Fig. 5

Non-DNA-damaging agents are effective in prophylactic vaccination, which mostly correlates with condensation of nucleoli (CON) and cell death. (a) Human osteosarcoma U2OS cells expressing photoactivatable (PA)GFP-H2A were used to evaluate hits’ chromatin-damaging activity after activation by laser of a portion of the nucleus. Segmentation of nuclei in colors (each color represents each nucleus that has been automatically tracked over time) and photoactivated region (PR) in blue are overlayed on representative cells treated with 10 µM doxorubicin (DOXO) or left untreated (CTR). Photoactivated PAGFP-H2A was subsequently monitored by time-lapse confocal microscopy for 60 min and tracked over time. (b) Partial nuclear photoactivation was performed as described above, cells were then treated with 10 µM etoposide (ETO), 10 µM aclarubicin (ACLA), 10 µM bisantrene (BISA), 10 µM BMH21, 10 µM CX5461, 5 µM plicamycin (PLICA), 10 µM doxorubicin (DOXO), or left untreated (CTR). Representative images of the first (0 min) and last (60 min) timepoint are displayed, with a segmentation overlay of nuclei in colors (each color represents each nucleus that has been tracked over time) and in the PR in blue. (c) Quantification of the fluorescence intensity of PAGFP-H2A in the PR expressed as fold change (FC) to the initial timepoint (T0) after each indicated treatment is reported over time. (d) Quantification of the fluorescence intensity of PAGFP-H2A expressed as FC to T0 in the PR after each treatment is reported in a bar chart corresponding to the last timepoint (60 min). Scale bars equal 10 µm. (e) Mouse fibrosarcoma MCA205 cells were treated in vitro with 4 µM mitoxantrone (MTX), 15 µM aclarubicin (ACLA), 5 µM BMH21, 15 µM CX5461, 10 µM bisantrene (BISA) or 50 µM plicamycin (PLICA). After harvesting, dying cells were subcutaneous (s.c.) injected into the left flank of immunocompetent syngeneic C57Bl/6 mice (n = 10 mice per group), while the control group was injected with PBS. Ten days later, animals were rechallenged with living MCA205 cells in the contralateral flank of the mice and tumor size was regularly measured. The course of tumor volume curves is depicted. (f) Tumor-free survival is shown for each group. (g) Vaccinated tumor free mice (ACLA n = 9, BMH21 n = 5, CX5461 n = 10, BISA n = 5, PLICA n = 4, MTX n = 9) as well as naïve mice (n = 5) were rechallenged by s.c. injection of living MCA205 cells in the left flank of the mice, with simultaneously s.c. injection of living mouse melanoma B16-F10 in the right flank. Tumor growth was monitored over time and tumor volumes at endpoint of each group are displayed in a dot plot for B16-F10, and in (h) for MCA205. Values in tumor growth curves are expressed as mean ± SEM, and median of tumor sizes are reported for the endpoint dot plot with p-values calculated with pairwise Mann-Whitney test versus control. TumGrowth (https://github.com/kroemerlab) was used to analyze in vivo data. Statistical significance of tumor-free survival was calculated with log-rank test. (i) The clustered heatmap summarizes the relative effect of each evaluated phenotype, i.e. vaccination efficacy (Vacc. efficacy), condensation of nucleoli (CON), death, nucleolar size, chromatin damage (Chro. Damage), phosphorylation of eIF2α (PeIF2α), dsDNA and γH2AX, assessed throughout the study for ACLA, BMH21, BISA, CX5461, PLICA, etoposide (ETO), oxaliplatin (OXA), dactinomycin (DACT), doxorubicin (DOXO) and the untreated (CTR) group. (j) A Pearson correlation matrix for the aforementioned effects is displayed. Pearson correlation coefficients are indicated in the lower triangular matrix, while being represented by circles whose size and color are mapped to their value in the upper triangular matrix