Organoids from primary human prostate cancer exhibit heterogeneity in the states of tumor-associated epithelial cells

Prostate cancer (PCa) is the most commonly diagnosed malignant tumour in men and the second leading cause of cancer-related mortality worldwide. Despite the high long-term survival rates in localised PCa, metastatic disease remains largely incurable even after intensive multimodal therapy. Our limited understanding of the drivers of aggressive behaviour in advanced PCa and the molecular pathways that underlie their failure to respond to various drug therapies underscores the need to study tumours in vitro so that more effective therapies can be evaluated. Tumour-derived organoids represent an advanced in vitro model for studying cancer and are increasingly being used in the development of new therapeutic strategies, serving as valuable tools for advancing precision medicine in oncology. Our goal was to characterise patient-derived organoids from our prostate samples cohort of the FMRP-USP, which accurately model the original patient tumours. Our approach involved establishing longer-term organoid cultures from biopsy samples in Matrigel. For this, we used 9 fresh prostatectomy tissue samples that were processed by mechanical dissection and enzymatic digestion, then filtered and cultured in advanced DMEM/F12 medium with supplements (GlutaMAX, B27, N-acetylcysteine, recombinant EGF, FGF-10 and FGF-2, A-83-01, SB202190, nicotinamide, DHT, PGE2, Y-27632, Noggin, and R-spondin). Nine patients were aged between 50 and 75 years and diagnosed with high-risk PCa with ISUP grade of 4 or 5 (Ethics Committee Number 88216025.3.0000.5440). The organoids were characterized using morphological 3D imaging and histological (H&E staining, IHC and IF) approaches and compared to the primary sample of the same tumor to confirm their cancer-modelling capacity and suitability for the downstream assays. We successfully established long-term organoid culture for up to 2 months. We verified through characterisation that the organoids presented a morphology and tumour immune profile similar to the tissues of origin. In summary, we have successfully established and characterised long-term patient-derived PCa organoids, which closely recapitulate the original tumours, providing a robust platform for future pre-clinical studies and precision oncology applications.