Introduction: Extracellular vesicles (EV), recovered from a liquid biopsy, are emerging as a putative diagnostic test for cancer. To date, ultracentrifugation has been accepted as the gold standard for EV isolation despite its lengthy processing time and low recovery rate. Thanks to the recent technological advances in microfluidics, several microfluidic devices for EV isolation have been developed and are showing promise with respect to improved recovery rates, the ability to extract EVs from small sample volumes, and shorter processing time compared to ultracentrifugation. Among the techniques, immunoaffinity based microfluidic isolation using antibodies against tetraspanins, such as anti-CD63, has been widely applied for EV isolation in general, however, anti-CD63 is not specific to cancer-associated EVs and some cancer cells secrete exosomes with low expression of CD63, thus limiting exosome isolation for cancer study. Alternatively, one of the phospholipids, phosphatidylserine (PS), usually expressed in the inner leaflet of the lipid bilayer of the cells, has also been shown to be expressed on the outer surface of cancer-associated EVs during its vesiculation procedure. Here, we present a new exosome isolation device (newExoChip), conjugated with PS-specific protein, to isolate cancer-associated exosomes from plasma

Methods: The newExoChip, having 30x60 circular channels conjugated with PS-specific protein, isolates cancer-associated exosomes in a Ca2+ dependent manner. The isolated exosomes using the device were easily released using Ca2+ chelation and the released exosomes has been analyzed quantitatively and qualitatively using nanoparticle tracking analysis (NTA) and western blot analysis, respectively. For clinical studies, we obtained non-small cell lung cancer (NSCLC) blood samples and each blood sample was centrifuged and the 30-100µl of plasma supernatant was flowed through the device using a syringe pump.

Results: The newExoChip achieves 84.58% capture efficiency for NSCLC-derived exosomes compared to 38.43% for healthy exosomes and isolates averagely 47.4% more A549-derived exosomes than anti-tetraspanin devices (anti-CD63, 9, and 81). From a clinical study using 4 NSCLC blood samples, we recovered 1.47x109 EVs per 1 milliliter and 75.03% of vesicles was within the exosomal size range (30-150nm). The isolated exosomes from clinical samples were characterized by western blot using CD9 and flotillin-1, revealing exosomal protein expressions.

Discussion and conclusion: Our results demonstrated that the isolated and released vesicles using the newExoChip show conventional features of exosomes, such as exosomal marker expressions and the exosomal size. We believe that the newExoChip facilitates the isolation of a specific subset of exosomes, allowing us to explore the undiscovered roles of exosomes in cancer.

Citation Format: Yoon-Tae Kang, Emma Purcell, Colin Palacios-Rolston, Ting-Wen Lo, Nithya Ramnath, Shruti Jolly, Sunitha Nagrath. Microfluidic isolation (newExoChip) and profiling of cancer-associated exosomes using extracellular vesicular lipid-protein binding affinity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 751.