Figure 8: Loss of Ascl1 in established NEPC results in modest tumor control and increased tumor heterogeneity.

a. Schematic of Ascl1 doxycycline (dox)-inducible in vivo platform. RPM-Ascl1^KO organoids infected with inducible mScarlet (Ctrl) or Ascl1-P2A-mScarlet (Ascl1) vectors were transplanted OT into mice fed dox-chow (primary recipient host, 1°). Tumor volume was monitored by ultrasound. Upon primary tumor establishment, mice were randomized into dox ON (maintained) or dox OFF (withdrawal) groups. b. Survival curves of Ctrl or Ascl1 induced OT tumors following dox-maintenance (ON groups) or withdrawal groups (OFF groups). Statistics derived from log-rank (Mantel-Cox) test comparing primary Ascl1 ON to primary Ascl1 OFF groups. Ctrl ON n=7, Ctrl OFF n=8, Ascl1 ON n=11, Ascl1 OFF n=13 independent mice. c. Schematic of SQ Ascl1 doxycycline (dox)-inducible in vivo platform (secondary recipient host, 2°). Ascl1 ON primary tumors were dissociated for flow cytometry to enrich for RPM-NEPC cells used for transplantation assays into the flanks of secondary recipient mice fed dox-chow. Tumor volume was monitored by caliper. Upon tumor establishment, mice were randomized into dox ON (maintained) or dox OFF (withdrawal) groups. d. Survival curves of Ctrl or Ascl1 induced secondary tumors following dox-maintenance (ON groups) or withdrawal groups (OFF). Stats derived from Log-rank (Mantel-Cox) test. Ascl1 ON n=5, Ascl1 OFF n=7 independent mice per group. e. Serial sections from secondary transplanted mice (SQ) stained for the indicated markers by H&E and IHC. f. Representative NEUROD1 IHC within a secondary transplant containing mostly NEPC histology. Data representative of n=5 independent tumors. g. (Left) Average percent marker positive nuclei or (right) cells across biologically independent secondary SQ Ascl1 ON (n=5) or OFF (n=4) tumors. Statistics derived from two-sided t-test. Error bars indicate mean and standard deviation. All scale bars depicted in the figure panels.