Biology subjects

Crescitelli, R.

Publications and source records attributed to Crescitelli, R..

4 recordsLinked to original sources

bioRxiv2025

Proteomic and lipidomic profiling of immune cell-derived subpopulations of extracellular vesicles

IntroductionExtracellular vesicles (EVs) are a heterogeneous group of membrane-enclosed vesicles released by cells. They play important roles in intercellular communication and contribute to several physiological and pathological processes. Cells release subpopulations of EVs with distinct biogenesis and functions, however, we currently have few markers to differentiate them. This study, therefore, aimed to determine proteomic and lipidomic differences among four EV subpopulations of varying sizes and densities. MethodsLarge and small EVs (L-EVs and S-EVs) were isolated from two immune cell lines by differential ultracentrifugation at 16,500 x g and 118,000 x g, respectively. The crude EVs were then further separated by density cushion centrifugation. EVs were isolated from the interphase between 1.079-1.146 and 1.146-1.185 g/ml, hereafter referred to as low density (LD) and high density (HD), respectively. This resulted in four subpopulations of EVs: L-EVs LD, L-EVs HD, S-EVs LD, and S-EVs HD. The morphology, size, and yield of EVs were determined by nanoparticle tracking analysis, electron microscopy, and western blot. The proteome and lipidome of the four subpopulations of EVs were determined with mass spectrometry. ResultsA total of 5364 proteins were quantified in the dataset. L-EV and S-EVs as well as LD and HD were well separated. Briefly, L-EVs LD were enriched in mitochondrial proteins such as the TIMM/TOMM complex, MICOS, and ATP5 proteins. In contrast, L-EVs HD were enriched in proteins associated with the cytoskeleton, such as KIF proteins. Furthermore, S-EVs LD were enriched in tetraspanins and ESCRT machinery proteins, while S-EVs HD were enriched in histones, CCT proteins, and proteins from the complement pathway. Proteins such as flotillins, RABs, annexins, and integrins were enriched in two or several subpopulations. Furthermore, 107 lipids were quantified, and the most abundant lipids in EVs were phosphatidylcholine (PC), sphingomyelin, and phosphatidylethanolamine (PE). The most profound difference was that PE was less abundant in L-EVs LD as compared to the other EV subtypes, and ceramides were enriched in L-EVs as compared to S-EVs. ConclusionThis study demonstrates that the proteome and lipidome differ in EV subpopulations separated based on size and density. Furthermore, it validates several protein groups that have previously been suggested to be enriched in either S-EVs or L-EVs.

molecular biology↗

bioRxiv2024

Extracellular vesicles isolated from frozen and fresh human melanoma tissue are similar in purity and protein composition

Extracellular vesicles (EVs) isolated from tumor tissues represent a precious source of EVs because they are enriched in disease-associated biomarkers and are highly concentrated. However, a fundamental question is whether freezing of tissues influences the EVs integrity and function and whether non-EVs are co-isolated with the EVs. In this work, we isolated EVs from metastatic melanoma tissue both immediately after tissue resection and after being frozen for at least two weeks. Specifically, the samples were divided into two parts: one was immediately processed for EV isolation (hereafter called fresh), and the other was frozen on dry ice and stored for two weeks before being processed for EV isolation (hereafter called frozen). Large and small EVs were isolated through ultracentrifugation, pooled, and further purified on an iodixanol density cushion. The EVs were analyzed by transmission electron microscopy, nanoparticle tracking analysis, and protein quantification as well as by quantitative mass spectrometry. The results did not show any significant difference between EVs isolated from fresh and frozen tissue. Importantly, there was no enrichment of either intracellular proteins or mitochondrial proteins in EVs isolated from frozen tissues vs. fresh tissues. Moreover, there were no significant differences in the quantity of proteins like MT-CO2, Cox6, SLC24A22, HLA-DR, and Erlin2, which were previously identified as potential markers of melanoma, ovarian cancer, and breast cancer. Overall, this study supports the use of frozen tissues as a source of EVs for research purposes because frozen tissue-derived EVs do not differ in purity or protein composition compared to their fresh counterparts.

cell biology↗

bioRxiv2023

Heat inactivation of foetal bovine serum causes protein contamination of extracellular vesicles

Both the release of extracellular vesicles (EVs) in cell cultures and the cargo that these EVs carry can be influenced by cell culture conditions such as the presence of foetal bovine serum (FBS). Although several studies have evaluated the effect of removing FBS-derived EVs by ultracentrifugation (UC), less is known about the influence of FBS heat inactivation on the cell-derived EVs. To assess this, three protocols based on different combinations of EV depletion by UC and heat inactivation were evaluated, including FBS that was ultracentrifuged but not heat-inactivated, FBS that was heat inactivated before EV depletion, and FBS that was heat inactivated after EV depletion. The FBS samples were then added to the culture media of three melanoma cell lines, and after 72 h both large and small EVs were isolated by differential UC. We demonstrated by transmission electron microscopy, protein measurement, and quantification of the number of particles that heat inactivation performed after EV depletion reduced the purity of small EVs but had no effect on large EV purity. Quantitative mass spectrometry analysis of FBS-derived small EVs showed that the EV protein content was different when FBS was heat inactivated after EV depletion compared to EVs isolated from FBS that was not heat inactivated or that was heat inactivated prior to EV depletion. Moreover, several of the quantified proteins were wrongly attributed to be of human origin because the EVs were of obvious bovine origin. Our results demonstrated that proteins of bovine origin coming from FBS-derived EVs could mistakenly be attributed to human cell-derived EVs in EV proteomic studies. Moreover, we concluded that heat inactivation performed after EV depletion induced the release of proteins that might contaminate EV samples, and the recommendation is therefore to always perform heat inactivation prior to EV depletion.

cell biology↗

bioRxiv2017

A subgroup of mitochondrial extracellular vesicles discovered in human melanoma tissues are detectable in patient blood

Extracellular vesicles (EVs), including exosomes and microvesicles, are secreted from all cells, and convey messages between cells in health and disease. However, the diversity of EV subpopulations are only beginning to be explored. Since EVs have been implicated in tumor microenvironmental communication, we started to determine the diversity of EVs specifically in this tissue. To do this, we isolated EVs directly from patient melanoma metastatic tissues. Using EV membrane isolation and mass spectrometry analysis, we discovered enrichment of mitochondrial membrane proteins in the melanoma tissue-derived EVs, compared to non-melanoma-derived EVs. Specifically, EVs positive for a combination of the two mitochondrial inner membrane proteins MT-CO2 (mitochondrial genome) and COX6c (nuclear genome) were detected in the plasma of melanoma patients, and in ovarian and breast cancer patients. Furthermore, this subpopulation of EVs, contains active mitochondrial enzymes. Our findings show that tumor tissues are enriched in EVs with mitochondrial proteins and enzymatic activity, and these EVs can be detected in blood.

cancer biology↗