(592b) Quantifying Enzymatic Exosomes for Cancer Companion Diagnostics Using Magnetic Nanoporous Membrane (MNM)-Based Activity Assay

Small extracellular vesicles (sEVs) are particles ranging in size from 20 to 200 nm in diameter that source surface protein signatures from their cell of origin. Over the years, many active surface proteins such as membrane-type matrix metalloproteinases (MT-MMPs) and a-disintegrin and metalloproteinases (ADAMs) have been identified to be highly expressed in cancers. Increased metalloproteinase (MP) expression has been observed prior to tumor metastasis to remodel distal extracellular matrices (ECM) readying the site for invasion and proliferation. These active proteinases have come to serve as strong biomarkers for cancers, yet present methods hold several limitations in their abilities to detect relevant populations in biosamples for non-invasive liquid biopsy. The current state-of-the-art affinity-based assays as well as existing activity-based assays lack the sensitivity to detect the low concentrations of target enzymatic disease sEVs in biofluids (sub-picomolar in blood). Many MPs must also be activated within the cell before exhibiting relevant enzymatic behaviors. The minute structural changes in the activation event limits the specificity of affinity-based assays due to antibodies only being able to capture non-enzymatic epitopes of the target protein.

Our lab has been developing an activity-based assay meant for the detection of low concentration enzymatic sEVs with surface-bound ADAM10 in biofluids for use as a non-invasive companion diagnostic tool to monitor cancer progression and metastasis. The base activity assay relies on a highly specific peptide substrate target for ADAM10 designed to fluoresce once broken to proteolytic activity. This yields an activity assay that can produce readings in <1 hour. sEVs from biofluids or cell-cultured media are first isolated, and buffer exchanged with >90% capture using a tangential-flow asymmetric nanoporous membrane. These sEVs are incubated with superparamagnetic nanobeads functionalized with capture antibodies for various cell-surface protein biomarkers. Using a magnetic nanoporous membrane (MNM), the bound sEVs are trapped by the strong field gradient present at the sharp edges of the MNM pore edge at >99% capture. This, in effect, concentrates a low sEV concentration sample into a monolayer at the surface of the membrane thus amplifying an otherwise low signal response to the activity assay. The membrane system has been designed to operate in a 96-well plate so that the signal may be read using a standard lab-grade plate reader. Due to the strong anchoring of the target vesicles to the membrane surface and nondestructive nature of the activity assay, the individual sample can be drained, washed, and repeatedly assayed for surface features. The sEVs may be subsequently released or lysed for interrogation via other assays.

Zhang, C. et al. Electrodeposited magnetic nanoporous membrane for high-yield and high-throughput immunocapture of extracellular vesicles and lipoproteins. Commun Biol 5, 1-10 (2022).

Chen, X., Zhang, Y. & Guan, X. Simultaneous detection of multiple proteases using a non-array nanopore platform. Nanoscale 13, 13658-13664 (2021).