Extended Data Fig. 4: Protective role of intestinal caspases -3 and -7 in the cell death of ileal IEC against colon cancer.
a-b, Vaccination of naïve C57BL/6 J (a) or BALB/c (b) mice using ileal or colonic IEC which were exposed to OXA to protect against syngeneic transplantable colon cancers (such as MC38 or CT26, respectively (Fig. 3a-b)) or irrelevant syngeneic tumors (such as MCA205 (a) and 4T1 (b), respectively. Tumor growth curves showing one representative experiment out of 3 yielding similar results. MCA205 n = 5/group. 4T1 naïve n = 6, ileum-OXA n = 6, colon OXA n = 5. Mean ± SEM is depicted. c-d, Dying ileal IEC were harvested at 6 h post-OXA i.p. and separated by a FACS cell sorting based on Annexin V/7AAD expression or vaccination with IEC subjected to three freeze-thaw cycles (F/T) before being inoculated as a vaccine in naive mice as described in Fig. 3a. A representative flow cytometric analysis is shown (c) and tumor growth kinetics of MC38 in recipients vaccinated with the four cell subsets are depicted (d). Naïve n = 6, IEC annV-7AAD- n = 5, IEC annV+7AAD- n = 5, IECAnnV+7AAD + n = 6, IEC F/T n = 10. Mean ± SEM is depicted. e, Vaccination as in Fig. 3a but using an IEC cell suspension enriched in villi versus crypt cells. Naïve n = 5, Villi IEC OXA n = 10, Crypt IEC OXA n = 10. Mean ± SEM is depicted. f, Representative FACS dot plot of the IEC fraction from Lgr5−EGFP-IRES-creERT2 mice used for lineage-tracing of Lgr5-expressing stem cells of the small intestine. One representative sorting data out of two performed. g, Id. as in Fig. 3a but vaccination has been performed using ileal IEC composed of either LGR5+ (purity > 95%) or LGR5-. Tumor size at day 21 in 2 pooled experiments is shown, each dot representing one mouse. Naïve n = 13, LGR5+ n = 12, LGR5- n = 11. Means ± SEM are represented. Statistics: mixed-effect modeling with a specific software (Methods) for longitudinal tumor growth analysis (a, b, d, e) and Mann-Whitney U test used to compare two independent groups (after Kruskal-Wallis was implemented for multiple groups) (g). Significant two-tailed p-values are shown in the figures.
