Isolation and Profiling of Circulating Tumor-Associated Exosomes Using Extracellular Vesicular Lipid-Protein Binding Affinity Based Microfluidic Device

Abstract

Extracellular vesicles (EVs) are emerging as a potential diagnostic test for cancer. Owing to the recent advances in microfluidics, on-chip EV isolation is showing promise with respect to improved recovery rates, smaller necessary sample volumes, and shorter processing times than ultracentrifugation. Immunoaffinity-based microfluidic EV isolation using anti-CD63 is widely used; however, anti-CD63 is not specific to cancer-EVs, and some cancers secrete EVs with low expression of CD63. Alternatively, phosphatidylserine (PS), usually expressed in the inner leaflet of the lipid bilayer of the cells, is shown to be expressed on the outer surface of cancer-associated EVs. A new exosome isolation microfluidic device (^new ExoChip), conjugated with a PS-specific protein, to isolate cancer-associated exosomes from plasma, is presented. The device achieves 90% capture efficiency for cancer cell exosomes compared to 38% for healthy exosomes and isolates 35% more A549-derived exosomes than an anti-CD63-conjugated device. Immobilized exosomes are then easily released using Ca²⁺ chelation. The recovered exosomes from clinical samples are characterized by electron microscopy and western-blot analysis, revealing exosomal shapes and exosomal protein expressions. The ^new ExoChip facilitates the isolation of a specific subset of exosomes, allowing the exploration of the undiscovered roles of exosomes in cancer progression and metastasis.

Keywords: cancer-associated exosomes; exosome isolation; extracellular vesicles; lung cancer; melanoma; microfluidics.