Vaccination With Irradiated Induced Pluripotent Stem Cells Genetically Engineered To Produce GM-CSF Confers Potent T Cells-Mediated Antitumor Immunity

Development of a novel therapeutic modality targeting cancer stem cells (CSCs) holds great promise for the eventual eradication of cancer. It was demonstrated that CSCs shared antigenic similarities with embryonic stem (ES) cells and the vaccination using ES cells could generate antitumor immunity. However, the use of ES cells raises potential immunological and ethical problematic issues. Recently, by the forced ectopic expression of defined transcription factors, autologous somatic cells were successfully reprogrammed into induced pluripotent stem (iPS) cells that closely resemble ESCs. We hypothesized that novel cell vaccines using mouse iPS cells genetically engineered to express the immunostimulatory cytokine of GM-CSF would cross-react CSC cells to induce antitumor immunity against poorly immunogenic syngeneic LLC mouse lung cancer cells, which would resolve such problematic issues. Our results of in vitro assays demonstrated that non-transmissible recombinant Sendai virus-mediated mouse GM-CSF gene transfer to iPSCs (iPS/GM-CSF) was effective to produce abundant GM-CSF in vitro and iPS/GM-CSF cells maintained their stemness in terms of morphology and antigenicity as evidenced by the expression of SSEA-1,Oct3/4 and alkaline phosphatase compared with unmodified iPS cells. Prophylactic iPSCs vaccine studies revealed that wild-type female mice subcutaneously vaccinated with irradiated iPS (ir.iPS) cells on weeks 1, 2, and 3 before the tumor challenge with LLC cells significantly suppressed the LLC tumor growth compared with untreated mice (p<0.05). Additionally, mice vaccinated with ir.iPS/GM-CSF cells significantly inhibited the tumor growth compared with mice treated with ir.iPS/GFP cells (p<0.05), showing that genetic manipulation of iPS cells with GM-CSF encoding gene potentiated the antitumor effect. Of note, no serious adverse events were observed with lack of liver and kidney dysfunctions as evidenced by biochemical analysis. Furthermore, therapeutic vaccinations with repeated ir.iPS/GM-CSF cells significantly inhibited the pre-established LLC tumor growth compared with untreated mice (p<0.05), with unaltered body weight. To address the effectors of the observed antitumor effects by ir.iPS/GM-CSF cells, we performed in vivo depletion experiments. The antitumor effects observed in mice treated with iPS/GM cells were significantly abrogated when CD4⁺ T cells- or CD8⁺ T cells were depleted, showing that iPS cells-based vaccines effectively generated T cells-mediated antitumor immunity. Lastly, we performed a cDNA microarray analysis to detect comparably expressed genes for the putative CSCs-associated antigens as a target of iPS cells-based vaccines on LLC cells, iPS cells, ir.iPS cells and ir.iPS/GM-CSF cells. Several sperm- or cell surface- specific antigens were predominantly expressed and shared between LLC cells and these iPS cell fractions, indicating that LLC cells and iPS cells may share CSCs-associated antigens. In conclusion, our results collectively demonstrate iPS cells-based vaccine can induce both prophylactic and therapeutic antitumor immunity in syngeneic mouse models, and indicate that this novel vaccine strategy may eliminate CSCs that shared antigenic similarities as a promising modality for cancer immunotherapy.