Abstract 6493: Optimized methods for studies of extracellular vesicles in kidney cancer
Authors: Zieren Richard C. , Dong Liang , Pierorazio Phillip M. , Pienta Kenneth J. , de Reijke Theo M. , Amend Sarah R. 
Published: September 2020

Abstract
Background: Extracellular vesicles (EVs) are small membranous vesicles. All cells can secrete EVs into ECM and biofluids. The EV membrane helps to protect protein or nucleotide cargos, that can be transferred from the parental to recipient cells in an autocrine or paracrine manner. Thus, EVs are a mode of intercellular communication and valuable source of (cancer) biomarkers. The majority of human cancer EV research studies EVs from cell culture, blood or urine. Cell culture EVs are reproducible but lack sensitivity. Benign cells contribute to the total EV population found in blood and urine and thus lack tumor-specificity. We optimized EV isolation directly from renal cell carcinoma (RCC) tissue, which can aid RCC biomarker discovery. We isolated from human RCC and healthy kidney tissue. The isolated EV-product was evaluated in correspondence with the latest guidelines MISEV-guidelines (Minimal Information for Studies of Extracellular Vesicles).
Methods: Following radical nephrectomy, three technical replicates and three biological replicates were obtained for both normal and clear cell RCC. The tissues were processed and used to incubate media. EVs were isolated using a combination of differential centrifugation, filtration, and ultracentrifugation. Vesicle yield and size distribution of each sample were measured using two methods; the conventional Nanoparticle Tracking Analysis (NanoSight), and the novel method Nano Flowcytometry (NanoFCM). Transmission Electron Microscopy (TEM) was used to determine presence of intact vesicles in the EV samples. Western blot was used to detect presence of EV protein markers (CD81, CD63, Flotillin-1), and absence of cellular debris (calnexin).
Results: The normalized vesicle counts for the technical replicates of normal kidney EVs were 1.44 × 1010 ± 2.51 × 109 p/mL (mean ± SD) measured by NanoSight, and 1.67 × 1010 ± 6.89 × 109 p/mL (mean ± SD) measured by NanoFCM. Vesicle counts for replicates RCC EVs measured by NanoSight were 1.80 × 1010 ± 3.59 × 109 p/mL (mean ± SD), and they were 1.68 × 1010 ± 4.18 × 109 p/mL (mean ± SD) measured by NanoFCM. We observed an acceptable biological variance in EV counts of less than 10-fold among different patients. Comparing NanoSight with NanoFCM, the differences in measured vesicle counts of identical samples were less than 5-fold. TEM confirmed presence of small EVs in all technical and biological replicates. Protein analyses demonstrate presence of CD81, CD63, and Flotillin-1 in all samples. Furthermore, calnexin was absent in all samples.
Conclusions: We optimized a method for EV isolation from human kidney cancer and normal kidney tissue. Our protocol consistently results in high vesicle counts, as we confirmed using a standard method and a new method. Particle concentrations of both methods were similar, but the NanoFCM performs better in assessing particle size distribution. We confirmed the presence of intact vesicles and EV proteins, and the absence of cellular contamination. This optimized protocol can contribute to RCC-biomarker discovery and aid biological kidney cancer research.
Citation Format: Richard C. Zieren, Liang Dong, Phillip M. Pierorazio, Kenneth J. Pienta, Theo M. de Reijke, Sarah R. Amend. Optimized methods for studies of extracellular vesicles in kidney cancer [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 6493.