IUC24411-77 In search of metastasis-initiating cells in prostate cancer

Abstract Background This study aimed to define the extent and complexity of tumor dissemination in patients with d’Amico high-risk prostate cancer (PCa), with a focus on identifying putative metastasis-initiating cells. Methods Tumor-draining vein blood (TDVB) and peripheral blood (PB) samples were collected from 118 patients with high-risk localized PCa undergoing radical prostatectomy, alongside PB from 16 patients with newly diagnosed metastatic PCa. Circulating tumor cells (CTCs) were enumerated, phenotyped, and morphologically analyzed using 2 immunofluorescence protocols: (1) epithelial/EMT protocol (pan-keratin/vimentin/DAPI/CD45/CD31/αSMA/CD29), capturing general dissemination and (2) EGFR/AR protocol (pan-keratin/EGFR/AR/DAPI/CD45/CD31), focused on identifying metastasis-related and druggable CTC phenotypes. Imaging flow cytometry enabled single-cell image acquisition. Statistical analyses were conducted in IBM SPSS Statistics, and representative images were shared via CTC Atlas. Results Over 2,500 CTCs were analyzed. The epithelial/EMT protocol defined 4 major CTC phenotypes (K+V-, K+V+, K-V+, K-V-), each exhibiting notable morphological diversity. CTCs were more abundant in TDVB, primarily of epithelial phenotype, while EMT-like, smaller CTCs were enriched in PB and associated with shorter time to biochemical recurrence. Platelet-coated epithelial CTCs in TDVB correlated with high serum TGF-β levels and disease progression, suggesting platelet-mediated modulation of CTC phenotype and metastatic potential. The EGFR/AR protocol identified a rare EGFRhigh/ARneg CTC population associated with aggressive disease. This phenotype was also observed in matched tumor tissue and bone metastases and was characterized by dedifferentiation, disrupted protein trafficking, and impaired vascularization. EGFRhigh/ARneg CTCs were enriched after androgen deprivation therapy and showed potential tropism for bone, potentially linked to tissue stiffness. Conclusions Dual CTC phenotyping reveals distinct patterns of tumor dissemination and identifies a metastasis-associated EGFRhigh/ARneg CTC phenotype with clinical relevance. These findings contribute to a deeper understanding of CTC biology and support the use of phenotypic profiling for patient stratification and treatment guidance in PCa. Funding This work was funded by National Science Center (#2017/26/D/NZ5/01088, #2020/39/B/NZ5/01258) and Ministry of Science and Higher Education, Poland (#NdS-II/SP/0398/2023/01). See www.CTCAtlas.org for related data.