Fig. 4: High tumor initiating potential of pPDH⁺ cells from intestinal adenomas.

a Immunofluorescence for pPDH and ChgA of intestinal adenomas from Apc^min mice. A representative image of 24 adenomas from 6 mice is shown. Scale bar, 25 μm. b Immunofluorescence for pPDH and Ki67 of intestinal adenomas from Apc^min mice. A representative image of four adenomas from two mice is shown. Scale bar, 25 μm. c MALDI-MSI experiment showing relative abundance of hexose-phosphate in pPDH+ areas compared to pPDH− areas of an intestinal adenoma (pPDH staining of a consecutive section is shown, image is representative of three independent acquired sections from a single adenoma). Bar plot represents the fold change (average log2FC) of indicated metabolites of pPDH+ compared to pPDH− areas (one sample t-test p values are shown). n corresponds to a 100 μm (10 pixels of 10 μm size) of either pPDH+ or pPDH− clusters of cells. Data are presented as mean ± SEM. Scale bars, 100 µm. d Two photon microscopy image of a full-grown organoid from a single adenoma cell infected with the Pdk1-mCherry reporter. Organoids from three different APC^min mice were infected (a representative image is shown). Scale bar, 50 μm. e qPCR analysis showing RNA expression of indicated genes on mCherry sorted cells from two APC^min-Pdk1-mCherry organoid lines (n = 2). Data are presented as mean ± SEM. f Single mCherry^low and mCherry^high cells FACS-sorted from Apc^min-derived organoids expressing the Pdk1-mCherry reporter were plated in EN medium and organoids formed counted at day 7. The bar plot shows the quantification of three independent sorting experiments of two different clones. Error bars indicate SD. g mCherry expression in a subcutaneous tumor from MC38-Pdk1-mCherry cells (a representative image of six different tumors is shown). Scale bar, 50 μm. h mCherry^high and mCherry^low sorted cells were injected at the indicated cell dilutions in the flanks of C57BL6/J mice to analyze tumor-initiating potential. i Intestinal crypts from Apc^min mice were grown in EN medium in the absence or presence of 10 mM DCA and organoids counted at day 7 (n = 4 independent experiments). Data are presented as mean ± SEM. Two tailed t-test was used to determine statistical significance between groups (p = 0.028). j Quantification of the size of the organoids from (i). Error bars represent SD (p < 0.0001). Two tailed t-test was used to determine statistical significance between groups (*p < 0.05; **p < 0.01; ***p < 0.001). Source data are provided as a Source Data file.