Studies On Inhibitory Effects Of Monoclonal Antibodies Targeting Liver Cancer Stem Cells On Growth And Metastasis Of Liver Cancer

Department of Cell and Molecular Biology, Cancer Institute (Hospital), Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing100021, ChinaLiver cancer is the fifth most common cancer in the world. The mortality rate of liver cancer ranks third in China, and recurrence and metastasis are the main reason leading to poor prognosis of liver cancer. The recurrence rate of advanced liver cancer is60%after radiotherapy and chemotherapy. Studies have reported that cancer stem cells play a key role in recurrence and metastasis. Therapy targeting liver cancer stem cells implicates to develop novel effective therapy in liver cancer.Cancer cells were obstained from fresh liver cancer tissues using enzymatic digestion and primary culture. In vivo passaging was conducted in SCID mice. Liver cancer stem cell markers were detected by flow cytometry. Liver cancer cell line for cancer stem cell research was selected by serm-free suspension culture with PKH26staining, flow cytometry and immunofluorescence. Proliferation analysis was used to screen functional mAbs.11C9and4F11were choosed for further functional studies. The expression of the antigen of mAb11C9and4F11were decteted by flow cytometry and immunofluorescence; functions in vitro were identified by cell proliferation and Transwell experiment; functions in vivo were examined by treating xenograft tumor. The molecular weight of antigens of mAb11C9and4F11was identified by Western blot. Liver cancer stem cells-related functional genes were identified by immuno-affinity and MALDI-TOP-PMF. The results of mass spectrometry were verified by immunoprocipitation and immunofluorescence.Cancer cells were successfully separated from fresh liver cancer tissues, and primary culture. Flow cytometry results showed that the expressions of liver cancer stem cell markers ESA, CD133, CD24, CD44and AFP were significantly different in different liver cancer tissues. MHCC97H cells could form spheres by culturing them with serum-free medium, and PKH26staining demonstrated that each MHCC97H sphere was proliferated and differentiated from one single cancer stem cell. Cisplatin resistance of sphere cells was significantly higher than that of MHCC97H cells(IC50:69.7ug/ml vs22. lug/ml)。The positive rates of CD90with sphere cells were significantly higher than those with MHCC97H cells(23.8%vs2.7%)。 CD90co-stained with PKH26on MHCC97H sphere, identifying CD90+MHCC97H cells could be liver cancer stem cells. mAbs3G7、4F11、11C9、15B7、15D2co-stained with CD90on MHCC97H cells, and the positive rate of4F11with CD90+MHCC97H cells was42.3%;The positive rate of11C9with CD90+MHCC97H cells was21.6%. mAb11C9significantly inhibited the proliferation of spheres(the inhibit rate:23.2%), while had no significant inhibit effect on the proliferation of MHCC97H cells. The positive rates of11C9with sphere cells were significantly higher than those with MHCC97H cells[11.9%vs3.8%]. mAb11C9co-stained with PKH26and CD90on MHCC97H sphere, proving that mAb11C9recognizing CD90+liver cancer stem cells. Compared with MHCC97H cells and11C9-cells,11C9+cells possess more ability with cisplatin resistance(5-FU, IC50:289.37ug/ml vs101.25ug/ml vs71.26ug/ml), sphere formation(spheres:(6±3)vs(5±3)vs(3±3)). invasion(invasion cells:(111±14)vs(87±14)vs(57±4)) and migration(migration cells:(156±15)vs(130±16)vs(114±11)). The inhibition of sphere cells proliferation by mAb11C9is significantly higher than that of MHCC97H cells. mAb11C9combined with5-FU treatment of xenograft tumor significantly inhibited cancer recurrence. Antigen reconized by mAb11C9was A, identified by MALDI-TOP-PMF. Immunoprecipitation and immunoflurescence verified the result. The positive rates of4F11with sphere cells were significantly higher than those with MHCC97H cells(12.5%vs2.7%). mAb4F11co-stained with PKH26and CD90on MHCC97H sphere, proving that mAb4F11identifying CD90+liver cancer stem cells. The inhibition of sphere cells proliferation by mAb4F11is significantly higher than that of MHCC97H cells[29.4%vs12.0%], and mAb4F11extremely suppressed sphere formation ability of MHCC97H cells(the inhibit rate:58.0%) and significantly inhibited invasion and migration in vitro(48.6%;47.6%). Antigen reconized by mAb4F11was B, C and D, identified by MALDI-TOP-PMF.Our study provided evidences for the existence of liver cancer stem cell in liver cancer tissues. CD90+MHCC97H cells are liver cancer stem cells. mAb11C9significantly inhibite the proliferation of cells rich liver cancer stem cells; Cells recognized by mAb11C9possess the characteristics of cancer stem cells; mAb11C9combined with5-FU treatment of xenograft tumor significantly inhibited cancer recurrence. The results suggested that mAb11C9could be antibody drug candidate for cancer stem cell targeted therapy of liver cancer; the resisitance of some liver cancers could be related with11C9+liver cancer stem cells. Therapy targeting the target antigen A of mAb11C9combined with chemotherapy maybe could effectively inhibite liver cancer recurrence. mAb4F11significantly inhibite the proliferation of cells rich liver cancer stem cells, the invasion and migration of cancer cells, which could be antibody drug candidate for cancer stem cell targeted therapy of liver cancer. The study would provide noval candidate targets and drugs for liver cancer stem cells target therapy, sametime provide new ideas for the treatment with liver cancer recurrence.