Extracellular vesicles isolated from human renal cell carcinoma tissues disrupt vascular endothelial cell morphology via azurocidin

Jingushi, Kentaro; Uemura, Mamoru; Ohnishi, Naomi; Nakata, Wataru; Fujita, Kazutoshi; Naito, Takuya; Fujii, Risa; Saichi, Naomi; Nonomura, Norio; Tsujikawa, Kazutake; Ueda, Koji

doi:10.1002/ijc.31080

Cited by 70 publications

(76 citation statements)

References 38 publications

(76 reference statements)

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“…In 2017, Vella et al used frozen human brain tissue to study EVs [31]. More recently, Jang et al studied EVs from metastatic human melanoma and Jingushi et al isolated EVs from human kidney tumors [21,32]. Although Jingushi et al reported successful EV isolation from RCC, we found it necessary to optimize available protocols to reduce non-EV contaminants to acceptable levels.…”

Section: Discussionmentioning

confidence: 96%

Extracellular vesicle isolation from human renal cancer tissue

et al. 2020

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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.

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Section: Discussionmentioning

confidence: 96%

Extracellular vesicle isolation from human renal cancer tissue

et al. 2020

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“…Since numerous publications have reported tissue-derived EVs without thorough protein characterization of EV-depleted cellular markers and specific EV subtype markers 12,15,16,[49][50][51][52] , we compared the BH and bdEV proteomes with established EV-related databases. Substantial concordance between our data and the databases was clear (Tabe 3).…”

Section: Discussionmentioning

confidence: 99%

Influence of species and processing parameters on recovery and content of brain tissue-derived extracellular vesicles

Huang

Cheng

Turchinovich

et al. 2020

Preprint

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Extracellular vesicles (EVs) are involved in a wide range of physiological and pathological processes by shuttling material out of and between cells. Tissue EVs may thus lend insights into disease mechanisms and also betray disease when released into easily accessed biological fluids.Since brain-derived EVs (bdEVs) and their cargo may serve as biomarkers of neurodegenerative diseases, we evaluated modifications to a published, rigorous protocol for separation of EVs from brain tissue and studied effects of processing variables on quantitative and qualitative outcomes.To this end, size exclusion chromatography (SEC) and gradient density ultracentrifugation were compared as final separation steps in protocols involving stepped ultracentrifugation. bdEVs were separated from brain tissues of human, macaque, and mouse. Effects of tissue perfusion and a model of post-mortem interval (PMI) before final bdEV separation were probed.MISEV2018-compliant EV characterization was performed, and both small RNA and protein profiling were done. We conclude that the modified, SEC-employing protocol achieves EV separation efficiency roughly similar to a protocol using gradient density ultracentrifugation, while decreasing operator time and, potentially, variability. The protocol appears to yield bdEVs of higher purity for human tissues compared with those of macaque and, especially, mouse, suggesting opportunities for optimization. Where possible, perfusion should be performed in animal models. The interval between death/tissue storage/processing and final bdEV separation can also affect bdEV populations and composition and should thus be recorded for rigorous reporting. Finally, different types of EVs obtained through the modified method reported herein display characteristic RNA and protein content that hint at biomarker potential. To conclude, this study finds that the automatable and increasingly employed technique of SEC can be applied to tissue EV separation, and also reveals more about the importance of species-specific and technical considerations when working with tissue EVs. These results are expected to enhance the use of bdEVs in revealing and understanding brain disease. Results Protocol comparison: bdEV separationThe tissue processing and bdEV separation protocol previously published by several members of the author team (Vella et al., JEV, 2017) 13 was followed through the 2,000 x g centrifugation step ( Figure 1). After enrichment of large EVs (lEVs) by filtration and 10,000 x g centrifugation, supernatant was subjected to SDGU (as previously published) or SEC. Where indicated, SEC fractions were then concentrated by UC or ultrafiltration (UF) (Figure 1). Comparison of bdEV particle count, morphology, and protein markers in different fractions of SDGU and SEC: human and mouse tissueParticle yield per 100 mg tissue input (human or mouse) was determined by nanoparticle tracking analysis (NTA). Particle yield was highest for F2 from SDGU and F7-10 from SEC+UC. Particle concentration was below the reliable range of meas...

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“…In-Gel Digestion [33][34][35][36][37] FASP [38,39] Combined method In-Gel Digestion [28,35,36,[40][41][42][43][44][45][46][47][48][49][50][51] In-Sol Digestion [52][53][54] FASP [51,[55][56][57][58][59] Plasma/ Serum Ultracentrifuge In-Sol Digestion [60] FASP [56] Combined method In-Gel Digestion [43] In-Sol Digestion [44,52] FASP [51,57,58] secreted by MCF7, 30 proteins have been known to participate in breast cancer growth, metastasis, and chemoresistance. The most abundant MDA-MD-231 derived exosomal protein is β-actin, which is frequently used as a housekeeping gene in the exosome.…”

Section: Cell Ultracentrifugementioning

confidence: 99%

Exploring the key communicator role of exosomes in cancer microenvironment through proteomics

Kim

Cho

2019

Proteome Sci

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There have been many attempts to fully understand the mechanism of cancer behavior. Yet, how cancers develop and metastasize still remain elusive. Emerging concepts of cancer biology in recent years have focused on the communication of cancer with its microenvironment, since cancer cannot grow and live alone. Cancer needs to communicate with other cells for survival, and thus they secrete various messengers, including exosomes that contain many proteins, miRNAs, mRNAs, etc., for construction of the tumor microenvironment. Moreover, these intercellular communications between cancer and its microenvironment, including stromal cells or distant cells, can promote tumor growth, metastasis, and escape from immune surveillance. In this review, we summarized the role of proteins in the exosome as communicators between cancer and its microenvironment. Consequently, we present cancer specific exosome proteins and their unique roles in the interaction between cancer and its microenvironment. Clinically, these exosomes might provide useful biomarkers for cancer diagnosis and therapeutic tools for cancer treatment.

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Extracellular vesicles isolated from human renal cell carcinoma tissues disrupt vascular endothelial cell morphology via azurocidin

Cited by 70 publications

References 38 publications

Extracellular vesicle isolation from human renal cancer tissue

Extracellular vesicle isolation from human renal cancer tissue

Influence of species and processing parameters on recovery and content of brain tissue-derived extracellular vesicles

Exploring the key communicator role of exosomes in cancer microenvironment through proteomics

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