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Preparation of functional monoclonal antibodies against human liver cancer stem cells
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Abstract:
Abstract Objective:To prepare functional monoclonal antibodies(McAbs) against liver cancer stem cells, so as to provide candidate antibody drugs for stem cell-targeted therapy of liver cancer. Methods: Human liver cancer stem-like cells (hLCSLCs) were separated from human hepatocarcinoma tissues and were used to immunize BALB/c nude mice. Spleen cells from hLCSLCs-immunized mice were fused with SP2/0 cells to prepare large monoclonal antibody library. Hybridoma McAbs recognizing hLCSLCs were screened and identified by immunofluorescence, sphere formation culture and in vivo tumor formation assays. hLCSLCs side population cells (hLCSLCs-SP) were sorted by flow cytometry. The effects of hybridoma McAbs on self-renewal and proliferation of hLCSLCs-SP were identified by serum-free suspension culture and CCK-8 assay. Results: A total of 2 964 McAb clones were obtained by fusing immunized spleen cells with SP2/0 cells, and 237 McAbs could interact with hLCSLCs as detected by fixed-cell immunofluorescence; 116 of the 237 McAbs interacted with the membrane of hLCSLCs, and 33 McAbs specifically reacted with hLCSLCs-SP but not with non-hLCSLCs-SP, with positive rates being 2%-5%. Six of the 33 McAbs co-stained with CD133 on hLCSLCs-SP. Further investigation showed that the positive rates of these 6 McAbs were 3%-26% with sphere cells after serum-free suspension culture, which were significantly higher than those with hLCSLCs-SP. Tumor formation rate was 100% when 1×104 hybridoma clone 15D2-positive hLCSLCs were injected into nude mice. Functional study showed that 4 of these 6 McAbs significantly suppressed the proliferation and sphere formation ability of hLCSLCs-SP, with the inhibitory rates being 24%-42% and 13%-50%, respectively. Conclusion: We have successfully constructed the large McAb library against hLCSLCs, from which 4 hybridoma McAbs can specifically react with hLCSLCs-SP, laying a foundation for cancer stem cell-based antibody-targeted therapy for liver cancer.
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Project Supported:
Project supported by the Major Basic Research and Development Program (973 Program) of China（No. 2009CB521804）
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