Skip to main content
. 2025 Apr 6;44(25):2091–2102. doi: 10.1038/s41388-025-03365-5

Fig. 2. Tankyrase and the transcription factor CDX2 drive intrinsic WNT signaling in tumoroids.

Fig. 2

a Schematic overview of the WNT pathway and WNT pathway inhibitors used in this study. Axin2 levels of KPN organoids (b) and tumoroids (c) cultured after 72 h of R-spondin withdrawal (EN) or LGK-974 (5 µM, S7143, Selleckhem, USA) treatment. Fold change (FC) relative to standard culture conditions (ENR). d Volcano plot highlighting expression of Cdx2 among transcripts differentially expressed in KPN tumoroids vs KPN organoids. KPN tumoroids represent 3 independent experiments with 2 different tumoroid lines (single cell clones) derived from different mice (N = 6). KPN organoids represent 2 independent experiments with 2 different organoid lines (bulk) derived from different mice (N = 4). e Representative IHC staining for CDX2 in KPN organoids and tumoroids. f Levels of Axin2 in KPN tumoroids after 72 h treatment with IWR-1 (10 µM, S7086, Selleckhem, USA). Fold change relative to standard culture conditions (ENR). Dots represent 3 independent experiments (N = 3). g Levels of Cdx2 in KPN organoids and KPN tumoroids, and KPN tumoroids with Cdx2 overexpression (OE). Fold change relative to expression levels in KPN organoids. Dots represent N = 6 independent experiments. h Levels of Axin2 in untransduced KPN tumoroids and tumoroids with Cdx2 OE after 72 h treatment with standard culture conditions (ENR) or IWR-1 (10 µM). Fold change relative to untransduced tumoroids cultured under standard culture conditions (ENR). Dots represent 3 independent experiments (N = 3). i Geometrical mean of WNT reporter fluorescence in KPN organoids after 48 h treatment with CHIR (5 µM) relative to untreated KPN parental cells lacking Cdx2 KO. Dots represent N = 3 independent experiments. Data analyzed with unpaired one-tailed Student’s t test. j Levels of Axin2 in untransduced KPN organoids and organoids with Cdx2 KO after 48 h treatment with standard culture conditions (ENR) or CHIR (5 µM). Fold change relative to untransduced organoids cultured under standard culture conditions (ENR). Dots represent 3 independent experiments (N = 3). Data analyzed with unpaired one-tailed Student’s t test. k Representative western blot image for β-catenin protein levels in KPN tumoroids and tumoroids with Cdx2 OE after 48 h treatment with IWR-1 (10 µM) or KPN organoids and organoids with Cdx2 KO after 48 h treatment with CHIR (5 µM). Values below image indicate the mean grayscale values of β-catenin relative to GAPDH levels from 3 independent experiments (N = 3). l GSEA of intrinsic WNT target genes (geneset derived from [61]) on pre-ranked (log2 fold change) mode for all differentially expressed genes in APN tumors (N = 3) compared to KPN tumors (N = 7) [9]. m Single cell RNA sequencing data for Lgr5 and Tacstd2 in KPN tumoroids (N = 2). n Flow Cytometry for LY6a or TROP2 expression (PE) measured on 561 nm laser. Data are mean ± s.e.m from N = 3 independent experiments, unless otherwise specified, and analyzed with unpaired two-tailed Student’s t test.