Fig. 1

Prophylactic autophagosome vaccination delayed 4T1 tumor growth, improved overall survival, and increased intratumoral CD3 + CD8+ infiltration. a Mice were vaccinated in both inguinal lymph nodes with 4T1 autophagosome-enriched vaccine plus poly-I:C, vaccine alone, adjuvant alone, or left untreated. Animals were boosted subcutaneously after two weeks. After another two weeks, sera or spleens were harvested at Day 0 for in vitro antibody and T cell assays or animals were challenged with live 4T1 tumor cells for survival endpoints, tumor bearing sera, and immunohistochemistry. b Upon challenge, reduced average tumor growth was observed in combination vaccine + poly-I:C pretreated animals with maximum separation occurring at Day 22 versus poly-I:C alone (P = 0.04) and Day 27 versus naïve animals (P = 0.002) by Dunnett’s multiple comparisons test. Data were pooled from five independent experiments with error bars plotted as the standard error of the mean. c Overall survival was improved in combination treatment versus all other groups (P = 0.02) by Gehan-Breslow-Wilcoxon test. Data were pooled from three independent experiments. d-e Zinc and alcohol fixed day 30 4T1 tumors were stained for six color immunohistochemistry with tyramide signal amplification. d Three color representative image showing CD8+ (red), F4/80 (green), and DAPI (blue). e Fifteen 20× fields were imaged for each of 4 to 6 tumors per group and quantified for CD3 + CD8+ per mm² (fields from individual tumors colored separately). Higher numbers of CD3 + CD8+ infiltrates were seen in the fields from vaccine + poly-I:C pretreated tumors versus all other groups (P < 0.0001) by t-test. Lines plotted are the median and interquartile range