Generation of a monoclonal antibody recognizing the CEACAM glycan structure and inhibiting adhesion using cancer tissue-originated spheroid as an antigen. Homo sapiens

Spheroids cultured directly from tumours can better reflect in vivo tumour characteristics than two-dimensional monolayer culture or three-dimensional culture of established cell lines. In this study, we generated antibodies by directly immunizing mice with cancer tissue-originated spheroids from colorectal cancer. We performed phenotypic screening via recognition of the surface of the spheroids and inhibition of their adhesion to extracellular matrices to identify a monoclonal antibody, clone 5G2. The antibody inhibited cell migration in two-dimensional culture and promoted cell detachment. Western blotting and immunohistochemistry detected the 5G2 signal in many colorectal cancer spheroids, as well as patient tumours, but the signal was reduced in various cell lines. The expression pattern also changed when spheroids were cultured in two-dimensional conditions. We found that 5G2 recognized the N-glycan structure, including Lea and Lec, and their major carrier proteins were CEACAM5 and CEACAM6. Translocation of integrin 4 from the lateral membrane to the extracellular matrix contact interface in the spheroids was delayed by pre-incubation with 5G2. Thus, cancer tissue-originated spheroids can be a useful antigen for generating novel anti-cancer antibodies as the glycan structure is lost in cell lines but retained in cancer tissue-originated spheroids. Overall design: Gene expression was compared in cancer tissue-originated spheroids (CTOSs) under floating conditions, under gel embeded conditions, CTOS-derived xenograft tumors, and corresponding microdissected patient tumors. One experiment was performed for each sample.

直接从肿瘤组织培养获得的肿瘤球体（cancer tissue-originated spheroids, CTOS），相较于二维单层培养或已建立细胞系的三维培养体系，更能精准反映体内肿瘤的生物学特性。本研究以结直肠癌患者来源的肿瘤球体直接免疫小鼠以制备抗体，并通过识别球体表面及抑制其与细胞外基质黏附的表型筛选策略，成功筛选得到单克隆抗体5G2克隆。该抗体可抑制二维培养环境下的细胞迁移，并促进细胞脱附。蛋白质印迹（Western blotting）与免疫组织化学检测结果显示，5G2信号可在多数结直肠癌球体及患者肿瘤组织中被检出，但在多种细胞系中信号强度显著降低。当肿瘤球体被转换为二维培养条件时，其蛋白表达模式亦发生显著改变。本研究发现，5G2可识别包含Lea与Lec的N-聚糖（N-glycan）结构，其主要载体蛋白为CEACAM5与CEACAM6。经5G2预孵育后，肿瘤球体中整合素4（integrin 4）从侧膜向细胞外基质接触界面的转位过程被显著延迟。综上，由于细胞系中该糖链结构发生丢失，而患者来源肿瘤球体仍保留该糖型，因此肿瘤球体可作为制备新型抗肿瘤抗体的高效抗原。整体实验设计：本研究对漂浮培养条件下、凝胶包埋培养条件下的患者来源肿瘤球体（CTOSs）、CTOS来源的异种移植瘤以及对应显微切割的患者肿瘤组织开展基因表达对比分析，所有样本均仅进行单次实验。