Large Extracellular Vesicle Characterization and Association with Circulating Tumor Cells in Metastatic Castrate Resistant Prostate Cancer

Abstract

Liquid biopsies hold potential as minimally invasive sources of tumor biomarkers for diagnosis, prognosis, therapy prediction or disease monitoring. We present an approach for parallel single-object identification of circulating tumor cells (CTCs) and tumor-derived large extracellular vesicles (LEVs) based on automated high-resolution immunofluorescence followed by downstream multiplexed protein profiling. Identification of LEVs >6 µm in size and CTC enumeration was highly correlated, with LEVs being 1.9 times as frequent as CTCs, and additional LEVs were identified in 73% of CTC-negative liquid biopsy samples from metastatic castrate resistant prostate cancer. Imaging mass cytometry (IMC) revealed that 49% of cytokeratin (CK)-positive LEVs and CTCs were EpCAM-negative, while frequently carrying prostate cancer tumor markers including AR, PSA, and PSMA. HSPD1 was shown to be a specific biomarker for tumor derived circulating cells and LEVs. CTCs and LEVs could be discriminated based on size, morphology, DNA load and protein score but not by protein signatures. Protein profiles were overall heterogeneous, and clusters could be identified across object classes. Parallel analysis of CTCs and LEVs confers increased sensitivity for liquid biopsies and expanded specificity with downstream characterization. Combined, it raises the possibility of a more comprehensive assessment of the disease state for precise diagnosis and monitoring.

Molecular Characterization of Prostate Cancers in the Precision Medicine Era

“…In addition, they are also indicators of therapy response in many cancer types, including PCa [ 161 ]. Proteomic studies performed in PCa cell lines and patients showed that EVs are a source of intracellular proteins and may be useful to improve PCa diagnosis [ 162 ]. Currently, the major limitation in the use of EVs in clinical practice is the lack of a standardized method for the detection and isolation of EVs, due to their small sizes and low densities.…”

“…The selection of the isolation method to use is related to the downstream application. Recently, a new method based on imaging mass cytometry has been proposed to study and characterize large EVs in parallel to CTCs in metastatic PCa through a multiplexed protein profiling [ 162 ]. However, this is a preliminary approach that needs further investigation.…”

Cell-Secreted Vesicles: Novel Opportunities in Cancer Diagnosis, Monitoring and Treatment

“…Levels of LEV in the blood are associated with tumor load and bone marrow involvement in AVPC. The association of LEV levels with the CTC count improves the assessment of disease state [143]. In another recent study, the lipid profile of the urinary EVs of patients with stage 2 and 3 prostate cancer showed five metabolites (three ceramides, one glycerophospholipid and one acyl carnitine) that were significantly different between the two stages.…”

Extracellular Vesicles in Advanced Prostate Cancer: Tools to Predict and Thwart Therapeutic Resistance

“…A recent study described the possibility to purify circulating tumor cells and LOs in order to confer a higher sensitivity to the liquid biopsy assay in the case of CRPC. CTCs and LEVs could be discriminated based on size, morphology and DNA content [124].…”