Presentation Authors: Fabrice Lucien-Matteoni*, Rochester, MN, Janice Gomes, Harmenjit Brar, London, Canada, Matthew Lowerison, Mario Cepeda, Vidhu Joshi, Yohan Kim, Paras Shah, Rochester, MN, Stephen Pautler, Nicholas Power, London, Canada, Haidong Dong, Stephen Boorjian, Bradley Leibovich, Rochester, MN
Introduction: Next-generation biomarkers are emerging as valuable tools to improve cancer diagnostics, disease stratification and treatment monitoring. Our group has developed an innovative &[laquo]liquid biopsy&[raquo] based on enumeration of submicron cell fragments called extracellular vesicles (EVs) that bear tissue and cancer-specific biomarkers. This approach relies on the use of nanoscale flow cytometry (nFC) allowing high-throughput multi-parametric detection and enumeration of particles of events between 100-1000 nm in diameter. Despite the growing interest for developing EV-based blood tests, there is still an unmet need to optimize pre-analytical procedures and analytical parameters. Our team has established the standard operating procedures required for accurate detection of EVs from patient plasmas.
Methods: We utilized the A50-Micro Plus nanoscale flow cytometer (Apogee FlowSystems Inc.) to identify and measure 100-1000nm sized EVs. Silica and polystyrene beads were used to determine resolution limits of the nFC and established optimal acquisition parameters for EV enumeration. Plasmas from healthy volunteers and cancer patients were used to standardize pre-analytical conditions (plasma isolation, storage, handling) and to assess the performance of the nFC.
Results: A50-Micro Plus was capable of detecting EVs from 110 to 1000 nm in a linear manner by using light-scatter and fluorescence detection. Platelet-free plasma, storage temperature (-80C), dilution range (1/15-1/60) are critical considerations to ensure integrity and accurate enumeration of EVs. We used the standard operating procedure to enumerate prostate cancer-derived EVs in prostate cancer patients and unveiled a blood signature which identifies patients with clinically significant prostate cancers.
Conclusions: We have established a workflow to develop EV-based liquid biopsies using nanoscale flow cytometry. In prostate cancer, we have identified an EV signature that may enhance selective identification of patients with clinically significant prostate cancer and decrease unnecessary tissue biopsies in individuals with absent or low-risk disease. This technique has the potential to facilitate the development of next-generation peripheral blood tests to allow personalized treatment protocols for patients with urogenital cancers.
Source of Funding: Movember Foundation, Mayo Clinic