AUTHOR=Damisch Elisabeth , Brunner Elena , Nommensen Lukas , Neumann Lucy , Fotakis Georgios , Trajanoski Zlatko , Sopper Sieghart , Schäfer Georg , Puhr Martin , Heidegger Isabel , Kruithof-de Julio Marianna , Sampson Natalie 

TITLE=Optimised dissociation and multimodal profiling of prostate cancer stroma reveal fibromuscular cell heterogeneity with clinical correlates

JOURNAL=Frontiers in Cell and Developmental Biology

VOLUME=Volume 13 - 2025

YEAR=2025

URL=https://www.frontiersin.org/journals/cell-and-developmental-biology/articles/10.3389/fcell.2025.1653780

DOI=10.3389/fcell.2025.1653780

ISSN=2296-634X

ABSTRACT=BackgroundDynamic remodelling of the tumour microenvironment (TME) plays a central role in prostate cancer (PCa) progression, immune evasion and therapy resistance. However, the co-existence of both tumour-promoting and tumour-restraining stromal elements necessitates extensive characterisation of the TME for effective targeting. Fibromuscular cell heterogeneity in PCa remains poorly characterised, in part due to challenges in isolating cells embedded within the desmoplastic stroma. This study therefore aimed to better characterise fibroblast and smooth muscle cell (SMC) populations as the major tissue-resident stromal cell subtypes within the PCa TME.MethodsA PCa single-cell RNA sequencing (scRNA-seq) dataset was re-analysed to define fibromuscular subtypes. Due to low fibroblast yields, an optimised tissue dissociation protocol was developed and benchmarked against two commercial kits via flow cytometry, immunostaining of clinical specimens and ex vivo culture. Dimensionality reduction and clustering were applied to the CD31− stromal fraction using a multiparameter surface marker panel. Annotation of the resulting clusters based on their surface marker profile was supported by integrating scRNA-seq and immuno-histological findings.ResultsThe optimised protocol yielded over twice the viable cells/mg tissue compared to two commercial kits, preserved surface marker integrity, enhanced successful cultivation of mesenchymal cells and recovered diverse stromal subpopulations from benign and malignant samples. Dimensionality reduction and clustering of flow cytometry counts identified 11 distinct CD31− stromal populations. Integration with transcriptomic data and immunofluorescence of clinical specimens identified spatially- and prognostically-distinct fibroblast subtypes, including inflammatory and myofibroblastic cancer-associated fibroblasts, pericytes linked to poor prognosis and a novel SMC subset associated with stromal activation.ConclusionThis study presents a robust workflow for improved isolation and characterisation of fibromuscular stromal cells in PCa. The multimodal approach enabled refined characterisation of phenotypically distinct and clinically-relevant stromal subpopulations within their spatial context providing a foundation for future TME-targeted therapies.