Abstract 6481: Multiplexed single-cell pharmacotranscriptomics and spatial tumor biology reveals targetable reciprocal cancer-stromal interactions in prostate cancer

Abstract Prostate cancer (PCa) remains a leading cause of cancer-related deaths, with castration-resistant metastatic disease posing significant therapeutic challenges. The tumor microenvironment, particularly cancer-associated fibroblasts (CAFs), plays a crucial role in cancer development and in its progression to an aggressive and lethal stage. Previously, we identified a specific CAF subtype expressing fibroblast activation protein and platelet-derived growth factor receptor beta (FAP+/PDGFRβ+) as a strong predictor of poor patient prognosis, associated with PTEN loss and PI3K/pAKT activation in adjacent tumor cells. This study aimed to functionally characterize and pharmacologically inhibit this lethal cancer-stroma interaction phenotype. We first developed a 3D co-culture system using patient-derived primary CAFs and human PCa cells that recapitulates the lethal tumor microenvironment phenotype ex vivo and in vitro, providing robust models for functional studies. Utilizing 96-plex single-cell RNA sequencing with antibody barcoding of live cells, we analyzed the transcriptomic landscape of primary CAFs and assessed the impact of mono- and co-culture conditions on both cell types. Each patient-derived CAF model possessed a unique transcriptional profile, indicating patient-specific CAF characteristics. In 3D co-culture with PCa cells, CAFs exhibited upregulation of FAP and degradome genes, suggesting cancer-induced reprogramming of CAFs to remodel the extracellular matrix and support malignant progression. Concurrently, co-cultured CAFs induced pAKT upregulation in PCa cells, implicating stromal cells in promoting cancer cell proliferation. Hence, we functionally confirmed reciprocal cancer-stroma interactions in co-cultured models, mirroring changes seen in human tumor tissues of aggressive prostate cancers. To investigate potential therapeutic interventions, we applied a 96-plex single-cell pharmacotranscriptomic platform to evaluate drug responses using a library of 30 cancer drugs and key pathway inhibitors. Our findings indicate that dual inhibition of PI3K-AKT signaling in cancer cells and stromal signaling in CAFs could pave the way for combinatorial therapies targeting the reciprocal interactions between PCa cells and CAFs. This approach highlights the opportunity to develop precision cancer medicine strategies aimed at disrupting critical cell interactions within the tumor microenvironment in a tailored fashion. Citation Format: Astrid Murumägi, Frida Rantanen, Alice Dini, Mariliina Arjama, Katja Välimäki, Elina Multamäki, Tuomas Mirtti, Antti Rannikko, Teijo Pellinen, Daniela Ungureanu, Olli Kallioniemi. Multiplexed single-cell pharmacotranscriptomics and spatial tumor biology reveals targetable reciprocal cancer-stromal interactions in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6481.