Development of novel cancer cell vaccine using GM-CSF gene-transuduced IPS cells targeting cancer stem cells

我们的体外实验结果表明，非传染性重组仙台病毒介导的小鼠GM- csf基因转染iPS （iPS/GM）细胞可在体外产生丰富的GM- csf （200~ng/106个细胞/24小时），iPS/GM- csf细胞在形态和抗原性方面保持了干细胞性，SSEA-1、Oct3/4和碱性磷酸酶的表达水平与未修饰的iPS细胞相似。我们在免疫活性小鼠体内的研究结果表明，使用辐照的iPS/GM-CSF （ir.iPS/GM-CSF）细胞进行预防性和治疗性疫苗治疗的小鼠，可显著抑制皮下移植的同基因LLC小鼠左侧低免疫原性肺癌的肿瘤生长。值得注意的是，在上述治疗过程中，没有观察到严重的不良事件，生化分析证明没有肝脏和肾脏功能障碍，也没有体重减轻，这表明使用iPS细胞作为疫苗细胞的相对耐受性。此外，体内消耗实验表明，在小鼠体内观察到抗肿瘤作用。通过消耗CD4+T细胞或CD8+T细胞，iPS/GM-CSF细胞被显著破坏，表明其部分依赖于T细胞介导的细胞抗肿瘤免疫。总之，这是第一个证明正常成纤维细胞衍生的iPS细胞疫苗（包括iPS/GM细胞）具有治疗性抗肿瘤功效的报告，这可能意味着这些使用iPS细胞的新型疫苗策略可以诱导CSCs相关抗原特异性抗肿瘤免疫，是一种有希望的预防或治疗抗癌方式

Our results of in vitro assays demonstrated that non-transmissible recombinant Sendai virus-mediated mouse GM-CSF gene transfer to iPS (iPS/GM) cells was effective to produce abundant GM-CSF in vitro（200～ng/106cells/24 hrs）and iPS/GM-CSF cells maintained their stemness in terms of morphology and antigenicity as evidenced by the expression levels of SSEA-1,Oct3/4 and alkaline phosphatase similar to those seen in unmodified iPS cells. Our results of in vivo studies using immunocompetent mice demonstrated that mice treated with both prophylactic and therapeutic vaccination using irradiated iPS/GM-CSF (ir.iPS/GM-CSF) cells significantly suppressed the tumor growth of subcutaneously transplanted syngeneic LLC mouse poorly immunogenic lung cancer in the left flank. Of note, during these treatments described above, no serious adverse events were observed with lack of liver and kidney dysfunctions as evidenced by biochemical analysis as well as absence of loss of body weight, suggesting its relative tolerability of use of iPS cells as vaccine cells. Furthermore, in vivo depletion assays showed that the antitumor effect observed in mice treated with ir.iPS/GM-CSF cells was significantly abrogated by depleting CD4+T or CD8+T cells, demonstrating its partial dependence on T cell-mediated cellular antitumor immunity. In conclusion, this is the first report that demonstrated a therapeutic antitumor efficacy of normal fibroblast-derived iPS cell vaccines including iPS/GM cells, providing possible implications that these novel vaccine strategy using iPS cells could induce CSCs associated antigens-specific antitumor immunity and be a promising prophylactic or therapeutic anticancer modality