Pancreatic cancer (PC) has a poor prognosis and displays resistance to immunotherapy. A better understanding of tumor-derived extracellular vesicle (EV) effects on immune responses might contribute to improved immunotherapy. EVs derived from Capan-2 and BxPC-3 PC cells isolated by ultracentrifugation were characterized by atomic force microscopy, Western blot (WB), nanoparticle tracking analysis, and label-free proteomics. Fresh PBMCs from healthy donors were treated with PC- or control-derived heterologous EVs, followed by flow cytometry analysis of CD8+ and CD4+ lymphocytes. The proteomics of lymphocytes sorted from EV-treated or untreated PBMCs was performed, and the IFN-γ concentration was measured by ELISA. Notably, most of the proteins identified in Capan-2 and BxPC-3 EVs by the proteomic analysis were connected in a single functional network (p= 1 × 10−16) and were involved in the “Immune System” (FDR: 1.10 × 10−24and 3.69 × 10−19, respectively). Interestingly, the treatment of healthy donor-derived PBMCs with Capan-2 EVs but not with BxPC-3 EVs or heterologous control EVs induced early activation of CD8+ and CD4+ lymphocytes. The proteomics of lymphocytes sorted from EV-treated PBMCs was consistent with their activation by Capan-2 EVs, indicating IFN-γ among the major upstream regulators, as confirmed by ELISA. The proteomic and functional analyses indicate that PC-EVs have pleiotropic effects, and some may activate early immune responses, which might be relevant for the development of highly needed immunotherapeutic strategies in this immune-cold tumor.
胰腺癌预后不良且对免疫治疗存在抵抗。深入理解肿瘤源性细胞外囊泡对免疫应答的影响，可能有助于改善免疫治疗效果。本研究通过超速离心法分离Capan-2和BxPC-3胰腺癌细胞来源的EVs，并采用原子力显微镜、蛋白质印迹、纳米颗粒追踪分析及无标记蛋白质组学技术进行表征。将健康供体外周血单个核细胞分别与胰腺癌来源或对照异源EVs共培养后，通过流式细胞术分析CD8+和CD4+淋巴细胞。对EV处理组与未处理组PBMCs分选的淋巴细胞进行蛋白质组学分析，并采用ELISA检测IFN-γ浓度。值得注意的是，蛋白质组学分析显示Capan-2和BxPC-3 EVs中鉴定到的大多数蛋白质构成单一功能网络（p=1×10−16），并参与"免疫系统"相关功能（错误发现率分别为1.10×10−24和3.69×10−19）。有趣的是，Capan-2 EVs（而非BxPC-3 EVs或异源对照EVs）处理健康供体PBMCs可诱导CD8+和CD4+淋巴细胞早期活化。EV处理组淋巴细胞蛋白质组学特征与其被Capan-2 EVs激活的状态一致，显示IFN-γ是主要上游调控因子之一，该结果经ELISA验证。蛋白质组学与功能分析表明，胰腺癌EVs具有多效性作用，其中部分可能激活早期免疫应答，这对开发这种免疫冷肿瘤亟需的免疫治疗策略具有重要意义。
肿瘤(癌症)患者之家