(295e) Quantifying Enzymatic Small Extracellular Vesicles (sEVs) for Metastatic Cancer Staging Via Magnetic Nanoporous Membrane (MNM) Activity Assay

Cancer metastasis is the leading cause of death for cancer patients. Active surface proteins like membrane-type matrix metalloproteinases (MT-MMPs) and a-disintegrin and metalloproteinases (ADAMs) have been identified to be highly expressed in later stages of disease. Additionally, increased metalloproteinase (MP) expression has been observed prior to tumor metastasis to remodel distal extracellular matrices (ECM) readying the site for invasion and proliferation. Small extracellular vesicles (sEVs) – ranging from 20-200 nm – have been shown to display parent cell membrane enzymes on their lipid bilayer surface. Affinity assays offer little metastatic information captured by the activity level of the enzyme. Alternatively, existing bulk plasma activity assays for these enzymes lack sensitivity and are prone to interfering agents found in plasma.

In this work, we overcome these limitations with a Magnetic Nanoporous Membrane (MNM) diagnostic technology which uses superparamagnetic beads to rapidly isolate and enrich specific tumor cell-originated sEVs. A rapid sample incubation (30-minutes) is performed using 0.22-um filtered plasma with antibody-functionalized nanobeads. The bound sEVs are trapped by the strong field gradient present at the sharp edges of the MNM pore edge at >99% capture. A controlled wash is applied to remove non-specifically bound entities allowing for direct characterization from minimally pretreated blood plasma. The activity assay utilizes molecular beacons synthesized from enzyme-specific substrates that fluoresce upon enzymatic digestion. The platform is capable of running up to 12 wells simultaneously and can be read-out on a custom-made 96-well plate using a standard laboratory fluorescence plate reader. This assay has an overall turn-around time of two to three hours.

We establish the localization of the majority of ADAM10 enzymatic activity from cancer cells to the surface of sEVs. ADAM10+ sEV activity normalized to sEV concentration is indicative of metastatic potential in prostate cancer cell lines. Interestingly, we find that lysed sEV sample produces significantly less activity signal, suggesting that the activity of ADAM10 is conferred by its specific conformation on the sEV lipid bilayer. ADAM10+ sEV activity hence produces significant separation as a general metastatic staging biomarker in mCRC patients. The findings recapitulate existing understandings of ADAM10 in cancer metastasis and focus the field towards the sEV-bound partition. Work on the MNM well-plate platform is ongoing with significant promise in improving sensitivity down by one to two orders of magnitude compared to the bulk assay (sub-femtomolar). Our group is currently working on selective pin-down of cancer-specific EGFR+ sEVs in a larger clinical cohort of mCRC patients.