Nanoscale flow cytometry to distinguish subpopulations of prostate extracellular vesicles in patient plasma.

OBJECTIVE: To determine if prostate-derived extracellular vesicles (EVs) present in patient plasma samples are of exocytotic origin (exosomes) or released by the cell membrane (microparticles/microvesicles). Both malignant and normal prostate cells release two types of EVs into the circulation, exosomes, and microparticles/microvesicles which differ in size, origin, and mode of release. Determining what proportion of prostate-derived EVs are of exosomal versus microparticle/microvesicle EV subtype is of potential diagnostic significance. MATERIALS AND METHODS: Multi-parametric analytical platforms such as nanoscale flow cytometry (nFC) were used to analyze prostate derived extracellular vesicles. Plasmas from prostate cancer (PCa) patient plasmas representing benign prostatic hyperplasia (BPH), low grade prostate cancer (Gleason Score 3 + 3) and high grade prostate cancer (Gleason Score ≥4 + 4) were analyzed for various exosome markers (CD9, CD63, CD81) and a prostate specific tissue marker (prostate specific membrane antigen/PSMA). RESULTS: By using nanoscale flow cytometry, we determine that prostate derived EVs are primarily of cell membrane origin, microparticles/microvesicles, and not all PSMA expressing EVs co-express exosomal markers such as CD9, CD63, and CD81. CD9 was the most abundant exosomal marker on prostate derived EVs (12-19%). There was no trend observed in terms of more PSMA + CD9 or PSMA + CD63 co-expressing EVs versus increasing grade of prostate cancer. CONCLUSION: The majority of prostate derived EVs present in plasmas are from the cell membrane as evidenced by their size and most importantly, lack of co-expression of exosomal markers such as CD9/CD63/CD81. In fact, CD81 was not present on any prostate derived EVs in patient plasmas whereas CD9 was present on a minority of prostate derived EVs. The addition of an exosomal marker for detection of prostate-derived EVs does not provide greater clarity in distinguishing EVs released by the prostate.

Immunoaffinity based methods are superior to kits for purification of prostate derived extracellular vesicles from plasma samples.

BACKGROUND: The ability to isolate extracellular vesicles (EVs) such as exosomes or microparticles is an important method that is currently not standardized. While commercially available kits offer purification of EVs from biofluids, such purified EV samples will also contain non-EV entities such as soluble protein and nucleic acids that could confound subsequent experimentation. Ideally, only EVs would be isolated and no soluble protein would be present in the final EV preparation. METHODS: We compared commercially available EV isolation kits with immunoaffinity purification techniques and evaluated our final EV preparations using atomic force microscopy (AFM) and nanoscale flow cytometry (NFC). AFM is the only modality capable of detecting distinguishing soluble protein from EVs which is important for downstream proteomics approaches. NFC is the only technique capable of quantitating the proportion of target EVs to non-target EVs in the final EV preparation. RESULTS: To determine enrichment of prostate derived EVs relative to non-target MPs, anti-PSMA (Prostate Specific Membrane Antigen) antibodies were used in NFC. Antibody-based immunoaffinity purification generated the highest quality of prostate derived EV preparations due to the lack of protein and RNA present in the samples. All kits produced poor purity EV preparations that failed to deplete the sample of plasma protein. CONCLUSIONS: While attractive due to their ease of use, EV purification kits do not provide substantial improvements in isolation of EVs from biofluids such as plasma. Immunoaffinity approaches are more efficient and economical and will also eliminate a significant portion of plasma proteins which is necessary for downstream approaches.

Prostate extracellular vesicles in patient plasma as a liquid biopsy platform for prostate cancer using nanoscale flow cytometry.

BACKGROUND: Extracellular vesicles released by prostate cancer present in seminal fluid, urine, and blood may represent a non-invasive means to identify and prioritize patients with intermediate risk and high risk of prostate cancer. We hypothesize that enumeration of circulating prostate microparticles (PMPs), a type of extracellular vesicle (EV), can identify patients with Gleason Score≥4+4 prostate cancer (PCa) in a manner independent of PSA. PATIENTS AND METHODS: Plasmas from healthy volunteers, benign prostatic hyperplasia patients, and PCa patients with various Gleason score patterns were analyzed for PMPs. We used nanoscale flow cytometry to enumerate PMPs which were defined as submicron events (100-1000nm) immunoreactive to anti-PSMA mAb when compared to isotype control labeled samples. Levels of PMPs (counts/µL of plasma) were also compared to CellSearch CTC Subclasses in various PCa metastatic disease subtypes (treatment naïve, castration resistant prostate cancer) and in serially collected plasma sets from patients undergoing radical prostatectomy. RESULTS: PMP levels in plasma as enumerated by nanoscale flow cytometry are effective in distinguishing PCa patients with Gleason Score≥8 disease, a high-risk prognostic factor, from patients with Gleason Score≤7 PCa, which carries an intermediate risk of PCa recurrence. PMP levels were independent of PSA and significantly decreased after surgical resection of the prostate, demonstrating its prognostic potential for clinical follow-up. CTC subclasses did not decrease after prostatectomy and were not effective in distinguishing localized PCa patients from metastatic PCa patients. CONCLUSIONS: PMP enumeration was able to identify patients with Gleason Score ≥8 PCa but not patients with Gleason Score 4+3 PCa, but offers greater confidence than CTC counts in identifying patients with metastatic prostate cancer. CTC Subclass analysis was also not effective for post-prostatectomy follow up and for distinguishing metastatic PCa and localized PCa patients. Nanoscale flow cytometry of PMPs presents an emerging biomarker platform for various stages of prostate cancer.