Single-cell analysis uncovers preserved prostate cancer lineages and universally altered pathways in Matrigel-free patient-derived organoids

Patient-derived organoids (PDOs) offer new opportunities to model various cancers. However, their application in prostate cancer (PCa) has been hampered by poor take-rates and overgrowth of benign cell types. Initial experiments with 136 samples highlighted the limitations of existing culture conditions, while 30 additional samples were subsequently used to explore the effects of niche factors, carbon source, and extracellular matrix (ECM) composition on organoid outcome. Single-cell RNA sequencing (scRNA-seq) reveals that Matrigel-free PDOs exhibit cellular heterogeneity and preserve patient-specific PCa cell populations with active AR signaling, while enriching in intermediate club cell populations. In contrast, Matrigel fails to maintain primary PCa cells and produces in vitro basal-like benign transcriptomic profiles that are divergent from patient samples. Furthermore, we redefine cell type-signatures, identifying RNA- and protein-based biomarkers discriminating tumor versus all other cell types ex vivo, and show that expression of laminin-binding integrins is a hallmark of Matrigel-derived organoids. Finally, integrating previously-published datasets with our new data, we generate the first Prostate PDO single-cell atlas (PPScA). The PPScA captures a spectrum of cellular identities and malignancies, while revealing pathways universally altered in PDOs as compared to primary PCa tissues. Altogether, our study represents a significant advancement in the field, providing methodological improvements and novel cellular biology insights.