Littérature scientifique sur le sujet « EV subpopulation »

Anaplastic lymphoma kinase (ALK) translocation is an actionable mutation in lung adenocarcinoma. Nonetheless tumour consists of heterogeneous cell subpopulations with diverse phenotypes and genotypes, and cancer cells can actively release extracellular vesicles (EVs) to modulate the phenotype of other cells in the tumour microenvironment. We hypothesized that EVs derived from a drug-resistant subpopulation of cells could induce drug resistance in recipient cells. We have established ALK-translocated lung adenocarcinoma cell lines and subclones. The subclones have been characterized and the expression of EV-RNAs determined by quantitative polymerase chain reaction. The effects of EV transfer on drug resistance were examined in vitro. Serum EV-RNA was assayed serially in two patients prescribed ALK-tyrosine kinase inhibitor (ALK-TKI) treatment. We demonstrated that the EVs from an ALK-TKI-resistant subclone could induce drug resistance in the originally sensitive subclone. EV-RNA profiling revealed that miRNAs miR-21-5p and miR-486-3p, and lncRNAs MEG3 and XIST were differentially expressed in the EVs secreted by the resistant subclones. These circulating EV-RNA levels have been found to correlate with disease progression of EML4-ALK-translocated lung adenocarcinoma in patients prescribed ALK-TKI treatment. The results from this study suggest that EVs released by a drug-resistant subpopulation can induce drug resistance in other subpopulations and may sustain intratumoural heterogeneity.

Biomaterials composed of extracellular matrix (ECM) provide both mechanical support and a reservoir of constructive signaling molecules that promote functional tissue repair. Recently, matrix-bound nanovesicles (MBVs) have been reported as an integral component of ECM bioscaffolds. Although liquid-phase extracellular vesicles (EVs) have been the subject of intense investigation, their similarity to MBV is limited to size and shape. Liquid chromatography–mass spectrometry (LC-MS)–based lipidomics and redox lipidomics were used to conduct a detailed comparison of liquid-phase EV and MBV phospholipids. Combined with comprehensive RNA sequencing and bioinformatic analysis of the intravesicular cargo, we show that MBVs are a distinct and unique subpopulation of EV and a distinguishing feature of ECM-based biomaterials. The results begin to identify the differential biologic activities mediated by EV that are secreted by tissue-resident cells and deposited within the ECM.

Abstract Introduction: Aqueous humor (AH), the clear fluid in front of the eye, maintains the pressure and vitality of ocular tissues. This fluid is accessible via the cornea which enables use of AH as a liquid biopsy for intraocular disease. Retinoblastoma (Rb), an intraocular cancer in children, is unique in that direct tumor biopsy is prohibited, thus liquid biopsy has great clinical potential. cfDNA in the AH as a biomarker for Rb patients has been investigated and extracellular vesicles (EVs) are detectable in the AH from adults. However, AH EVs in Rb have previously never been explored. We know very little about the heterogeneity of AH EV populations in ocular cancers. Materials and Methods: 27 AH samples from 19 patients from 13 tumor-free eyes and 11 Rb eyes were collected. Rb eyes were further divided into treatment-naïve (Rb_Tn) and treatment-active (Rb_Tx) subgroups. Unprocessed AH samples were subjected to Nanoparticle Tracking Analysis (NTA) (Nanosight NS300) for size distribution and concentration, and to Single Particle-Interferometric Reflectance Imaging Sensor (SP-IRIS) (Exoview R100) for fluorescent-based immunophenotyping of EV marker expression (CD63, CD81, and CD9). Results: NTA demonstrated the concentration of AH EVs is 3.11 x 109-1.38 x 1010 vesicles/mL; majority sized 76.8-103 nm. Study group comparisons showed that non-tumor containing eyes had a smaller nanoparticle mean size compared to Rb containing eyes (P = 0.002). More EVs were significantly detected in Rb_Tn containing eyes compared to Rb_Tx containing eyes (P = 0.022), suggesting the possible presence of tumor-derived EVs in Rb_Tn which were subsequently eliminated by treatment. SP-IRIS revealed distinct patterns of tetraspanin expression of AH small EVs (sEVs). Mono-CD63+ EVs were identified to be the most dominant sEV subpopulation from AH across non-tumor and Rb_Tx eyes. However, more diverse sEV subpopulation profile was detected in Rb_Tn AH samples. Significantly lower percentage of mono-CD63+ EVs could be determined in Rb_Tn eyes compared to Rb_Tx eyes (70.3% vs. 96.1%, P = 0.001). A significant accumulation of CD9/CD63 (4.3% vs. 1.1%, P = 0.035), CD63/CD81 (20.7% vs. 2.0%, P = 0.012) and CD9/CD63/CD81 (4.8% vs. 0.8%, P = 0.022) subpopulations in Rb_Tn was also observed. An enriched mono-CD63+ sEV subpopulation identified in AH indicates this is a potential AH-specific biomarker. In the setting of Rb there was a more heterogeneous population of sEVs which normalized with treatment. Conclusions: Small EVs are readily detectable in unprocessed AH with a dominant mono-CD63+ subpopulation in AH regardless of pediatric eye disease states. Tetraspanin colocalization analysis indicates the clearance of retinoblastoma-derived EV heterogeneity by chemotherapy. These novel finding suggests a potential clinical application of measurement of sEV subpopulations in AH samples to monitor response to therapy. Citation Format: Chen-Ching Peng, Deborah Im, Shreya Sirivolu, Bibiana Reiser, Aaron Nagiel, Paolo Neviani, Liya Xu, Jesse L. Berry. Clearance of tumor-derived extracellular vesicle heterogeneity in aqueous humor after chemotherapy in retinoblastoma eyes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3416.

ObjectiveTo develop a reliable and fast assay to quantify the α-synuclein (α-syn)–containing extracellular vesicles (EVs) in CSF and to assess their diagnostic potential for Parkinson disease (PD).MethodsA cross-sectional, multicenter study was designed, including 170 patients with PD and 131 healthy controls (HCs) with a similar distribution of age and sex recruited from existing center studies at the University of Washington and Oregon Health and Science University. CSF EVs carrying α-syn or aggregated α-syn were quantified using antibodies against total or aggregated α-syn, respectively, and highly specific, sensitive, and rapid assays based on the novel Apogee nanoscale flow cytometry technology.ResultsNo significant differences in the number and size distribution of total EVs between patients with PD and HCs in CSF were observed. When examining the total α-syn–positive and aggregated α-syn–positive EV subpopulations, the proportions of both among all detected CSF EVs were significantly lower in patients with PD compared to HCs (p < 0.0001). While each EV subpopulation showed better diagnostic sensitivity and specificity than total CSF α-syn measured directly with an immunoassay, a combination of the 2 EV subpopulations demonstrated a diagnostic accuracy that attained clinical relevance (area under curve 0.819, sensitivity 80%, specificity 71%).ConclusionUsing newly established, sensitive nanoscale flow cytometry assays, we have demonstrated that total α-syn–positive and aggregated α-syn–positive EVs in CSF may serve as a helpful tool in PD diagnosis.Classification of EvidenceThis study provides Class III evidence that total and aggregated α-syn–positive EVs in CSF identify patients with PD.

Application of mesenchymal stromal cells (MSC) has been proposed for solid organ transplantation based on their potent immunomodulatory effects. Since side effects from the injection of large cells cannot be excluded, the hypothesis rises that extracellular vesicles (EV) may cause immunomodulatory effects comparable to MSC without additional side effects. We used MSC-derived EV in a rat renal transplant model for acute rejection. We analysed peripheral blood leukocytes (PBL), kidney function, graft infiltrating cells, cytokines in the graft, and alloantibody development in animals without (allo) and with EV application (allo EV). There was no difference in kidney function and in the PBL subpopulation including Tregs between allo and allo EV. In the grafts T- and B-cell numbers were significantly higher and NK-cells lower in the allo EV kidneys compared to allo. TNF-αtranscription was lower in allo EV grafts compared to allo; there was no difference regarding IL-10 and in the development of alloantibodies. In conclusion, the different cell infiltrates and cytokine transcription suggest distinct immunomodulatory properties of EV in allotransplantation. While the increased T- and B-cells in the allo EV grafts may represent a missing or negative effect on the adaptive immune system, EV seem to influence the innate immune system in a contrary fashion.

Abstract The goal is to reverse cancer immune evasion at the level of the sentinel lymph node (SLN). Reduced expansion of CD8+ T cells and other innate immune effector cells in the cancer draining SLN is associated with progression and resistance to checkpoint inhibitors. Cancer-derived extracellular vesicles (EVs) that are PD-L1+ suppress immune recognition at the level of the SLN. The experimental goal is to remodel the SLN to overcome cancer EV-associated immune suppression and induce immune rejection of the tumor. We developed three methodologies for this project: a) Collection of draining lymph fluid to characterize EVs shed by 4T1 syngeneic breast tumors growing in the mammary fat pad. b) Chromatographic separation and characterization of the repertoire of EVs shed by tumors into the tumor microenvironment interstitial space using western blotting, mass spectrometry, and electron microscopy. c) Nanoparticle (NP) delivery of purified populations of EVs to the tumor draining SLN in combination with cytokine chemoattractants for innate immune cells recruitment. We characterized the in vivo interstitial fluid (IF) content of a GFP-4T1 syngeneic murine cancer model to study resident IF EVs transit to the draining lymph node. GFP labeling confirmed the IF EV tumor cell origin. Molecular analysis revealed an abundance of IF EV-associated proteins specifically involved in mitophagy and secretory autophagy. A set of proteins required for sequential steps of fission-induced mitophagy preferentially populated the CD81+/PD-L1+ IF EVs; including PINK1 and ARIH1 E3 ubiquitin ligase (required for Parkin-independent mitophagy), DRP1 and FIS1 (mitochondrial pinching), and VPS35, SEC22b, and Rab33b (vacuolar sorting). SLN immune cell populations could be massively remodeled by introducing hydrogel NPs which have a controlled release of T-cell and dendritic cell chemoattractant to the subcapsular sinus. NPs were successfully used to deliver concentrated packages of EVs subpopulations to the SLN. Introduction of the large CD81-/VEGF+/PD-L1- EV subpopulation (amphisome characteristics) to the SLN augmented tumor growth, angiogenesis, and metastasis, even when cytokine induction was used to remodel the SLN. In marked contrast, introduction of the CD81+/PD-L1+ EV subpopulation (containing mitophagy components) to the SLN in combination with NP release of chemoattractants, induced immune rejection of the syngeneic breast cancer, reducing tumor growth, and blocking metastasis. These findings demonstrate that different populations of EVs have opposite effects on cancer immune evasion at the level of the SLN and that EV-mediated immune suppression can be reversed by SLN remodeling to augment dendritic and CD8+ T cells. Citation Format: Marissa Howard, James Erickson, Amanda Haymond, Alessandra Luchini, Fatah Kashanchi, Lance Liotta. Reversing extracellular vesicle induced tumor immune suppression at the sentinel lymph node: Role of secretory autophagy and mitophagy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3516.

Abstract. Objective of the work was to conduct a comparative assessment of the immune-modulating effect of the combined use of Yersinia pestis EV NIIEG vaccine strain with Polyoxidonium and Ingaron preparations on a BALB/c mouse model.Materials and methods. Mice of the BALB/c line were immunized subcutaneously with Yersinia pestis EV NIIEG culture at a dose of 2.5 104 m.c. (1st group), in combination with Ingaron at a dose of 150 IU (2nd group) or with Polyoxidonium at a dose of 4 мg (3rd group), the 4th group is intact mice. On days 3, 7, 21 and 90 after immunization, the subpopulation composition of lymphocytes, the production of mediators of the cellular response (INF-ɣ and IL-10), the titers of specific antibodies to the capsular antigen of plague microbe (F1), the nuclear apparatus of lymphocytes, and the nature of histological changes in the organs of mice were determined. Characterization of immunogenic (protective) activity of the combined use of Y. pestis EV NIIEG with immune-modulators against Y. pestis 231 in experiments on BALB/c mice was performed on the 21st day after immunization through determining the number of dead animals and their average life expectancy.Results and discussion. The combined administration of Y. pestis EV NIIEG vaccine strain with Polyoxidonium or Ingaron to experimental animals allowed us to establish differences in the response of the immune system of biomodels, due to the mechanism of action of a specific immune-modulator. It has been established that both Polyoxidonium and Ingaron combined with Y. pestis EV NIIEG enhance the response of immune-competent cells in experimental animals, contribute to the activation of the humoral response and the production of mediators of the cellular response, do not have a damaging effect on the tissue of the macroorganism. At the same time, the efficacy of using combined vaccination of Y. pestis EV NIIEG with immune-modulators in the inoculation test is confirmed for Polyoxidonium only.

Within tumors, Cancer Stem Cell (CSC) subpopulation has an important role in maintaining growth and dissemination while preserving high resistance against current treatments. It has been shown that, when CSCs are eliminated, the surrounding Differentiated Cancer Cells (DCCs) may reverse their phenotype and gain CSC-like features to preserve tumor progression and ensure tumor survival. This strongly suggests the existence of paracrine communication within tumor cells. It is evidenced that the molecular crosstalk is at least partly mediated by Extracellular Vesicles (EVs), which are cell-derived membranous nanoparticles that contain and transport complex molecules that can affect and modify the biological behavior of distal cells and their molecular background. This ability of directional transport of small molecules prospects EVs as natural Drug Delivery Systems (DDS). EVs present inherent homing abilities and are less immunogenic than synthetic nanoparticles, in general. Currently, strong efforts are focused into the development and improvement of EV-based DDS. Even though EV-DDS have already reached early phases in clinical trials, their clinical application is still far from commercialization since protocols for EVs loading, modification and isolation need to be standardized for large-scale production. Here, we summarized recent knowledge regarding the use of EVs as natural DDS against CSCs and cancer resistance.

The early detection of cutaneous melanoma, a potentially lethal cancer with rising incidence, is fundamental to increasing survival and therapeutic adjustment. In stages II–IV especially, additional indications for adjuvant therapy purposes after resection and for treatment of metastatic patients are urgently needed. We investigated whether the fatty acid (FA) and protein compositions of small extracellular vesicles (sEV) derived from the plasma of stage 0–I, II and III–IV melanoma patients (n = 38) could reflect disease stage. The subpopulation of sEV expressing CD81 EV marker (CD81sEV) was captured by an ad hoc immune affinity technique from plasma depleted of large EV. Biological macromolecules were investigated by gas chromatography and mass spectrometry in CD81sEV. A higher content of FA was detectable in patients with respect to healthy donors (HD). Moreover, a higher C18:0/C18:1 ratio, as a marker of cell membrane fluidity, distinguished early (stage 0–I) from late (III–IV) stages’ CD81sEV. Proteomics detected increases in CD14, PON1, PON3 and APOA5 exclusively in stage II CD81sEV, and RAP1B was decreased in stage III–IV CD81sEV, in comparison to HD. Our results suggest that stage dependent alterations in CD81sEV’ FA and protein composition may occur early after disease onset, strengthening the potential of circulating sEV as a source of discriminatory information for early diagnosis, prediction of metastatic behavior and following up of melanoma patients.

Eukaryotic and prokaryotic cells in physiological and pathological conditions form membrane-bound extracellular vesicles, known as EVs. The ability of these submicron structures to transfer their cargoes (miRNA, DNA, protein, cytokines, receptors, etc.) into recipient cells is described. Recent data revealed that platelet-derived extracellular vesicles (PEVs) crosstalk promotes cancer growth and metastasis formation. Moreover, they exert immunosuppressive activities on phagocytes. This EV subpopulation is the most abundant amongst all types in circulation. According to the authors’ best knowledge, there is no information regarding the impact of PEVs on canine lymphocytes. The aim of this study was to evaluate the influence of PEVs on lymphocyte proliferation, phenotype and cytokine production in vitro. In the study, it was demonstrated (i) that PEVs interact differently with lymphocyte subsets and are preferentially associated with T-lymphocytes PBMC, while (ii) they are rarely detected in association with B-lymphocytes, and there is evidence that (iii) PEV uptake is observed after 7 h of co-culturing with lymphocytes. In addition, obtained data support the notion that PEVs do not influence in vitro lymphocyte proliferation, differentiation and cytokine production in a canine model.

Nonostante l'incidenza dell'osteoartrosi (OA) del ginocchio, una delle maggiori cause di disabilità a livello mondiale, sia in crescita, la sua diagnosi precoce è ancora impossibile. L'attuale processo diagnostico è basato sull'esame clinico del paziente e sull'imaging dell'articolazione ma questi esami sono di norma prescritti quando l'OA è in uno stadio avanzato. In aggiunta, questo approccio non riesce a rilevare l'attivazione di processi biologici nell'instaurarsi dell'OA, come l'infiammazione. Per questi motivi la ricerca si è recentemente focalizzata sullo studio di biomarcatori che rilevino le alterazioni biologiche precoci nei tessuti articolari durante lo sviluppo dell'OA. In questa prospettiva, le vescicole extracellulari (EVs) isolate dai liquidi biologici tramite biopsie stanno acquisendo maggiore importanza in quanto esse rispecchiano lo stato metabolico della cellula di origine. Recentemente è stato dimostrato che le EVs consistono in numerose sottopopolazioni con caratteristiche fisico-chimiche ed effetti biologici diversi. Quindi, il primo obiettivo di questo progetto di dottorato è stato la ricerca della tecnica migliore per isolare e separare sottopopolazioni di EVs dal liquido sinoviale (LS) in base alla loro dimensione. Ho confrontato centrifugazione differenziale, cromatografia ad esclusione dimensionale, cromatografia liquida ad alta prestazione ed il frazionamento campo-flusso a flusso asimmetrico (AF4). Quest'ultima tecnica si è rivelata la più promettente, quindi ho sviluppato un nuovo protocollo di separazione delle EVs all'Istituto Italiano di Tecnologia di Genova. Prima di tutto ho ottimizzato i flussi nello strumento fino ad essere in grado di isolare particelle con un raggio compreso fra i 20 e i 700 nm, suddivise in 4 sottopopolazioni. Ho inoltre ottenuto il profilo dimensionale delle EVs e l'abbondanza relativa delle 4 sottopopolazioni. Successivamente ho caratterizzato le vescicole quantificando il potenziale Z e la concentrazione proteica e di acidi nucleici, effettuando un'analisi di microscopia elettronica per confermare la morfologia delle EVs e valutando la presenza di marcatori specifici ed il loro contenuto proteico, anche grazie all'immunomicroscopia elettronica. L'ultima parte del progetto è stata svolta presso l'Università di Göteborg, in Svezia. Alla mancanza di marcatori precoci dell'OA, si aggiunge l'indisponibilità di approcci terapeutici efficaci che invertano i processi degenerativi nei tessuti articolari artrosici. Sono stati proposti diversi trattamenti biologici per i quali però la traslazione in clinica non è semplice. In questo scenario, le piattaforme di screening per nuovi farmaci possono accelerare lo sviluppo di nuove terapie e la microfluidica è uno degli approcci utilizzati per creare queste piattaforme. Il secondo obiettivo del progetto di dottorato è stato quindi focalizzato sullo sviluppo di un modello microfluidico paziente specifico usato come piattaforma di screening per trattamenti innovativi per l'OA. Il device consiste in un chip microfluidico multi-compartimento che permette la coltura separata di fibroblasti sinoviali e condrociti primari in un ambiente 3D rilevante ed in presenza di LS, tutti isolati dallo stesso paziente. Il modello è stato disegnato per valutare l'effetto di trattamenti biologici, aggiunti al sistema simulando un'iniezione intra-articolare. Per ricreare un ambiente cartilagineo, ho ottimizzato idrogeli commerciali a base di acido ialuronico e/o collagene 1 che sono stati crosslinkati enzimaticamente o con luce UV; i condrociti coltivati in queste matrici hanno mostrato un'espressione più elevata di marcatori cartilaginei rispetto a matrici standard (es. fibrina). Successivamente ho ottimizzato l'ambiente del modello, valutando l'effetto di LS sano o artrosico sulle cellule. Infine, ho quantificato l'effetto antiinfiammatorio di cellule mesenchimali stromali da tessuto adiposo o midollo osseo.
Despite the increasing incidence of knee osteoarthritis (OA), a world-leading cause of disability, its early diagnosis is still unattainable. The current diagnostic process is mainly based on the patients' clinical examination and the joint imaging. However, prescription for examination occur when the OA is already in an advanced stage. In addition, with this approach the biological processes activated during the OA, such as inflammation, are not considered. For these reasons the research is focusing on the finding of biological markers that can reflect the early biological alterations occurring in the joint. In this view, extracellular vesicles (EVs) isolated from biofluids with liquid biopsies are gaining importance as their content reflect the metabolic state of the origin cells. Contrarily to the classical view, it has been demonstrated that EVs include many subpopulations with different physicochemical features and biological roles. Thus, the first aim of this PhD project was to find the most effective technique to isolate and separate different size EV subpopulations from the synovial fluid (SF). To this end, I compared differential centrifugation, size exclusion chromatography, high performance liquid chromatography (HPLC) and asymmetrical flow field-flow fractionation (AF4). The AF4 resulted the most promising one, so I developed a new EV separation protocol at the Italian Institute of Technology in Genoa. Firstly, the flow rates in the AF4 were optimized until being able to isolate particles with a radius ranging from 20 up to more than 700 nm, that were gathered in 4 different subpopulations. I also obtained the EV profile and the relative percentage of each subpopulation. Then I characterized the EVs belonging to each subset by quantifying the Z potential, the protein and nucleic acid concentrations, by performing electron microscopy analysis to confirm the EV morphology and by evaluating the presence of EV-specific markers and the protein content, also with immune EM. The last part of the project was performed at the University of Gothenburg, Sweden. In addition to the lack of early biomarkers, no effective therapies able to revert the degeneration processes in the arthritic tissues are available, and the current approaches mainly aim at managing the pain. Different biological approaches have been proposed to fill this gap, but their clinical translation is not straightforward. In this scenario, the development of drug screening platforms can accelerate this translation, and microfluidics represents a promising approach. Hence, the second aim of my PhD project was focused on the development of a patient-specific microfluidic model to be used as drug screening platform for the evaluation of OA innovative treatments. The system consisted in a multi-channel microfluidic device that allowed the compartmentalized co-culture of primary and patient-matched synovial fibroblasts and chondrocytes in a 3D relevant hydrogel with synovial fluid interposed. The device was designed to allow the addition of biological treatments, mimicking an intra-articular injection, and the evaluation of their biological effects. To recreate a relevant cartilaginous compartment, I optimized commercially available hydrogels based on hyaluronic acid and/or type I collagen that were crosslinked enzymatically or via UV light. The chondrocytes cultured in these hydrogels showed higher expression of chondrocyte-specific markers. Then, I optimized the OA microenvironment within the model, evaluating the beneficial effect of the SF on the articular cells, that behaved differently when cultured with healthy or arthritic SF. Finally, the anti-inflammatory capabilities of adipose and bone-marrow mesenchymal stromal cells (MSCs) were assessed. The model effectively supported the injection of MSCs and the evaluation of their anti-inflammatory effects on the arthritic articular cells.