Generation of a monoclonal antibody recognizing the CEACAM glycan structure and inhibiting adhesion using cancer tissue-originated spheroid as an antigen.
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.
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.
Yumi
Y
Sato
Biotechnology Research Institute for Drug Discovery (BRD), National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan.
Hiroaki
H
Tateno
Laboratory of Proteome Research, National Institute of Biomedical Innovation, Health and Nutrition. 7-6-8 Saito-Asagi, Ibaraki-shi, Osaka 567-0085, Japan.
Jun
J
Adachi
Department of Biochemistry Osaka Medical Center for Cancer and Cardiovascular Diseases, 1-3-3 Nakamichi, Higashinari-ku, Osaka 537-8511, Japan.
Hiroaki
H
Okuyama
Department of Biochemistry Osaka Medical Center for Cancer and Cardiovascular Diseases, 1-3-3 Nakamichi, Higashinari-ku, Osaka 537-8511, Japan.
Hiroko
H
Endo
Laboratory of Proteome Research, National Institute of Biomedical Innovation, Health and Nutrition. 7-6-8 Saito-Asagi, Ibaraki-shi, Osaka 567-0085, Japan.
Takeshi
T
Tomonaga
Department of Biochemistry Osaka Medical Center for Cancer and Cardiovascular Diseases, 1-3-3 Nakamichi, Higashinari-ku, Osaka 537-8511, Japan.
Masahiro
M
Inoue