Figure 1: Rapid establishment of genetically-defined prostate cancer with prostate organoids transplanted into immunocompetent syngeneic hosts.

a. Schematic of timeline required to establish, propagate, edit, and select for organoids harboring mutations in cancer-associated genes prior to transplantation into immunocompetent hosts for tumor establishment. b. Representative microscopy of Pten^−/−; Trp53^−/−; cMyc^T58A (PtPM) organoids, and stereoscopic and fluorescence images of orthotopic (OT) prostate tumors with prostate adenocarcinoma (PRAD) histology. Tumor images are representative of n=22 independent mice. H&E, hematoxylin and eosin. c. Top: Representative microscopy of Rb1^−/−; Trp53^−/−; cMyc^T58A (RPM) organoids, and stereoscopic and fluorescence images of OT prostate tumors and lung metastases. Bottom: Representative histological assessment of RPM-PRAD and RPM-neuroendocrine prostate cancer (NEPC) primary tumor or metastases histology at varying magnifications. Primary and metastatic histology are representative of n=25 independent mice. LN, lymph node (iliac). d. Phospho-Histone H3 (Ser10; pHH3) positive tumor cells per total tumor area (μm²). Each data point represents the average number of pHH3+ cells per individual tumor subset by tumor histology (PRAD vs NEPC) and experimental end point. PtPM-Early (<4 weeks), n=14; PtPM-Late (>6 weeks), n=8; RPM-PRAD, n=11; RPM-NEPC, n=14. Statistics derived using one-way ANOVA with Tukey’s multiple comparisons correction. Error bars denote mean and standard deviation. e. Survival of mice transplanted with the indicated cell numbers of PtPM, RPM, and Pten^−/−; Rb1^−/−; cMyc^T58A (PtRM) ex-vivo edited organoids. PtPM 1k, n=5; PtPM 250k, n=5; RPM 250k, n=14; PtRM 250k, n=8. Statistics derived from the Log-rank (Mantel-Cox) test for each pair-wise comparison. f. Metastatic disease penetrance of the indicated organoid transplants. Regional metastases include dissemination into the iliac lymph nodes. Distal metastases include dissemination to kidney, pancreas, liver, or lungs. Statistics derived from two-sided Fisher’s exact test. Number of biological replicates indicated within the figure. Scale bars indicated within each figure panel.