Next-generation liquid biopsy for circulating tumor cell detection in metastatic prostate cancer

Prostate cancer (PCa) is the most frequently diagnosed malignancy among Canadian men, and metastatic disease (mPCa) carries a poor prognosis. Liquid biopsy that analyzing circulating tumor cells (CTCs) provide a minimally invasive tool for disease assessment, yet commonly used CTC markers such as EpCAM show variable expression in PCa, especially in advanced or treatment-resistant settings. More reliable biomarkers are needed to improve CTC detection and patient stratification. We use monoclonal antibodies against STEAP1, a transmembrane protein highly overexpressed in PCa but minimally present in normal tissues, and integrated them into an imaging flow cytometry (imFC) CTC assay to detect and characterize CTCs. CTCs from localized and metastatic PCa patients were isolated using Ficoll density gradients and stained with DAPI, CD45, EpCAM, and STEAP1 antibodies. High-content images were analyzed through a computational pipeline incorporating machine-learning classification to identify and classify intact CTCs, CTC fragments, and tumor-derived extracellular vesicles (EVs) internalized by immune cells. STEAP1⁺ CTCs were detected in most metastatic patients, including individuals who lacked detectable EpCAM⁺ CTCs, highlighting the limitations of EpCAM-only assays. Patients with active disease exhibited higher STEAP1⁺ CTC counts compared with those with localized or stable disease. Additionally, STEAP1⁺ extracellular vesicles and CTC fragments were observed within CD45⁺ immune cells, suggesting broader tumor-immune interactions measurable through this platform. We further extended this workflow to a clinical study evaluating treatment-associated changes in abundance of CTC expressed with different PCa biomarkers, PSMA, STEAP1, and STEAP2 in patients receiving Pluvicto therapy. In this study, CTCs were enriched using the Parsortix microfluidic capture platform. Early results indicate that STEAP-family markers remain detectable in patients with low or fluctuating PSMA expression, supporting their value in monitoring therapeutic response. Together, these findings position STEAP1, STEAP2 and PSMA as robust biomarkers for next-generation CTC-based liquid biopsy assays and demonstrate their potential to enhance diagnostic sensitivity and treatment monitoring in mPCa.