DataSheet_2_SILAC-based quantitative proteomics and microscopy analysis of cancer cells treated with the N-glycolyl GM3-specific anti-tumor antibody 14F7.xlsx

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.

癌症免疫疗法是一种可特异性靶向并治疗癌症的极具前景的治疗策略。单克隆抗体杀伤细胞的常见机制包括抗体依赖性细胞介导的细胞毒性（antibody-dependent cell-mediated cytotoxicity）、补体依赖性细胞毒性（complement-dependent cytotoxicity）以及细胞凋亡（apoptosis），此外还有其他已被文献报道的杀伤机制。14F7是一种靶向肿瘤相关抗原NeuGc GM3的抗体，此前有研究显示该抗体可通过不诱导细胞凋亡的途径杀伤癌细胞，另有研究表明该抗体可诱导肿瘤细胞形成巨大膜损伤，并使细胞骨架发生显著改变。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（LC-MS）以及活细胞成像技术，探究人源化14F7对HeLa细胞的作用效果。实验结果显示，14F7并未杀伤HeLa细胞，但可引起细胞蛋白质表达谱发生改变（质谱数据已上传至ProteomeXchange数据库，检索标识符为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的表达水平显著下调，这一结果为阐释14F7如何在高表达NeuGc GM3的细胞中诱导膜损伤提供了线索。本研究鉴定得到的差异表达谱，有助于进一步阐明14F7不同寻常的杀伤机制。