Fig 2: Dclk1+pancreatic ductal adenocarcinoma (PDAC) cells have remarkable spheroid- and tumor-forming potential.(A) Scheme of FACS and 3D culture of Dclk1+ and Dclk1- PDAC cells from DKPF mice. (B) FACS-sorting of PDAC cells on the basis of Epcam and Dclk1 expression in DKPF mice. (C and D) Representative images of tumor spheroids derived from sorted Dclk1- (C) and Dclk1+ (D) PDAC cells at day 7. (E) Size of spheroids were compared (mean ± SEM; Dclk1+, n = 5; Dclk1-, n = 5; n: number of mice). Statistical significance of the differences is indicated as ***p<0.001, Student’s t-test. (F) The number of spheroids larger than 20 µm were compared (mean ± SEM; Dclk1+, n = 5; Dclk1-, n = 5; n: number of mice). Statistical significance of the differences is indicated as ***p<0.001, Student’s t-test. (G and H) Tumor-forming assay of Dclk1+ or Dclk1- PDAC cells. (G) Macroscopic image of the NOD/SCID mice after subcutaneous transplantation of Dclk1+ or Dclk1- PDAC cells into the flank. A thousand of Dclk1+ PDAC cells developed subcutaneous tumors whereas Dclk1- PDAC cells did not. (H) Dilution series showed 100, 500, or 1000 Dclk1+ PDAC cells developed subcutaneous tumors in 16.7%, 25.0%, and 75.0% of NOD/SCID mice, whereas same or larger numbers of Dclk1- PDAC cells did not. Statistical significance of the differences is indicated as p<0.05, chi-squared test. (I–K) Histological analysis of primary xenografts derived from FACS-sorted Dclk1+ PDAC cells. (I) Hematoxylin and Eosin (H and E) staining. (J) Immunostaining for Krt19. (K) Immunostaining for Dclk1. Scale bar, 200 µm (I), 50 µm (J and K). (L) Increasing curve of subcutaneous tumor (mean ± SEM; Dclk1+, n = 3; Dclk1-, n = 4; n: number of mice). Statistical significance of the differences is indicated as *p<0.05, Student’s t-test. (M) Passaged tumor-forming assay of Dclk1+ or Dclk1- xenograft cells collected by FACS. A thousand of Dclk1+ xenograft cells developed subcutaneous tumors whereas Dclk1- xenograft cells did not. (N–P) Histological analysis of passaged xenografts derived from FACS-sorted Dclk1+ primary xenograft cells. (N) Hematoxylin and Eosin (H and E) staining. (O) Immunostaining for Krt19. (P) Immunostaining for Dclk1. Scale bar, 200 µm (N), 50 µm (O and P).Figure 6—source data 1.Growth of pancreatic ductal adenocarcinoma (PDAC) xenograft.Measured value of increasing curve of subcutaneous tumor derived from Dclk1+ PDACs cells sorted by FACS.

Fig 3: Analysis of murine-derived autologous pancreatic cancer organoid/immune cell co-cultures. Flow cytometric analysis quantifying the percentage of (A) zombie-positive (dead) EpCAM+PD-L1+ tumor, and (B) CD8+IFN?+ and CD8+IL-2+ cells in co-cultures. (C,D) CTL proliferation as measured by CFSE T cell proliferation assay. (E) Co-cultures were fractionated into EpCAM+, MDSC+, and CD8+ cell populations by magnetic separation. Quantitative RT-PCR was performed using RNA extracted from (F) MDSC, (G) CD8, and (H) EpCAM fractions. * p < 0.05; n = 4 individual co-cultures per group.

Fig 4: Mutant KMT2C results in enrichment of the MYC gene signature in prostate epithelial cells. a Schematic overview of the magnetic bead-based enrichment procedure of murine prostate epithelial cells. Sequence from left to right: 19-week-old mice were sacrificed and prostates were dissected. Tissue was enzymatically and mechanically dissociated to generate single cell suspensions. Cells were labelled with biotinylated anti-EpCAM antibody and retrieved from the bulk population using streptavidin-coated magnetic beads. The sorting efficiency was verified via flow cytometry. b Unsupervised hierarchical clustering and heatmap of significant differentially expressed genes between Pten?/? and Pten?/?Kmt2cSET?/? prostate epithelial cells. 5 biological replicates were included per group. Number of genes deregulated with an adj. P value < 0.05 and log2FC = 1 / = -1 are shown on the left. c HALLMARK gene sets enriched in Pten?/?Kmt2cSET?/? versus Pten?/? groups at an FDR < 0.25. Dotted lines: adj. P value = -log10(0.05). d Gene expression levels of Myc based on normalized counts from RNA-Seq analysis of Pten?/? and Pten?/?Kmt2cSET?/? prostate epithelial cells. Individual biological replicates are shown. Data are plotted as mean ± standard deviation, and the P value was determined by unpaired two-tailed Student’s t-test. e–h fGSEA plots of Pten?/?Kmt2cSET?/? versus Pten?/? groups showing an enrichment of MYC target genes (HALLMARK_MYC_TARGETS_V1) (e), genes involved in EMT (HALLMARK_EPITHELIAL_MESENCHYMAL_TRANSITION) (f), genes upregulated in metastasis of solid tumours (RAMASWAMY_METASTASIS_UP) (g) and genes upregulated in prostate cancer lymph node metastasis versus primary prostate cancer (PCa LN Metastasis UP, see also Supplementary Materials and Methods) (h)

Fig 5: Human gastric organoid/macrophage co-cultures.A Acridine orange assay using gastric organoids treated with either vehicle or histamine. B Shift in F458 (red)/F488 (green) fluorescence ratio was measured in the organoid cultures treated with vehicle or histamine. C Quantitative RT-PCR measuring changes in Shh expression in the gastric organoids treated with vehicle (control) or histamine (Hist). *P < 0.05 compared to control, n = 4 cultures generated from four individual patients. D Bright-field micrographs showing tissue-derived human fundic gastric organoids (huFGOs), PBMC-derived macrophages, and representative co-culture. E Magnetic separation was used to isolate epithelial and immune cells from treated human gastric organoid/macrophage co-cultures. F RT-PCR demonstrating expression of Shh in EpCAM positive and CD68 in macrophage fractions. Data are shown as the mean ± SEM.