Figure 6.

Rafoxanide has anti-cancer vaccination effect on CRC. (A). Experimental protocol of the tumor vaccination model. CT26 cells were treated with 5μM rafoxanide for 48 h. Then the cells were collected and subcutaneously (s.c.) injected (1 × 10⁶ cells per mouse) into the left flank of immunocompetent BALB/c mice (vaccination). An equal volume of PBS was used as control (sham). One week later (day 0), all mice were re-challenged with living CT26 cells (1 × 10⁶ per mouse) into the right flank to test the elicitation of immunological memory. Tumor growth was monitored until sacrifice (day 18). (B). Representative dot-plots showing the percentages of AV- and/or PI-positive CT26 cells treated with either DMSO (vehicle) or 5 μM rafoxanide for 48 h. Rafoxanide-treated CT26 cells were collected and used for in vivo vaccination as indicated in A. (C). Representative images of immunocompetent BALB/c mice vaccinated with either rafoxanide-treated succumbing CT26 cells or PBS (sham) and then re-challenged with living CT26 cells as indicated in A. Mice were sacrificed 18 days after the re-challenge. Arrows indicate CT26-derived tumors growing at the re-challenge site. (D). Evolution of tumor incidence over time as a Kaplan–Meier curve in mice treated as indicated in A. Shown is the percentage of tumor-free mice pooled from 2 independent experiments (n = 8 mice per group). Statistical significance was calculated by log-rank (Mantel-Cox) test. *** p < 0.001, as compared to mice vaccinated with PBS (sham). The right inset indicates the percentage of mice that were tumor-free at the end of the protocol (day 18). Data are derived from two independent experiments and the number of animals globally employed for these determinations is indicated. (E). Evolution of tumor growth over time. Tumor volume is represented as mean + SD. Statistical significance was calculated using the two-tailed Student’s t-test. * p < 0.05, as compared to mice vaccinated with PBS (sham).