Exosomal microRNA in serum is a novel biomarker of recurrence in human colorectal cancer

Background:

Functional microRNAs (miRNAs) in exosomes have been recognised as potential stable biomarkers in cancers. The aim of this study is to identify specific miRNAs in exosome as serum biomarkers for the early detection of recurrence in human colorectal cancer (CRC).

Methods:

Serum samples were sequentially obtained from six patients with and without recurrent CRC. The miRNAs were purified from exosomes, and miRNA microarray analysis was performed. The miRNA expression profiles and copy number aberrations were explored using microarray and array CGH analyses in 124 CRC tissues. Then, we validated exosomal miRNAs in 2 serum sample sets (90 and 209 CRC patients) by quantitative real-time RT–PCR.

Results:

Exosomal miR-17-92a cluster expression level in serum was correlated with the recurrence of CRC. Exosomal miR-19a expression levels in serum were significantly increased in patients with CRC as compared with healthy individuals with gene amplification. The CRC patients with high exosomal miR-19a expression showed poorer prognoses than the low expression group (P<0.001).

Conclusions:

Abundant expression of exosomal miR-19a in serum was identified as a prognostic biomarker for recurrence in CRC patients.     

Trop-2 is up-regulated in invasive prostate cancer and displaces FAK from focal contacts

In this study, we show that the transmembrane glycoprotein Trop-2 is up-regulated in human prostate cancer (PCa) with extracapsular extension (stages pT3/pT4) as compared to organ-confined (stage pT2) PCa. Consistent with this evidence, Trop-2 expression is found to be increased in metastatic prostate tumors of Transgenic Adenocarcinoma of Mouse Prostate mice and to strongly correlate with α5β1 integrin levels. Using PCa cells, we show that Trop-2 specifically associates with the α5 integrin subunit, as binding to α3 is not observed, and that Trop-2 displaces focal adhesion kinase from focal contacts. In support of the role of Trop-2 as a promoter of PCa metastatic phenotype, we observe high expression of this molecule in exosomes purified from Trop-2-positive PCa cells. These vesicles are then found to promote migration of Trop-2-negative PCa cells on fibronectin, an α5β1 integrin/focal adhesion kinase substrate, thus suggesting that the biological function of Trop-2 may be propagated to recipient cells. In summary, our findings show that Trop-2 promotes an α5β1 integrin-dependent pro-metastatic signaling pathway in PCa cells and that the altered expression of Trop-2 may be utilized for early identification of capsule-invading PCa.     

随机光学重构显微镜在外泌体观察中的应用

目的探讨随机光学重构显微镜（STORM）在外泌体观察中的应用价值,并介绍外泌体在随机光学重构显微镜下的成像原理及方法。方法实验中在肾性甲旁亢患者原代培养的甲状旁腺细胞的培养液上清中加入EXOQUICK-TC外泌体沉淀剂来获取细胞培养上清中的外泌体,将外泌体膜表面的特异性跨膜蛋白CD63作免疫荧光标记后利用随机光学重构显微镜对外泌体行超高分辨率成像并测量外泌体直径。结果实验中随机光学重构显微镜成功的对继发性甲旁亢甲状旁腺细胞原代培养液上清中的外泌体行单分子定位、直径测量及超高分辨率成像。结论因随机光学重构显微镜具有独特的光学特性并突破了光学衍射极限的限制,较传统光学显微镜,能获得20~50 nm的分辨率,可对外泌体行单分子精确定位、直径测量、超高分辨率成像。基于STORM的成像优势,相信STORM及其他超高分辨率成像技术将在外泌体及外泌体参与的生物学过程的研究中发挥重要作用。     

Exosomes play roles in sequential processes of tumor metastasis

Overwhelming evidence demonstrates that exosomes, a series of biologically functional small vesicles of endocytic origin carrying a variety of active constituents, especially tumor-derived exosomes, contribute to tumor progression and metastasis. This review focuses on the specific multifaceted roles of exosomes in affecting sequenced four crucial processes of metastasis, through which cancer cells spread from primary to secondary organs and finally form macroscopic metastatic lesions. First, exosomes modulate the primary tumor sites to assist cancer growth and dissemination. In this part, five main biological events are reviewed, including the transfer of oncogenic constituents, the recruitment and activation of fibroblasts, the induction of angiogenesis, immunosuppression and epithelial-mesenchymal transition (EMT) promotion. In Step 2, we list two recently disclosed mechanisms during the organ-specific homing process: the exosomal integrin model and exosomal epidermal growth factor receptor (EGFR)/miR-26/hepatocyte growth factor (HGF) model. Subsequently, Step 3 focuses on the interactions between exosomes and pre-metastatic niche, in which we highlight the specific functions of exosomes in angiogenesis, lymphangiogenesis, immune modulation and metabolic, epigenetic and stromal reprogramming of pre-metastatic niche. Finally, we summarize the mechanisms of exosomes in helping the metastatic circulating tumor cells escape from immunologic surveillance, survive in the blood circulation and proliferate in host organs.     

M1表型的小胶质细胞外泌体对血脑屏障功能的影响

目的 研究M1表型的小胶质细胞外泌体（M1 microglia-derived exosome,M1-exo）对体外血脑屏障（blood-brain barrier,BBB）功能及血管内皮细胞间紧密连接蛋白表达的影响。方法 用脂多糖（lipopolysaccharide,LPS）刺激小鼠小胶质细胞来源的细胞系BV2细胞,流式技术检测其向M1型极化情况,分离提取外泌体。用小鼠脑微血管内皮细胞系b.End3细胞与原代培养的小鼠星形胶质细胞构建体外BBB模型,随机分为3组：b.End3细胞正常培养组（b.End3组）、b.End3细胞+25 μg/mL正常BV2细胞来源外泌体（BV2-derived exosome,BV2-exo）组（b.End3+BV2-exo组）、b.End3细胞+25 μg/mL M1表型的小胶质细胞来源外泌体组（b.End3+M1-exo组）。按实验分组将不同来源外泌体与BBB模型共培养,检测各组的跨膜电阻（trans-endothelial electrical resistance,TEER）、荧光黄通过率,免疫印迹法（Western blot,WB）检测紧密连接复合物蛋白Claudin-1、Occludin、ZO-1及JAM蛋白的表达。结果 ①小胶质细胞向M1型极化成功,其细胞标志物CD16/32阳性率较对照组明显升高（P=0.023）;②与M1-exo共培养后,体外BBB模型的TEER明显下降（P=0.000）,对荧光黄的透过率明显增加（P=0.000）;③与b.End3组和b.End3 + BV2-exo组相比,b.End3+M1-exo组的Claudin-1、Occludin及ZO-1蛋白的表达水平明显下降。结论 M1-exo可以破坏BBB的完整性,影响其功能。     

外泌体在肿瘤体外诊断中的临床研究和应用前景

外泌体由于富含生物信息分子,提供了潜在的生物标志物,在肿瘤体外诊断中显示出良好的临床应用前景。阐述了外泌体的功能、在体外诊断中的优势及检测方法,并深入探讨了外泌体在肺癌、肠癌、卵巢癌、胃癌、胰腺癌、膀胱癌、骨与软组织肉瘤等肿瘤体外诊断中的价值和临床获批情况,展望了外泌体在体外诊断领域的发展趋势。     

Modeling putative therapeutic implications of exosome exchange between tumor and immune cells

Development of effective strategies to mobilize the immune system as a therapeutic modality in cancer necessitates a better understanding of the contribution of the tumor microenvironment to the complex interplay between cancer cells and the immune response. Recently, effort has been directed at unraveling the functional role of exosomes and their cargo of messengers in this interplay. Exosomes are small vesicles (30–200 nm) that mediate local and long-range communication through the horizontal transfer of information, such as combinations of proteins, mRNAs and microRNAs. Here, we develop a tractable theoretical framework to study the putative role of exosome-mediated cell–cell communication in the cancer–immunity interplay. We reduce the complex interplay into a generic model whose three components are cancer cells, dendritic cells (consisting of precursor, immature, and mature types), and killer cells (consisting of cytotoxic T cells, helper T cells, effector B cells, and natural killer cells). The framework also incorporates the effects of exosome exchange on enhancement/reduction of cell maturation, proliferation, apoptosis, immune recognition, and activation/inhibition. We reveal tristability—possible existence of three cancer states: a low cancer load with intermediate immune level state, an intermediate cancer load with high immune level state, and a high cancer load with low immune-level state, and establish the corresponding effective landscape for the cancer–immunity network. We illustrate how the framework can contribute to the design and assessments of combination therapies.Immunotherapeutic approaches have recently emerged as effective therapeutic modalities (1) exemplified by immune checkpoint blockade with anti–CTLA-4 to activate T-cells and induce tumor cell killing, which has been shown to be effective for some cancers but not others (2). A better understanding of the intricate interplay between cancer and the immune system, and of mechanisms of immune evasion and of hijacking of the host response by cancer cells, is relevant to the development of effective immunotherapeutic approaches (3–6).The immune-based suppression of tumor development and progression is mediated through nonspecific innate immunity and antigen-specific adaptive immunity (7). However, cancer cells can inhibit the immune response, thus evading suppression in multiple ways (8) (see below for details), and additionally hijack the immune system to their advantage (3, 4). The challenge to understand the tumor–immune interplay stems from the dynamic nature, and complexity and heterogeneity of both the cancer cells and the immune system and their interactions through the tumor microenvironment (9).Here we consider immune cells as consisting of macrophages (10), natural killer cells (11), cytotoxic T cells (12), helper T cells (13), and regulatory T cells (3). These various immune cells are produced, activated, and perform their functions separated by space and time, which contributes to additional complexity (14). Among the immune cells, dendritic cells (DCs) are the most efficient antigen-presenting cells to bridge innate immunity with adaptive immunity (15). DCs also secrete cytokines that promote the antitumor functions of both natural killer cells and macrophages (16, 17). We consider the tumor microenvironment as comprised of a heterogeneous population of cancer cells (18), stromal cells (19), and tumor-infiltrating immune cells (20). The interactions among these cell types contribute to tumor development and progression. Tumor-associated macrophages and cancer-associated fibroblasts regulate tumor metabolism and engender an immune-suppressive environment by secreting TGF-β and other cytokines (21). Fluctuations in energy sources and oxygen within a tumor contribute to malignant progression and cell phenotypic diversity (22, 23).Though secreted factors play critical roles in cell–cell communications, here we focus on the additional role of cell–cell communication mediated by the exchange of special extracellular lipid vesicles called exosomes (24). These nanovesicles of ∼30–200 nm are formed in the multivesicular bodies and then released from the cell into the extracellular space (25). The exosomes carry a broad range of cargo, including proteins, microRNAs, mRNAs, and DNA fragments, to specific target cells at a remote location (26). Membrane markers assign the exosomes to specific targeted cells. Notably, upon entering the target cell, the exosomes induce modulation of cell function and even identity switch (phenotypic, epigenetic, and even genetic) (27). Exosomes have recently emerged as playing an important role in the immune system interaction with tumors (28, 29). Tumor-derived exosomes can promote metastatic niche formation by influencing bone marrow-derived cells toward a prometastatic phenotype through upregulation of c-Met (29). DCs have been shown to induce tumor cell killing through release of exosomes that contain potent tumor-suppressive factors such as TNF and through activation of natural killer cells, cytotoxic T cells, and helper T cells (24, 30–32). However, tumor-derived exosomes (Tex) can directly inhibit the differentiation of DCs in bone marrow (33), which strongly inhibits the dendritic cell-mediated immune response to the tumor. In addition, Tex can also directly inhibit natural killer cells (34).Mathematical models have been devised to study the complex interactions of cancer and immune system, including those that consider spatial heterogeneity (as reviewed in ref. 35) and those that consider spatially homogeneous populations (as reviewed in ref. 36). Cancer–immunity models have been constructed to investigate the effects of therapy (37–39), cancer dormancy (40), and interactions with time delay (41). Other types of modeling methods have also been applied. For example, tumor growth has also been fitted to experimental data by artificial neural networks (42); a detailed network of cancer immune system has been modeled by multiple subset models (43).In this study, we have developed an exosome exchange-based cancer–immunity interplay (ECI) model, to incorporate the special role of DCs and exosome-mediated communications. Distinct from the previous approaches, our modeling strategy is adapted from methodology used in studies of gene regulatory circuits, allowing us to check the multistability features of the system (44). We find that, by including exosome exchange, the cancer–immunity interplay can give rise to three quasi-stable cancer states, which may be associated with the elimination/equilibrium/escape phases proposed in the immunoediting theory (45). The ECI model is also capable of explaining tumorigenesis by considering the time evolution of immune responses. Guided by the treatment simulations, we assess the effectiveness of various therapeutic protocols with and without time delay and noise.