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

The application of patient-derived organoids (PDOs) in prostate cancer (PCa) research has been hampered by poor take-rates and benign overgrowth. We highlight the limitations of existing culture conditions and identify extracellular matrix (ECM) composition as a determinant of organoid outcome. Single-cell RNA sequencing (scRNA-seq) reveals that Matrigel-free PDOs preserve patient-specific PCa cells with active AR signaling, and enrich in intermediate cells. In contrast, Matrigel fails to maintain primary PCa cells and produces in vitro basal-like cells divergent from patient samples. Furthermore, we redefine cell type-signatures, identify biomarkers discriminating tumor versus all other cell types, and show that expression of laminin-binding integrins is a hallmark of Matrigel-derived organoids. Finally, integrating previously published datasets with our data, we generate a Prostate PDO single-cell atlas (PPScA). The PPScA captures a spectrum of cellular identities, and reveals pathways universally altered in vitro. Our study provides methodological improvements for short-term cultures and cellular biology insights.