Characterization of virus-mediated immunogenic cancer cell death and the consequences for oncolytic virus-based immunotherapy of cancer

摘要

( ) Cell viability of Ad-infected cells (MOI 10 –10 ) at days 1, 2, 3, 5, and 6 was measured using AlamarBlue™ viability assay. Cell viability is represented as percentage relative to non-infected control cells. Data are presented as mean ± SEM (  = 3). ( HOS cells and ( ) A549 cells were infected with Ad at MOI 10. Cell index values as a measure of cell viability were measured by the xCELLigence system. Analysis of ( ) Caspase-3/7 and ( ) Caspase-8 in Ad-infected (MOI 10 –10 ) HOS and A549 cells at 6 h and 24 h was performed using Caspase-3/7ApoTox-Glo™ Triplex and Caspase-Glo® 8 assays. Caspase activity is represented as percentage relative to non-infected control cells. Data are presented as mean ± SEM (  = 3). ( Mitochondrial membrane potential was measured by flow cytometry after infection with Ad (MOI = 10) at days 1, 2 and 3. ( ) Phosphorylated RIP3 (p-RIP3) was detected in Ad-infected (MOI 10) HOS and A549 cells by Western blot 6, 24 and 48 h after infection. Densitometric analysis of fold change in p-RIP3 post Ad-infection in ( ) HOS and ( ) A549 compared to un-infected control (  = 3). ( HOS and A549 cells cultured on glass slides were infected with Ad (MOI = 10) for 48 h and phosphorylated MLKL (p-MLKL) was detected by antibody staining (red). Cell nuclei were stained with Hoechst 33342 (blue). The multicolor fluorescent analyses were carried out in three individual experiments. Representative images from one experiment is shown. HOS and A549 cells expressing GFP-ASC were infected with the Ad (MOI 10) for 48 h. ASC specks were quantified by flow cytometry ( ) ( -test, *  p n = 3) and representative images ( ) were taken from cells cultured on glass chamber slides. ( Lysates were harvested from Ad-infected HOS and A549 cells (MOI = 50) at 6, 24 and 48 h. Full-length GSDMD , truncated and active GSDMD (lower band, indicated by arrow) and mature IL-1β were detected by Western blot. Vinculin was used as internal loading control. HOS and A549 cells expressing eGFP-LC3 cells were infected with Ad (MOI = 10) and monitored by fluorescence microscopy. ( Images were acquired at 48 h. ( The cell lysates of Ad-infected HOS and A549 cells (MOI = 50) were harvested at 6, 24, and 48 h for analysis of the non-lipidated form of LC3 (LC3-I), lipidated form (LC3-II) and the cargo-loading adaptor protein SQSTM1/p62. The housekeeping membrane-cytoskeletal protein vinculin was used as gel loading control. ( Densitometric analysis of ratio of LC3-II/LC3-I and SQSTM1/p62 degradation Ad-infection in HOS and A549 compared to un-infected control. Statistical analysis for normally distributed data were performed by means of one-way ANOVA with Dunnet’s post hoc test and for not normally distributed data were performed by means of Kruskal–Wallis test with Dunn’s post hoc test (*  p n = 3).