Abstract 253: Investigating key molecular players in putative stem cell subpopulations of prostate cancer

Abstract Background: Androgen deprivation therapy is the standard treatment for prostate cancer (PCa). Nevertheless, despite initial effectiveness, pre-existing cancer stem cell (CSC) populations invariably lead to incurable castration-resistant prostate cancer. CSCs are a subset of cancer cells possessing self-renewal properties, driving tumor progression and regrowth. CD44+ PCa cells exhibit more stemness features and are enriched in tumorigenic and metastatic progenitor cells. Here, we aim to explore different subpopulations in tumors based on levels of CD44 expression and investigate whether they have distinct molecular properties and functional characteristics. Material and Method: The number of CD44+ cells was evaluated in a tissue microarray from primary prostate cancer patient samples (EMPACT cohort) with clinical follow-up. Flow cytometry sorted the CRPC model LAPC9 tumor into CD44 high (CD44-H) and CD44 low (CD44-L) cells. RNA sequencing was performed on these subpopulations to explore their transcriptomic profiles. CD44 expression was validated at the cDNA and protein levels using qPCR and Western blot (WB) analysis. Sorted cells were maintained as organoids in vitro. To trace the dynamics of CD44-H and CD44-L subpopulations in vivo, LAPC9 tumor was labeled by fluorescent and luciferent markers. The CD44 status was determined by flow cytometry when tumor growth. Results: Patients with elevated expression of CD44 (higher number of CD44+ cells) at the time of surgery exhibited a greater propensity for clinic progression (5-year progression-free survival: 75% vs 95%, P=0.03). Furthermore, CD44-H cell ratio increased in the LAPC9 after castration, indicating that CD44-H cells can survive after treatment. Subsequent WB analysis confirmed that CD44 was highly expressed in sorted CD44-H cells, validating the flow cytometry results. Analysis of the CD44 RNA-seq data showed CD44 transcript variants CD44v10 (CD44-201) and CD44v7-10 (CD44-209) were notably upregulated in CD44-H cells. In organoid culture, sorted CD44-H cells displayed enhanced formation, indicating they possessed higher proliferative capacity and clonogenicity than CD44-L cells. Furthermore, when GFP-labeled CD44-H cells and RFP-labeled CD44-L cells were recombined in vivo, CD44-H cells exhibited robust proliferation, while CD44-L cells transitioned to a CD44hi state to facilitate tumor growth. Moreover, those GFP and RFP subpopulations displayed the same CD44hi cell ratio at the endpoint. Conclusion: Elevated CD44 expression is related to an increased risk in primary PCa. In addition, CD44-H cells exhibit high tumorigenicity in vitro and in vivo and can convert to each other. Moreover, the ratio of CD44-H cells in tumor at endpoint is consistent despite the starting CD44 expression status. Furthermore, the upregulation of CD44v10 and v7-10 in the CD44-H subpopulation may promote tumor formation and metastasis. Citation Format: Wanli Cheng, Marta De Menna, Francesco Bonollo, Panagiotis Chouvardas, George N. Thalmann, Sofia Karkampouna, Marianna Kruithof Julio. Investigating key molecular players in putative stem cell subpopulations of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 253.