Fig. 2
Oxaliplatin-induced ICD provides a successful anti-PDAC vaccination approach. a Confocal microscopy showing the induction of the ICD marker, CRT, in KPC cells in the presence of PBS, Cis (100 µM), OX (50 µM), and DOX (1 µM) for 4 h. The cell nuclei, surface membrane and CRT were detected by Hoechst 33342, Alexa Fluor® 488-Conjugated Wheat Germ Agglutinin, and Alexa Fluor® 647-conjugated anti-CRT antibody staining, respectively. Scale bar is 20 μm. b CRT surface detection by flow cytometry, using the same conditions and reagents as in a (3 independent experiments). c Animal experimentation using 2 rounds of vaccination one week apart, followed by injecting live KPC cells SC on the contralateral side. The details of the animal vaccination experiment are provided in the methods section. Tumors were collected on day 29 for IHC and flow cytometry analysis. d Spaghetti curves to show KPC tumor growth in the contralateral flank. e Tumor collection was performed after euthanizing the animal to conduct IHC. Representative images are shown for the IHC staining of CD8 (upper panel) and Foxp3 (lower panel) T cells. The tumor tissues were also analyzed by flow cytometry to determine the CD8/Tregs ratio (see experimental section for details) (right panel). f IHC staining for cleaved caspase-3 (CC-3) and IFN-γ to demonstrate recruitment of cytotoxic T cells in response to ICD. Scale bar in IHC is 100 μm. g The 3 surviving animals in the OX-treated group, described in c, received orthotopic implant of live KPC cells on day 74. Animals maintained their tumor-free status up to 132 days, whereupon they were euthanized for collecting the immune splenocytes to perform an adoptive transfer experiment. IV injection of the immune splenocytes into the tail vein of B6/129 mice prevented the growth of KPC cells, implanted SC. The controls included IV administration of non-immune splenocytes or saline. The same experiment was also carried out in mice receiving SC injection of B16 melanoma cells. In this case, there was no interference in tumor growth by immune splenocytes, demonstrating the antigen specificity of the adoptive transfer response (Supplementary Fig. 3). The results are expressed as mean ± SEM. *p < 0.05; **p < 0.01, (ANOVA)
