Mouse procedures
NPp53 mice were maintained on a mixed C57BL/6-129Sv background and have been previously described7. Tamoxifen induction was performed in mice at 3–5 months of age by oral delivery of tamoxifen (MilliporeSigma; 100 mg kg–1 day–1 in corn oil) for 4 consecutive days as previously described58. The survival time of tumour-bearing mice in this study ranged from 228 to 435 days after tamoxifen induction (Supplementary Table 1). Mice were housed under specific pathogen-free conditions in individually ventilated autoclaved cages with irradiated feed and automated reverse-osmosis watering, under a 12-h dark–12-h light cycle with temperatures at 20–26 °C and humidity between 30 and 70%. All procedures followed protocols approved by the Institutional Animal Care and Use Committee (IACUC) at Columbia University Irving Medical Center.
Establishment and maintenance of mouse prostate organoids
Tumour tissues from NPp53 mice were cut into two pieces, with half fixed in 10% formalin for paraffin embedding and the other half used for organoid establishment. Tissues were minced with scissors in 0.2% collagenase IV (Thermo Fisher Scientific, 17104019) and incubated at 37 °C for 30 min, followed by neutralization with 1:10 Hank’s buffer (Stemcell Technologies, 37150) supplemented with 10 µM Y-27632 (Stemcell Technologies) and 5% charcoal-stripped fetal bovine serum (CS-FBS; Gemini, 100-119). After centrifugation at 1,000 rpm for 10 min, pellets were incubated with prewarmed TrypLE (Thermo Fisher Scientific, 12605010) at 37 °C for 10 min. The cell suspension was then neutralized 1:10 with PBS, passed through a 100-µm cell strainer (Corning, 352360) and spun down at 1,000 rpm for 10 min. Before plating, cell numbers were counted in a TC20 automated cell counter (Bio-Rad).
Cells were resuspended in organoid culture medium supplemented with 10 µM Y-27632, 10 µM A83-01 (Tocris 2939) and 5% Matrigel (Corning 354234) and plated at a seeding density of approximately 50,000 cells per well in 96-well ultralow attachment microplates (Corning 3474). Organoid culture medium consisted of hepatocyte culture medium (Corning, 355056), 5% CS-FBS, 1× GlutaMAX supplement (Thermo Fisher Scientific, 35050061), 5 ng ml–1 EGF, 100 µg ml–1 primocin (Invivogen, ant-pm-1) and 100 nM DHT, as previously described59. For heterogeneous neuroendocrine organoids, such as NPPO-1 and NPPO-5, organoid culture medium was replaced every 4 days. For maintenance of homogeneous neuroendocrine organoids, such as NPPO-1NE, NPPO-2, NPPO-4 and NPPO-6, we used neuroendocrine organoid culture medium, which was identical to the organoid culture medium except that no EGF was added; the medium was replaced every 4 days. For maintenance of non-neuroendocrine NPPO organoids (NPPO-1nonNE, NPPO-7, NPPO8 and NPPO9), we used the standard organoid culture medium. In organoid experiments involving CRISPR–Cas9-mediated targeting of Nsd2, oncohistone H3.3K36M expression or NSD2i treatment, DHT was removed from the culture medium starting at day 0, and organoids were maintained in the absence of DHT for subsequent analyses.
For passaging, organoids were collected by centrifugation at 1,000 rpm for 1 min, followed by the addition of 1 ml pre-warmed TrypLE for 10 min at 37 °C for cell dissociation. After neutralization with 10 ml PBS, cells were spun down and counted, with approximately 50,000 cells plated per well in 96-well ultralow attachment microplates (Corning, 3474). To generate cryopreserved stocks, organoids were frozen in 90% CS-FBS and 10% DMSO and stored in liquid nitrogen. We considered neuroendocrine organoid lines to be successfully established when they could be stably passaged, cryopreserved and recovered without loss of neuroendocrine phenotypes. Details regarding establishment of the TKO organoids (Extended Data Fig. 4a) from Ptenfl/fl;Rb1fl/fl;Trp53fl/fl;mT:mG (PtRP) prostate epithelial cells are described in a separate manuscript (in preparation). Organoid cultures routinely tested negative for mycoplasma contamination.
Human prostate tumour organoids
MSKPCa2, MSKPCa10, MSKPCa14, WCM154 and WCM1262 organoids have been previously described5,36,37. Human prostate tumour organoids were maintained in 80% Matrigel in human neuroendocrine culture medium, which was replaced every other day. Human neuroendocrine culture medium consisted of human hepatocyte culture medium (LifeNet Health LifeSciences, MED-HHCM-500ML), human hepatocyte culture medium supplement (LifeNet Health LifeSciences, MED-HHCMS), 5% CS-FBS, 1× GlutaMAX, 5 ng ml–1 EGF (Thermo Fisher Scientific, PHG0311), 100 µg ml–1 primocin and 10 nM DHT.
H&E staining
For tissue processing and embedding, organoids were fixed in 10% formalin (Fisher Scientific, SF100-4) for 1 h, washed once with PBS, placed in rat tail collagen I (Corning, 354249) and incubated at 37 °C for 30 min. The collagen button was then put into a biopsy cassette (Fisher Scientific, 15182705E) and fixed in 10% formalin for 24 h. After replacing the formalin with 70% ethanol, the cassettes were put into an automated tissue processor for tissue processing and embedding.
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