Extended Data Fig. 2: Transplantation of 3D, but not 2D, cultured mouse organoids maintain PRAD histology and marker profiles.
From: The neuroendocrine transition in prostate cancer is dynamic and dependent on ASCL1
a. Schematic representation of steps taken to establish OT prostate tumors in mice from edited organoids grown in matrigel (3D) or monolayer culture (2D) conditions. b. Representative brightfield images of the indicated organoids seeded in monolayer growth. Images taken 5 days post seeding. Images representative of n = 2 independent experiments with similar results. c. Western blot validation of knockout efficiency 5 days post electroporation with Cas9 in complex with purified sgRNA. As in panel a, edited organoids were seeded in matrigel or monolayer culture prior to lysis and western validation. Data representative of n = 2 independent experiments with similar results. RB1, cMYC, HSP90, and P53 were blotted on a single gel. EGFP, AR, and PTEN were blotted on a second gel with an independent HSP90 blot shown in Source Extended Data. d. Representative H&E stains (low and high magnification images) of established mouse models (Hi-MYC), human prostate cancer, and organoid OT-derived prostate tumors and are representative of n = 5 tumor samples. Data related to panels a-c. OT prostate tumors derived from transplantation of organoids grown in (top) monolayer or (bottom) traditional 3D matrigel conditions. e. Representative Synaptophysin (SYP) or ASCL1 IHC of tumors isolated from mice transplanted with RP organoids grown in (top) monolayer or (bottom) traditional 3D matrigel conditions. Data representative of n = 2 tumors per stain. f. Percentage of mice with tumors per genotype and organoid growth conditions. Sample size (mice) per genotype indicated within the figure panel. g. Survival of mice OT transplanted with 250k dissociated organoids grown in matrigel. Sample size (mice) per cohort indicated within the figure legend. h. Representative H&E stains of n = 1 mouse that developed OT tumors following transplantation of PtRM organoids grown in matrigel. i. Representative phospho-histone H3 IHC stains of PtPM or RPM prostate tumors isolated 4 weeks post OT. Histological classification performed using serial sectioned H&E. Dotted line represents the boundary of PRAD and NEPC. Images related to quantification shown in Fig. 1d. j. Average percentage of KRT8+ or KRT5+ tumor cells relative to total detected cells (DAPI+ nuclei) within RPM tumors 5 weeks post OT. Data represent the average positive cell number per tumor. Data derived from n = 5 RPM OT tumors. k. Average percentage of total ASCL1+ cells relative to total detected cells (DAPI+ nuclei) within RPM OT tumors at the indicated time points. Each data point represents the average marker positive cell per mouse tumor. 2–3 weeks, n = 4; 4–5 weeks, n = 6; 6–8 weeks, n = 8; and 10 weeks, n = 11 tumors. Statistics derived using one-way ANOVA with Tukey’s multiple comparisons correction. l. Percentage of cells staining positively for nuclear ASCL1 from tumors harvest 4 to 5 weeks post OT. Data represent the average positive cell number per tumor. Data derived from n = 4 (PtPM) and n = 5 (RPM) OT tumors per group. For panels j-l, error bars indicate mean and s.d. For panels j, l statistics derived using two-sided Student's t-test. All scale bars denoted within the figure panel.
