From Biomarkers to Roles in Pathogenesis: 4 Studies Using TRPS in EV Research

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Tunable Resistive Pulse Sensing (TRPS) publication watch: Q4 2022

1. Respiratory cell EVs facilitate SARS-CoV-2 infection

Research has shown that viral infections can modify the host cell EV content or even hijack small EV biogenesis systems. SARS-CoV-2 entry to respiratory cells is mediated by changes in conformation of viral Spike (S) protein, its cleavage by host proteases (e.g., TMPRSS2), and binding to human angiotensin-converting enzyme 2 (ACE2) receptor. However, the roles of EVs specifically during SARS-CoV-2 entry or infection are unknown. Primary human nasal epithelial cells (HNECs) were initially used in this study, as they present important characteristics of in vivo human respiratory epithelia, including three major cell types and mucus. EVs were isolated from HNEC-produced mucus, using tunable resistive pulse sensing (TRPS) and NP150 and NP800 nanopores to determine EV size distribution and the concentration of small (sEVs) and large EVs (lEVs). Results showed a significant higher amount of sEVs compared to lEVs; thus, studies were continued with the sEV population.  

Western blot analysis revealed that HNEC mucus-derived sEVs contained full lengths of two SARS-CoV-2 entry factors – ACE2 and TMPRSS2 – and when these sEVs were applied to HNECs in increasing doses, SARS-CoV-2 infection showed a dose-dependent increase. Pre-incubation of SARS-CoV-2 particles and VCap-sEVs (an alternative in vitro source of sEVs) at the time of infection, and not 2 hours post-infection, resulted in a significant increase of SARS-CoV-2 infection of Calu-3 cells, indicating facilitation of early step of SARS-CoV-2 entry by sEVs. Immunoprecipitation and Proteinase K treatments confirmed that there was no fusion in the interaction between SARS-CoV-2 particles and VCap-sEVs but did require unaltered surface integrity in sEVs. Finally, western blot analysis of fragments sizes of S protein after incubation with HNECs mucus-derived sEVs concluded that sEVs induced cleavage and activation of S protein in SARS-CoV-2, facilitating its entry.1

2. EV-miRNAs biomarkers for one of the deadliest skin cancers

To move away from relying on just the pathological examination of skin lesions potentially associated with melanoma cancer, reliable non-invasive biomarkers are needed to improve diagnosis, staging, and risk assessment, and to predict the response to therapy. For this, EVs were isolated from plasma of patients with stage III or IV melanoma or healthy individuals, using the Exoid (TRPS) and a nanopore 150 (NP150) to determine particle size distribution and concentration. Among 65 miRNAs differentially expressed in pEV-miRNA expression profiles, 4 miRNA candidates could be used to discriminate metastatic melanoma patients from healthy controls, with successful validation and performance in independent cohorts.2

3. A novel approach to enhance bioactivity of mesenchymal stem cell EVs

MSC cell priming or modification of the MSC culture environment can further help confer desired function-specific traits in the cell’s secretome, including EVs. Shien et al. investigated the effects of priming avian MSCs with Chinese herb Polygonum multiflorum (PM) on EV production and the benefit of EVs to skin fibroblasts and hair follicle cells. After purifying avian MSC EVs with qEV10 35 nm columns and profiling them in size and concentration with the Exoid (TRPS), it was shown that PM-treated avian MSCs released significantly more, but smaller, EVs compared to untreated cells. When PM-treated MSC EVs were applied to fibroblasts,  proliferation increased, markers of skin remodelling increased (wound healing, expression of collagen, elastin) and the expression of anti-inflammatory genes increased, whilst hair follicle cells also increase cell proliferation (expression of proliferation-associated genes).3  

4. Leukemia cell EVs modulate tumour microenvironment to promote its growth  

The survival and proliferation of chronic lymphocytic leukemia (CLL) cells heavily depends on the highly immune-suppressive tumour microenvironment. When studied in vitro, EVs have shown a role in progression, invasion and resistance to treatment. Here, Gargiulo et al. use a multifaceted approach to determine the role of EVs in CLL pathogenesis in vivo.  

Small EVs (sEVs) were isolated from the spleen of healthy mice and those with ongoing CLL. Following sEV profiling of concentration and size distribution with the Exoid (TRPS) and NP100 nanopore, a 10-fold sEV enrichment was found in CLL mice compared to healthy controls.

Analysis of sEVs’ proteome and miRNAome further revealed CCL sEVs were relatively depleted in proteins involved in the positive regulation of immune processes (i.e., lymphocyte activation, migration, and cytokine production). CCL sEVs were also relatively enriched in miRNAs involved in unfavourable clinical parameters (i.e., miR-150 and miR-155) or a miRNA associated with hypoxia (miR-210). By using CLL spleen-isolated sEVs to inject mice and analyse the gene expression of subsets of lymphocytes, it was found that only CD8+ T-cells showed downregulation of genes involved in immune response and upregulation of genes associated with inhibition of immune response, leading to cell exhaustion, decreased granzyme B production, cytokine secretion and tumour cell lysis.4

References

  1. Berry, F., Morin-Dewaele, M., Majidipur, A., Jamet, T., Bartier, S., Ignjatovic, E., Toniutti, D., Gaspar Lopes, J., Soyeux-Porte, P., Maillé, P., Saldana, C., Brillet, R., Ahnou, N., Softic, L., Couturaud, B., Huet, É., Ahmed-Belkacem, A., Fourati, S., Louis, B.,…Bruscella, P. (2022). Proviral role of human respiratory epithelial cell-derived small extracellular vesicles in SARS-CoV-2 infection. Journal of Extracellular Vesicles, 11, e12269. https://doi.org/10.1002/jev2.12269
  1. Sabato C, Noviello T, Covre A,  Coral S, Caruso F, Besharat Z, Splendiani E, Masuelli L, Battistelli C, Vacca A, Catanzaro G, Po A, Anichini A, Maio M, Ceccarelli M, Di Giacomo A, and E Ferretti. A novel microRNA signature for the detection of melanoma by liquid biopsy. Journal of Translational Medicine (2022) 20:469 https://doi.org/10.1186/s12967-022-03668-1
  1. Shieh J, Chin Y, Chiu H, Hsieh Y, Cheng H, Gu H, and F Chang. Bio-Pulsed Stimulation Effectively Improves the Production of Avian Mesenchymal Stem Cell-Derived Extracellular Vesicles That Enhance the Bioactivity of Skin Fibroblasts and Hair Follicle Cells. Int. J. Mol. Sci. 2022, 23, 15010. https://doi.org/10.3390/ijms232315010
  1. Gargiulo, E., Viry, E., Morande, P. E., Largeot, A., Gonder, S., Xian, F., Ioannou, N., Benzarti, M., Kleine Borgmann, F. B., Mittelbronn, M., Dittmar, G., Nazarov, P. V., Meiser, J., Stamatopoulos, B., Ramsay, A. G., Moussay, E., & Paggetti, J. (2022). Extracellular vesicle secretion by leukemia cells in vivo promotes CLL progression by hampering antitumor T-cell responses. Blood Cancer Discovery. https://doi.org/10.1158/2643-3230.BCD-22-0029  

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