Figure 1. Assessing the optimal vaccination schedule, followed by successful prophylactic treatment of breast cancer and melanoma in mice.

(A) Optimal vaccination was set to C+I vaccination for four weeks, as assessed by % IgG binding to DB7, without a significant increase in non-specific MEF binding (n=3 control animals, n=4 iPSC primed animals, n=4 C+I primed 2 week, and n=4 C+I primed 4 week animals, mean±s.e.m., ANOVA with Tukey’s multiple comparison test). (B) Representative FACS plot of serum IgG binding of PBS 4-week, iPSC 4-week, C+I 2-week, or C+I 4-week vaccinated mice to embryonic fibroblasts, iPSCs, and DB7 cancer cells. As a control sample for differentiated cells, a partly differentiated cell culture was included in the analysis. This is shown by IgG positive and negative cells, indicating that the IgG binding is specific to the undifferentiated portion of the analyzed cells. C+I 4-week vaccinated mice showed the best IgG binding to DB7 breast cancer cells. (C) Schema showing vaccine preparation consists of sorting murine iPSCs for pluripotency, irradiation, resuspension in adjuvant solution, and subcutaneous injection in the flank, sites 1 to 4. (D) Vaccination of FVB mice with the C+I vaccine resulted in a complete rejection of the cancer cells in 7 out of 10 mice by four weeks and overall reductions in DB7 tumor size (n=10 per group; representative images; left panel). Quantification of the data presented in right panel. (E) Vaccination of C57BL/6 mice with the C+I vaccine resulted in significant reduction of melanoma sizes initiated by the aggressive B16F0 melanoma cell line by week two (n=8 PBS, n=9 iPSC primed, n=10 CpG primed, and n=9 C+I primed). (F) Quantification of the tumor size data presented in panel E. Data in D and F expressed as mean±s.e.m., ANOVA with Tukey’s multiple comparison test, *p<0.05, **p<0.001, ***p<0.001, ****p<0.0001).