Figure 5.

Combination of tumor-derived vaccines harboring immunomodulators enhance antitumor response. (A) Experimental design for in vivo experiment, Tumors were injected on day 0, following injections of irradiated immunomodulatory B16-derived cells on days 1, 4, and 7. On day 28, animals were sacrificed, performing analysis of lymphocytes in tumors and spleens by flow cytometry and immunofluorescence. Tumor-free mice were rechallenged on day 30 and monitored for more than 250 days. (B) Tumor growth and survival curves in C57BL/6 mice bearing subcutaneous B16F10 tumors, mice were treated with PBS or 1 × 10⁶ cells of different combinations of irradiated immunomodulatory B16-derived cells. These cumulative survival curves and tumor progression graph represent three independent experiments (n = 10 mice per group) the log-rank Mantel–Cox test was used to compare survival curves between different groups. (C) An analysis of tumor infiltrate lymphocytes and lymphocytes in spleens using flow cytometry reveals that the ratio of CD8/Tregs was increased by all immunomodulatory combinations. (D) Immunofluorescence of tumors reveals CD8 enrichment in animals treated with immunomodulatory vaccines. (This image is representative of all conditions challenged with double and triple combinations since no significant differences were observed among these groups.) (E) Histological analysis with hematoxylin–eosin of tumor and spleens of animals treated with the immunomodulatory vaccines. (This image is representative of all conditions challenged with double and triple combinations since no significant differences were observed among these groups.) and non-treated mice. Data are shown as the mean ± SEM. The ratio of CD8/Tregs equal to CD8 positive cells divided by FOXP3 positive cells (CD8^+Teff/Treg ratio). Results are pooled from three independent experiments (n = 10 per experimental condition for each experiment).