Extended Data Fig. 2: Smad4 restoration in vitro and in murine liver and lung metastases, and SMAD4 expression in human metastatic recurrence.
(a) Proliferation assays of KC-shRen and KC-shSmad4 cells +/-Dox +/-TGFβ in vitro (mean + SEM; n = 3 and 4 independent cell lines per genotype, respectively). (b) Brightfield images of KC-shRen and KC-shSmad4 cells after TGFβ treatment +/-Dox for 6 days. Data represent 3 independent cell lines per genotype. (c) H&E staining of liver and lung metastases generated by intrasplenic or tail vein injections of KC-shSmad4 cell lines. Data represent 4 independent mice per organ. (d) Percent-area quantification of liver metastasis burden at day 0 of Dox withdrawal after intrasplenic injections of KC-shSmad4 cell (n = 8 independent mice). Different color shading indicates independent cell lines. (e) H&E staining of liver and lung metastases from KC-shSmad4 cells without (Smad4 OFF) or with Smad4 restoration (Smad4 ON). Data are representative of 6 independent mice per organ. (f) Schematic of orthotopic experiments with KC-shRen and KC-shSmad4 cells for analysis of metastasis burden. Created in BioRender. Tsanov, K. (2025) https://BioRender.com/yihgg0q. (g) Analysis of liver metastasis burden after orthotopic injections of KC-shRen or KC-shSmad4 cells with or without subsequent Dox withdrawal. (Left) Percent-area quantifications at endpoint (mean + SEM; n = 5, 3, 8 and 8 independent mice for groups shown, left to right). (Right) Representative macroscopic images of tumor-bearing livers. (h) Analysis of lung metastasis burden after orthotopic injections of KC-shRen or KC-shSmad4 cells with or without subsequent Dox withdrawal. (Left) Percent-area quantifications at endpoint (mean + SEM; n = 4, 5, 8 and 8 independent mice for groups shown, left to right). (Right) Representative macroscopic images of tumor-bearing lungs. Different color shading in (g-h) indicates independent cell lines, arrowheads highlight metastatic tumors, and insets show mKate2 reporter. (i) (Left) Schematic of metastasis recurrence studies in human PDAC25,26. Created in BioRender. Tsanov, K. (2025) https://BioRender.com/dt9i4jd. (Right) Fraction of patients whose tumors stained SMAD4-positive or -negative by IHC, according to site of recurrence. Exact numbers are indicated on each bar. Statistical analyses were two-way ANOVA (a), unpaired two-sided t-test (g, h), and Fisher’s exact test (i).
