Hostname: page-component-8448b6f56d-t5pn6 Total loading time: 0 Render date: 2024-04-24T10:48:33.275Z Has data issue: false hasContentIssue false
  • English
  • Français

Electron Microscopy-Based Comparison and Investigation of the Morphology of Exosomes Derived from Hepatocellular Carcinoma Cells Isolated at Different Centrifugal Speeds

Published online by Cambridge University Press:  13 February 2020

Jing-Huan Deng Open the ORCID record for Jing-Huan Deng [Opens in a new window] ,
Zhong-Jie Li ,
Zi-Xuan Wang ,
Ji Feng ,
Xue-Jing Huang  and
Zhi-Ming Zeng
Jing-Huan Deng*
Affiliation:
The Guangxi Colleges and Universities Key Laboratory of Prevention and Control of Highly Prevalent Diseases, School of Public Health, Guangxi Medical University, 22 Shuangyong Road, Nanning, Guangxi Zhuang Autonomous Region530021, China
Zhong-Jie Li
Affiliation:
Department of Toxicology, School of Public Health, Guangxi Medical University, Nanning, Guangxi Zhuang Autonomous Region530021, China
Zi-Xuan Wang
Affiliation:
Department of Toxicology, School of Public Health, Guangxi Medical University, Nanning, Guangxi Zhuang Autonomous Region530021, China
Ji Feng
Affiliation:
Department of Toxicology, School of Public Health, Guangxi Medical University, Nanning, Guangxi Zhuang Autonomous Region530021, China
Xue-Jing Huang
Affiliation:
Department of Environmental Hygiene, School of Public Health, Guangxi Medical University, Nanning, Guangxi Zhuang Autonomous Region530021, China
Zhi-Ming Zeng
Affiliation:
Department of Medical Oncology, The First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi Zhuang Autonomous Region530021, China
*
*Author for correspondence: Jing-Huan Deng, E-mail: 502696499@qq.com
Get access

Abstract

Exosomes derived from hepatocellular carcinoma (HCC) cells are nanovesicles and are involved in the occurrence and development of HCC, they also serve as important carriers and drug targets of nanodrug delivery systems. The external shape and internal structure of exosomes are important indexes of identification, and isolated intact morphology is crucial to biological function integrity. However, given their susceptibility to various influencing factors, the external shape and internal structure of exosomes derived from HCC cells remain incompletely studied. In this study, exosomes purified from HCC cells were isolated at different centrifugation speeds and examined via multiple electron microscopy (EM) techniques. The results demonstrate that exosomes possess a nearly spherical shape and bilipid membranous vesicle with a concave cavity structure containing electron-dense and coated vesicles, suggesting the possible existence of subpopulations of exosomes with specific functions. The exosomes isolated at ultracentrifugation (UC) speed (≥110,000×g) presented irregular and diverse external morphologies, indicating the effect on the integrity of the exosomes. Transforming growth factor signaling bioactive substances (TGF-β1, S100A8, and S100A9) can be found in exosomes by performing Western blotting, showing that the internal content is associated with metastasis of HCC. These findings show that EMelectron microscopy and UC speed can affect exosome characteristics, including external shape, internal structure, and content of bioactive substances. The electron-dense and coated vesicles that had been discovered in exosomes might become new additional morphological features, which could help to improve the interpretation of experimental results and widen our understanding of exosome morphology.

Keywords

centrifugal speedelectron microscopyexosomehepatocellular carcinomamorphology
Type
Biological Applications
Information
Microscopy and Microanalysis , Volume 26 , Issue 2 , April 2020 , pp. 310 - 318
Copyright
Copyright © Microscopy Society of America 2020

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

Abd El Gwad, A, Matboli, M, El-Tawdi, A, Habib, EK, Shehata, H, Ibrahim, D & Tash, F (2018). Role of exosomal competing endogenous RNA in patients with hepatocellular carcinoma. J Cell Biochem 119(10), 8600–8610.CrossRefGoogle ScholarPubMed
Baietti, MF, Zhang, Z, Mortier, E, Melchior, A, Degeest, G, Geeraerts, A, Ivarsson, Y, Depoortere, F, Coomans, C, Vermeiren, E, Zimmermann, P & David, G (2012). Syndecan-syntenin-ALIX regulates the biogenesis of exosomes. Nat Cell Biol 14(7), 677–685.CrossRefGoogle ScholarPubMed
Balletta, A, Lorenz, D, Rummel, A, Gerhard, R, Bigalke, H & Wegner, F (2013). Human mast cell line-1 (HMC-1) cells exhibit a membrane capacitance increase when dialysed with high free-Ca(2+) and GTPgammaS containing intracellular solution. Eur J Pharmacol 720(1–3), 227–236.CrossRefGoogle ScholarPubMed
Basso, D, Bozzato, D, Padoan, A, Moz, S, Zambon, CF, Fogar, P, Greco, E, Scorzeto, M, Simonato, F, Navaglia, F, Fassan, M, Pelloso, M, Dupont, S, Pedrazzoli, S, Fassina, A & Plebani, M (2014). Inflammation and pancreatic cancer: Molecular and functional interactions between S100A8, S100A9, NT-S100A8 and TGFbeta1. Cell Commun Signal 12, 20.CrossRefGoogle ScholarPubMed
Bobrie, A, Krumeich, S, Reyal, F, Recchi, C, Moita, LF, Seabra, MC, Ostrowski, M & Thery, C (2012). Rab27a supports exosome-dependent and -independent mechanisms that modify the tumor microenvironment and can promote tumor progression. Cancer Res 72(19), 4920–4930.CrossRefGoogle ScholarPubMed
Chen, L, Guo, P, He, Y, Chen, Z, Chen, L, Luo, Y, Qi, L, Liu, Y, Wu, Q, Cui, Y, Fang, F, Zhang, X, Song, T & Guo, H (2018). HCC-derived exosomes elicit HCC progression and recurrence by epithelial-mesenchymal transition through MAPK/ERK signalling pathway. Cell Death Dis 9(5), 513.CrossRefGoogle ScholarPubMed
Chen, L, Xiang, B, Wang, X & Xiang, C (2017). Exosomes derived from human menstrual blood-derived stem cells alleviate fulminant hepatic failure. Stem Cell Res Ther 8(1), 9.CrossRefGoogle ScholarPubMed
Chernyshev, VS, Rachamadugu, R, Tseng, YH, Belnap, DM, Jia, Y, Branch, KJ, Butterfield, AE, Pease, LF 3rd, Bernard, PS & Skliar, M (2015). Size and shape characterization of hydrated and desiccated exosomes. Anal Bioanal Chem 407(12), 3285–3301.CrossRefGoogle ScholarPubMed
Colombo, M, Moita, C, van Niel, G, Kowal, J, Vigneron, J, Benaroch, P, Manel, N, Moita, LF, Thery, C & Raposo, G (2013). Analysis of ESCRT functions in exosome biogenesis, composition and secretion highlights the heterogeneity of extracellular vesicles. J Cell Sci 126(Pt 24), 5553–5565.CrossRefGoogle ScholarPubMed
Colombo, M, Raposo, G & Thery, C (2014). Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles. Annu Rev Cell Dev Biol 30, 255–289.CrossRefGoogle ScholarPubMed
Crescitelli, R, Lasser, C, Szabo, TG, Kittel, A, Eldh, M, Dianzani, I, Buzas, EI & Lotvall, J (2013). Distinct RNA profiles in subpopulations of extracellular vesicles: Apoptotic bodies, microvesicles and exosomes. J Extracell Vesicles 2, 20677.Google ScholarPubMed
Gardiner, C, Di Vizio, D, Sahoo, S, Thery, C, Witwer, KW, Wauben, M & Hill, AF (2016). Techniques used for the isolation and characterization of extracellular vesicles: Results of a worldwide survey. J Extracell Vesicles 5, 32945.CrossRefGoogle ScholarPubMed
Hakulinen, J, Sankkila, L, Sugiyama, N, Lehti, K & Keski-Oja, J (2008). Secretion of active membrane type 1 matrix metalloproteinase (MMP-14) into extracellular space in microvesicular exosomes. J Cell Biochem 105(5), 1211–1218.CrossRefGoogle ScholarPubMed
Hosseini-Beheshti, E, Pham, S, Adomat, H, Li, N & Tomlinson Guns, ES (2012). Exosomes as biomarker enriched microvesicles: Characterization of exosomal proteins derived from a panel of prostate cell lines with distinct AR phenotypes. Mol Cell Proteomics 11(10), 863–885.CrossRefGoogle ScholarPubMed
Jeppesen, DK, Hvam, ML, Primdahl-Bengtson, B, Boysen, AT, Whitehead, B, Dyrskjot, L, Orntoft, TF, Howard, KA & Ostenfeld, MS (2014). Comparative analysis of discrete exosome fractions obtained by differential centrifugation. J Extracell Vesicles 3, 25011.CrossRefGoogle ScholarPubMed
Jiang, K, Dong, C, Yin, Z, Li, R, Wang, Q & Wang, L (2018). The critical role of exosomes in tumor biology. J Cell Biochem 120, 1–13.Google Scholar
Johnstone, RM, Adam, M, Hammond, JR, Orr, L & Turbide, C (1987). Vesicle formation during reticulocyte maturation. Association of plasma membrane activities with released vesicles (exosomes). J Biol Chem 262(19), 9412–9420.Google Scholar
Lee, Y, El Andaloussi, S & Wood, MJ (2012). Exosomes and microvesicles: Extracellular vesicles for genetic information transfer and gene therapy. Hum Mol Genet 21(R1), R125–R134.CrossRefGoogle Scholar
Li, P, Kaslan, M, Lee, SH, Yao, J & Gao, Z (2017). Progress in exosome isolation techniques. Theranostics 7(3), 789–804.CrossRefGoogle ScholarPubMed
Li, SP, Lin, ZX, Jiang, XY & Yu, XY (2018). Exosomal cargo-loading and synthetic exosome-mimics as potential therapeutic tools. Acta Pharmacol Sin 39(4), 542–551.CrossRefGoogle ScholarPubMed
Lin, XJ, Fang, JH, Yang, XJ, Zhang, C, Yuan, Y, Zheng, L & Zhuang, SM (2018). Hepatocellular carcinoma cell-secreted exosomal microRNA-210 promotes angiogenesis in vitro and in vivo. Mol Ther Nucleic Acids 11, 243–252.CrossRefGoogle ScholarPubMed
Meyer, GK, Neetz, A, Brandes, G, Tsikas, D, Butterfield, JH, Just, I & Gerhard, R (2007). Clostridium difficile toxins A and B directly stimulate human mast cells. Infect Immun 75(8), 3868–3876.CrossRefGoogle Scholar
Morishita, M, Takahashi, Y, Matsumoto, A, Nishikawa, M & Takakura, Y (2016). Exosome-based tumor antigens-adjuvant co-delivery utilizing genetically engineered tumor cell-derived exosomes with immunostimulatory CpG DNA. Biomaterials 111, 55–65.CrossRefGoogle ScholarPubMed
O'Driscoll, L (2015). Expanding on exosomes and ectosomes in cancer. N Engl J Med 372(24), 2359–2362.CrossRefGoogle ScholarPubMed
Pick, H, Alves, AC & Vogel, H (2018). Single-vesicle assays using liposomes and cell-derived vesicles: From modeling complex membrane processes to synthetic biology and biomedical applications. Chem Rev 118(18), 8598–8654.CrossRefGoogle ScholarPubMed
Qu, Z, Wu, J, Wu, J, Luo, D, Jiang, C & Ding, Y (2016). Exosomes derived from HCC cells induce sorafenib resistance in hepatocellular carcinoma both in vivo and in vitro. J Exp Clin Cancer Res 35(1), 159.CrossRefGoogle ScholarPubMed
Rackov, G, Garcia-Romero, N, Esteban-Rubio, S, Carrion-Navarro, J, Belda-Iniesta, C & Ayuso-Sacido, A (2018). Vesicle-mediated control of cell function: The role of extracellular matrix and microenvironment. Front Physiol 9, 651.CrossRefGoogle ScholarPubMed
Raposo, G & Stoorvogel, W (2013). Extracellular vesicles: Exosomes, microvesicles, and friends. J Cell Biol 200(4), 373–383.CrossRefGoogle ScholarPubMed
Santangelo, L, Battistelli, C, Montaldo, C, Citarella, F, Strippoli, R & Cicchini, C (2017). Functional roles and therapeutic applications of exosomes in hepatocellular carcinoma. BioMed Res Int 2017, 2931813.CrossRefGoogle ScholarPubMed
Shao, H, Chung, J, Balaj, L, Charest, A, Bigner, DD, Carter, BS, Hochberg, FH, Breakefield, XO, Weissleder, R & Lee, H (2012). Protein typing of circulating microvesicles allows real-time monitoring of glioblastoma therapy. Nat Med 18(12), 1835–1840.CrossRefGoogle ScholarPubMed
Shao, H, Im, H, Castro, CM, Breakefield, X, Weissleder, R & Lee, H (2018). New technologies for analysis of extracellular vesicles. Chem Rev 118(4), 1917–1950.CrossRefGoogle ScholarPubMed
Shen, S, Kong, J, Qiu, Y, Yang, X, Wang, W & Yan, L (2019). Identification of core genes and outcomes in hepatocellular carcinoma by bioinformatics analysis. J Cell Biochem 120(6), 10069–10081.CrossRefGoogle ScholarPubMed
Skog, J, Wurdinger, T, van Rijn, S, Meijer, DH, Gainche, L, Sena-Esteves, M, Curry, WT Jr., Carter, BS, Krichevsky, AM & Breakefield, XO (2008). Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers. Nat Cell Biol 10(12), 1470–1476.CrossRefGoogle ScholarPubMed
Sokolova, V, Ludwig, AK, Hornung, S, Rotan, O, Horn, PA, Epple, M & Giebel, B (2011). Characterisation of exosomes derived from human cells by nanoparticle tracking analysis and scanning electron microscopy. Colloid Surface B 87(1), 146–150.CrossRefGoogle ScholarPubMed
Stoorvogel, W, Kleijmeer, MJ, Geuze, HJ & Raposo, G (2002). The biogenesis and functions of exosomes. Traffic 3(5), 321–330.CrossRefGoogle ScholarPubMed
Street, JM, Barran, PE, Mackay, CL, Weidt, S, Balmforth, C, Walsh, TS, Chalmers, RT, Webb, DJ & Dear, JW (2012). Identification and proteomic profiling of exosomes in human cerebrospinal fluid. J Transl Med 10, 5.CrossRefGoogle ScholarPubMed
Tatischeff, I, Larquet, E, Falcon-Perez, JM, Turpin, PY & Kruglik, SG (2012). Fast characterisation of cell-derived extracellular vesicles by nanoparticles tracking analysis, cryo-electron microscopy, and Raman tweezers microspectroscopy. J Extracell Vesicles 1, 19179.Google ScholarPubMed
Thery, C, Amigorena, S, Raposo, G & Clayton, A (2006). Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol. Chapter 3, Unit 3.22. doi:10.1002/0471143030.cb0322s30.CrossRefGoogle ScholarPubMed
Thery, C, Ostrowski, M & Segura, E (2009). Membrane vesicles as conveyors of immune responses. Nat Rev Immunol 9(8), 581–593.CrossRefGoogle ScholarPubMed
Thery, C, Zitvogel, L & Amigorena, S (2002). Exosomes: Composition, biogenesis and function. Nat Rev Immunol 2(8), 569–579.CrossRefGoogle ScholarPubMed
van der Pol, E, Hoekstra, AG, Sturk, A, Otto, C, van Leeuwen, TG & Nieuwland, R (2010). Optical and non-optical methods for detection and characterization of microparticles and exosomes. J Thromb Haemost 8(12), 2596–2607.CrossRefGoogle ScholarPubMed
Wang, S, Chen, G, Lin, X, Xing, X, Cai, Z, Liu, X & Liu, J (2017). Role of exosomes in hepatocellular carcinoma cell mobility alteration. Oncol Lett 14(6), 8122–8131.Google ScholarPubMed
Wu, Z, Zeng, Q, Cao, K & Sun, Y (2016). Exosomes: Small vesicles with big roles in hepatocellular carcinoma. Oncotarget 7(37), 60687–60697.CrossRefGoogle ScholarPubMed
Yang, N, Li, S, Li, G, Zhang, S, Tang, X, Ni, S, Jian, X, Xu, C, Zhu, J & Lu, M (2017). The role of extracellular vesicles in mediating progression, metastasis and potential treatment of hepatocellular carcinoma. Oncotarget 8(2), 3683–3695.CrossRefGoogle ScholarPubMed
Yuana, Y, Koning, RI, Kuil, ME, Rensen, PC, Koster, AJ, Bertina, RM & Osanto, S (2013). Cryo-electron microscopy of extracellular vesicles in fresh plasma. J Extracell Vesicles 2, 21494.CrossRefGoogle ScholarPubMed
Zabeo, D, Cvjetkovic, A, Lasser, C, Schorb, M, Lotvall, J & Hoog, JL (2017). Exosomes purified from a single cell type have diverse morphology. J Extracell Vesicles 6(1), 1329476.CrossRefGoogle ScholarPubMed
Zarovni, N, Corrado, A, Guazzi, P, Zocco, D, Lari, E, Radano, G, Muhhina, J, Fondelli, C, Gavrilova, J & Chiesi, A (2015). Integrated isolation and quantitative analysis of exosome shuttled proteins and nucleic acids using immunocapture approaches. Methods 87, 46–58.CrossRefGoogle ScholarPubMed
Zeringer, E, Barta, T, Li, M & Vlassov, AV (2015). Strategies for isolation of exosomes. Cold Spring Harb Protoc 2015(4), 319–323.CrossRefGoogle ScholarPubMed
Zlotogorski-Hurvitz, A, Dayan, D, Chaushu, G, Korvala, J, Salo, T, Sormunen, R & Vered, M (2015). Human saliva-derived exosomes: Comparing methods of isolation. J Histochem Cytochem 63(3), 181–189.CrossRefGoogle ScholarPubMed