Exosomes in the ascites of ovarian cancer patients: origin and effects on anti-tumor immunity

This study was performed to identify the origin of the ascites-derived exosomes from patients with ovarian cancer and to observe the effect of exosomes on anti-tumor immunity. Exosomes were isolated from the ascites of patients with ovarian epithelial cancer by ultracentrifugation plus density gradient centrifugation. The origin of exosomes was identified by immunoelectronmicroscopy (IEM). The growth curve of the tumor cell line SKOV3 cultured with or without exosomes was analyzed. The apoptosis of autogeneic tumor cells (ATCs) and SKOV3 cells affected by exosomes was measured by flow cytometry (FCM) and light phase contrast microscopy. The cytotoxic effect of the peripheral blood mononuclear cells (PBMCs) stimulated by exosomes and/or dendritic cells (DCs) on ovarian cancer cells was measured using a CCK-8 assay. The levels of IFN-γ released by PBMCs stimulated by exosomes and/or DCs were measured by ELISA. The apoptosis of PBMCs and DCs affected by exosomes was measured by FCM and light microscopy. Whether the mature process of DCs was affected by exosomes was studied by FCM. The ratio of CD4+ T cell and CD8+ T cell were measured by FCM. FasL and TRAIL molecules on exosomes were detected by western blot analysis. The human FasL antagonistic antibody was used to block the apoptosis of DCs and PBMCs induced by exosomes. The receptors of TRAIL DR4 and DR5 on PBMCs and DCs were detected by FCM. In 41 patients examined, we isolated exosomes from the ascites of 35 patients. We detected TCR, CD20, HLA-DR, B7-2, HER2/neu, CA125 and Histone H2A on exosomes. We found that exosomes might impair the cytotoxic activity of PBMCs when DCs are present. We found that exosomes had no effect on the growth and apoptosis of SKOV3 cells. However, exosomes may induce apoptosis of precursors, mature DCs and PBMCs. We found that FasL and TRAIL were present in the exosome suspension and addition of an anti-FasL antibody may decrease the percentage of apoptosis of DCs and PBMCs. We conclude that exosomes exist in ascites of 85.4% of patients with ovarian cancer. Moreover, these exosomes may be of multi-origin. Exosomes had no effect on the growth and apoptosis of tumor cells but impaired the cytotoxic activity of PBMCs in the presence of DCs. Exosomes also may induce apoptosis of the precursors of DCs, DCs and PBMCs. FasL and TRAIL on exosomes may partly account for the apoptosis of cells of the immune system.     

外泌体在癌症诊治中的作用与机制

外泌体是一种新型的癌症生物标志物，它由所有体液中各种活细胞分泌的双层纳米囊泡构成，含有丰富的蛋白质、DNA、mRNA和非编码RNA。目前外泌体被认为是细胞间通讯的另一种机制，参与细胞间交换蛋白质、脂质和遗传物质。越来越多的研究表明，外泌体在肿瘤的发生、生长、进展、转移、耐药性和免疫逃逸中发挥重要作用。在本文中，我们根据外泌体生物学的最新进展，详细阐述了外泌体影响肿瘤之间通信的具体机制，并报道了外泌体可能成为癌症诊断中有前途的生物标志物，并代表癌症治疗的新靶点。     

Role of exosomes in treatment of hepatocellular carcinoma

Exosomes are nanovesicles that may play a role in intercellular communication by acting as carriers of functional contents such as proteins, lipids, RNA molecules and circulating DNA from donor to recipient cells. In addition, exosomes may play a potential role in immunosurveillance and tumor pathogenesis and progression. Recently, research has increasingly focused on the role of exosomes in hepatocellular carcinoma (HCC), the most common primary liver malignancy. We herein review data on emerging experimental and clinical studies focused on the role of exosomes in the pathogenesis, diagnosis, progression and chemotherapy response of patients with HCC. Beyond their diagnostic value in HCC, exosomes are involved in different mechanisms of HCC tumor pathogenesis and progression including angiogenesis and immune escape. Moreover, exosomes have been demonstrated to change the tumor microenvironment to a less tolerogenic state, favoring immune response and tumor suppression. These results underline a practical and potentially feasible role of exosomes in the treatment of patients with HCC, both as a target and a vehicle for drug design. Future studies will need to further elucidate the exact role and reliability of exosomes as screening, diagnostic and treatment targets in patients with HCC.     

间充质干细胞来源外泌体在恶性肿瘤中的研究进展

间充质干细胞（mesenchymal stem cells,MSCs）是存在于各种组织中的多能基质细胞。外泌体为细胞间的通讯载体,能在细胞间传递脂质、核酸以及蛋白质等生物活性分子。MSCs分泌的外泌体（mesenchymal stem cell-derived exosomes,MSC-EXO）为肿瘤微环境（tumor microenvironment,TME）的主要组成部分,并且在肿瘤的发生发展、血管生成及转移过程中发挥重要作用。本文旨在对MSCs来源的外泌体在癌症研究及其对肿瘤的作用机制予以综述,为适当利用修饰的MSC-EXO作为肿瘤治疗的策略提供新思路。      

外泌体在急性髓系白血病中的作用及临床意义

外泌体是活细胞分泌的一种纳米级的脂质双层囊泡,内含核酸(DNA、mRNA、microRNA、lncRNA 等)、蛋白质、脂质等生物大分子。外泌体作为细胞间通信的载体,参与正常细胞的调控,同时参与疾病的发生发展。急性髓系白血病（acute myeloid leukemia,AML）是骨髓造血细胞克隆性增殖的一种恶性血液病,其特征为骨髓造血干细胞恶性增殖、分化受阻和凋亡抑制。有研究表明,外泌体通过构建肿瘤微环境、抑制骨髓造血、促进血管形成、促进AML细胞增殖、抑制AML细胞凋亡,干扰细胞免疫反应等途径参与急性髓系白血病的发生发展。通过监测外周血或者其他体液中的外泌体,可以提高AML的诊断效率、耐药监测及预后判断等。本文就外泌体在AML中的作用及临床意义的最新研究进行综述,为外泌体在AML中研究提供理论基础及新思路。     

外泌体miRNAs在乳腺癌肿瘤微环境中的研究进展

乳腺癌是全球女性最常发生的恶性肿瘤,患者死亡的主要原因是复发、转移和耐药性的出现。研究已经证明,外泌体介导癌细胞与肿瘤微环境之间的信息交流,外泌体携带的miRNAs通过差异表达于乳腺癌细胞,在微环境中影响癌基因表达的调控,介导乳腺癌细胞的信号通路,调节癌细胞周期进程以及重塑肿瘤相关成纤维细胞等,从而促进乳腺癌的发生、发展和转移;另外外泌体介导中和、药物外排和免疫系统抑制三种主要机制导致耐药性。未来,各种类型乳腺癌中差异表达的miRNAs有望成为临床诊断和预后的相关生物标志物,及抗肿瘤治疗的新靶点。     

Exosomal microRNAs derived from colon cancer cells promote tumor progression by suppressing fibroblast TP53 expression

The tumor microenvironment offers favorable conditions for tumor progression, and activated fibroblasts, known as cancer‐associated fibroblasts, play a pivotal role. TP53‐deficient cancer cells are known to induce strong fibroblast activation. We aimed to elucidate the oncogenic role of exosomes derived from TP53‐deficient colon cancer cells in fibroblast proliferation and tumor growth. Cancer cell‐derived exosomes (CDEs) were isolated from the conditioned media of cancer cells using a sequential ultracentrifugation method. The effects of exosomes on tumor growth were evaluated using human cell lines (TP53‐WT colon cancer, HCT116; TP53‐mutant colon cancer, HT29; and fibroblasts, CCD‐18Co and WI‐38) and an immune‐deficient nude mouse xenograft model. HCT116 (HCT116^{sh p53}) cells deficient in TP53 accelerated cocultured fibroblast proliferation compared to TP53‐WT HCT116 (HCT116^{sh control}) cells in vitro. Exosomes from HCT116^{sh p53} cells suppressed TP53 expression of fibroblasts and promoted their proliferation. Xenografts of HCT116^{sh p53} cells grew significantly faster than those of HCT116^{sh control} cells in the presence of co‐injected fibroblasts, but this difference was diminished by CDE inhibition. Microarray analysis identified upregulation of several microRNAs (miR‐1249‐5p, miR‐6737‐5p, and miR‐6819‐5p) in TP53‐deficient CDEs, which were functionally proven to suppress TP53 expression in fibroblasts. Exosomes derived from TP53‐mutant HT29 cells also suppressed TP53 expression in fibroblasts and accelerated their growth. The proliferative effect of HT29 on cocultured fibroblasts was diminished by inhibition of these miRNAs in fibroblasts. Our results suggest that CDEs play a pivotal role in tumor progression by fibroblast modification. Cancer cell‐derived exosomes might, therefore, represent a novel therapeutic target in colon cancer.     

调节性T细胞参与胰腺癌免疫微环境重塑的基础与转化研究进展

调节性T细胞（regulatory T cells/Treg cells）是发挥负性免疫调节功能的一类CD4+T细胞,主要通过抑制多种效应性T细胞的活性和功能,维持机体获得性免疫系统的平衡,防止自身免疫性疾病的发生。Treg细胞也是免疫抑制性肿瘤微环境的主要组成成分,其在肿瘤局部微环境中,对抗肿瘤免疫应答过程,发挥免疫抑制作用,并协助肿瘤细胞参与免疫逃逸,进而影响肿瘤的恶性演进过程。本文对Treg细胞在胰腺癌免疫微环境重塑过程中发挥功能的机制及其临床转化研究进展进行综述,旨在加深对胰腺癌免疫抑制微环境的认识,为胰腺癌的免疫调节治疗提供新思路。      

Pattern response of dendritic cells in the tumor microenvironment and breast cancer

Breast cancer (BC) is the most common malignant neoplasm and the cause of death by cancer among women worldwide. Its development, including malignancy grade and patient prognosis, is influenced by various mutations that occur in the tumor cell and by the immune system’s status, which has a direct influence on the tumor microenvironment and, consequently, on interactions with non-tumor cells involved in the immunological response. Among the immune response cells, dendritic cells (DCs) play a key role in the induction and maintenance of anti-tumor responses owing to their unique abilities for antigen cross-presentation and promotion of the activation of specific lymphocytes that target neoplasic cells. However, the tumor microenvironment can polarize DCs, transforming them into immunosuppressive regulatory DCs, a tolerogenic phenotype which limits the activity of effector T cells and supports tumor growth and progression. Various factors and signaling pathways have been implicated in the immunosuppressive functioning of DCs in cancer, and researchers are working on resolving processes that can circumvent tumor escape and developing viable therapeutic interventions to prevent or reverse the expression of immunosuppressive DCs in the tumor microenvironment. A better understanding of the pattern of DC response in patients with BC is fundamental to the development of specific therapeutic approaches to enable DCs to function properly. Various studies examining DCs immunotherapy have demonstrated its great potential for inducing immune responses to specific antigens and thereby reversing immunosuppression and related to clinical response in patients with BC. DC-based immunotherapy research has led to immense scientific advances, both in our understanding of the anti-tumor immune response and for the treatment of these patients.     

Exosome‐mediated regulation of tumor immunology

Exosomes are representative extracellular vesicles (EV) derived from multivesicular endosomes (MVE) and have been described as new particles in the communication of neighborhood and/or distant cells by serving as vehicles for transfer between cells of membrane and cytosolic proteins, lipids, and nucleotides including micro (mi) RNAs. Exosomes from immune cells and tumor cells act in part as a regulator in tumor immunology. CD8⁺ T cells that show potent cytotoxic activity against tumor cells reside as an inactive naïve form in the T‐cell zone of secondary lymphoid organs. Once receiving tumor‐specific antigenic stimulation by dendritic cells (DC), CD8⁺ T cells are activated and differentiated into effector CTL. Subsequently, CTL circulate systemically, infiltrate into tumor lesions through the stromal neovasculature where mesenchymal stromal cells, for example, mesenchymal stem cells (MSC) and cancer‐associated fibroblasts (CAF), abundantly exist, destroy mesenchymal tumor stroma in an exosome‐mediated way, go into tumor parenchyma, and attack tumor cells by specific interaction. DC‐derived and regulatory T (Treg) cell‐derived exosomes, respectively, promote and inhibit CTL generation in this setting. In this review, we describe the roles of exosomes from immune cells and tumor cells on the regulation of tumor progression.     

外来体源性miRNA在恶性肿瘤中的研究进展

外来体(exosomes)是由细胞内多泡体与细胞膜融合后,释放到细胞外的一种直径约30-100nm的膜性囊泡。外来体广泛存在于血清、尿液、乳液、唾液等细胞外液体中。而miRNA则是一类内源性非编码小分子RNA,通过与靶mRNA特异的碱基配对,引起靶mRNA翻译抑制,从而在转录后水平调节基因的表达。外来体中含有各种蛋白质、miRNA以及mRNA,并能将其携带的miRNA递送至受体细胞而发挥生物学作用。近年来,关于外来体源性miRNA可作为新的肿瘤标志物、参与细胞间信息传递及作为肿瘤靶向治疗等方面的研究成为热点,本文主要对外周血中的外来体源性miRNA在恶性肿瘤中的研究进展进行综述。     

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.     

Recent advances in extracellular vesicles in gastrointestinal cancer and lymphoma

Extracellular vesicles (EVs) are intercellular communication agents that transfer microRNAs (miRNAs), other non-coding RNAs (ncRNAs), messenger RNAs (mRNAs), proteins, lipids, metabolites, and other molecules from donor cells (e.g., cancer cells) to recipient cells (e.g., stromal cells). In 2007, miRNAs were reported to be abundant among the ncRNAs present in EVs. Since then, many studies have investigated the functions of miRNAs and have attempted to apply these molecules to aid in the diagnosis and treatment of cancer. Research on EVs has expanded, particularly in the field of cancer, because cancer cells heavily secrete EVs. The cargo of these EVs, especially those in small EVs, such as exosomes, is assumed to work cooperatively and significantly in the tumor microenvironment and to promote metastasis. In this review, we first summarize recent studies on EVs in gastrointestinal cancer and highlight studies on human satellite II RNAs, which are a type of ncRNA found in EVs that possess repetitive sequences. Second, since several recent studies have revealed that phospholipids, which are components of EV membranes, play important roles in intercellular communication and the generation of lipid mediators in the tumor microenvironment, we review the reported roles of these molecules and discuss their potential use in the design of new cancer treatments.     

The role of exosomes and miRNAs in drug‐resistance of cancer cells

Chemotherapy, one of the principal approaches for cancer patients, plays a crucial role in controlling tumor progression. Clinically, tumors reveal a satisfactory response following the first exposure to the chemotherapeutic drugs in treatment. However, most tumors sooner or later become resistant to even chemically unrelated anticancer agents after repeated treatment. The reduced drug accumulation in tumor cells is considered one of the significant mechanisms by decreasing drug permeability and/or increasing active efflux (pumping out) of the drugs across the cell membrane. The mechanisms of treatment failure of chemotherapeutic drugs have been investigated, including drug efflux, which is mediated by extracellular vesicles (EVs). Exosomes, a subset of EVs with a size range of 40–150 nm and a lipid bilayer membrane, can be released by all cell types. They mediate specific cell‐to‐cell interactions and activate signaling pathways in cells they either fuse with or interact with, including cancer cells. Exosomal RNAs are heterogeneous in size but enriched in small RNAs, such as miRNAs. In the primary tumor microenvironment, cancer‐secreted exosomes and miRNAs can be internalized by other cell types. MiRNAs loaded in these exosomes might be transferred to recipient niche cells to exert genome‐wide regulation of gene expression. How exosomal miRNAs contribute to the development of drug resistance in the context of the tumor microenvironment has not been fully described. In this review, we will highlight recent studies regarding EV‐mediated microRNA delivery in formatting drug resistance. We also suggest the use of EVs as an advancing method in antiresistance treatment.     

外泌体传输microRNA在肿瘤研究中的进展*

外泌体是一类可以在细胞间传递直径为30～100 nm的内吞衍生囊泡,可包含与其来源和功能相关的蛋白质和RNA 等物质。外泌体作为一种天然的载体,已被视为一种新型的药物传输系统用于肿瘤的治疗。microRNA（miRNA ）是一类新型的RNA调控基因,不仅仅存在于细胞内,亦存在于细胞外,这些细胞外miRNA 可作为分泌型信号分子影响受体细胞表型,并一定程度上反映出供体细胞内的分子改变,具有一定的诊断及潜在的治疗用途。肿瘤患者血液中外泌体高于正常人,并能够包裹肿瘤相关miRNA 行使生物学功能,因此机体通过外泌体传输特异性miRNA 可能在肿瘤的发病过程中扮演着重要的角色。本文将对外泌体作为miRNA 的传输载体在肿瘤发生发展以及肿瘤治疗等方面的研究进行综述。      

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。     

The cancer exosomes: Clinical implications,applications and challenges

The exosome is a small functional vesicle enriched in selected proteins, lipids and nucleic acids, displaying distinct molecular heterogeneity. Exosomes released can transform the extracellular matrix microenvironments, transmit signals and molecules to recipient cells and trigger changes in their pathophysiological functions. Tumor-derived exosomes mediate the interactions of tumor cells and microenvironment significantly, and they stimulate tumor growth and development through specific signaling pathways related to metastasis, therapeutic resistance and immunosuppression. Exosome biogenesis from tumors often represents abundant biological information, and novel and efficient isolation and detection methods of exosomes provide a promising approach for tumor diagnosis and prognosis estimation. Moreover, exosome can even be developed as therapeutic agents for multiple disease models based on effective material transport characteristics and biofilm specificity. This review reports the clinical implications and challenges of exosomes in cancer progression, therapy resistance, metastasis and immune escape, and underlying cancerogenic pathological phenotypes including fibrosis and viral infection.     

Immune evasion mechanisms and therapeutic strategies in gastric cancer

Gastric cancer (GC) is a malignancy with a high incidence and mortality. The tumor immune microenvironment plays an important role in promoting cancer development and supports GC progression. Accumulating evidence shows that GC cells can exert versatile mechanisms to remodel the tumor immune microenvironment and induce immune evasion. In this review, we systematically summarize the intricate crosstalk between GC cells and immune cells, including tumor-associated macrophages, neutrophils, myeloid-derived suppressor cells, natural killer cells, effector T cells, regulatory T cells, and B cells. We focus on how GC cells alter these immune cells to create an immunosuppressive microenvironment that protects GC cells from immune attack. We conclude by compiling the latest progression of immune checkpoint inhibitor-based immunotherapies, both alone and in combination with conventional therapies. Anti-cytotoxic T-lymphocyte-associated protein 4 and anti-programmed cell death protein 1/programmed death-ligand 1 therapy alone does not provide substantial clinical benefit for GC treatment. However, the combination of immune checkpoint inhibitors with chemotherapy or targeted therapy has promising survival advantages in refractory and advanced GC patients. This review provides a comprehensive understanding of the immune evasion mechanisms of GC, and highlights promising immunotherapeutic strategies.     

HPV蛋白、miRNAs和外泌体在宫颈癌炎症作用中的研究进展

人乳头瘤状病毒(Human papilloma virus,HPV)感染是导致宫颈癌发生发展最重要的危险因素。目前有研究表明,HPV蛋白直接或间接参与慢性炎症发生,进而促进肿瘤发展。非编码RNA(miRNAs)是基因表达的调控因子,也是炎症通路的关键参与者,miRNAs的异常表达可能与宫颈癌的炎性反应有关。外泌体是细胞分泌到细胞外的囊泡,这些囊泡可以作为运载体与受体结合。最近研究发现外泌体参与了炎症过程,影响免疫反应。在这篇综述中,我们讨论了HPV蛋白、miRNAs和外泌体在宫颈癌炎症作用中的研究进展。     

外泌体在胰腺癌中的研究现状与进展

外泌体是一种双层脂质膜连接囊泡样小体，存在于各种体液中，参与细胞及肿瘤微环境之间的物质运输和信号传递。外泌体含有多种生物活性分子，包括脂质、蛋白质、DNA、mRNA以及非编码RNA，可以通过这些活性分子影响肿瘤的发生和发展，甚至可以影响肿瘤的治疗。胰腺癌是一种常见的恶性肿瘤，侵袭性强，预后较差，死亡率高。胰腺癌来源的外泌体是胰腺肿瘤微环境中的重要组成部分，促使胰腺癌细胞成功逃避细胞凋亡的重要因素，并且可以促进肝脏转移微环境的形成。近年来，与胰腺癌相关的外泌体逐渐成为新的研究热点，研究发现外泌体有望成为早期胰腺癌筛查的新型生物学标志，并将为胰腺癌靶向治疗提供可行的技术基础。