Characterizing the Heterogeneity of Small Extracellular Vesicle Populations in Multiple Cancer Types via an Ultrasensitive Chip

Abstract

Identifying small extracellular vesicle (sEV) subpopulations based on their different molecular signatures could potentially reveal the functional roles in physiology and pathology. However, it is a challenge to achieve this aim due to the nanosized dimensions of sEVs, low quantities of biological cargo each sEV carries, and our incomplete knowledge of identifying features capable of separating heterogeneous sEV subpopulations. Here, a sensitive, multiplexed, and nano-mixing-enhanced sEV subpopulation characterization platform (ESCP) is proposed to precisely determine the sEV phenotypic heterogeneity and understand the role of sEV heterogeneity in cancer progression and metastasis. The ESCP utilizes spatially patterned antitetraspanin-functionalized micro-arrays for sEV subpopulation sorting and nanobarcode-based surface-enhanced Raman spectroscopy for multiplexed read-outs. An ESCP has been used for investigating sEV phenotypic heterogeneity in terms of canonical sEV tetraspanin molecules and cancer-associated protein biomarkers in both cancer cell line models and cancer patient samples. Our data explicitly demonstrate the selective enrichment of tetraspanins and cancer-associated protein biomarkers, in particular sEV subpopulations. Therefore, it is believed that the ESCP could enable the evaluation and broader application of sEV subpopulations as potential diagnostic disease biomarkers.

Emerging micro-nanotechnologies for extracellular vesicles in immuno-oncology: from target specific isolations to immunomodulation

“…The recently developed high throughput small EV subpopulation characterization platform (ESCP) demonstrated successful multiplex phenotypic detection of EV-related biomarkers utilizing nanoscopic flow and barcoding for multiplexed detection. 145 Their results emphasize the implication of EV subpopulation isolation for investigating the illusive pathology of tumor cells and immunomodulation capabilities.…”

C5b-9 Membrane Attack Complex Formation and Extracellular Vesicle Shedding in Barrett’s Esophagus and Esophageal Adenocarcinoma

“…50 µL of cell culture supernatant sample and 20 µL SERS nanotags were sequentially added to each well of the microfluidic device. Following previously optimized conditions ( 22 ), alternating current was applied for sample incubation (i.e., frequency = 500 Hz, amplitude = 800 mV, incubation time = 45 min) and for labelling with SERS nanotags (500 Hz, 800 mV, 20min), respectively. The alternating current induced a nanoscopic fluid flow on the surface of the antibody-functionalized asymmetric gold electrodes to stimulate EVs capture and labelling.…”

Microfluidic platforms for extracellular vesicle isolation, analysis and therapy in cancer

“…A different study recently reported the use of SERS-based microfluidics to characterize small EV subpopulations. 76 A microarray device was developed with three integrated electrodes functionalized with anti-CD63, -CD9, and -CD81 antibodies to create tetraspanin specific "hot spots" (Fig. 8D).…”