Extracellular vesicles (EVs) derived from tumor cells have the potential to provide a muchneeded source of non-invasive molecular biomarkers for liquid biopsies. However, current methods for EV isolation have limited specificity towards tumor-derived EVs that limit their clinical use. Here, we present an approach called immunomagnetic sequential ultrafiltration (iSUF) that consists of sequential stages of purification and enrichment of EVs (nonspecifically and specifically) in 2h. In iSUF, EVs present in different volumes of biofluids (0.1 mL to 100 mL) can be significantly enriched (up to 1000 times), with 99.9 % removal of contaminating proteins (e.g., albumin). The yields of cell culture media (CCM), serum, and urine EVs corresponded to 98% ± 3.6%, 94% ± 2.0% and 95% ± 2.0%, respectively (p > 0.05). The final step of iSUF enables the separation of tumor-specific EVs by incorporating immunomagnetic beads specific to a target subpopulation of EVs.Serum from a small cohort of clinical samples from metastatic breast cancer patients and healthy donors were processed by the iSUF platform and the isolated EVs from patients showed significantly higher expression levels of breast cancer biomarkers (i.e., HER2, CD24, and miR21).CD24 and miR21 biomarkers at significantly higher levels compared to healthy controls (p < 0.05).
Materials and Methods
MaterialsHollow fiber cartridges (molecular weight cut off, MWCO: 500 kDa, material: polysulfone, surface area: 28 cm 2 ) were supplied by Repligen (Rancho Dominguez, CA).Amicon® ultra-15 centrifugal filter units (MWCO: 10, 30 kDa) were purchased from MilliporeSigma (Burlington, MA). Streptavidin-coated magnetic particles (3.0-3.9 μm) were obtained from Spherotech (Lake Forest, IL). For capturing EVs, Cetuximab was 6 purchased from ImClone LLC (Branchburg, NJ), EpCAM, CD63, HER2 were obtained from R&D Systems (Minneapolis, MN). Antibodies were biotinylated using an EZ-Link™ micro Sulfo-NHS-biotinylation kit (ThermoFisher Scientific, Waltham, MA). For surface marker EV detection, CD24 was obtained from Novus Biological LLC (Centennial, CO), and HER2 was obtained from Cell Signaling Technology (Danvers, MA).
Cell culture and supernatant collectionU-251 glioblastoma (GBM), MCF-7 breast, and A375 melanoma cancer cell lines were supplied by American Type Culture Collection (ATCC, Manassas, VA). Cell lines were cultured in their recommended culture medium 35 containing 10% FBS and 1% penicillin-streptomycin at 37°C in a 5% CO2 incubator. For isolation of EVs from CCM, U251, MCF7, and A375 were grown in T75 flasks to 90% cell confluence, followed by washing the cells twice with PBS. Culture medium with 10 % EV-depleted FBS was added to cells for 24 h. CCM was centrifuged at 1, 000 x g for 5 min at room temperature (RT) to discard cell debris before further processing. EV-depleted FBS was prepared by using the permeate of FBS filtered by tangential flow filtration (TFF) (MWCO: 300 kDa). The concentration of EVs present in the purified FBS was not detectable when a tunable resistive puls...