Extended Data Fig. 2: Immunogenic chemotherapy triggers putative cancer stem cell (CSC) appearance.
From: Type I IFNs promote cancer cell stemness by triggering the epigenetic regulator KDM1B
(a) Schematic representation of the ‘donor’-‘receiving’ cell coculture experimental protocol. (b) Flow cytometry analysis showing the induction of cell death upon oxaliplatin treatment (OXP, 300 µM, 24 h) in MCA205 cells with the illustrated genetic background. Data are presented as mean ± s.e.m. and individual data points, n = 3 independent experiments. (c) Multiparametric flow cytometry analysis of CSC surface markers in MCA205 cells treated with OXP alone or combined with the AIM2 inhibitor thalidomide (AIM2 inh, 10 µg ml-1) or inhibitors of the RIG-I pathway amlexanox (RIG-I inh#1, 5 µM), BX795 (RIG-I inh#2, 100 nM) and MRT67307 (RIG-I inh#3, 500 nM). The histograms represent the percentage (mean ± s.e.m. and individual data points; the number of independent experiments) of CD133+CD24+CD44+high (CD44H) and CD133+CD24+CD44+low (CD44L) cells. (d) Flow cytometry analysis of doxorubicin (DOX) efflux ability in MCA205 cells left untreated (gray) or exposed to DOX (2.5 μM, 48 h). The two DOXlow (orange) and DOXhigh (red) cell subsets display high and low capability to efflux DOX and Hoechst 33342 (one representative experiment out of three independent experiments). (e) Representative pictures of FACS-isolated DOX+low and DOX+high cells in standard culture conditions and under treatment with different chemotherapeutics (DOX+low cells). MCA205 cells were firstly treated with 2.5 μM DOX for 48 h, and then FACS-isolated based on their low or high positivity for red fluorescence. DOX+low and DOX+high sorted cells were then left untreated (control, CTR) or treated with OXP (30 μM), DOX (2.5 μM) or mitoxantrone (MTX, 0.04 μM) for 48 h. Representative pictures from one representative experiment out of two yielding similar results of CTR, DOX+high and treated DOX+low cells are shown. The percentage of counted cells is indicated for each condition, as determined by cell counts on pictures using ImageJ software. Scale bar, 100 μm. (f) Ex vivo flow cytometric analysis of the percentage of NANOG+ MCA205 cells grown in C57Bl/6 J mice treated intratumorally with vehicle (PBS) or 2.9 mg/kg DOX or 2.5 mg/kg cisplatin (CDDP). Data are presented as mean FC ± s.e.m. and individual data points over PBS treatment for 10 mice/group from 2 experimental replicates. (b,c,f) Ordinary one-way ANOVA test followed by Bonferroni’s correction.
