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.患者来源类器官（Patient-derived organoids, PDOs）为各类癌症的建模提供了全新机遇。然而其在前列腺癌（prostate cancer, PCa）中的应用却受限于较低的接种成功率与良性细胞过度增殖问题。针对136份样本开展的初步实验揭示了现有培养体系的局限性，后续又利用额外30份样本探究了微环境因子、碳源与细胞外基质（extracellular matrix, ECM）组成对类器官培养效果的影响。单细胞RNA测序（Single-cell RNA sequencing, scRNA-seq）结果显示，不含基质胶（Matrigel）的患者来源类器官呈现细胞异质性，能够保留携带活跃雄激素受体（AR）信号的患者特异性前列腺癌细胞群，同时富集中间性棒状细胞群。与之相反，基质胶（Matrigel）无法维持原代前列腺癌细胞，并会产生与患者样本转录组特征迥异的体外类基底良性转录组谱。此外，本研究重新定义了细胞类型特征谱，筛选出可在离体条件下区分肿瘤细胞与其余所有细胞类型的RNA与蛋白质生物标志物，并证实层粘连蛋白结合整合素的表达是基质胶来源类器官的标志性特征。最后，本研究整合已发表数据集与本团队的新增数据，构建了首个前列腺患者来源类器官单细胞图谱（Prostate PDO single-cell atlas, PPScA）。该图谱覆盖了一系列细胞身份与恶性表型，并揭示了相较于原代前列腺癌组织，患者来源类器官中普遍发生改变的信号通路。综上，本研究为该领域带来了重要进展，不仅实现了培养方法的优化，还提供了全新的细胞生物学认知。