Sato Yumi, Tateno Hiroaki, Adachi Jun, Okuyama Hiroaki, Endo Hiroko, Tomonaga Takeshi, Inoue Masahiro
Department of Biochemistry Osaka Medical Center for Cancer and Cardiovascular Diseases, 1-3-3 Nakamichi, Higashinari-ku, Osaka 537-8511, Japan.
Division of Molecular Virology and Oncology, Cancer Research Institute, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan.
Sci Rep. 2016 Apr 21;6:24823. doi: 10.1038/srep24823.
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 primary-cultured living spheroids from human 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 failed to detect in various cell lines examined. We found that 5G2 recognized the Le(a) and Le(c) on N-glycan, and their major carrier proteins were CEACAM5 and CEACAM6. Pre-incubation of the spheroids with 5G2 impaired translocation of integrin β4 from the lateral membrane to the contact interface between the extracellular matrix when embedded in it. As we successfully obtained a functional antibody, which antigen was glycan structures and lost in cell lines, cancer tissue-originated spheroids can be a useful antigen for generating novel anti-cancer antibodies.
直接从肿瘤培养的球体比已建立细胞系的二维单层培养或三维培养能更好地反映体内肿瘤特征。在本研究中，我们通过用人结直肠癌原代培养的活球体直接免疫小鼠来产生抗体。我们通过识别球体表面和抑制其与细胞外基质的黏附进行表型筛选，以鉴定出单克隆抗体克隆5G2。该抗体在二维培养中抑制细胞迁移并促进细胞脱离。蛋白质印迹法和免疫组织化学在许多结直肠癌球体以及患者肿瘤中检测到5G2信号，但在所检测的各种细胞系中未检测到。我们发现5G2识别N -聚糖上的Le(a)和Le(c)，其主要载体蛋白是CEACAM5和CEACAM6。当球体嵌入细胞外基质时，用5G2对其进行预孵育会损害整合素β4从侧膜向细胞外基质接触界面的转运。由于我们成功获得了一种功能性抗体，其抗原是聚糖结构且在细胞系中缺失，源自癌症组织的球体可成为产生新型抗癌抗体的有用抗原。
