Study On Antibody-targeted Therapy For Hepatic Cancer Stem Cells

Currently, the incidence of liver cancer ranked five in the world, and it is the first three causes of cancer death in a variety of tumors, China also has a high morbidity and mortality.The high drug-resistance to traditional therapy and it’s metastasis, recurrence, making the majority of patients with poor prognosis. Among them, the presence of stem cells in liver cancer is considered to have a close relationship with the drug-resistance, metastasis and recurrence of liver cancer.Immunology in mice with human hepatic cells within a ball figure is performed in our laboratory in the preliminary period to prepared anti-human monoclonal antibody library which is containing2964stem cell monoclonal antibodies.116hybridoma monoclonal antibodies were acquired after immunofluorescence screening and another34mAb were identified by co-expression with liver cancer stem cell marker CD133.According to the literature, stem cells can be enriched in serum-free culture with a sphere suspension growth. So we selected hepatoma cell lines MHCC97H cells as a model with the stem cell marker CD90, using flow fluorescence cytometry to detect the co-expression of monoclonal antibody and CD90in MHCC97H parental cells and sphere cells,18mAbs were selected according to the feature that the mAb can be enriched in the sphere cells compared with parent cells and the co-expression with CSC marker CD90were enriched as well.In order to test wether the identified18monoclonal antibodies have the ability to recognize different liver cancer stem cell subsets including CD90, we chose Be17402-V3cells with the CSC marker ESA for another screening, using the same method as mentioned previously to detect the co-expression of monoclonal antibody and ESA in Be17402-V3parental cells and sphere cells,15mAbs were selected according to the feature that the mAb can be enriched in the sphere cells compared with parent cells and the co-expression with CSC marker ESA were enriched as well. From those, we selected mAb15B7as an aim to study it in depth in Be17402-V3cellsThe outcome of study found that the expression of monoclonal antibody15B7has increased1.2times in sphere cells compared with Be17402-V3sphere cells and immunofluorescence detected in Be17402-V3viable cells and fixed cells shows that mAb15B7recognized target antigen in the cell membrane and cytoplasm in both,the distribution of antigen expression in the cytoplasm is higher than membrane;flow cytometry detect viable cells and immunofluorescence detect fixed cells results show that mAb15B7is able to identify24%of ESA+stem cells.Meanwhile, the sorting of15B7+cells also show the higher sphere forming ability and invasion ability than15B7"and parental cells; more importantly,the tumorigenicity of15B7+in nude mice which is the "gold standard" of sorting cells with the CSC characteristics, displayed that15B7+cells have the higher tumorigenicity compared with parental cells, proving mAb15B7is an anti-stem cell monoclonal antibody. Further, we conducted varies functional experiments with purified monoclonal antibody15B7to detect its function. Sphere forming ability inhibition experiments show that the spheres of mAb15B7group (91.7±17.0) was significantly lower than the negative control group(135.7±12.7), the sphere forming inhibition rate for Be17402-V3cells is32.4%, P<0.5; invasion inhibition experiments showed that mAb15B7invasive cells (143.1±37.7)/field were significantly lower than the number of cells in negative control group (206.4±16.8)/vision, the invasion inhibition rate for Be17402-V3cell is30.7%,P<0.5; all of those experiments proved that mAb15B7is an anti-CSC antibody with significantly inhibition ability for self-renewal capacity and invasion ability.Treatment in vivo with antibody and combined chemotherapy in nude mouse demonstrated that15B7mAb can inhibit the growth of gastric cancer xenograft (ration as8.4%、53.1%、67.4%for low, moderate and high dosage) with dosage dependent manner;15B7combined with chemotherapy treatment showed excellent result with87.2%of inhibition rate which is not only inhibition growth of xenograft, but recurrence of tumor caused by chemotherapy, which exhibited huge benefit and provide a new direction and candidate medicine for targeting treatment for cancer stem cells. Antigen recognized by15B7mAb was A protein, identified by MODI-TOP PMF, which reported in the literature is associated with tumor prognosis. The immuno-fluorescence confocal method validated this conclusion, and other verification works is in progress.To develope a silver lining of cancer biologocal-theraputic rigemen,the hepatic carcinoma cell line Be17402-V3was intervened with combination of monoclonal antibodies15D2and9A9to monitor its subsequent functions change including self-renewal,invading and drug-resistance abilities, which was contrast with sole antibody affecting.The expression percentige of cancer stem cell marker,9A9,15D2recognized on spheres is2.6times,2.0times,4.1times the number of proportion on parent cells.The co-expression antigen of combined antibodies and ESA is more than the sum of antigen located on (9A9+ESA) and (15D2+ESA).The combined antibodies have notable inhibitory ratio than sole antibody with equal concentration on spheres forming and invading ability/which is same as drug-resistance,that combine antibodies group IC50:0.32ug/ml,9A9IC50:0.58ug/ml,15D2IC50:0.56ug/ml. And all of those results were identified in lung cancer stem cell line SPCA-1as well.In summary,the study screened out15mAbs that recognize CSC marker CD90and ESA simultaneously, mAb15B7selected from liver cancer stem cells shows powerful function to cancer cells, and there is significant inhibition of tumor growth in vivo.Its targeted antigen was identified which may be a new promising target in the future.Combination of multi-targeted antibodies is capable of inhibiting cancer stem cells highly featured functions effectively in vitro that may responsible for tumor recurrence and improving outcome significantly.