| Liver cancer is one of the most popular cancers today. The mortality rate of liver cancer ranks third in our countriy. Lack of effective treatments, its 5-years survival rate is very low now. Studies have reported that cancer stem cells play a key role in recurrence and metastasis. With building up cancer stem cell theory, human has realized that the tumorigenesis and progression of cancer is associated with abnormal expression of gene and protein in cancer stem cells. Great efforts have been focused on the research of specific gene and protein which target the cancer stem cells may be the key of effective cancer therapy. To provide some potential therapeutic agents and functional targets for liver cancer target treatment, this study characterized and isolated liver cancer stem-like cells (hLCSLCs) from human liver cancer specimens and then we established a liver cancer McAb library and identified functional McAbs targeted liver cancer stem cells by functional assay.The hLCSLCs were obtained from fresh liver cancer tissue using enzymatic digestion and short-term primary culture. Serum-free medium suitable for suspension sphere formation of hLCSLCs was selected by culturing them with serum-free medium suitable for suspension sphere formation of hLCSLCs was selected by culturing them with serum-free suspension culture medium with heparin, albumin or hydrocortisone. Numbers of side population (SP) cells, and expressions of CD133 and CD90 in the hLCSLCs and sphere cells were examined by flow cytometry assay. Tumor formation ability of hLCSLCs and sphere cells were assessed by tumor formation assay. hLCSLCs were used to immunize the BALB/c mice. The spleen cells of the immunized mouse were fused with SP2/0 cells and cultured with methyl cellulose selective medium to construct McAb library. By double immunofluorescence staining, clones were identified which can recognize the SP cells stained by CD133+(the marker of liver cancer stem cells). ELISA was used to identify the heavy and light chain isotypes of antibodies. Floating culture assay and proliferation analysis were used to screen functional McAbs. The relative antigen was identified by western blot. So as to ascertain the function, we purified the 15B7 mAb. Subsequently we located the expression of the antigen recognized by 15B7 mAb on hLCSLCs by immunofluorescence. And the functional antibodies were identified by cell proliferation, sphere formation, migration, invasion, the cell cycle and apoptosis assay in vitro. To assess the impact of antibodies on hLCSLCs, application of purified 15B7 mAb was used to treat hLCSLCs xenograft in vivo. At last we acquired one liver cancer stem cells-related functional gene by 15B7. One mAbs were finally produced for antigen identification by immuno-affinity and MALDI-TOP-PMF.hLCSLCs were successfully obtained from human liver cancer tissues, with SPcells being 0.9%, CD 133 positive cells being 0.8% and CD90 positive cells being 12.7% in the hLCSLCs. The tumor formation rate was 100% when 2000 SP cells were subcutaneously injected into nude mice. Serum-free medium containing heparin was more suitable for sphere formation of hLCSLCs, with SP cells being 4.6%, CD133+ cells being 9.7%, and CD90+ cells being 48% in these sphere cells. The tumor formation rate was 100% when 10 000 sphere cells were injected into nude mice. The result showed that hLCSLCs contained a high percentage of liver cancer stem cells. A total of 2964 McAbs 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 237 McAbs interacted with membrance of hLCSLCs. Using double immunofluorescence staining,33 McAbs co-stained with CD 133 on hLCSLCs-SP. Subclass of 33 clones was identified, including IgM, IgA, IgG1 and IgG3. It demonstrated that the library was diverse. By floating culture system and proliferation analysis, it showed that 14 clones inhibited hLCSLCs spheres formation and the inhibition rate was from 2% to 62% in which there were 8 clones inhibition rate was over 10%. Tumor formation rate was 100% when 2×104 hybridoma clones 15D2 and 3G7-positive hLCSLCs were injected into nude mice. Another 6 clones can inhibit the growth of hLCSLCs SP cells and hLCSLCs-sphere cells. The inhibition rate was over 10%. Among them, we chose 15B7 clone for further identification. The CCK-8 cell viable assay result:15B7 McAb significantly suppressed the proliferation of hLCSLCs,hLCSLCs-SP,hLCSLC-sphere cells, with the inhibitory rates being 13.8%,7.1% and 9.3%. We analized the effects of 15B7 mAbs on hLCSLCs migration and invasion in vitro, which the inhibition rates was 30.9%(P<0.05) and 15.7%(P>0.05). To further study the mechanism of I5B7mAb on hLCSLCs proliferation, we assayed the cell cycle after purified 15B7 mAb was to used to treat which lead to G1 arrest and apoptosis. When purified 15B7 mAb was to used to treat hLCSLCs xenograft in vivo a significant tumor growth inhibition was observed with the inhibitory rates being 59.2%,60.5% and 8.4% for the high, middle and low dosage of 15B7 mAb respectively.15B7 antibody treatment can prolong the survival of nude mice. To further study the mechanism of 15B7 mAb on hLCSLCs proliferation in vivo, we assayed the Ki67 mAb, CyclinDl mAb, Caspase3 mAb. Immunohistochemistry assay that dose treatmentgroup could suppress cell proliferation, dose treatmentgroup up-regulated cyclinDl. And the change of the Caspase3 protein expression level is significantly up-regulatation. Finally produced for antigen identification by immuno-affinity and MALDI-TOP-PMF was POTE-actin by 15B7mAb. Together, these results demonstrated that 15B7 mAb was a novel potential functional target for liver cancer therapy.We have successfully established a method for isolating hLCSLCs from human liver cancer tissues. Serum-free suspension medium containing heparin can effectively enrich liver cancer stem cells. We established a high capacity and variable McAb library and identified McAbs which can significantly inhibit liver cancer stem cells growth and sphere formation in vitro and vivo. We received 2 functional McAbs which could spctifically inhibit cancer stem cells growth.At least obtained two McAbs recognized their positive cells may be liver cancer stem cells. And one of these antibodies,15B7 mAb and related antigen-POTE-actin may be applied in a potential candidate drug and target molecular for therapy of liver cancer. |
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