Extracellular vesicle isolation from human renal cancer tissue

Abstract

Renal cell carcinoma is a lethal disease that is often discovered incidentally. New non-invasive biomarkers are needed to aid diagnosis and treatment. Extracellular vesicles (EVs), membranous vesicles secreted by all cells, are a promising potential source for cancer biomarkers, but new methods are required that are both sensitive and specific for cancer identification. We have developed an EV isolation protocol optimized for kidney tumor and normal kidney tissue that yields a high vesicle concentration, confirmed by nanoparticle tracking analysis (NanoSight) and by nanoscale flow cytometry (NanoFCM). Using Western blot, we confirmed presence of EV markers CD81, CD63, flotillin-1, and absence of cellular debris, calnexin. Transmission electron microscopy images demonstrate intact membranous EVs. This new method improves existing protocols with additional steps to reduce contaminants in the EV product. Characterization of our isolation product confirms successful isolation of EVs with minimal contamination. The particle yields of our protocol are consistent and high as assessed by both standard and novel methods. This optimized protocol will contribute to biomarker discovery and biological studies of EVs in renal cancer.

Comprehensive evaluation of methods for small extracellular vesicles separation from human plasma, urine and cell culture medium

“…Separated EVs were assessed by transmission electron microscopy (TEM) as described previously (Zieren et al., 2020). First, 10 μl of each sample was adsorbed to an ultra‐thin carbon coated 400 mesh copper grid that was glow discharged (EMS GloQube™) by floatation for 2 min.…”

“…Nanoparticle tracking analysis (NTA) (NanoSight NS300, Malvern Pananalytical) was used to measure the particle counts and size distributions of EV preparations separated from different sample types using different methods, as described previously (Zieren et al., 2020). Briefly, 10 μl of each EV sample was diluted with PBS according to the detection range (20–100 particles/frame).…”

Characterization of CD147, CA9, and CD70 as Tumor-Specific Markers on Extracellular Vesicles in Clear Cell Renal Cell Carcinoma

“…To date, there are only few data on the isolation of exosomes from kidney tissue. Zieren et al reported the isolation of exosomes based on collagenase treatment, differential centrifugation, and filtration with 800 nm and 450 nm filters, followed by ultracentrifugation [46]. In contrast, our protocol was based on a density gradient without mechanical filters.…”

“…In addition to the size, the source of exosomes plays an important role in the selection of the isolation method. Our isolation protocol was optimized in order to obtain the best possible ratio of purity and concentration while being cost-and time-efficient [46][47][48].…”

Exosomal PD-L1 Derived From Osteosarcoma is Associated With Pulmonary Metastasis for Patients With Osteosarcoma

“…Exosomes are secreted as the vesicles of a size range of 30 to 150 nm diameter, which are enclosed by the cell membrane. Inward budding of late endosomes develop into intracellular multivesicular endosomes and RNA, DNA, proteins and other small moleculars are encapsulated into exosomes during the formation of exosome [1,2]. Various types of cells such as immune cells, broblast and endothelial cells, release exosomes into the extracellular space and microenviroment, involving the tumor pathogenesis [3,4].…”