Prostate Organoid Culture and Xenograft Modeling

The lobes of the prostate were isolated from TMPRSS2^{Cre‐ERT2‐IRES‐GFP}; Pten^fl/fl mice, minced into small pieces (∼1 mm³), digested at 37°C on a shaking platform and sorted by GFP expression to isolate prostate luminal cells. Cells were resuspended in phosphate buffered saline (PBS) mixed with Matrigel (BD Biosciences, volume ratio = 1:1), and the suspensions were plated into 24‐well plates covered with prostate organoid culture. Afimoxifene (4‐OHT, Sigma–Aldrich, H7904) was added to the culture to induce CreERT2 and Pten deletion. Then, the packaged lentivirus was added to the organoids, and infected cells were selected by puromycin. The numbers, size and formation efficiency of the organoids were determined on day 10. Urogenital sinus mesenchyme (UGSM) cell isolation and the organoid transplantation assay were performed as previously described.¹⁷ A total of 1×10⁶ UGSM cells were mixed with 1×10⁶ organoid cells stably expressing luciferase and then used to generate xenografts. The cells were resuspended in Matrigel (BD Biosciences, 1:1) and then injected into the VPs of 6‐week‐old male NSG mice. Two months later, a NightOWL II LB 983 Imaging System (Berthold Technologies) was used to conduct BLI and determine the tumour volumes in mice.
Biopsy specimens from PCa patients were minced into small pieces (∼1 mm³) and digested at 37°C on a shaking platform in 5 ml of 5 mg/ml type II collagenase (Invitrogen) in Advanced DMEM/F12 (ADMEM/F12) for 2 h. Dissociated cells were washed and resuspended in PBS mixed with Matrigel (BD Biosciences, volume ratio = 1:1), and the suspensions were plated into 24‐well plates covered with prostate organoid culture. Human PCa organoids were weekly passaged at a 1:3 passage ratio, digested by TrypLE (Sigma–Aldrich) at 37°C for 5 min. The organoids were cultured and collected for Western blotting (WB). The reagents used for the organoid culture are listed in the Supporting information.