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METHODS:

N (N kx3. 1 CreERT2, R26 LSL-YFP/LSL-YFP), NP (N kx3. 1 CreERT2; P ten flox/flox, R26 LSL-YFP/LSL-YFP) and NPK (N kx3. 1 CreERT2; P ten flox/flox; K ras LSL-G12D/+, R26 LSL-YFP/LSL-YFP) were crossed with conditional Cripto knockout (CRIPTO flox/flox) to generate NC, NPC and NPKC. N mice mirror normal epithelium, NP mimic the first stage of the disease, with high-grade prostatic intraepithelial neoplasia (PIN)/carcinoma lesions with local invasive epithelium, while NPK have invasive prostate adenocarcinoma with metastasis. In vivo experiments’ workflow is the following: castration of 8-weeks old mice and induction after 1 month with 5 daily injections of tamoxifen, after 2.5 weeks mice were treated with weekly testosterone (10 weeks). Single cells were isolated from prostate tissue and the YFP+/-population recovered by FACS sorting and cultured as organoids.

RESULTS …