Abstract:Large oncosomes (LO) are atypically large (1-10μm diameter) cancer-derived extracellular vesicles (EVs), originating from the shedding of membrane blebs and associated with advanced disease. We report that 25% of the proteins, identified by a quantitative proteomics analysis, are differentially represented in large and nano-sized EVs from prostate cancer cells. Proteins enriched in large EVs included enzymes involved in glucose, glutamine and amino acid metabolism, all metabolic processes relevant to cancer. G… Show more
“…L-EVs were isolated from U2OS cells (osteosarcoma cell line) stably transfected with a GFP-conjugated histone H2B (U2OS-H2B-GFP). GFP signal was detected in purified L-EVs (Figure S1A, B) in line with previous identification of histones in EVs [24,32]. In L-EVs stained with Hoechst, a fluorescent dye that binds to the minor groove of dsDNA (Fig.…”
Section: Resultssupporting
confidence: 89%
“…In order to determine if we could use L-EVs and S-EVs as surrogates for LO and Exo, we tested expression of specific markers of LO and Exo, obtained by floatation of L-EVs and S-EVs in discontinuous density gradients [24]. L-EVs expressed LO markers CK18 and HSPA5 [24], and S-EVs expressed CD81, which is typically enriched in Exo [3], suggesting that we can use L- and S-EVs as surrogates for LO and Exo (Figure 3(b)).10.1080/20013078.2018.1505403-F0001Figure 1. Most extracellular DNA is packaged into L-EVs . (a) Tunable resistive pulse sensing (TRPS, qNano) using two different pore membranes (NP4000 and NP200) identified as L-EVs (left) and S-EVs (right) derived from PC3 cells.…”
Section: Resultsmentioning
confidence: 99%
“…For isolation of S-EVs, the supernatant obtained after the 10,000 g spin was subjected to additional centrifugation at 100,000 g for 60 min. In select experiments, L-EV and S-EV preparations were further subjected to density gradient centrifugation using discontinuous iodixanol density gradient (Optiprep™), following deposition of EVs at the bottom of the tubes, as described previously [24]. Briefly, 60%, 50%, 40%, 30%, 25%, 15%, 10% and 5% solutions were made by diluting the stock solution of Optiprep™ (60% aqueous iodixanol, Sigma) in 0.25 M sucrose/0.9 M NaCl/120 mM HEPES, pH 7.4.…”
Section: Methodsmentioning
confidence: 99%
“…L-EVs and S-EVs were isolated from human plasma samples (1 ml) by differential centrifugation as described above and previously reported [24]. The blood samples were collected in BD Vacutainer™ ACD tubes and processed within 2 h of phlebotomy.…”
Cancer-derived extracellular vesicles (EVs) are membrane-enclosed structures of highly variable size. EVs contain a myriad of substances (proteins, lipid, RNA, DNA) that provide a reservoir of circulating molecules, thus offering a good source of biomarkers. We demonstrate here that large EVs (L-EV) (large oncosomes) isolated from prostate cancer (PCa) cells and patient plasma are an EV population that is enriched in chromosomal DNA, including large fragments up to 2 million base pair long. While L-EVs and small EVs (S-EV) (exosomes) isolated from the same cells contained similar amounts of protein, the DNA was more abundant in L-EV, despite S-EVs being more numerous. Consistent with in vitro observations, the abundance of DNA in L-EV obtained from PCa patient plasma was variable but frequently high. Conversely, negligible amounts of DNA were present in the S-EVs from the same patients. Controlled experimental conditions, with spike-ins of L-EVs and S-EVs from cancer cells in human plasma from healthy subjects, showed that circulating DNA is almost exclusively enclosed in L-EVs. Whole genome sequencing revealed that the DNA in L-EVs reflects genetic aberrations of the cell of origin, including copy number variations of genes frequently altered in metastatic PCa (i.e. MYC, AKT1, PTK2, KLF10 and PTEN). These results demonstrate that L-EV-derived DNA reflects the genomic make-up of the tumour of origin. They also support the conclusion that L-EVs are the fraction of plasma EVs with DNA content that should be interrogated for tumour-derived genomic alterations.
“…L-EVs were isolated from U2OS cells (osteosarcoma cell line) stably transfected with a GFP-conjugated histone H2B (U2OS-H2B-GFP). GFP signal was detected in purified L-EVs (Figure S1A, B) in line with previous identification of histones in EVs [24,32]. In L-EVs stained with Hoechst, a fluorescent dye that binds to the minor groove of dsDNA (Fig.…”
Section: Resultssupporting
confidence: 89%
“…In order to determine if we could use L-EVs and S-EVs as surrogates for LO and Exo, we tested expression of specific markers of LO and Exo, obtained by floatation of L-EVs and S-EVs in discontinuous density gradients [24]. L-EVs expressed LO markers CK18 and HSPA5 [24], and S-EVs expressed CD81, which is typically enriched in Exo [3], suggesting that we can use L- and S-EVs as surrogates for LO and Exo (Figure 3(b)).10.1080/20013078.2018.1505403-F0001Figure 1. Most extracellular DNA is packaged into L-EVs . (a) Tunable resistive pulse sensing (TRPS, qNano) using two different pore membranes (NP4000 and NP200) identified as L-EVs (left) and S-EVs (right) derived from PC3 cells.…”
Section: Resultsmentioning
confidence: 99%
“…For isolation of S-EVs, the supernatant obtained after the 10,000 g spin was subjected to additional centrifugation at 100,000 g for 60 min. In select experiments, L-EV and S-EV preparations were further subjected to density gradient centrifugation using discontinuous iodixanol density gradient (Optiprep™), following deposition of EVs at the bottom of the tubes, as described previously [24]. Briefly, 60%, 50%, 40%, 30%, 25%, 15%, 10% and 5% solutions were made by diluting the stock solution of Optiprep™ (60% aqueous iodixanol, Sigma) in 0.25 M sucrose/0.9 M NaCl/120 mM HEPES, pH 7.4.…”
Section: Methodsmentioning
confidence: 99%
“…L-EVs and S-EVs were isolated from human plasma samples (1 ml) by differential centrifugation as described above and previously reported [24]. The blood samples were collected in BD Vacutainer™ ACD tubes and processed within 2 h of phlebotomy.…”
Cancer-derived extracellular vesicles (EVs) are membrane-enclosed structures of highly variable size. EVs contain a myriad of substances (proteins, lipid, RNA, DNA) that provide a reservoir of circulating molecules, thus offering a good source of biomarkers. We demonstrate here that large EVs (L-EV) (large oncosomes) isolated from prostate cancer (PCa) cells and patient plasma are an EV population that is enriched in chromosomal DNA, including large fragments up to 2 million base pair long. While L-EVs and small EVs (S-EV) (exosomes) isolated from the same cells contained similar amounts of protein, the DNA was more abundant in L-EV, despite S-EVs being more numerous. Consistent with in vitro observations, the abundance of DNA in L-EV obtained from PCa patient plasma was variable but frequently high. Conversely, negligible amounts of DNA were present in the S-EVs from the same patients. Controlled experimental conditions, with spike-ins of L-EVs and S-EVs from cancer cells in human plasma from healthy subjects, showed that circulating DNA is almost exclusively enclosed in L-EVs. Whole genome sequencing revealed that the DNA in L-EVs reflects genetic aberrations of the cell of origin, including copy number variations of genes frequently altered in metastatic PCa (i.e. MYC, AKT1, PTK2, KLF10 and PTEN). These results demonstrate that L-EV-derived DNA reflects the genomic make-up of the tumour of origin. They also support the conclusion that L-EVs are the fraction of plasma EVs with DNA content that should be interrogated for tumour-derived genomic alterations.
“…Given that our EV isolation procedure (filtration through 0.22 microns, and ultracentrifugation at 10,000 × g) removed most of the large EVs from the sample, and enrich in small EVs (mostly exosomes and small microvesicles), this difference could be underestimated in our samples. Importantly, in agreement with our result, it has already been reported that prostate cancer cells release large EVs named oncosomes with a size between 1 and 10 microns [31] that contain a distinct protein cargo [32]. They have also been detected in circulation in models of PCa and shown that their abundance correlate with tumoral progression [31].…”
Urine contains extracellular vesicles (EVs) that concentrate molecules and protect them from degradation. Thus, isolation and characterisation of urinary EVs could increase the efficiency of biomarker discovery. We have previously identified proteins and RNAs with differential abundance in urinary EVs from prostate cancer (PCa) patients compared to benign prostate hyperplasia (BPH). Here, we focused on the analysis of the metabolites contained in urinary EVs collected from patients with PCa and BPH. Targeted metabolomics analysis of EVs was performed by ultra-high-performance liquid chromatography–mass spectrometry. The correlation between metabolites and clinical parameters was studied, and metabolites with differential abundance in PCa urinary EVs were detected and mapped into cellular pathways. We detected 248 metabolites belonging to different chemical families including amino acids and various lipid species. Among these metabolites, 76 exhibited significant differential abundance between PCa and BPH. Interestingly, urine EVs recapitulated many of the metabolic alterations reported in PCa, including phosphathidylcholines, acyl carnitines, citrate and kynurenine. Importantly, we found elevated levels of the steroid hormone, 3beta-hydroxyandros-5-en-17-one-3-sulphate (dehydroepiandrosterone sulphate) in PCa urinary EVs, in line with the potential elevation of androgen synthesis in this type of cancer. This work supports urinary EVs as a non-invasive source to infer metabolic changes in PCa.
Microplasts are large extracellular vesicles originating from migratory, invasive, and metastatic cancer cells. Here, to gain insight into the role of microplasts in cancer progression, we performed a proteomic and transcriptomic characterization of microplasts isolated from MCF-7 breast cancer cells treated with macrophage-conditioned medium. These cells were found to be viable, highly migratory, and metabolically active, indicating that microplasts derived from these cells are not apoptotic bodies. Transcriptomic/proteomic analyses identified 10273 mRNAs and 821 proteins in microplasts. Interestingly, 377 microplast mRNAs coded for corresponding microplast proteins. Microplast mRNAs and proteins were mainly associated with binding and catalytic activities. Microplasts showed enrichment of mRNAs involved in transcription regulation and proteins involved in processes such as cell-cell adhesion and translation. Pathway analysis showed enrichment of ribosomes and carbon metabolism. These results suggest a close resemblance between microplasts and parent cells, with mRNA and protein cargo relevant in intercellular signaling.
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