Extracellular Vesicles In Patients Research Articles

#212 The clinical significance of circulating piRNAs in extracellular vesicles in patients with idiopathic membranous nephropathy

Abstract Background and Aims Of various small RNAs, many miRNAs have been studied as potential biomarkers for various renal diseases. However, there are few literatures about P-element-induced wimpy testis-interacting RNAs (piRNAs), which are the most recently discovered class of small RNAs. Therefore, we investigated the clinical significance of piRNAs in idiopathic membranous (IMN) patients. Method We prospectively enrolled 60 participants, including those with IMN (n = 19) and idiopathic nephrotic syndrome (INS; n = 21) and healthy volunteers (HVs; n = 20) in this study. Using RNA sequencing, we assessed the serum extracellular vesicle (EV)-piRNA profiles of all participants. We investigated whether the piRNAs could be helpful to discriminate IMN from INS and predict the treatment response of patients with IMN. Based on achievement of clinical remission, IMN patients were divided in to two groups: a well-responding (IMN-W) and refractory (IMN-R) group [IMN-W (n = 9) vs IMN-R (n = 10)]. Results We found 11 piRNAs that were upregulated and 6 that were downregulated in patients with IMN when compared to HVs. There were 22 miRNAs that were upregulated and 23 that were downregulated in patients with IMN when compared to the INS. Among these miRNAs, we found 5 piRNAs, whose levels were significantly up- or downregulated in patients with IMN compared to HVs and patients with INS. There were 50 upregulated and 11 downregulated piRNAs in patients with IMN-R when compared to patients with IMN-W. Of these piRNAs, the expression level of piRNA-42775 and piRNA-36743 were found to be inversely correlated with proteinuria on kidney biopsy. Meanwhile, a positive correlation was observed between renal function and expression of piRNA-43108. Conclusion Patients with IMN have a distinct EV-piRNA expression profile compared with INS and HVs. In addition, circulating EV-piRNAs could be useful to predict the treatment response of IMN patients.

Profiling plasma alterations of extracellular vesicles in patients with acutely decompensated cirrhosis and bacterial infection.

Extracellular vesicles (EVs) modulate inflammation, coagulation and vascular homeostasis in decompensated cirrhosis. To characterize the profile of plasmatic EVs in patients with decompensated cirrhosis and bacterial infections and evaluate the association between EVs and the development of hemostatic complications. We measured the levels of EVs using high-sensitivity flow cytometry and phospholipid-dependent clotting time (PPL) in a prospective cohort of hospitalized patients with acutely decompensated cirrhosis with versus without bacterial infections. A separate cohort of patients with bacterial infections without cirrhosis was also enrolled. We measured endothelium-, tissue factor (TF)-bearing, platelet- and leukocyte-derived EVs. In patients with infections, EVs were reassessed upon resolution of infection. Bleeding and thrombotic complications were recorded during 1-year follow-up. Eighty patients with decompensated cirrhosis were recruited (40 each with and without bacterial infections). Electron microscopy confirmed the presence of plasma EVs. Despite no difference in total EVs and PPL, patients with cirrhosis and infection had significantly higher TF+ EVs, P-Selectin+ EVs (activated platelet-derived), CD14+ EVs (monocyte/macrophages derived) and CD14+ TF+ EVs versus those with cirrhosis without infection. Upon infection resolution, levels of these EVs returned to those without infection. Patients with infections showed a significant association between reduced P-Selectin+ EVs and bleeding complications (HR 8.0 [95%CI 1.3-48.1]), whereas high levels of leukocyte-derived EVs (CD45+) and CD14+ EVs were significantly associated with thrombotic complications (HR 16.4 [95%CI 1.7-160] and 10.9 [95%CI 1.13-106], respectively). Results were confirmed in a validation cohort. Bacterial infections are associated with particular alterations of plasma EVs profile in decompensated cirrhosis. Bacterial infections trigger the release of EVs originating from various cell types, which may tip the precarious hemostatic balance of patients with acutely decompensated cirrhosis towards hyper- or hypocoagulability.

Characterising the HLA‐I immunopeptidome of plasma‐derived extracellular vesicles in patients with melanoma

AbstractExtracellular vesicles (EVs) frequently express human leukocyte antigen class I (HLA‐I) molecules. The immunopeptidomes presented on EV HLA‐I are being mapped to provide key information on both specific cancer‐related peptides, and for larger immunopeptidomic signatures associated with disease. Utilizing HLA‐I immunoisolation and mass spectrometry, we characterised the HLA‐I immunopeptidome of EVs derived from the melanoma cancer cell line, ESTDAB‐026, and the plasma of 12 patients diagnosed with advanced stage melanoma, alongside 11 healthy controls. The EV HLA‐I immunopeptidome derived from melanoma cells features T cell epitopes with known immunogenicity and peptides derived from known tumour associated antigens (TAAs). Both T cell epitopes with known immunogenicity and peptides derived from known TAAs were also identifiable in the melanoma patient samples. Patient stratification into two distinct groups with varying immunological profiles was also observed. The data obtained in this study suggests for the first time that the HLA‐I immunopeptidome of EVs derived from blood may aid in the detection of important diagnostic or prognostic biomarkers and also provide new immunotherapy targets.

Gut microbiome-derived bacterial extracellular vesicles in patients with solid tumours

IntroductionGut microbiome–derived nanoparticles, known as bacterial extracellular vesicles (bEVs), have garnered interest as promising tools for studying the link between the gut microbiome and human health. The diverse composition of bEVs, including their proteins, mRNAs, metabolites, and lipids, makes them useful for investigating diseases such as cancer. However, conventional approaches for studying gut microbiome composition alone may not be accurate in deciphering host–gut microbiome communication. In clinical microbiome research, there is a gap in the knowledge on the role of bEVs in solid tumor patients. ObjectivesAnalyzing the functionality of bEVs using (meta)genomics and proteomics could highlight the unique aspects of host–gut microbiome interactions in solid tumor patients. Therefore, we performed a comparative analysis of the proteome and microbiota composition of gut microbiome-derived bEVs isolated from patients with solid tumors and healthy controls. MethodsAfter isolating bEVs from the feces of solid tumor patients and healthy controls, we performed spectrometry analysis of their proteomes and next-generation sequencing (NGS) of the 16S gene. We also investigated the gut microbiomes of feces from patients and controls using 16S sequencing and machine learning to classify the samples into patients and controls based on their bEVs and fecal microbiomes. ResultsSolid tumor patients showed decreased microbiota richness and diversity in both the bEVs and feces. However, the bEV proteomes were more diverse in patients than in the controls and were enriched with proteins associated with the metabolism of amino acids and carbohydrates, nucleotide binding, and oxidoreductase activity. Metadata classification of samples was more accurate using fecal bEVs (100%) compared with fecal samples (93%). ConclusionOur findings suggest that bEVs are unique functional entities. There is a need to explore bEVs together with conventional gut microbiome analysis in functional cancer research to decipher the potential of bEVs as cancer diagnostic or therapeutic biomarkers.

Lipoprotein apheresis affects the concentration of extracellular vesicles in patients with elevated lipoprotein (a)

Lipoprotein apheresis (LA) is a therapeutic option for hyperlipoproteinemia(a) (hyper-Lp(a)) and atherosclerotic cardiovascular disease (ASCVD). LA improves blood rheology, reduces oxidative stress parameters and improves endothelial function. The underlying molecular mechanisms of LA beneficial effects are unknown, but it has been suggested that LA exhibits multiple activities beyond simply removing lipoproteins. We hypothesized that LA removes not only lipoproteins, but also extracellular vesicles (EVs). To test this hypothesis, we performed a prospective study in 22 patients undergoing LA for hyper-Lp(a) and ASCVD. Different EVs subtypes were measured before and directly after LA, and after 7 days. We used calibrated flow cytometry to detect total particle concentration (diameter > ~ 100 nm), total lipoproteins concentration (diameter > 200 nm, RI > 1.51), total EV concentration (diameter > 200 nm, RI < 1.41), concentrations of EVs derived from erythrocytes (CD235a+; diameter > 200 nm, RI < 1.41), leukocytes (CD45+; diameter > 200 nm, RI < 1.41) and platelets (CD61+, PEVs; diameter > 200 nm, RI < 1.41). LA reduced the concentrations of all investigated EVs subtypes and lipoproteins. Lp(a) concentration was lowered by 64.5% [(58% – 71%); p < 0.001]. Plasma concentrations of EVs > 200 nm in diameter derived from platelets (CD61 +), leukocytes (CD45+) and erythrocytes (CD235a+) decreased after single LA procedure by 42.7% [(12.8–54.7); p = 0.005], 42.6% [(29.7–54.1); p = 0.030] and 26.7% [(1.0–62.7); p = 0.018], respectively, compared to baseline. All EV subtypes returned to the baseline concentrations in blood plasma after 7 days. To conclude, LA removes not only Lp(a), but also cell-derived EVs, which may contribute to LA beneficial effects.

Molecular characterization of small extracellular vesicles in patients with pancreatic cancer treated with neoadjuvant chemotherapy followed by stereotactic body radiation (SBRT).

685 Background: Currently, pancreatic cancer (PanC) is one of the serious gastrointestinal diseases, and more than 90% of the patients die within five years of diagnosis. Therefore, to improve PanC-related mortality, new therapeutic and diagnostic/prognostic measures are urgently needed. In this substudy, we examined the usefulness of plasma small extracellular vesicles (sEV) to discover molecular biomarkers associated with treatment response and overall survival from the plasma samples collected on a prospective clinical trial assessing the efficacy of SBRT following chemotherapy in pancreatic cancers (NCT03600623). Methods: PanCpatients (n=22) with locally advanced and borderline inoperable disease were recruited at the University of Alabama Comprehensive Cancer Center. They were administered either FOLFIRINOX (5-FU, folinic acid, oxaliplatin, and irinotecan) or paclitaxel (gemcitabine-nab-paclitaxel) for 2 months followed by SBRT (33 Gray in 5 fractions). The primary objective of this single-center pilot study was to evaluate the safety and tolerability of this treatment regimen. Further, blood was collected at baseline and, at the end of chemotherapy and radiotherapy. sEV were isolated from archived plasma samples by an established ultracentrifugation method and characterized for size and concentration by nanoparticle tracking analyses (NTA), shape and size by transmission electron microscope (TEM), and surface expression of exosomal tetraspanin markers (CD63, CD9, and CD81) and a PanC marker (CA19-9) by nano-flow cytometry. Lastly, sEV in longitudinal plasma samples were characterized for the expression of specific PanC-related miRNAs (miR196a-5p, miR155-5, miR194-5p, miR301-3p, miR21-5p, miR1246, and miR34a-5p) by real time-PCR (RT-PCR). Results: Neoadjuvant FOLFIRINOX and gemcitabine-nab-paclitaxel followed by SBRT were safe and well tolerated by most patients. NTA data showed that the ultracentrifugation method yielded highly pure sEV (with average diameter of &lt;200 nm) from archived baseline and longitudinal plasma samples. TEM analysis further confirmed the shape and size of the isolated sEV. Nano-flow cytometry showed the expression of exosomal markers, CD63, CD9, and CD81, as well as PanC marker CA19-9 on the surfaces of sEV. The expression of various PanC-related miRNAs in sEV was heterogenous and correlated with corresponding clinical parameters, including treatment response and overall survival. Conclusions: Neoadjuvant chemotherapy in combination with SBRT is safe and tolerable regimen to treat patients with locally advanced and borderline PanC. Further, sEV in the plasma of PanC patients could serve as useful prognostic and predictive markers.

Novel mediator in anaphylaxis: decreased levels of miR-375-3p in serum and within extracellular vesicles of patients.

Anaphylaxis is among the most severe manifestations of allergic disorders, but its molecular basis remains largely unknown and reliable diagnostic markers are not currently available. MicroRNAs (miRNAs) regulate several pathophysiological processes and have been proposed as non-invasive biomarkers. Therefore, this study aims to evaluate their involvement in anaphylactic reaction and their value as biomarkers. Acute (anaphylaxis) and baseline (control) serum samples from 67 patients with anaphylaxis were studied. Among them, 35 were adults with drug-induced anaphylaxis, 13 adults with food-induced anaphylaxis and 19 children with food-induced anaphylaxis. The circulating serum miRNAs profile was characterized by next-generation sequencing (NGS). For this purpose, acute and baseline samples from 5 adults with drug-induced anaphylaxis were used. RNA was extracted, retrotranscribed, sequenced and the readings obtained were mapped to the human database miRBase_20. In addition, a system biology analysis (SBA) was performed with its target genes and revealed pathways related to anaphylactic mediators signaling. Moreover, functional and molecular endothelial permeability assays were conducted with miR-375-3p-transfected cells in response to cAMP. A total of 334 miRNAs were identified, of which 21 were significant differentially expressed between both phases. Extracellular vesicles (EVs) were characterized by Western blot, electron microscopy and NanoSight. A decrease of miR-375-3p levels was determined by qPCR in both serum and EVs of patients with anaphylaxis (****p<.0001). Precisely, the decrease of miR-375-3p correlated with the increase of two inflammatory cytokines: monocyte chemoattractant protein-1 (MCP-1) and granulocyte macrophage colony-stimulating factor (GM-CSF). On the other hand, functional and molecular data obtained showed that miR-375-3p partially blocked the endothelial barrier maintenance and stabilization by disassembly of cell-cell junctions exhibiting low Rac1-Cdc42 levels. These findings demonstrate a differential serum profile of circulating miRNAs in patients with anaphylaxis and exhibit the miR-375-3p modulation in serum and EVs during drug- and food-mediated anaphylactic reactions. Furthermore, the in silico and in vitro studies show a negative role for miR-375-3p/Rac1-Cdc42 in the endothelial barrier stability.

RNA Profiles of Tear Fluid Extracellular Vesicles in Patients with Dry Eye-Related Symptoms.

Currently, diagnosing and stratifying dry eye disease (DED) require multiple tests, motivating interest in a single definitive test. The purpose of this study was to investigate the potential for using tear fluid extracellular vesicle (EV)-RNA in DED diagnostics. With a role in intercellular communication, nanosized EVs facilitate the protected transport of diverse bioactive molecules in biofluids, including tears. Schirmer strips were used to collect tears from 10 patients presenting with dry eye-related symptoms at the Norwegian Dry Eye Clinic. The samples comprised two groups, five from patients with a tear film break-up time (TBUT) of 2 s and five from patients with a TBUT of 10 s. Tear fluid EV-RNA was isolated using a Qiagen exoRNeasy Midi Kit, and the RNA was characterized using Affymetrix ClariomTM D microarrays. The mean signal values of the two groups were compared using a one-way ANOVA. A total of 26,639 different RNA transcripts were identified, comprising both mRNA and ncRNA subtypes. Approximately 6% of transcripts showed statistically significant differential abundance between the two groups. The mRNA sodium channel modifier 1 (SCNM1) was detected at a level 3.8 times lower, and the immature microRNA-130b was detected at a level 1.5 times higher in the group with TBUT 2 s compared to the group with TBUT 10 s. This study demonstrates the potential for using tear fluid EV-RNA in DED diagnostics.

Levels of small extracellular vesicles in patients treated with hyperbaric oxygenation.

Hyperbaric oxygen (HBO2) therapy involves the inhalation of pure oxygen in a pressure chamber under increased ambient pressure. Recent research indicates that circulating small extracellular vesicles (sEVs) play important roles in human physiology and pathology. Therefore, the objective of this pilot study was to monitor the impact of HBO2 therapy on the levels of circulating sEVs in the serum of patients with necrotizing soft-tissue infections (NSTI), aseptic bone necrosis (ABN) or idiopathic sudden sensory neural hearing loss (ISSNHL). Serum-derived sEVs were isolated and quantified in 80 patients before and after HBO2 therapy applied for NSTI, ISSNHL and ABN patients as well as in normal controls who received neither HBO2 therapy nor steroids. We observed a significant increase of circulating sEVs in patients with ISSNHL after HBO2 therapy (p < 0.05), as well as significantly elevated levels of sEVs after HBO2 therapy compared to patients with NSTI (p < 0.05) and ABN (p < 0.01). The increase in the levels of sEVs in ISSNHL may be evidence for both the intended reduction of inflammation as a result of steroid therapy and the inhibitory effect of oxidative stress induced by HBO2 therapy. Thus, sEVs released during HBO2 therapy might play an important biological role in mediating the response to therapy and might be a promising approach to gain further insights into the therapeutic efficacy of HBO2 therapy.

Procoagulant effect of extracellular vesicles in patients after transcatheter aortic valve replacement or transcatheter aortic valve replacement with percutaneous coronary intervention.

Patients with severe aortic stenosis (AS) after replacement of the transcatheter aortic valve (TAVR) are more likely to develop thrombotic complications such as cerebral embolism and artificial valve thrombosis. However, the mechanism is not yet well defined. We aimed to explore the plasma extracellular vesicles (EVs) levels and their role in the induction of procoagulant activity (PCA) in patients receiving TAVR alone or TAVR with percutaneous coronary intervention (PCI). EVs were analyzed with flow cytometer. Markers of platelet and endothelial cell activation were quantified using selective enzyme-linked immunosorbent assay (ELISA) kits. Procoagulant activity (PCA) was assessed by clotting time, purified clotting complex assays, and fibrin production assays. Our results confirmed that EVs with positive phosphatedylserin (PS+EV), platelet EVs (PEVs) and positive tissue factor EVs (TF+EVs) were higher in patients following TAVR than before TAVR, particularly in TAVR with PCI. Furthermore, endothelial-derived EVs (EEVs) were also higher in patients after TAVR with PCI than pre-TAVR, however, the EEVs levels in TAVR alone patients were gradually reduce than pre-TAVR. In addition, we further proved that total EVs contributed to dramatically shortened coagulation time, increased intrinsic/extrinsic factor Xa and thrombin generation in patients after TAVR, especially in TAVR with PCI. The PCA was markedly attenuated by approximately 80% with lactucin. Our study reveals a previously unrecognized link between plasma EV levels and hypercoagulability in patients after TAVR, especially TAVR with PCI. Blockade of PS+EVs may improve the hypercoagulable state and prognosis of patients.

Annexin-V positive extracellular vesicles level is increased in severe COVID-19 disease.

To evaluate extracellular vesicles levels in a cohort of SARS-CoV-2's patients hospitalized in an intensive care unit with and without COVID-19 associated thromboembolic events. In this study, we aim to assess endothelial and platelet membrane-derived extracellular vesicles levels in a cohort of SARS-CoV-2 patients with and without COVID-19-associated thromboembolic events who were hospitalized in an intensive care unit. Annexin-V positive extracellular vesicles levels were prospectively assessed by flow cytometry in one hundred twenty-three critically ill adults diagnosed with acute respiratory distress syndrome associated with a SARS-CoV-2 infection, ten adults diagnosed for moderate SARS-CoV-2 infection and 25 healthy volunteers. On our critically ill patients, thirty-four patients (27.6%) had a thromboembolic event, Fifty-three (43%) died. Endothelial and platelet membrane-derived extracellular vesicles were drastically increased in SARS-CoV-2 patients hospitalized in the ICU compared to healthy volunteers. Moreover a slighty higher small/large ratio for platelets membrane-derived extracellular vesicles in patients was linked to thrombo-embolic events. A comparison between total annexin-V positive extracellular vesicles levels in severe and moderate SARS-CoV-2 infection and healthy controls showed a significant increase in patients with severe infection and their sizes could be considered as biomarkers of SARS-CoV-2 associated thrombo-embolic events.

Predicting ICANS by means of plasma CAR‐T cell derived extracellular vesicles in patients undergoing infusion of anti‐CD19 CAR‐T cells

Introduction: Immune effector cell-associated neurotoxicity syndrome (ICANS) is a life-threatening adverse effect of anti-CD19 chimeric antigen receptor (CAR) T-cell therapy that usually occurs within 5–7 days after cell infusion. Although several clinical and biochemical parameters have been associated with ICANS, it is still a matter of debate how to predict its onset at the patient level. We here tested the hypothesis that CAR-T cell derived extracellular vesicles (EV) carrying the engineered CAR protein and produced early after CAR-T cell activation can be used as predictive biomarker of ICANS. Purposely, we measured plasma CAR+ EV in lymphoma patients underwent anti-CD19 CAR-T cell therapy. Methods: Seventy-one patients with aggressive r/r B-cell lymphomas were admitted to the advanced cell therapy unit of IRCCS AOU of Bologna (NCT04892433) for anti-CD19 CAR-T cell infusion. Included patients received tisa-cel (n = 27), axi-cel (n = 34), or brexu-cel (n = 10) after a median number of 3 prior lines of treatment (2–11); median age was 62 years (19–76) and no patients had CNS disease at the time of CAR-T cell infusion. Twenty out of 71 patients (28%) had ICANS of any grade: 5 patients (7%) ICANS grade 1, 7 patients (10%) ICANS grade 2 and 8 patients (11%) ICANS grade ≥3 (3 patients ICANS grade 3, 3 patients ICANS grade 4 and 2 patients ICANS grade 5 with diffuse cerebral edema). ICANS was classified according to Lee et al. The median time from CAR-T cell infusion to ICANS onset was 5 days (3–12). Available plasma samples at day +1 after CAR-T cell infusion were analyzed for CAR+ EV by FACS analysis. Data analysis was performed with Prism software v9.1.3 (GraphPad). Results: CAR+ EV were already detectable +1 day after CAR-T cell infusion in 58 patients. The median onset of ICANS was at day +5 (3–12). Patients with ICANS of any grade showed higher CAR+ EV level compared to no-ICANS ones (p < 0.0001). CAR+ EV anticipated the median ICANS onset of 2 to 11 days. CAR+ EV ROC analysis showed that a concentration >187.5 CAR+ EV/μl at day +1 after infusion predicts ICANS onset with sensitivity of 100% and specificity of 83.33% (p < 0.0001). Conclusions: These findings lead us to hypothesize that the plasma level of CAR+ EV mirrors target engagement by CAR-T cells, and their massive release is related to ICANS. Thus, CAR+ EV level could be considered a putative early predictor of ICANS onset; further analyses in larger cohorts are warranted to confirm this finding. Keywords: Cellular therapies, Diagnostic and Prognostic Biomarkers Conflicts of interests pertinent to the abstract. P. L. Zinzani Consultant or advisory role: Secura Bio, Celltrion, Gilead, Janssen-Cilag, BMS, Servier, Sandoz, MSD, AstraZeneca, Takeda, Roche, EUSA Pharma, Kyowa Kirin, Novartis, ADC Therapeutics, Incyte, BeiGene Other remuneration: Speakers bureau: Celltrion, Gilead, Janssen-Cilag, BMS, Servier, MSD, AstraZeneca, Takeda, Roche, EUSA Pharma, Kyowa Kirin, Novartis, Incyte, BeiGene F. Bonifazi Consultant or advisory role: Novartis, Gilead

Abstract 3327: Proteomic profiling of extracellular vesicles in patients with biliary tract cancers

Abstract Background and Aim: Biliary tract cancers (BTCs) have a poor prognosis. One of the reasons for the prognosis is the difficulty of early diagnosis. The absence of a sufficient biomarker to screen for BTCs in high-risk populations makes the patient to be recognized in advanced status. Therefore, it is urgent to discover a biomarker for early detection of BTCs. This study analyzed extracellular vesicle (EV) peptides to find a new serum biomarker of BTCs. Methods: Plasma samples were obtained from 5 healthy people as control, 10 from patients with benign biliary diseases such as choledocholithiasis, sclerosing cholangitis, and gallbladder polyp, and 29 from BTCS patients. The EV peptides have been obtained from 2.5ml of patient plasma. The EV peptide samples were analyzed by liquid chromatography-data-dependent acquisition-tandem mass spectrometry (LC-DDA-MS/MS). The acquired peptides and proteins were investigated by searching in Fragpipe-MSFragger. Characterization was performed using the EVs database of previous studies. After that, Differential Expression Protein (DEP) analysis and Pathway/GO analysis are performed to know the biological process. Results: Total 25,382 peptides and 3,313 proteins were defined (PSM, peptide-level FDR 1%). Most of the gene ontology biological processes were related to vesicle-mediated transport. On DEP analysis, total 72 proteins were up-regulated, and 29 proteins were down-regulated. Compared to cancer samples, 9 proteins were up-regulated to both healthy and benign samples and 2 were down-regulated. According to biological process/pathway analysis, up-regulated 9 proteins were mostly related to cytoskeleton/ECM and Hemostasis. Down-regulated 2 proteins were related to endocytosis and immunity. Conclusions: By analyzing EV peptides, a total of 11 proteins have been identified. With validation experiments, we expect to finalize biomarker candidates. Citation Format: Dong Uk Kim, Sunghee Park, Sung Yong Han, Daye Jeon, Hyeji Lee, Jonghyun Lee, Hyeong Seok Nam, Dong Wook Lee, Chang-Min Cho, Min Kyu Jung, Jun Heo, Un-Beom Kang, Seong-Jun Park, Sunghyun Huh, Yourae Shin. Proteomic profiling of extracellular vesicles in patients with biliary tract cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3327.

Comparison of Different Isolation Methods for Plasma-Derived Extracellular Vesicles in Patients with Hyperlipidemia.

Extracellular vesicles are commonly found in human body fluids and can reflect current physiological conditions of human body and act as biomarkers of disease. The quality of isolated extracellular vesicles facilitates the early diagnosis of various diseases accompanied by hyperlipidemia. Nonetheless, there are no reports on which special methods are suitable for isolating extracellular vesicles from the plasma of patients with hyperlipidemia. Thus, this study compared three different research-based extracellular vesicle isolation approaches, namely ultracentrifugation (UC), polyethylene glycol (PEG) precipitation, and size exclusion chromatography (SEC), and determined which of them was the most effective method. We selected blood samples from 12 patients with clinically diagnosed hyperlipidemia and isolated plasma-derived extracellular vesicles using three methods. The morphology of the isolated extracellular vesicles was observed using transmission electron microscopy, while the concentration was detected by asymmetric flow field-flow fractionation and multi-angle light scattering. Marker proteins were identified by Western blotting, and protein composition was evaluated by silver staining. Both determined the contaminations in the extracellular vesicle samples. The results showed that the three methods can be successfully used for the isolation of extracellular vesicles. The extracellular vesicles isolated by UC were larger in size, and the yield was much lower. Although the yield of extracellular vesicles isolated by PEG precipitation was greatly improved, the contamination was increased. Of the three methods, only the SEC-isolated extracellular vesicles were characterized by high yield and low contamination. Therefore, our data suggested that the SEC was a more ideal method for isolating extracellular vesicles from the plasma of patients with hyperlipidemia.

Bone Marrow Plasma Proteome Analysis By Differential Ultracentrifugation and Tandem Mass Spectrometry Allow for Characterization of Leukemia-Derived Extracellular Vesicles in Patients with Acute Lymphoblastic Leukemia

In recent years, the interplay of leukemic cells with the bone marrow niche has emerged as a potential contributing factor for the survival, therapy resistance and relapse-initiating capacity of leukemic cell subsets in ALL. This is known as early, environment-mediated drug resistance (EMDR). The role for leukemia-derived extracellular vesicles (LEVs) in this interplay remains largely unexplored. In vitro studies and PDX models have demonstrated that LEVs are internalized by bone marrow stromal cells (BMSC), leading to BMSC reprogramming and metabolic switch to glycolysis, with beneficial effects on leukemic cell survival as a result. This suggests that LEVs could also play a role in leukemic cell survival in ALL during chemotherapy, however, studies examining LEVs in patients are warranted. We performed fractionation by differential ultracentrifugation and subsequent mass spectrometry analysis of bone marrow plasma samples (n=12) collected at time of diagnosis (n=6, one sample was processed at 30, 60 and 120 min after sampling to assess the potential effect of contamination with platelet-derived EVs), during (n=2) and after induction therapy (n=4) in patients with B-cell precursor ALL (n=4). We isolated four fractions: A (2,000g pellet): Enriched in platelets/apoptotic bodies, M (20,000g pellet, enriched in microvesicles: large, membrane-derived EVs), E (150,000g pellet, enriched in exosomes), and S (150,000g supernatant, mostly soluble proteins), as well as R (raw untreated plasma), and we subjected these to mass spectrometry-based quantitative proteomics in 3 technical replicates. We examined protein composition and enriched pathways in the fractions and investigated associations of EV protein levels with the size of the leukemic cell fraction in the bone marrow. Flowcytometric data was used to confirm cell distributions/surface marker expression. Examining the protein composition of the five fractions (A, M, E, S, and R) showed that although the highest number of proteins was found in the A fraction (2803 proteins), the M fraction (2477 proteins) had the highest proteome diversity (Figure 1). Focusing on the M fraction, we compared diagnostic with follow up samples (blast percentage day 0: 92, 95, and 72, day 15: 0.4 and 1.3, day 29: 0.05, 0.2, 0, and 0.001, one day 0 sample excluded due to poor readout quality). We found significant upregulation of B-cell associated proteins at diagnosis, incl. HLA-DR, CD19, and CD22, along with the stem cell marker CD34, while T-cell and platelet-associated proteins did not show this pattern. Flow cytometry demonstrated only very few healthy B-cells in the samples at time of diagnosis (5, 0.8, and 0.6% vs. 25, 3, 32 and 21% on day 29) and showed all the leukemic clones to be CD34positive, confirming the BCP-ALL leukemic origin of the EVs. Examining the proteins most closely correlated to the percentage of blasts across all the samples, we found several proteins associated with poor survival, cell migration/adhesion and chemoresistance in acute leukemia, including CD97 (r=0.85, p=0.006), PLXNB2 (r=0.72, P=0.037), and ITGA4 (r=0.6, p=0.097). Gene set enrichment analysis of the proteins showing the most prominent differences between day 0 and day 15/29 using Reactome identified a variety of metabolic processes, but also oncogenic MAPK signaling (NES 1.94, p-adj=0.005), integrin signaling (NES 1.94, p-adj=0.003), and regulation of expression of SLITs and ROBOs (NES 2.1, p-adj=3x10-7) as enriched among the day 0 samples. Interestingly, we recently found genes within the latter two selectively upregulated in leukemias with poor induction therapy response (Modvig et al, Molecular Oncology, 2022). Taken together, this suggests that LEVs indeed could play a role in leukemic cell adherence and EMDR in the bone marrow niche. In conclusion, LEV content can be reliably characterized in bone marrow plasma of patients with ALL through differential ultracentrifugation and mass spectrometry-based proteomics. LEV protein composition reflects processes such as leukemic adhesion/migration, survival and drug resistance, carefully suggesting a role for LEVs in EMDR, but larger studies in patients are warranted. The identification of LEVs and key pathways in the leukemic-stromal cell interplay/EMDR can lead to development of new drugs blocking this cross-talk, potentially contributing to an improved relapse-free survival in patients with ALL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Clinical Significance of Circulating piRNAs in Extracellular Vesicles in Patients With Idiopathic Membranous Nephropathy

The Clinical Significance of Circulating piRNAs in Extracellular Vesicles in Patients With Idiopathic Membranous Nephropathy

Microbiome profiling of nasal extracellular vesicles in patients with allergic rhinitis

BackgroundNasal microbiota is crucial for the pathogenesis of allergic rhinitis (AR), which has been reported to be different from that of healthy individuals. However, no study has investigated the microbiota in nasal extracellular vesicles (EVs). We aimed to compare the microbiome composition and diversity in EVs between AR patients and healthy controls (HCs) and reveal the potential metabolic mechanisms in AR. MethodsEosinophil counts and serum immunoglobulin E (IgE) levels were measured in patients with AR (n = 20) and HCs (n = 19). Nasal EVs were identified using transmission electron microscopy and flow cytometry. 16S rRNA sequencing was used to profile the microbial communities. Alpha and beta diversities were analyzed to determine microbial diversity. Taxonomic abundance was analyzed based on the linear discriminant analysis effect size (LEfSe). Microbial metabolic pathways were characterized using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUst2) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. ResultsEosinophils, total serum IgE, and IgE specific to Dermatophagoides were increased in patients with AR. Alpha diversity in nasal EVs from patients with AR was lower than that in HCs. Beta diversity showed microbiome differences between the AR and HCs groups. The microbial abundance was distinct between AR and HCs at different taxonomic levels. Significantly higher levels of the genera Acetobacter, Mycoplasma, Escherichia, and Halomonas were observed in AR patients than in HCs. Conversely, Zoogloea, Streptococcus, Burkholderia, and Pseudomonas were more abundant in the HCs group than in the AR group. Moreover, 35 microbial metabolic pathways recognized in AR patients and HCs, and 25 pathways were more abundant in the AR group. ConclusionPatients with AR had distinct microbiota characteristics in nasal EVs compared to that in HCs. The metabolic mechanisms of the microbiota that regulate AR development were also different. These findings show that nasal fluid may reflect the specific pattern of microbiome EVs in patients with AR.

Circulating small extracellular vesicles from patients with periodontitis contribute to development of insulin resistance.

Epidemiological studies have identified the role of periodontitis in the pathogenesis of type 2 diabetes, but the underlying mechanism is poorly understood. It is well-known that small extracellular vesicles are lipid bilayer vesicles derived from cells with a diameter around 30 to 200nm. The purpose of this study was to investigate whether periodontitis induced or exacerbated insulin resistance via circulating small extracellular vesicles. Plasma small extracellular vesicles from control and periodontitis rats were intravenously injected into type 2 diabetic rats. Insulin tolerance tests, glucose tolerance tests, and the activation of the insulin signaling pathway were measured to detect the effect of the plasma small extracellular vesicles on insulin sensitivity. In addition, circulating small extracellular vesicles from patients with periodontitis with or without diabetes were isolated and co-cultured with HepG2 cells. The ability of glucose uptake was assessed using the fluorescence of 2-NBDG via flow cytometry. The activation of insulin signaling pathway was examined via Western blotting. Real time quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression of enzyme related to glycolysis and gluconeogenesis. Small extracellular vesicles derived from the plasma of periodontitis rats further impaired glucose tolerance and insulin tolerance in diabetic rats and significantly reduced the activation of the insulin signaling pathway in liver tissues, as evidenced by the decreased levels of p-AKT and p-GSK3β and the reduced hepatic glycogen content. For small extracellular vesicles isolated from human plasma, the concentration of small extracellular vesicles in patients with type 2 diabetes combined with periodontitis was higher than that of the healthy control and periodontitis alone. Moreover, circulating small extracellular vesicles from patients with periodontitis significantly inhibited the glucose uptake capacity and inhibited insulin signaling of HepG2 cells. Periodontitis acted as a contributing factor to exacerbate insulin resistance of type 2 diabetic rats. Plasma small extracellular vesicles played a critical role in periodontitis aggravating insulin resistance.

Flow Cytometry Analysis of Blood Large Extracellular Vesicles in Patients with Multiple Sclerosis Experiencing Relapse of the Disease.

The number of people living with multiple sclerosis (MS) in developed countries is increasing. The management of patients is hindered by the absence of reliable laboratory tests accurately reflecting the disease activity. Extracellular vesicles (EVs) of different cell origin were reportedly elevated in MS patients. We assessed the diagnostic potential, with flow cytometry analysis, of fresh large EVs (lEVs), which scattered more light than the 590 nm silica beads and were isolated from the blood plasma of relapsing remitting MS patients. Venous blood was collected from 15 patients and 16 healthy controls (HC). The lEVs were isolated from fresh platelet-free plasma by centrifugation, labelled with antibodies and the presence of platelet (CD41+, CD36+), endothelial (CD105+), erythrocyte (CD235a+), leukocyte (CD45+, CD19+, CD3+) and phosphatidylserine (Annexin V+) positive lEVs was analyzed using standard flow cytometry. Cryo-electron microscopy was used to verify the presence of EVs in the analyzed plasma fractions. MS patients experiencing acute relapse had slightly reduced relative levels (% of positive lEVs) of CD105+, CD45+, CD3+, CD45+CD3+ or CD19+ labelled lEVs in comparison to healthy controls. An analysis of other markers or a comparison of absolute lEV counts (count of lEVs/µL) did not yield any significant differences. Our data do not support the hypothesis that the exacerbation of the disease in RRMS patients leads to an increased numbers of circulating plasma lEVs which can be monitored by standard flow cytometry.

MO619: Landscape RNA Profiling of Urinary Extracellular Vesicles in Patients with Diabetic Nephropathy

Abstract BACKGROUND AND AIMS Recent studies have revealed that urinary extracellular vesicles (uEV) are secreted with rich biological information useful for understanding the pathophysiological conditions and identifying potential biomarkers for kidney disease. Diabetic nephropathy (DN) has become the major cause of CKD worldwide, while the features of uEV in DN were poorly understood. The present study aims to provide the landscape and in-depth analysis of the transcriptome characteristics of uEV in DN. METHOD Urine was collected from a total of 40 subjects, including healthy controls (HC, n = 14), type 2 DM (T2DM, n = 16), and renal biopsy-confirmed DN (n = 10). uEV was isolated by differential centrifugation and total RNA was extracted for long RNA sequencing. A model based on CIBERSORT was calculated to trace the genetic source of uEV toward resident cells of urinary system. Differentially expressed genes (DEG) between DN and HC and DN and T2DM were applied to gene ontology (GO), Reactome pathway enrichment, as well as gene set enrichment analysis (GSEA). Correlation of the transcriptome profiling between uEV and kidney tissue in DN was also explored. RESULTS About 67%∼68% of transcripts in RNA-seq were mRNA, 11%∼12% were LncRNA and 7%∼10% were CircRNA, and the abundance of mRNA was greater than other RNA types. By CIBERSORT model applying marker genes of podocytes, epithelial cells of tubule of different segments, loop of Henle, and collecting duct, and bladder transitional epithelial cells. The uEV was shown to originate mainly from the collecting duct epithelial cells of the kidney and bladder transitional epithelial cells (Fig. 1a). Confocal laser microscopy showed the co-localization of CD63 and AQP2 (Fig. 1b) in kidney tissues. GO and Reactome analysis were further performed in upregulated mRNA between DN and T2DM and DN and HC to explore the mRNA profiling in terms of the biological significance. Impressively, ‘extracellular exosome’ as well as other terms associated with constitution of EV, ‘plasma membrane’ and ‘extracellular space’ were enriched by GO cellular component (CC) analysis in DN. Besides, Reactome analysis identified pathways of ‘immune system’, ‘extracellular matrix organization’ and ‘degradation of extracellular matrix’ reflecting the pathogenesis mechanism of DN (Fig. 1c). Additionally, microarray data of DN glomeruli and tubule from the GEO database were analyzed in comparison to uEV RNA-seq data. GSEA showed that great similarities were found in the top 10 terms of Reactome pathways (Fig. 2). CONCLUSION In conclusion, this study provides a landscape transcriptome profile, the genetic trace and the biological function terms of uEV in DN. uEV holds potentials in reflecting the transcriptome characteristics of renal tissue, which is of significance for understanding the pathogenesis of DN and biomarker discovery.