Distinct RNA profiles in subpopulations of extracellular vesicles: apoptotic bodies, microvesicles and exosomes

Abstract

IntroductionIn recent years, there has been an exponential increase in the number of studies aiming to understand the biology of exosomes, as well as other extracellular vesicles. However, classification of membrane vesicles and the appropriate protocols for their isolation are still under intense discussion and investigation. When isolating vesicles, it is crucial to use systems that are able to separate them, to avoid cross-contamination.MethodEVs released from three different kinds of cell lines: HMC-1, TF-1 and BV-2 were isolated using two centrifugation-based protocols. In protocol 1, apoptotic bodies were collected at 2,000×g, followed by filtering the supernatant through 0.8 µm pores and pelleting of microvesicles at 12,200×g. In protocol 2, apoptotic bodies and microvesicles were collected together at 16,500×g, followed by filtering of the supernatant through 0.2 µm pores and pelleting of exosomes at 120,000×g. Extracellular vesicles were analyzed by transmission electron microscopy, flow cytometry and the RNA profiles were investigated using a Bioanalyzer®.ResultsRNA profiles showed that ribosomal RNA was primary detectable in apoptotic bodies and smaller RNAs without prominent ribosomal RNA peaks in exosomes. In contrast, microvesicles contained little or no RNA except for microvesicles collected from TF-1 cell cultures. The different vesicle pellets showed highly different distribution of size, shape and electron density with typical apoptotic body, microvesicle and exosome characteristics when analyzed by transmission electron microscopy. Flow cytometry revealed the presence of CD63 and CD81 in all vesicles investigated, as well as CD9 except in the TF-1-derived vesicles, as these cells do not express CD9.ConclusionsOur results demonstrate that centrifugation-based protocols are simple and fast systems to distinguish subpopulations of extracellular vesicles. Different vesicles show different RNA profiles and morphological characteristics, but they are indistinguishable using CD63-coated beads for flow cytometry analysis.

141 Small RNA deep sequencing discriminates subsets of extracellular vesicles released by melanoma cells - evidence of unique microRNA cargos

“…Importantly, although our data was aligned with more than 80% to the HG19, we found the presence of a significant number of transcripts (>70%) that are un-annotated with known functions. As shown earlier by the presence of small RNA in the studied EV subpopulations, 6 we have here established a workflow for analyzing miRNA in these samples using small RNA sequencing data. This approach is unbiased, and can profile a large number of RNA species from the transcriptome, which is different from analyzing miRNAs using microarray systems with limited number of pre-known miRNA candidates.…”

“…The protocol has previously been described as "the modified protocol-2B" by Crescitelli, et al 6 Briefly, cells were pelleted by centrifugation at 300 £ g for 10 minutes and the supernatant was harvested to pellet down apoptotic bodies at 2,000 £ g for 20 minutes and microvesicles at 16,500 £ g for 20 minutes. The supernatant harvested from the 16,500 £ g centrifugation was filtered (0.2 mm pore size, Sarstedt, N€ umbrechtRommelsdorf, Germany) to remove particles greater than 200 nm.…”

“…2,3 Tumor cells, including melanoma cells, are known to secrete several subsets of extracellular vesicles (EVs), including apoptotic bodies, microvesicles and exosomes. [4][5][6][7][8][9][10] Further, EVs have been implicated in tumor microenvironment interactions, including angiogenesis, tumor cell proliferation and epithelial mesenchymal transition, [11][12][13] and malignant cells release different EV RNA molecules compared to normal cells. 14 Hence, describing the detailed molecular and genetic content of melanoma EVs is important to understand the complexity of cell-to-cell interaction in melanoma, including disease progression.…”

“…28 Recently, our group has demonstrated the presence of distinct RNA profiles in separate types of EVs released by different cell lines, including apoptotic bodies, microvesicles and exosomes. 6 We therefore hypothesized that different types of EVs might contain distinct group of small RNA species. We use small RNA sequencing (Ion Torrent platform) to define the RNA species present in apoptotic bodies, microvesicles and exosomes isolated from the melanoma cell line MML-1.…”

Exosomes in cancer theranostic: Diamonds in the rough

“…43,44 The exosome membrane composition is the same than that of the mother cell but present specific enrichments and they contain proteins, lipid and genetic material (Fig. 1).…”