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252 Background: In spite of significant outcome gains in recurrent and de novo metastatic PC, resistance inevitably develops to androgen deprivation (ADT) and chemotherapy. Resistance is supported by clonal heterogeneity due to complex genomic and epigenetic alterations within PC foci in the primary and metastases (mets) and by the enrichment of residual tumor foci with PC SCs that survive therapy. PCSCs maintain self-renewal capacity, adapt over time to different microenvironments and retain tumor propagating activity leading to castration resistant (CR) PC progression. To study these processes, we generated patient SC-derived PC Org from primary and mets specimens. Methods: We isolated cells from normal epithelium and PC foci of 4 histopathologically mapped radical prostatectomy specimens and 2 CRPC mets. We developed a 3D culture system utilizing single tumor initiating cells expressing CD29hi/CD49bhi/CD44hiand co-cultured with epithelial/mesenchymal growth factors. Org growth peaked at 2 weeks, and Org could be passaged up to 6 months without significant deviation from early passages. We evaluated Org morphology, molecular markers and biologic response (proliferation, viability and secondary Org-forming potential) in response to ADT, docetaxel (Doc), abiraterone (Abi), enzalutamide (E), BEZ325,and a BMI-1 inhibitor. Results: The Org, ~6 per region, mirror original foci in morphology (well-organized glandular structure with central E-Cad+ cells of variable nuclear-cytoplasmic ratios, AR and PSA expression, peripheral Vimentin+cells, surrounding basement membrane) and in molecular alterations (TMRSS2-Ets fusions/Erg-overexpression, PTEN loss, RB, CHD1, BMI-1, AR-Vs). ADT decreased growth and Org forming potential. ADT plus Doc, Abi or E further reduced viability and Org-forming potential; ADT plus BEZ325 only reduced viability. The BMI-1 inhibitor selectively blocked self-renewal and restored Doc growth inhibition of a Doc-resistant CRPC-derived Org. Conclusions: We established a personalized SC-derived PC Org system to study in real time the potential efficacy of therapeutic alternatives, as well as mechanisms of resistance.