Fig 2: Wnt-EarlyEx and Wnt1-LateEx tumors share features with different normal mammary cell types. (A) Cd49f/Epcam FACS profile of a representative Wnt1-EarlyEx tumor. (B) FACS population frequencies of Wnt1-EarlyEx tumors as measured from four independent primary tumors. Each measurement is represented as a cross. The error bars represent 1 s.d. (C) Cd49f/Epcam FACS profile of a representative Wnt1-LateEx tumor. (D) FACS population frequencies of Wnt1-LateEx tumors as measured from four independent primary tumors. Each measurement is represented as a cross. The error bars represent 1 s.d. (E) First two principle components of FACS-sorted Wnt1 tumor gene expression profiles.

Fig 3: Both Wnt1-LateEx tumor subpopulations have tumor-initiating potential. (A) Limiting dilution cell transplantation assay. Three primary tumors (two Wnt1-EarlyEx and one Wnt1-LateEx) were transplanted into five 6- to 8-week-old wild-type FVB female mice at each cell concentration and monitored for tumor growth over 120 days. (B) Cd49f/Epcam FACS profile of a representative Wnt1-LateEx CD49fpos/Epcamneg passaged tumor. (C) FACS population frequencies of Wnt1-LateEx CD49fpos/Epcamneg passaged tumors as measured from four independent transplanted tumors. Each measurement is represented as a cross. The error bars represent 1 s.d. (D) Cd49f/Epcam FACS profile of a representative Wnt1-LateEx CD49fpos/Epcampos passaged tumor. (E) FACS population frequencies of Wnt1-LateEx CD49fpos/Epcampos passaged tumors as measured from three independent transplanted tumors. Each measurement is represented as a cross. The error bars represent 1 s.d.

Fig 4: Ablating Lgr5+ stromal cells disrupts prostate homeostasis(A) Schematic illustration of experimental design.(B) Dot graphs show means ± SD of percentage of EGFP+ cells in Lin-CD49f-Sca-1+ proximal prostatic stromal cells of Lgr5-DTR-EGFP mice at day 3, day 9, week 3, and week 16 after treatment with diphtheria toxin (DT). Each dot represents the result from one mouse.(C) Dot graphs show means ± SD of AP weight normalized by body weight at day 9, week 3, and week 16 after DT treatment. Each dot represents the result from one mouse.(D) Transillumination images of the AP of Lgr5-DTR-EGFP and control mice at day 9 after DT treatment. Scale bars, 2 mm.(E) H&E staining of the AP of DT-treated Lgr5-DTR-EGFP and control mice at day 9 after DT treatment. Scale bars, 50 µm.(F) Dot graphs show means ± SD of total cell number of the AP of Lgr5-DTR-EGFP and control mice at day 9 after DT treatment. Each dot represents the result from one mouse.(G) Co-immunostaining of Ki67/Krt8/Krt5 in the AP of Lgr5-DTR-EGFP and control mice at day 3 after DT treatment. Scale bars, 50 µm.(H and I) Dot graphs show means ± SD of percentage of Ki67+ cells in Krt5+ prostatic basal cells (H) and Krt5- prostatic luminal cells (I) of the AP of Lgr5-DTR-EGFP and control mice at day 3, day 5, day 9, week 3, and week 16 after DT treatment. Each dot represents the result from one mouse.(J) Co-immunostaining of Krt5/Krt8/cleaved casp 3 in the AP of Lgr5-DTR-EGFP and control mice at day 3, day 5, day 9, week 3, and week 16 after DT treatment. Scale bars, 50 µm.(K) Co-immunostaining of aSMA/TH (tyrosine hydroxylase) in the AP of Lgr5-DTR-EGFP and control mice at day 3, day 5, day 9, week 3, and week 16 after DT treatment. Scale bars, 50µm.(L) Dot graphs show means ± SD of TH staining area normalized by prostatic stromal cell number in the AP of Lgr5-DTR-EGFP and control mice at day 3, day 5, day 9, week 3, and week 16 after DT treatment. Each dot represents result from one mouse.(M) Co-immunostaining of Krt8/Krt5 in the AP of Lgr5-DTR-EGFP and control mice at day 9 after DT treatment. Scale bars, 50µm. Pie graphs show percentage of Krt5+/Krt8- (red), Krt5+/Krt8+ (yellow) and Krt5-/Krt8+ (green) epithelial cells.(N) Bar graphs show percentage of Krt5+/Krt8- (red), Krt5+/Krt8+ (yellow) and Krt5-/Krt8+ (green) epithelial cells in the AP of Lgr5-DTR-EGFP and control mice at day 3, day 5, day 9, week 3, and week 16 after DT treatment.

Fig 5: Lgr5+ stromal cells are localized at the proximal ducts of mouse prostate(A) FACS plots of EGFP+ cells in Lin-CD49f+Sca-1+ basal cells, Lin-CD49f-Sca-1+ stromal cells, Lin-CD49fLowSca-1- distal (nonproximal) luminal cells, and Lin-CD49fLowSca-1+ proximal luminal cells of anterior (AP), dorsolateral (DLP), and ventral (VP) prostate lobes of 10-week-old adult Lgr5-DTR-EGFP mice. Dot graphs show means ± SD of the percentage of EGFP+ cells in basal (Ba), luminal (Lu), and stromal (Str) cells of each prostatic lobe from 4 independent experiments.(B) Schematic illustration of Lgr5-CreERT2;R26-LSL-EYFP (Lgr5-EYFP) mouse model and experimental design. Tmx, tamoxifen (5 mg/40 kg/day).(C) Co-immunostaining of EYFP/Krt5, EYFP/aSMA and EYFP/Vimentin in proximal and distal ducts of the AP of 10-week-old Lgr5-EYFP mice after tamoxifen treatment. Scale bars, 25 µm.(D) FACS plots of EGFP+ and tdTomato+ cells in Lin-CD49f+CD24Low basal cells, Lin-CD49fLowCD24+ luminal cells, and Lin-CD49f-CD24- stromal cells in the proximal APs of 10-week-old Pdgfrß-CreERT2;R26-LSL-tdTomato;Lgr5-DTR-EGFP (Pdgfrß-tdT;LDTR) and NG2-CreERT2;R26-LSL-tdTomato;Lgr5-DTR-EGFP (NG2-tdT;LDTR) mice. Pie graphs show percentage of EGFP+/tdTomato- (green), EGFP+/tdTomato+ (yellow), EGFP-/tdTomato+ (red), and EGFP-/tdTomato- (grey) stromal cells.(E) Representative image of OTLS three-dimensional (3D) microcopy of EYFP+ Lgr5-expressing cells in the AP of 10-week-old Lgr5-EYFP mice after tamoxifen treatment. Green: EYFP; red: TO-PRO-3 nuclear staining.(F) Quantification of EYFP+ Lgr5-expressing cell number along proximal-distal axis of AP lobe in OTLS 3D microcopy.(G) Representative image of OTLS 3D microcopy of EYFP+ Lgr5-expressing cells in the prostate and urethra of 10-week-old Lgr5-EYFP mice after tamoxifen treatment. Green: EYFP; red: TO-PRO-3 nuclear staining.