Hypomethylating treatment induces tumor specific CD8+ T-cell recognition to osteosarcoma / 中华骨科杂志

ABSTRACT

Objective To investigate feasible immunotherapy strategy using tumor specific cell against osteosarcoma,and to analyze the therapeutic effect of adoptive cellular infusion therapy on osteosarcoma.Methods Decitabine (DAC) was employed as a hypomethylating agent for the treatment in osteosarcoma cell lines HOS and U2OS.After treatment,the expression of cancer-testis antigen (CTA) was evaluated by PCR and Western Blot.In animal studies,human osteosarcoma cell line HOS,which was transfected by luciferase and HLA-A0201 in previous,was inoculated into immune deficient NOD-SCID mice to establish osteosarcoma xenografts.Ex-vivo expanded CTA specific homo CD8+T-ells were labeled with DiR and injected into the mice via the tail vein.In vivo imaging system was utilized to detect the distribution of administrated CD8+ T-cells.In addition,the progression of tumor xenografts was monitored.Moreover,mouse K7M2 osteosarcoma cell line was used to establish animal models in immune competent BALB/c mice.Immune competent models were utilized to evaluate the effectiveness of hypomethylating treatment in regarding to spontaneous immune attack against tumors.Flow cytometry was used to analyze the proportion of intratumoral lymphocytes and the status of these effector antitumor immune cells,and to reveal the effect of hypomethylating treatment in facilitating lymphocyte infiltration and activation.Results The expression of all the evaluated cancer/testis antigens were elevated in HOS and U2OS osteosarcoma cell lines after hypomethylating treatment with DAC.The proliferation of in vitro cultured osteosarcoma cells can be significantly suppressed after at least 5 d treatment with DAC.Besides,DAC alone controlled osteosarcoma cell proliferation.In immune deficient mouse models,hypomethylating pre-treatment resulted in successful T-cell homing to tumor sites.Moreover,the combination treatment with DAC and CTA specific T-cell adoptive transfer significantly suppressed tumor proliferation.In immune competent mouse models,hypomethylating treatment with DAC improved autologous T-cell infiltration into the tumor,and strengthened the activity of intratumoral CD8+ T-cells,elevated the secretion of IFN-gamma,granzyme B and perforin by CD8+ T-cells.Conclusion Hypomethylating treatment is able to suppress osteosarcoma cell proliferation,improve the expression of CTA in osteosarcoma cells,and consequently provide optimal environment for CTA specific T-cell adoptive therapy.