Protein Cargo Of Extracellular Vesicles Research Articles

Extracellular vesicles secreted from bone metastatic renal cell carcinoma promote angiogenesis and endothelial gap formation in bone marrow in a time-dependent manner in a preclinical mouse model.

Bone is a major metastatic site of renal cell carcinoma (RCC). Recently, it is well recognized that bone metastatic tumor cells remodel bone marrow vasculature. However, the precise mechanism underlying cell-cell communication between bone metastatic RCC and the cells in bone marrow remains unknown. Extracellular vesicles (EVs) reportedly play crucial roles in intercellular communication between metastatic tumor cells and bone marrow. Therefore, we conducted the current study to clarify the histological alteration in vascular endothelium in bone marrow induced by EVs secreted from bone metastatic RCC cells as well as association between angiogenesis in bone marrow and bone metastasis formation. We established a bone metastatic RCC cell line (786-O BM) by in vivo selection and observed phenotypic changes in tissues when EVs were intravenously injected into immunodeficient mice. Proteomic analysis was performed to identify the protein cargo of EVs that could contribute to histological changes in bone. Tissue exudative EVs (Te-EVs) from cancer tissues of patients with bone metastatic RCC (BM-EV) and those with locally advanced disease (LA-EV) were compared for in vitro function and protein cargo. Treatment of mice with EVs from 786-O BM promoted angiogenesis in the bone marrow in a time-dependent manner and increased the gaps of capillary endothelium. 786-O BM EVs also promoted tube formation in vitro. Proteomic analysis of EVs identified aminopeptidase N (APN) as a candidate protein that enhances angiogenesis. APN knockdown in 786-O BM resulted in reduced angiogenesis in vitro and in vivo. When parental 786-O cells were intracardially injected 12 weeks after treatment with786-O BM EVs, more bone metastasis developed compared to those treated with EVs from parental 786-O cells. In patient samples, BM-EVs contained higher APN compared to LA-EV. In addition, BM-EVs promoted tube formation in vitro compared to LA-EVs. EVs from bone metastatic RCC promote angiogenesis and gap formation in capillary endothelium in bone marrow in a time-dependent manner.

Quantitative proteomic analysis of extracellular vesicles in response to baculovirus infection of a Trichoplusia ni cell line.

Due to its outstanding suitability to produce complex biopharmaceutical products including virus-like particles and subunit vaccines, the baculovirus/insect cell expression system has developed into a highly popular production platform in the biotechnological industry. For high productivity, virus-cell communication and an efficient spreading of the viral infection are crucial, and, in this context, extracellular vesicles (EVs) might play a significant role. EVs are small particles, utilized by cells to transfer biologically active compounds such as proteins, lipids as well as nucleic acids to recipient cells for intracellular communication. Studies in mammalian cells showed that the release of EVs is altered in response to infection with many viruses, ultimately either limiting or fostering infection spreading. In this study we isolated and characterized EVs, from both uninfected and baculovirus infected Tnms42 insect cells. Via quantitative proteomic analysis we identified more than 3000 T. ni proteins in Tnms42 cell derived EVs, of which more than 400 were significantly differentially abundant upon baculovirus infection. Subsequent gene set enrichment analysis revealed a depletion of proteins related to the extracellular matrix in EVs from infected cultures. Our findings show a significant change of EV protein cargo upon baculovirus infection, suggesting a major role of EVs as stress markers. Our study might serve in designing new tools for process monitoring and control to further improve biopharmaceutical production within the baculovirus/insect cell expression system.

Proteomic Profiling of Extracellular Vesicles Released by Leptin-Treated Breast Cancer Cells: A Potential Role in Cancer Metabolism.

Tumor extracellular vesicles (EVs), as endocytic vesicles able to transport nucleic acids, proteins, and metabolites in recipient cells, have been recognized fundamental mediators of cell-to-cell communication in breast cancer. The biogenesis and release of EVs are highly regulated processes and both the quantity of EVs and their molecular cargo might reflect the metabolic state of the producing cells. We recently demonstrated that the adipokine leptin, whose circulating levels correlate with adipose tissue expansion, is an inducer of EV release from breast cancer cells. Here, we show a specific proteomic signature of EVs released by MCF-7 breast cancer cells grown in the presence of leptin (Lep-EVs), in attempt to find additional molecular effectors linking obesity to breast cancer biology. An analysis of the proteomic profile of Lep-EVs by LC-MS/MS revealed a significant enrichment in biological processes, molecular functions, and cellular components mainly related to mitochondrial machineries and activity, compared to protein content of EVs from untreated breast cancer cells. Metabolic investigations, carried out to assess the autocrine effects of these vesicles on breast cancer cells, revealed that Lep-EVs were able to increase ATP levels in breast cancer cells. This result is associated with increased mitochondrial respiration evaluated by Seahorse analyzer, supporting the concept that Lep-EVs can modulate MCF-7 breast cancer cell oxidative metabolism. Moreover, taking into account the relevance of tumor immune cell crosstalk in the tumor microenvironment (TME), we analyzed the impact of these vesicles on macrophage polarization, the most abundant immune component in the breast TME. We found that tumor-derived Lep-EVs sustain the polarization of M0 macrophages, derived from the human THP-1 monocytic cells, into M2-like tumor-associated macrophages, in terms of metabolic features, phagocytic activity, and increased expression of CD206-positive population. Overall, our results indicate that leptin by inducing the release of EV-enriched in mitochondrial proteins may control the metabolism of MCF-7 breast cancer cells as well as that of macrophages. Characterization of tumor-derived EV protein cargo in an obesity-associated milieu, such as in the presence of elevated leptin levels, might allow identifying unique features and specific metabolic mechanisms useful to develop novel therapeutic approaches for treatment of breast cancer, especially in obese patients.

Watermelon-Derived Extracellular Vesicles Influence Human Ex Vivo Placental Cell Behavior by Altering Intestinal Secretions.

During pregnancy, mother-to-fetus transfer of nutrients is mediated by the placenta; sub-optimal placental development and/or function results in fetal growth restriction (FGR), and the attendant risk of stillbirth, neurodevelopmental delay, and non-communicable diseases in adulthood. A maternal diet high in fruit and vegetables lowers the risk of FGR but the association cannot be explained fully by known macro- and micronutrients. This study investigates if dietary-derived extracellular vesicles (EVs) can regulate placental function. The study characterizes the microRNA and protein cargo of EVs isolated from watermelon, show they are actively internalized by human intestinal epithelial cells in vitro, use mass spectrometry to demonstrate that they alter the intestinal secretome and bioinformatic analyses to predict the likely affected pathways in cells/tissues distal to gut. Application of the watermelon EV-modified intestinal secretome to human placental trophoblast cells and ex vivo tissue explants affects the trophoblast proteome and key aspects of trophoblast behavior, including migration and syncytialization. Dietary-derived plant EVs can modify intestinal communication with distal tissues, including the placenta. Harnessing the beneficial properties of dietary-derived plant EVs and/or exploiting their potential as natural delivery agents may provide new ways to improve placental function and reduce rates of FGR.

Proteome profiling of Extracellular Vesicles in Pseudomonas aeruginosa endophthalmitis: Prognostic and therapeutic significance in a mouse model

Endophthalmitis is a sight-threatening infection and a serious consequence of complications during intraocular surgery or penetrating injury of which Pseudomonas aeruginosa is an important etiology. Extracellular vesicles (EVs) have evolved as a promising entity for developing diagnostic and therapeutic biomarkers due to their involvement in intracellular communication and pathogenesis of diseases. We aimed to characterise the protein cargo of extracellular vesicles, isolated from a murine (C57BL/6) model of P. aeruginosa endophthalmitis by LC-MS/MS at 24 h post infection (p.i). EVs were extracted by ultracentrifugation, characterized by Dynamic Light Scattering (DLS) and western blotting with tetraspannin markers, CD9 and CD81 and quantified by the ExoCet quantification kit. Multiplex ELISA was performed to estimate the levels of TNF-α, IL-6, IFN-γ and IL-1β. Proteomic analysis identified 2010 proteins (FDR ≤0.01) in EVs from infected mice eyes, of which 137 were differentially expressed (P-value ≤ 0.05). A total of 101 proteins were upregulated and 36 were downregulated. Additionally, 43 proteins were exclusive to infection set. KEGG and Gene Ontology revealed, Focal adhesion, Phagosome pathway, Complement cascade and IL-17 signalling pathway are crucial upregulated pathways involving proteins such as Tenascin, caveolin 1, caveolin 2, glutamine synthetase, microtubule-associated protein, C1, C8 and IL-17. Tenascin and caveolins are known to suppress anti-inflammatory cytokines further exacerbating the disease. The result of this study provides insight into the global extracellular vesicle proteome of P. aeruginosa endophthalmitis with their functional correlation and distinctive pattern of expression and tenascin, caveolin 1 and caveolin 2 are attractive biomarkers for P. aeruginosa endophthalmitis.

Proteomic Analysis of Extracellular Vesicles From Fasciola hepatica Hatching Eggs and Juveniles in Culture.

The identification of extracellular vesicles (EVs) in Fasciola hepatica has provided a new way to understand parasite-host communication. Most of the studies on EVs have focused on the adult stage of F. hepatica, but recently, the presence of EVs from different developmental stages has been reported. To better understand the potential role of EVs in the biology of the parasite and in the infection process, the protein cargo of EVs from embryonated eggs and newly-excysted juvenile (NEJs) flukes cultured up to 28 days, has been analyzed. EVs were isolated by size exclusion chromatography and evaluated by nanoparticle tracking analysis and transmission electron microscopy. LC-MS/MS proteomic analysis of EVs revealed the presence of 23 different proteins from embryonated egg-derived EVs and 29 different proteins from NEJ-derived EVs. Most of the identified proteins had been previously described in EVs from F. hepatica adults, including cytoskeletal proteins, glycolytic enzymes, stress-related proteins and tetraspanins. Nevertheless, EVs from hatching eggs and NEJs exhibited qualitative differences in composition, when compared to EVs form adults, including the absence of cathepsin cysteine peptidases. The differential content of the EVs released by the different developmental stages of the parasite reflect the intense activity of NEJs at this early stage, with several proteins involved in membrane traffic and cell physiology. This new set of identified proteins could help to understand key metabolic, biochemical and molecular mechanisms mediated by EVs that take place upon egg hatching and after parasite excystment.

Characterization and proteome profiling of extracellular vesicles in a murine model of Staphylococcus aureus endophthalmitis

Endophthalmitis is a vision-threatening complication of intraocular surgery or penetrating injury of which Staphylococcus aureus is an important etiological agent. Extracellular vesicles (EVs) hold a tremendous possibility for developing diagnostic and therapeutic biomarkers due to their role in the pathogenesis of various infections. The aim of this study was to characterise the protein cargo of EVs, isolated from a murine (C57BL/6) model of S. aureus endophthalmitis by LC–MS/MS. Contralateral eye injected with sterile media served as control and both eyes were enucleated after 24 h, followed by extraction of EVs by ultracentrifugation. EVs were characterized by DLS and western blotting with tetraspanin markers, CD9 and CD81 and quantified by ExoCet quantification kit. Proteomic analysis identified 1964 proteins (FDR ≤ 0.01) in EVs from infected mice eyes, of which 40 proteins varied significantly in their amounts in comparison to EVs obtained from control eyeballs (P-value ≤ 0.05). The results of this study provide insight into the global EV proteome of S. aureus endophthalmitis with their functional correlation and differential protein amounts between infected and control set. Annexin A5, cathepsin D and C5a play a pivotal role in disease pathogenesis and could possibly play a role as a prognostic marker in endophthalmitis.

Obese Adipose Tissue‐Derived Extracellular Vesicles increase Colorectal Cancer Cell proliferation and glycolysis

Colorectal cancer (CRC) is the second deadliest cancer worldwide and obesity is an established risk factor for both men and women. However, we still have limited knowledge regarding the molecular mechanisms that support obesity‐induced CRC. Obesity substantially modifies the adipose tissue (AT) and increases secretion of various factors including nanometer‐sized extracellular vesicles (EVs). EVs are lipid bilayer vesicles released from most types of cells and contain lipids, proteins, microRNAs and other cytoplasmic components of the parent cell. Considering their ability to travel to distant sites and deliver cargo to recipient cells, EVs could act as intercellular mediators that alter the tumorigenic potential of recipient cancer cells. Therefore, we hypothesized that obese AT secreted EVs carry cargo that enhances tumorigenicity of CRC cells. Here, we aimed to determine the functional and metabolic changes induced by obese human AT‐secreted EVs on human CRC cell lines. Visceral AT tissue from patients with obesity (BMI>35) undergoing bariatric surgery and non‐obese patients (BMI<30) undergoing anti‐reflux surgery was cultured ex vivo in EV‐free DMEM‐H/Ham’s F12 for 48 hrs. Conditioned media was collected and EVs were isolated by ultracentrifugation. EV size and concentration were measured through Nanoparticle Tracking Analysis (NTA). EV protein cargo was characterized by unbiased proteomics analysis using LC‐MS. Human CRC cell lines HCT‐116 and HT‐29 were treated with 10^7 EV/ml for 24 or 48 hrs and proliferation and mitochondrial/glycolytic ATP production were measured by CCK‐8 assay or Seahorse XF ATP Rate Assay, respectively. NTA reported our EV preps were on average 120 nm in size, suggesting we isolated exosomes and/or small microvesicles. Using KEGG Pathway Database and Panther Protein Classification Software, 25% of EV proteins secreted from human AT were associated with metabolism, with 13% of total metabolic proteins in EVs associating with glycolysis. Obese EVs exhibited increased expression of glycolytic enzymes pyruvate kinase, phosphoglycerate kinase 1, and G3P dehydrogenase. Obese EVs increased proliferation and glycolytic ATP production in CRC cell lines, both serving as indicators of cancer aggressiveness. Taken together, our results suggest that obese adipose EVs mediate the tumorigenic potential of CRC cells. Our study is significant since it aims to identify druggable pathways which support tumorigenesis in obese CRC patients.

Circadian regulation of protein cargo in extracellular vesicles.

The circadian clock controls many aspects of physiology, but it remains undescribed whether extracellular vesicles (EVs), including exosomes, involved in cell-cell communications between tissues are regulated in a circadian pattern. We demonstrate a 24-hour rhythmic abundance of individual proteins in small EVs using liquid chromatography–mass spectrometry in circadian-synchronized tendon fibroblasts. Furthermore, the release of small EVs enriched in RNA binding proteins was temporally separated from those enriched in cytoskeletal and matrix proteins, which peaked during the end of the light phase. Last, we targeted the protein sorting mechanism in the exosome biogenesis pathway and established (by knockdown of circadian-regulated flotillin-1) that matrix metalloproteinase 14 abundance in tendon fibroblast small EVs is under flotillin-1 regulation. In conclusion, we have identified proteomic time signatures for small EVs released by tendon fibroblasts, which supports the view that the circadian clock regulates protein cargo in EVs involved in cell-cell cross-talk.

Heterotopic ossification vs. fracture healing: Extracellular vesicle cargo proteins shed new light on bone formation

Fibrodysplasia ossificans progressiva (FOP) is an extremely rare disease in which bone tissue forms in extraskeletal sites, which is known as heterotopic ossification (HO). Extracellular vesicles (EVs) are small phospholipid-enclosed particles released by various cells which have an emerging, but not completely understood role in various (patho)physiological processes. In order to further study the pathophysiology of FOP we conducted a small observational study comparing the proteomic profiles of EV cargo, derived from pooled plasma of four patient groups: FOP patient (N = 1) during active disease phase (flare-up), FOP patients during remission (N = 2), patients after long bone fracture (N = 20) and healthy controls (N = 10). After isolation of EVs – their protein cargo was determined using liquid chromatography / mass spectrometry, after which a functional gene enrichment analysis was performed. Our results show a sizeable difference of the proteomics profiles in which EVs from the bone fracture group show significant activity of integrin interactions, Wnt, VEGF, IGF-1 and PDGF pathways; conversely, FOP patients' EVs indicate that HO occurs via processes of innate immunity and the Ephrin B signaling pathway. We hypothesize that the Ephrin B signaling (expressed in EVs) contributes to HO by aiding in mesenchymal stem cell recruitment and osteogenic differentiation, as well as by contributing to the inflammatory response, including macrophage chemotaxis and activation. This is, to our knowledge, the first published analysis of EV protein cargo in FOP.

Influences of age, race, and sex on extracellular vesicle characteristics.

Recent attention has focused on the use of extracellular vesicles (EVs) as biological indicators of health and disease. These small, nano-sized membrane bound vesicles are secreted from cells into the extracellular space and can be readily isolated from bodily fluids. EVs can carry various bioactive molecules as cargo including DNA, RNA, proteins, and lipids. These EVs can provide a snapshot of the cell of origin and a window of opportunity to assess normal physiological states as well as pathophysiological states. For EVs to further develop as potential biomarkers of disease, it is important to characterize whether these vesicles and their associated cargo are altered in the context of demographic factors. Here, we summarize the current literature on how demographics such as age, race, and sex affect the levels and cargo of EVs. Age and sex influence both EV cargo and concentration while race studies report differences mostly in EV protein cargo. This review also identifies areas of future research and important considerations for the clinical use of EVs as biomarkers.

Time-gated Raman spectroscopy and proteomics analyses of hypoxic and normoxic renal carcinoma extracellular vesicles

Extracellular vesicles (EVs) represent a diverse group of small membrane-encapsulated particles involved in cell–cell communication, but the technologies to characterize EVs are still limited. Hypoxia is a typical condition in solid tumors, and cancer-derived EVs support tumor growth and invasion of tissues by tumor cells. We found that exposure of renal adenocarcinoma cells to hypoxia induced EV secretion and led to notable changes in the EV protein cargo in comparison to normoxia. Proteomics analysis showed overrepresentation of proteins involved in adhesion, such as integrins, in hypoxic EV samples. We further assessed the efficacy of time-gated Raman spectroscopy (TG-RS) and surface-enhanced time-gated Raman spectroscopy (TG-SERS) to characterize EVs. While the conventional continuous wave excitation Raman spectroscopy did not provide a notable signal, prominent signals were obtained with the TG-RS that were further enhanced in the TG-SERS. The Raman signal showed characteristic changes in the amide regions due to alteration in the chemical bonds of the EV proteins. The results illustrate that the TG-RS and the TG-SERS are promising label free technologies to study cellular impact of external stimuli, such as oxygen deficiency, on EV production, as well as differences arising from distinct EV purification protocols.

Extracellular Vesicle Signatures and Post-Translational Protein Deimination in Purple Sea Urchin (Strongylocentrotus purpuratus) Coelomic Fluid-Novel Insights into Echinodermata Biology.

Simple SummaryThe purple sea urchin (Strongylocentrotus purpuratus) is a marine invertebrate that populates the east side of the Pacific Ocean from Mexico to Alaska, inhabiting intertidal and near-shore subtidal waters. Due to their ancient relationship with vertebrates, sea urchins are an important research model for developmental biology, cell biology, and immunology, as well as for understanding regenerative responses and ageing. This study assessed a specific protein modification called deimination/citrullination, which can alter protein function, allowing proteins to take on multiple and variable roles in different processes related to health and disease. This study also identified how extracellular vesicles, which are lipid blebs released from cells that participate in key processes for cell communication in health and disease, can carry proteins, including such modified protein cargo. This study may furthermore provide a platform for novel biomarker development to assess sea urchin health, which could be further applied, including for the monitoring of environmental changes.The purple sea urchin (Strongylocentrotus purpuratus) is a marine invertebrate of the class Echinoidea that serves as an important research model for developmental biology, cell biology, and immunology, as well as for understanding regenerative responses and ageing. Peptidylarginine deiminases (PADs) are calcium-dependent enzymes that mediate post-translational protein deimination/citrullination. These alterations affect protein function and may also play roles in protein moonlighting. Extracellular vesicles (EVs) are membrane-bound vesicles that are released from cells as a means of cellular communication. Their cargo includes a range of protein and RNA molecules. EVs can be isolated from many body fluids and are therefore used as biomarkers in physiological and pathological responses. This study assessed EVs present in the coelomic fluid of the purple sea urchin (Strongylocentrotus purpuratus), and identified both total protein cargo as well as the deiminated protein cargo. Deiminated proteins in coelomic fluid EVs were compared with the total deiminated proteins identified in coelomic fluid to assess putative differences in deiminated protein targets. Functional protein network analysis for deiminated proteins revealed pathways for immune, metabolic, and gene regulatory functions within both total coelomic fluid and EVs. Key KEGG and GO pathways for total EV protein cargo furthermore showed some overlap with deimination-enriched pathways. The findings presented in this study add to current understanding of how post-translational deimination may shape immunity across the phylogeny tree, including possibly via PAD activity from microbiota symbionts. Furthermore, this study provides a platform for research on EVs as biomarkers in sea urchin models.

MP39-02 NEW EXOSOMAL PROTEIN AND MRNA BIOMARKERS FOR RENAL CANCER

You have accessJournal of UrologyKidney Cancer: Basic Research & Pathophysiology I (MP39)1 Sep 2021MP39-02 NEW EXOSOMAL PROTEIN AND MRNA BIOMARKERS FOR RENAL CANCER Richard Zieren, David Clark, Liang Dong, Leandro Ferreira Moreno, Morgan Kuczler, Kengo Horie, Louis Vermeulen, Hui Zhang, Sarah Amend, Theo de Reijke, and Kenneth Pienta Richard ZierenRichard Zieren More articles by this author , David ClarkDavid Clark More articles by this author , Liang DongLiang Dong More articles by this author , Leandro Ferreira MorenoLeandro Ferreira Moreno More articles by this author , Morgan KuczlerMorgan Kuczler More articles by this author , Kengo HorieKengo Horie More articles by this author , Louis VermeulenLouis Vermeulen More articles by this author , Hui ZhangHui Zhang More articles by this author , Sarah AmendSarah Amend More articles by this author , Theo de ReijkeTheo de Reijke More articles by this author , and Kenneth PientaKenneth Pienta More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002054.02AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Renal cancer (RCC) accounts for over 73,750 new cases and 14,830 deaths annually in the US. Non-invasive biomarkers are needed to distinguish benign from RCC in small renal masses and to distinguish between clear cell RCC (ccRCC) and papillary RCC (pRCC) subtypes. Extracellular vesicles (EVs), such as exosomes, are a promising source of biomarkers for RCC. Compared with EVs enriched from plasma and urinary, EVs enriched from conditioned cell media (CCM) are readily available and their cellular origin is specific. In this study we aimed to select potential biomarkers by analyzing mRNA and protein cargo of EVs from various RCC and immortalized benign kidney epithelial cell lines and assess biological functions of the EV cargo. METHODS: We used the following cell lines; 786O, 769P, and Caki1 (all ccRCC); ACHN, and Caki2 (all pRCC); and HK2, and RPTEC (benign epithelial kidney cells). CCM EVs were enriched by a combination of differential centrifugation, ultrafiltration, and size exclusion chromatography. All CCM EVs were counted by NanoFCM, negative stained and imaged by TEM, and assessed for EV-markers (CD63, CD81, Flot1) by Western blot. The Qiagen miRNeasy kit was used to extract RNA from CCM EVs, the NanoString nCounter low RNA input kit was used for RNA amplification in combination with the Nanostring nCounter PanCancer Progression assay. CCM EVs were processed by Tandem Mass Tag mass spectrometry (TMT MS). RESULTS: Counts by NanoFCM demonstrated ACHN secreted the most EVs per million cells (1.4×10e7) and Caki-1 the least (5.6×10e4) EVs per million cells. Western blot expression of EV-associated markers CD81, CD63, CD9, and Flot1 was highly correlated with marker expression detected by TMT MS. In mRNA-analyses we found abundance of 74 unique mRNAs in benign EVs, 12 in ccRCC EVs, and 29 in pRCC EVs. By MS analysis we identified 1,726 proteins of which 186 proteins were enriched (> 1.5-fold change) in all types of EVs compared to their parental cell lysates. We found 83-124 proteins with increased abundance in pRCC EVs compared with benign EVs and 83-121 proteins with decreased abundance. In ccRCC EVs we found 95-141 increased abundance compared with benign EVs and 116-130 with decreased abundance. CONCLUSIONS: We compared CCM EVs derived from ccRCC, pRCC, and benign renal cell lines using a multi-omics approach. We used different experimental designs for the mRNA-analysis and mass spectrometry due to technical limitations, yet we were able to analyze exosomal cargos of benign and malign kidney cells. We identified several potential RCC-biomarkers that require further clinical validation. Source of Funding: Stichting Cure for Cancer (Amsterdam) © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e704-e704 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Richard Zieren More articles by this author David Clark More articles by this author Liang Dong More articles by this author Leandro Ferreira Moreno More articles by this author Morgan Kuczler More articles by this author Kengo Horie More articles by this author Louis Vermeulen More articles by this author Hui Zhang More articles by this author Sarah Amend More articles by this author Theo de Reijke More articles by this author Kenneth Pienta More articles by this author Expand All Advertisement Loading ...

Trastuzumab Modulates the Protein Cargo of Extracellular Vesicles Released by ERBB2+ Breast Cancer Cells

Cancers overexpressing the ERBB2 oncogene are aggressive and associated with a poor prognosis. Trastuzumab is an ERBB2 specific recombinant antibody employed for the treatment of these diseases since it blocks ERBB2 signaling causing growth arrest and survival inhibition. While the effects of Trastuzumab on ERBB2 cancer cells are well known, those on the extracellular vesicles (EVs) released from these cells are scarce. This study focused on ERBB2+ breast cancer cells and aimed to establish what type of EVs they release and whether Trastuzumab affects their morphology and molecular composition. To these aims, we performed immunoelectron microscopy, immunoblot, and high-resolution mass spectrometry analyses on EVs purified by differential centrifugation of culture supernatant. Here, we show that EVs released from ERBB2+ breast cancer cells are polymorphic in size and appearance and that ERBB2 is preferentially associated with large (120 nm) EVs. Moreover, we report that Trastuzumab (Tz) induces the expression of a specific glycosylated 50 kDa isoform of the CD63 tetraspanin and modulates the expression of 51 EVs proteins, including TOP1. Because these proteins are functionally associated with organelle organization, cytokinesis, and response to lipids, we suggest that Tz may influence these cellular processes in target cells at distant sites via modified EVs.

Environmental Plasticity of the RNA Content of Staphylococcus aureus Extracellular Vesicles.

The roles of bacterial extracellular vesicles (EVs) in cell-to-cell signaling are progressively being unraveled. These membranous spheres released by many living cells carry various macromolecules, some of which influence host-pathogen interactions. Bacterial EVs contain RNA, which may serve in communicating with their infected hosts. Staphylococcus aureus, an opportunistic human and animal pathogen, produces EVs whose RNA content is still poorly characterized. Here, we investigated in depth the RNA content of S. aureus EVs. A high-throughput RNA sequencing approach identified RNAs in EVs produced by the clinical S. aureus strain HG003 under different environmental conditions: early- and late-stationary growth phases, and presence or absence of a sublethal vancomycin concentration. On average, sequences corresponding to 78.0% of the annotated transcripts in HG003 genome were identified in HG003 EVs. However, only ~5% of them were highly covered by reads (≥90% coverage) indicating that a large fraction of EV RNAs, notably mRNAs and sRNAs, were fragmented in EVs. According to growth conditions, from 86 to 273 highly covered RNAs were identified into the EVs. They corresponded to 286 unique RNAs, including 220 mRNAs. They coded for numerous virulence-associated factors (hld encoded by the multifunctional sRNA RNAIII, agrBCD, psmβ1, sbi, spa, and isaB), ribosomal proteins, transcriptional regulators, and metabolic enzymes. Twenty-eight sRNAs were also detected, including bona fide RsaC. The presence of 22 RNAs within HG003 EVs was confirmed by reverse transcription quantitative PCR (RT-qPCR) experiments. Several of these 286 RNAs were shown to belong to the same transcriptional units in S. aureus. Both nature and abundance of the EV RNAs were dramatically affected depending on the growth phase and the presence of vancomycin, whereas much less variations were found in the pool of cellular RNAs of the parent cells. Moreover, the RNA abundance pattern differed between EVs and EV-producing cells according to the growth conditions. Altogether, our findings show that the environment shapes the RNA cargo of the S. aureus EVs. Although the composition of EVs is impacted by the physiological state of the producing cells, our findings suggest a selective packaging of RNAs into EVs, as proposed for EV protein cargo. Our study shedds light to the possible roles of potentially functional RNAs in S. aureus EVs, notably in host-pathogen interactions.

Phenotypic and Proteomic Analysis Identifies Hallmarks of Blood Circulating Extracellular Vesicles in NSCLC Responders to Immune Checkpoint Inhibitors.

Simple SummaryPurpose of this study was to investigate the prognostic and predictive role of blood circulating extracellular vesicles (EVs) in patients with advanced non-small cell lung cancer treated with immunotherapy. A newly optimized flow cytometry protocol was applied for identification and subtyping of blood circulating EVs in a total cohort of 59 NSCLC patients, which included 31 patients treated with anti-PD-1/PD-L1 agents and 28 patients treated with traditional chemotherapy. Our results show that pre-treatment concentration of blood circulating endothelial-derived EVs was correlated with overall survival and clinical response in patients treated with immunotherapy. Additionally, proteomic analysis of purified blood circulating EVs indicated differences in EV protein cargo between responders and non-responders to immunotherapy. These findings may pave the way to the identification of novel immunotherapy biomarkers in patients with advanced NSCLC.Immune checkpoint inhibitors (ICIs) induce durable clinical responses only in a subset of advanced non-small cell lung cancer (NSCLC) patients. There is a need to identify mechanisms of ICI resistance and immunotherapy biomarkers to improve clinical benefit. In this study, we evaluated the prognostic and predictive value of circulating endothelial and leukocyte-derived extracellular vesicles (EV) in patients with advanced NSCLC treated with anti-PD-1/PD-L1 agents. In addition, the relationship between total blood circulating EV proteome and response to ICIs was investigated. An optimized flow cytometry method was employed for the identification and subtyping of blood circulating EVs in 59 patients with advanced NSCLC. Blood samples were collected from patients receiving anti-PD-1/PD-L1 inhibitors (n = 31) or chemotherapy (n = 28). An exploratory proteomic analysis of sorted blood EVs was conducted in a subset of patients. Our results show that a low blood concentration of circulating endothelial-derived EVs before treatment was strongly associated to longer overall survival (p = 0.0004) and higher disease control rate (p = 0.045) in patients treated with ICIs. Interestingly, shotgun proteomics revealed that EVs of responders to anti-PD-1 therapy had a specific protein cargo before treatment. In addition, EV protein cargo was specifically modulated during immunotherapy. We identified a previously unknown association between circulating endothelial-derived extracellular vesicle concentration and immunotherapy-related clinical outcomes. We also observed differences in circulating extracellular vesicle proteome according to anti-PD-1-based treatment response in NSCLC patients. Overall, these results may contribute to the identification of novel circulating biomarkers for rational immunotherapy approaches in patients affected by NSCLC.

The Proteome and Citrullinome of Hippoglossus hippoglossus Extracellular Vesicles-Novel Insights into Roles of the Serum Secretome in Immune, Gene Regulatory and Metabolic Pathways.

Extracellular vesicles (EVs) are lipid bilayer vesicles which are released from cells and play multifaceted roles in cellular communication in health and disease. EVs can be isolated from various body fluids, including serum and plasma, and are usable biomarkers as they can inform health status. Studies on EVs are an emerging research field in teleost fish, with accumulating evidence for important functions in immunity and homeostasis, but remain to be characterised in most fish species, including halibut. Protein deimination is a post-translational modification caused by a conserved family of enzymes, named peptidylarginine deiminases (PADs), and results in changes in protein folding and function via conversion of arginine to citrulline in target proteins. Protein deimination has been recently described in halibut ontogeny and halibut serum. Neither EV profiles, nor total protein or deiminated protein EV cargos have yet been assessed in halibut and are reported in the current study. Halibut serum EVs showed a poly-dispersed population in the size range of 50–600 nm, with modal size of EVs falling at 138 nm, and morphology was further confirmed by transmission electron microscopy. The assessment of EV total protein cargo revealed 124 protein hits and 37 deiminated protein hits, whereof 15 hits were particularly identified in deiminated form only. Protein interaction network analysis showed that deimination hits are involved in a range of gene regulatory, immune, metabolic and developmental processes. The same was found for total EV protein cargo, although a far wider range of pathways was found than for deimination hits only. The expression of complement component C3 and C4, as well as pentraxin-like protein, which were identified by proteomic analysis, was further verified in EVs by western blotting. This showed that C3 is exported in EVs at higher levels than C4 and deiminated C3 was furthermore confirmed to be at high levels in the deimination-enriched EV fractions, while, in comparison, C4 showed very low detection in deimination-enriched EV fractions. Pentraxin was exported in EVs, but not detected in the deimination-enriched fractions. Our findings provide novel insights into EV-mediated communication in halibut serum, via transport of protein cargo, including post-translationally deiminated proteins.

Isolation of Proteins from Extracellular Vesicles (EVs) for Mass Spectrometry-Based Proteomic Analyses.

Extracellular vesicles (EVs) are freely circulating nano/micrometer-sized membrane-bound vesicles released by various cell types. Their cargo consists of proteins, lipids, metabolites, and different types of RNA molecules reflecting the origin of the secreting cell type or tissue. Since the EV cargo is constantly changing in response to pathological status or different environmental stressors, it has been extensively studied in the quest for biomarkers, especially in the cancer research. Mass spectrometry (MS)-based proteome analysis is a powerful tool to elucidate the protein cargo in EVs. This chapter describes and characterizes three MS-compatible lysis methods, namely by using urea, guanidium hydrochloride, and radioimmunoprecipitation buffer for isolating proteins from EVs.

Proteomic Characterization of EVs in Non-pathogenic Yeast Cells.

Most research on extracellular vesicles (EVs) from non-pathogenic fungi has been conducted in S. cerevisiae, taking advantage of the tools available for this model organism; but a few studies on EVs from yeasts of biotechnological interest are also available. Proteomic analyses in EVs from different yeast species and under different culture conditions are consistent in the identification of proteins related to glycolysis and cell wall biogenesis. Consequently, cell wall metabolism and biosynthesis appear as major functions of EVs. Additional functions have been proposed attending to the known biological activities identified on EVs proteomes, including interspecific antagonism, protection against antimicrobial agents, or clearance of aggregates of misfolded proteins (e.g. prion-like proteins). However, caution should be taken since some of these proteins might play a different role in the intracellular space or EVs (including some well known moonlighting proteins). It is also possible that many proteins appear in EVs as an indirect consequence of cellular metabolism and protein traffic, not related to a specific role in the extracellular space. These considerations become especially relevant in the context of the increasing detection power of proteomic technologies, leading in some cases to the identification of thousands of different proteins in the EVs proteome. Mutations in different secretory pathways have been related to differences in protein cargo of EVs, but no mutation has been found completely abolishing the production of EVs. Further work on the composition and biogenesis of EVs is required to better understand their biological significance.