Fig. 4: Visualization and targeting of the OnF program in CRC.
From: Oncofetal reprogramming drives phenotypic plasticity in WNT-dependent colorectal cancer
a, Schematic of the OnF phenotypic reporter structure. PGK, phosphoglycerate kinase promoter; NEO, neomycin resistance gene. b, Experimental flow diagram (top) and gene-set enrichment in GFPhigh versus GFP− cells sorted from VAKSPOnF (Villin-Cre AKSP) tumoroids expressing the OnF reporter (bottom) (n = 3 independent sorts). c, Representative pseudocolor plots from flow cytometry of VAKSP tumoroids co-expressing OnF–GFP (y axis) and stem/STAR–mCherry (x axis) phenotypic reporters. VAKSPOnF/STAR tumoroids were treated with dimethyl sulfoxide or FOLFIRI for 3 days before analysis. d, Quantitative representation of flow cytometry data from c. In c and d, n = 3 independent experiments; error bars = s.d.; P values are by paired, two-sided Student’s t-tests. e, Heatmap reporting log2(fold-change) in WNT (left) and YAP or AP-1 (right) target gene expression (n = 3 independent experiments). f, Summary of the VAKSP tumoroid models used for functional studies. g,i, Schematic of the experimental strategy to genetically target the OnF state, stem state or both, either transiently (g) or persistently (i), relevant to h–j. h–j, Growth rate of subcutaneous VAKSP tumors with indicated reporter combinations, in response to DT treatment. Mean tumor volume ± s.e.m. The dotted lines indicate saline treatment (all models, n = 6 tumors). h, Dashed lines indicate transient DT treatment (5 d; 3 doses on alternate days): OnFDTR (n = 11), STARDTR (n = 9), OnFDTR/STARDTR (n = 10). j, The solid lines indicate persistent DT treatment, on alternate days, throughout the experiment: OnFDTR (n = 12), STARDTR (n = 11), OnFDTR/STARDTR (n = 9). Bottom, DT dosing schedule; the dashed red line indicates treatment duration. k, Schematic of the experimental strategy used in l. l, Dotted line, vehicle (n = 8 tumors); solid lines, DT (n = 6), FOLFIRI (n = 7) and DT + FOLFIRI (n = 8). Mice received three doses per week on alternate days. Values are mean tumor volume ± s.e.m. In h, j and l, the P values were calculated using a mixed-effects linear model with Tukey’s adjustment for multiple comparisons (two sided). m–p, Heatmaps showing percentage viability of VAKSP tumoroids with the indicated drug combinations. A single dose of MRTX1133 and IAG933 was used in n and p, respectively. The effects of these single doses of each drug are highlighted in m (MRTX1133) and o (IAG933), respectively. IC, inhibitory concentration. q, Dot plot indicating the combination index from drug combinations in m–p. The P values were calculated using two-sided, paired Student’s t-tests. In m–q, n = 3 independent experiments. Illustrations in b, g, i and k created using BioRender.com.
