPV16-associated tumour cells inhibits growth of MHC class I-negative tumours

Loss or downregulation of MHC class I molecules on tumour cells is a common mechanism by which tumours can escape from T-cell mediated immune responses. In this study we have investigated the immunologic crossreactivity between murine tumour cell lines expressing human papilloma virus (HPV) 16-derived E6/E7 oncoproteins with distinct surface expression of MHC class I molecules. The aims of this study were to demonstrate whether immune responses capable of coping with MHC class I-positive tumours can also be effective against their MHC class I-deficient derivatives and whether it is possible to induce immunity against MHC class I-deficient tumours by cellular vaccines based on MHC class I-deficient tumour cell lines. Our data showed that immunization with MHC class I-deficient but not with MHC class I positive tumour cells inhibited the growth of MHC class I-deficient tumours. In vivo depletion studies revealed that the mechanisms underlying effective immune responses against MHC class I-negative tumours in animals immunized with MHC class I-deficient tumour cells involved natural killer cells. The presented findings are of particular clinical relevance in the sense of construction of vaccines directed against a broad spectrum of HPV-associated tumours.     

外泌体在结肠癌中的研究进展

外泌体是由细胞内多囊泡体与胞膜融合后向胞外分泌的直径为20～100 nm的囊泡样小体,其内包含的蛋白质、核酸、脂质等多种生物活性分子可以传递至靶细胞,介导细胞间沟通并改变靶细胞的功能状态。研究发现外泌体在结肠癌的发生、发展、诊疗以及耐药中发挥重要作用。本文就外泌体在结肠癌中的最新研究进展进行综述。     

Fusion of B lymphoblastoid and tumor cells expressing different antibiotic resistance genes facilitates selection of stable hybridomas

Abstract The development of tumor vaccines or generation of tumor-specific cytotoxic T lymphocytes (CTL) is limited by the fact that many tumor cells downregulate the expression of major histocompatibility complex (MHC) Class I and II molecules, as well as key co-stimulatory molecules such as CD80 and CD86. An immune response to a vaccine or in vitro stimulation of tumor-specific CTL requires antigen-presenting cells conveying tumor antigens in the context of a host's MHC antigens. We have used a retroviral vector (murine stem cell virus) encoding neomycin resistance to transduce three pediatric tumor cell lines (two neuroblastoma, one neuroepithelial tumor). An EBV transformed B lymphoblastoid cell line (BLCL) was transduced with a separate vector encoding puromycin resistance and green fluorescent protein, individual tumor lines were fused with the BLCL, and the resulting hybridomas were selected using both antibiotics. The resulting hybridoma cells expressed the neural antigen GD2 as well as MHC Class I, Class II, CD 80, and CD86. A similar strategy could be used to produce stable hybridomas for either vaccination or for CTL expansion.     

肿瘤外泌体对CD8+T细胞功能的影响及机制研究进展

外泌体（exosomes）是介导细胞间通讯的细胞外囊泡。它携带来源细胞的多种生物活性分子,并可将其输送给受体细胞,进而影响细胞功能。肿瘤来源外泌体可通过多种机制介导肿瘤的免疫逃逸。本文就肿瘤外泌体对肿瘤杀伤主力军CD8+T细胞的调控作用进行总结,分析其相关作用机制,以期为肿瘤免疫治疗的研发提供新的思路。     

Anti-tumor effect of the anti-KL-6/MUC1 monoclonal antibody through exposure of surface molecules by MUC1 capping

Human polymorphic epithelial mucin (MUC1) is a heavily glycosylated large protein that is frequently overexpressed on the surface of many human adenocarcinomas. Studies using monoclonal antibodies (mAb) identified MUC1 as a tumor-associated antigen that has been intensely studied as a target for cancer immunotherapy. We previously identified a mouse IgG(1) mAb that recognizes a sialylated sugar chain, designated as KL-6, classified in 'Cluster 9 (MUC1)'. Using the anti-KL-6 mAb, we investigated antitumor effects of anti-MUC1 mAb on breast cancer cell lines expressing MUC1 abundantly. We showed that anti-KL-6 mAb induced capping of MUC1 and facilitated E-cadherin-mediated cell-cell interaction in the breast cancer cell lines YMB-S and ZR-75-1S, which proliferate in suspension culture without aggregation. Moreover, anti-KL-6 mAb enhanced the cytotoxic activity of lymphokine-activated killer cells. These results indicate that the capping of MUC1 restores cell surface proteins, such as adhesion molecules and tumor antigens, to work in cell-cell interactions, leading to inhibition of tumor proliferation due to cell-cell adhesion and increased accessibility to effector cells that are needed to kill tumor cells.