DataSheet_1_SILAC-based quantitative proteomics and microscopy analysis of cancer cells treated with the N-glycolyl GM3-specific anti-tumor antibody 14F7.pdf

Cancer immunotherapy represents a promising approach to specifically target and treat cancer. The most common mechanisms by which monoclonal antibodies kill cells include antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity and apoptosis, but also other mechanisms have been described. 14F7 is an antibody raised against the tumor-associated antigen NeuGc GM3, which was previously reported to kill cancer cells without inducing apoptotic pathways. The antibody was reported to induce giant membrane lesions in tumor cells, with apparent changes in the cytoskeleton. Here, we investigated the effect of humanized 14F7 on HeLa cells using stable isotope labeling with amino acids in cell culture (SILAC) in combination with LC-MS and live cell imaging. 14F7 did not kill the HeLa cells, however, it caused altered protein expression (MS data are available via ProteomeXchange with identifier PXD024320). Several cytoskeletal and nucleic-acid binding proteins were found to be strongly down-regulated in response to antibody treatment, suggesting how 14F7 may induce membrane lesions in cells that contain higher amounts of NeuGc GM3. The altered expression profile identified in this study thus contributes to an improved understanding of the unusual killing mechanism of 14F7.癌症免疫治疗是一种极具前景的肿瘤特异性靶向治疗手段。单克隆抗体杀伤肿瘤细胞的常见机制包括抗体依赖细胞介导的细胞毒性（antibody-dependent cell-mediated cytotoxicity）、补体依赖的细胞毒性（complement-dependent cytotoxicity）以及细胞凋亡（apoptosis），此外已有研究报道了其他杀伤机制。14F7是一种针对肿瘤相关抗原NeuGc GM3制备的单克隆抗体，既往研究显示其可在不诱导细胞凋亡通路激活的前提下杀伤肿瘤细胞。已有研究表明，该抗体可诱导肿瘤细胞形成巨大膜损伤，并伴随细胞骨架的显著改变。本研究采用细胞培养氨基酸稳定同位素标记（stable isotope labeling with amino acids in cell culture，SILAC）结合液相色谱-质谱联用（LC-MS）技术与活细胞成像技术，探究了人源化14F7对海拉细胞（HeLa）的作用效果。实验结果显示，14F7并未杀伤海拉细胞，但可引起细胞蛋白质表达谱的改变（质谱数据已通过ProteomeXchange数据库上传，识别号为PXD024320）。研究发现，经抗体处理后，多种细胞骨架蛋白与核酸结合蛋白出现了显著下调，这为解析14F7在高表达NeuGc GM3的细胞中诱导膜损伤的机制提供了依据。本研究鉴定得到的差异表达谱，有助于进一步阐明14F7独特的肿瘤细胞杀伤机制。