Piro Anna, Cufaro Maria Concetta, Lanuti Paola, Brocco Davide, De Lellis Laura, Florio Rosalba, Pilato Serena, Pagotto Sara, De Fabritiis Simone, Vespa Simone, Catitti Giulia, Verginelli Fabio, Simeone Pasquale, Pieragostino Damiana, Del Boccio Piero, Fontana Antonella, Grassadonia Antonino, Di Ianni Mauro, Cama Alessandro, Veschi Serena
Department of Pharmacy, G. d'Annunzio University of Chieti-Pescara, 66100 Chieti, Italy.
Center for Advanced Studies and Technology (CAST), G. d'Annunzio University of Chieti-Pescara, 66100 Chieti, Italy.
Cancers (Basel). 2024 May 8;16(10):1795. doi: 10.3390/cancers16101795.
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 ( = 1 × 10) and were involved in the "Immune System" (FDR: 1.10 × 10 and 3.69 × 10, 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（PC）预后较差，且对免疫疗法表现出抗性。更好地了解肿瘤衍生的细胞外囊泡（EV）对免疫反应的影响可能有助于改善免疫疗法。通过超速离心从Capan-2和BxPC-3胰腺癌细胞中分离出的EV，采用原子力显微镜、蛋白质免疫印迹（WB）、纳米颗粒跟踪分析和无标记蛋白质组学进行表征。用来自PC或对照的异源EV处理健康供体的新鲜外周血单核细胞（PBMC），随后对CD8 +和CD4 +淋巴细胞进行流式细胞术分析。对经EV处理或未处理的PBMC中分选的淋巴细胞进行蛋白质组学分析，并通过酶联免疫吸附测定（ELISA）测量γ-干扰素（IFN-γ）浓度。值得注意的是，通过蛋白质组学分析在Capan-2和BxPC-3 EV中鉴定出的大多数蛋白质都连接在一个单一的功能网络中（= 1×10），并参与“免疫系统”（分别为错误发现率：1.10×10和3.69×10）。有趣的是，用Capan-2 EV而非BxPC-3 EV或异源对照EV处理健康供体来源的PBMC可诱导CD8 +和CD4 +淋巴细胞的早期激活。从经EV处理的PBMC中分选的淋巴细胞的蛋白质组学与其被Capan-2 EV激活的情况一致，表明IFN-γ是主要的上游调节因子之一，ELISA证实了这一点。蛋白质组学和功能分析表明，胰腺癌细胞外囊泡具有多效性作用，其中一些可能激活早期免疫反应，这可能与这种免疫冷肿瘤中急需的免疫治疗策略的开发有关。
