Fig 2: (A) Schema of modeling FPC in vivo. KRASG12D was induced for 8 weeks after orthotopic transplantation of CDKN2AWT/# iPSC-derived PDLOs in NSG mice. HE staining of identified engraftments with and without Dox treatment. Scale bar: 500 µm (overview), 100 µm (magnification); Hg lesion: high-grade lesion. The asterisk marks a glandular region, the pound sign a highly atypical and dysplastic region of the invasive PDAC-like tumor graft. (B) Summary of engraftment experiments. Left: Take rate was determined by identified engraftments per transplanted mice. Reporter expression was analyzed by mCherry and HA-tag IHC staining, and grafts were classified into normal ducts, low-grade lesions (non found), high-grade lesions, and PDAC-like tumors by the observed degree of atypia, dysplasia, and invasiveness. Right: Semi-quantitative marker expression: Each engraftment was classified into no (–), weak (+), moderate (++), or strong (+++) expression. The mean expression across the engraftments of one group is depicted. (C) CDKN2AWT/# PDLO grafts without oncogene induction formed epithelial duct-like tissue (KRT19, KRT7). Some EMT marker expression (VIM, N-CAD) was accompanied by moderate expression of CA19-9. MUC5AC was only expressed in one graft. (D) Two CDKN2AWT/# PDLO grafts 8 weeks after oncogene induction. The heterogeneous HA-tag expression in Hg lesion 1 correlated with increased dissemination and EMT, e.g., indicated by HA-tag and VIM staining (regions marked by arrows). This graft also strongly upregulated MUC5AC and CA19-9. The second invasive tumor graft (PDAC 1) with high levels of HA-tag expression was partially dedifferentiated (KRT19, KRT7, N-CAD) and only expressed CA19-9 and MUC5AC in some regions. All scale bars: 100 µm, if not stated differently.

Fig 3: TGF-ß augments KRASG12D-induced phenotypes in ACs and DUs(A) Expression of the cell proliferation marker Ki67 (red) in DUs (blue) and ACs (orange) with KRASG12D expression and TGF-ß treatment. Quantification of Ki67-positive organoids represented by box-and-whisker plots: range, 5%–95%; center lines indicate median values; gray dots represent individual measurement.(B and C) Organoid morphological changes induced by KRASG12D expression and TGF-ß treatment. Bar charts represent the mean and standard deviation of measurements from three independent cultures; gray dots represent individual measurements.(D) Organoid size changes induced by KRASG12D expression and TGF-ß treatment. Box-and-whisker plot representation as in (A).(E and F) Expression of pancreatic lineage markers in DUs (E) and ACs (F) altered by KRASG12D expression and TGF-ß treatment. Floating column charts represent RNA measurements from quantitative PCR (n = 3), hinges represent maximal and minimal values, central lines indicate mean values, and dots represent individual measurements.(G–J) Detection of KRT7 (G), AB (H), MUC2 (I), and MUC5AC (J) in organoids under different experimental conditions. Bar chart representation as in (C).Scale bars in main images, 50 µm; scale bars in insets, 25 µm. ***p < 0.001; **, 0.001 < p < 0.01; *, 0. 01 < p < 0.05; N.S., p > 0.05. All results represent the sum of three independent cultures.

Fig 4: Fabrication of epithelial sheets from hiPSC/SEAM-derived BST2+ cells(A) Schematic of the method of generating epithelial sheets from the cells.(B) Phase-contrast images of the sheet fabricated from sorted BST2+ (P1) and BST2- (P2) cells. n = three independent experiments. Scale bars; 100 µm.(C) Immunostaining of MUC5AC, K13, and K12 (green) in the sheet derived from P1–P2 cells. n = three independent experiments. Nuclei, red. Scale bars; 25 µm.(D) Gene expression analysis of ocular surface epithelial cell markers for sorted cells from P1–P2 (FACS). n = 7. *p < 0.05. Error bars show the standard deviation.(E) Gene expression analysis of ocular surface epithelial cell markers for the sheet derived from P1–P2 (Sheet). n = 7. *p < 0.05. Error bars show the standard deviation. See also Figure S4.

Fig 5: The GNAS mutant shows tropism for ductal-lineage epithelia(A) Immunoblot analysis for GNAS expression and phosphorylation of GNAS downstream effectors in DUs and ACs (n = 3, independent cultures).(B) Phase-contrast images of DUs and ACs expressing wild-type (WT) GNAS and GNASR201C. (-)DOX, without doxycline treatment; (+)DOX, with doxycycline treatment. Scale bar, 100 µm.(C) Change in size, measured as the total area of organoids expressing GNASWT or GNASR201C. Box-and-whisker plot, range 5%–95%; central lines, median values (n > 100, combination of three independent cultures).(D) Percentage of organoids surpassing the 99th percentile of control organoid (no transgene expression) size (n = 3 independent cultures).(E) Organoid cell number changes induced by GNASWT and GNASR201C as detected by CellTiter. Bar charts represent the mean and standard deviation of measurements from three independent cultures; gray dots represent individual measurements.(F) Immunofluorescent staining of polarity proteins in organoids without (-DOX) and with (+DOX) GNASR201C expression. ZO1, red; collagen IV, green; DAPI, blue. Scale bar, 100 µm.(G) Expression of SOX9 in organoids without (-DOX) and with (+DOX) GNASR201C expression. SOX9, red; DAPI, blue.(H) AB (pH 2.5) staining of organoids without (-DOX) and with (+DOX) GNASR201C expression. Scale bar, 100 µm.(I) Expression of E-cadherin (green) and MUC2 (red) in DUs without (-DOX) and with (+DOX) GNASR201C expression. Scale bar, 50 µm.(J) Percentages of DUs expressing MUC2 (top chart) or MUC5AC (bottom chart). Bar charts represent the mean and standard deviation of measurements from three independent cultures; gray dots represent individual measurements.(K and L) RNA expression of lineage markers in DUs (K) and ACs (L) altered by GNASR01C expression. Floating column charts represent RNA measurements from quantitative PCR (n = 3 technical repeats), hinges represent maximal and minimal values, central lines indicate mean values, and dots represent individual measurements. ***p < 0.001; **, 0.001 < p < 0.01; *, 0. 01 < p < 0.05; N.S., p > 0.05.