Extended Data Fig. 4: Elevated MYC activity drives adaptation to a WNT deficient environment.
From: MYC ecDNA promotes intratumour heterogeneity and plasticity in PDAC
a, Bar plot showing mean ± SD of number of passages at which organoid cultures (n = 9) passaged every week with a splitting ratio of 1:3 in -WR media reach extinction, compared to +WR media. The arrow indicates that the culture could be propagated indefinitely. b, Changes in relative expression levels of MYC of starved organoids (HSM), after culture in -WR, and +WR media for 8 h. Results shown as mean ± SD of three biological replicates (VR01, VR02, and VR23). P value was determined by Student’s two tailed t-test. GAPDH was used as a housekeeping control gene to normalise results. c, Immunoblot analysis of GFP-tag in whole cell lysate of VR01-O, VR06-O and VR23-O transduced with NTC (non-targeting control) and a GFP-tagged Myc ORF (open reading frame). GAPDH was used as loading control. d, Cell proliferation of parental (NTC) and MYC overexpressing (ORF) cultures over six passages in -WR medium. Cells were passaged weekly, and cell counts were measured at each passage. Results are expressed as the mean ± SD of three technical replicates for each condition. e, Bar plot showing the number of passages at which each organoid culture could be propagated in the presence of Wnt-C59 (100 nm, PORCN inhibitor). The arrows indicate that the culture could be propagated indefinitely (left). Representative brightfield images of parental (NTC) and MYC overexpressing cultures (ORF) cultivated in the absence (vehicle) or in the presence (Wnt-C59) of the PORCN inhibitor (right). f, Schematic representation of the experimental workflow. Created in BioRender. Corbo, V. (2025) https://BioRender.com/v05m748. g, Growth curve of MYC ecDNA+ (n = 2) and MYC ecDNA- (n = 4) organoids in -WR media. Culture growth is represented as number of domes (50 μl Matrigel/dome). h, Frequency of ecDNA+ metaphases for VR06-O cultured in +WR medium at early (n = 25) and late passages (n = 27). P value as determined by two-sided Chi-square (left). Copy number alterations on chromosome 8 (with a focus on MYC region) of VR06 late passage after few passages in depleted media (-WR). WGS coverage is displayed below the copy number level (right). i, Oncoplot displaying absence of mutations in genes involved in WNT pathway that could explain the acquisition of WR independence of WRi organoids. The arrows indicate WNT pathway genes commonly altered in cancers. j-l, Volcano plots showing differentially expressed genes between parental and WRi cultures. Upregulated genes are showed as red dots (padj < 0.05 and log2foldchange > 1). Downregulated genes are showed as blue dots (padj < 0.05 and log2foldchange < -1). P adjusted is calculated by two-sided Wald test with Benjamini-Hochberg correction. Canonical WNT target genes are indicated. m, Changes in the relative expression levels of LGR5 in WRi organoids compared to parental cultures (+WR). Results shown as mean ± SD of three technical replicates. Significance was determined by Two-way ANOVA with Tukey’s multiple comparisons test. HPRT1 was used as a control. ND, not determined. n, Bar plot showing the number of passages at which each organoid could be propagated in the presence of Wnt-C59 (left). Representative brightfield images of parental (+WR) and WRi organoids cultured in the presence of Wnt-C59 (100 nM, PORCN inhibitor) or appropriate vehicle (right).
