Efficiency of G2/M-related tumor-associated antigen-targeting cancer immunotherapy depends on antigen expression in the cancer stem-like population

The aim of this study was to establish a novel efficient cancer DNA vaccine approach. Many tumor-associated antigens (TAAs) have been reported; however, there is little information of the efficiency of each TAA. Normal cells barely undergo mitosis, whereas cancer cells divide frequently and grow well. Thus, G2/M-related antigens are cancer cell-specific and are regarded to be suitable candidates as targets of cancer immunotherapy. In this study, we compared the efficiencies of G2/M-related antigens including Birc5, Aurka, Nke2 and Plk1 by using a DNA vaccination model. Mice that had been immunized with G2/M-related antigens coding plasmid were challenged with CT26 colon cancer cells. Interestingly, Birc5- and Aurka-immunized mice showed an anti-tumor effect, whereas Nek2- and Plk1-immunized mice did not show any anti-tumor effect. We investigated the expression of G2/M-related antigens in cancer stem-like cell (CSC)/cancer-initiating cell (CIC) population to verify the difference in the anti-tumor effect. CSCs/CICs were isolated as side population (SP) cells using Hoechst 33342 dye from CT 26 cells. It was found that Birc5 and Aurka are expressed in both CSCs/CICs and non-CSCs/CICs (shared antigens), whereas Nek2 and Plk1 are expressed preferentially in non-CSCs/CICs (non-CSC antigens). Therefore, antigen expression in the CSC/CIC population might be related to the anti-tumor efficiency of cancer immunotherapy. Furthermore, we established a heat shock protein (Hsp90)-fused Birc5 plasmid to improve anti-cancer immunity. Birc5 fused to the N-terminal region of Hsp90 showed a stronger anti-tumor effect, whereas Birc5 fused to the C-terminal region of Hsp90 did not show enhancement compared with Birc5. These observations indicate that expression in the CSC/CIC population is essential to achieve tumor regression and that fusing antigens to the N-terminal region of Hsp90 enhances the anti-tumor effect.