Targeted Therapy For Hepatic Cancer Stem Cells

Hepatocellular carcinoma (HCC) is one of the most common malignant tumour in China. It’s recurrence, metastasis and drug-resistance result in the high mortality rate. Cancer stem cells (CSCs), which have the ability to self-renew and differentiate into various tumour cell types, are a special class of tumor cells. And it’s the root cause of the tumorigenesis, metastasis, recurrence and drug-resistance. Currently, the treatments are mainly target at the sensetive tumor daughter cells, but don’t effect on CSCs. It’s what lead to the recurrence and metastasis of tumor patients. Therefore, targeted therapy for CSCs could achieve better therapeutic effect.The monoclonal antibody library which is containing 2246 monoclonal antibodies was prepared by hybridoma technology. The mAb 28C10 were selected as an aim to study it in depth by the comparison of concentration, cell ELISA, function and Western blot.The immunofluorescence detected in Be17402-V13 and MHCC97-L viable cells and fixed cells shows that mAb 28C10 recognized target antigen in the cell membrane and cytoplasm in both. The result of flow cytometry detect viable cells shows that mAb 28C10 is able to recoganize hepatic cancer stem cells in preliminary. The positive expression of mAb 28C10’antigen was 72.0% in liver cancer specimens detected by immonohistochemistry. Meanwhile, the sorting of 28C10+ cells also shows the higher sphere forming ability, invasion ability and drug-resistance ability than 28C10- and parental cells. Besides that, the tumorigenicity of 28C10+ in nude mice displayed that 28C10+ cells have the higher tumorigenicity compared with 28C10+ and parental cells, proving mAb 28C10 is an anti-stem cell monoclonal antibody. Further, all of functional experiments in vitro proved that mAb 28C10 is an anti-CSC antibody with significantly inhibition ability for self-renewal capacity, invasion ability and drug-resistance ability in the Bel7402-V13 and MHCC97-L. Treatment in vivo with antibody and combined chemotherapy in nude mouse demonstrated that mAb 28C10 can inhibit the growth of xenograft tumor of HCC. The low-dose mAb 28C10 combined with chemotherapy treatment showed excellent result with 70.1% of inhibition rate. Antigen recognized by mAb 28C10 was HSP90a protein, identified by MALDI-TOF mass spectrometry, which reported in the literature is associated with tumor proliferation. The result of immuno-fluorescence confocal, western blot and co-immunoprecipitation all validated this conclusion. In addition, the positive expression of HSP90a was 82.52% in liver cancer specimens detected by immonohistochemistry, which was signification higher than those in the non-cancer tissues. And the expression of HSP90a was correlated with differentiation (P<0.05).In summary, mAb 28C10 is able to recognize liver cancer stem cells and inhibit the abilities of self-renewal, invasion and drug-resistance in vitro, besides, there is significant inhibition of tumor growth in vivo. Its targeted antigen was HSP90a which may be a new promising target in the future.