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Focused Ultrasound Hyperthermia Augments Release of Glioma-derived Extracellular Vesicles with Differential Immunomodulatory Capacity.

ABSTRACT: Background: Increasing evidence points to the critical role of extracellular vesicles (EVs) as molecular parcels that carry a diverse array of bioactive payloads for coordination of complex intracellular signaling. Focused ultrasound (FUS) hyperthermia is a technique for non-invasive, non-ionizing sublethal heating of cells in a near-instantaneous manner; while it has been shown to improve drug delivery and immunological recognition of tumors, its impact on EVs has not been explored to date. The goal of this study was to determine whether FUS impacts the release, proteomic profile, and immune-activating properties of tumor-derived EVs. Methods: Monolayered murine glioma cells were seeded within acoustically transparent cell culture chambers, and FUS hyperthermia was applied to achieve complete coverage of the chamber. Glioma-derived EVs (GEVs) were isolated for characterization by Nanoparticle Tracking Analysis, cryo-electron microscopy and mass spectrometry. An in vitro experimental setup was designed to further dissect the impact of GEVs on innate inflammation; immortalized murine dendritic cells (DCs) were pulsed with GEVs (either naïve or FUS hyperthermia-exposed) and assayed for production of IL-12p70, an important regulator of DC maturation and T helper cell polarization toward the interferon-?-producing type 1 phenotype. Results: We confirmed that FUS hyperthermia significantly augments GEV release (by ~46%) as well as shifts the proteomic profile of these GEVs. Such shifts included enrichment of common EV-associated markers, downregulation of markers associated with cancer progression and resistance and modulation of inflammation-associated markers. When DCs were pulsed with GEVs, we noted that naïve GEVs suppressed IL-12p70 production by DCs in a GEV dose-dependent manner. In contrast, GEVs from cells exposed to FUS hyperthermia promoted a significant upregulation in IL-12p70 production by DCs, consistent with a pro-inflammatory stimulus. Conclusion: FUS hyperthermia triggers release of proteomically distinct GEVs that are capable of facilitating an important component of innate immune activation, lending both to a potential mechanism by which FUS interfaces with the tumor-immune landscape and to a role for GEV-associated biomarkers in monitoring response to FUS.

SUBMITTER: Sheybani ND

PROVIDER: S-EPMC7330848 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Focused Ultrasound Hyperthermia Augments Release of Glioma-derived Extracellular Vesicles with Differential Immunomodulatory Capacity.

Sheybani Natasha D ND Batts Alec J AJ Mathew Alexander S AS Thim E Andrew EA Price Richard J RJ

Theranostics 20200612 16

<b>Background</b>: Increasing evidence points to the critical role of extracellular vesicles (EVs) as molecular parcels that carry a diverse array of bioactive payloads for coordination of complex intracellular signaling. Focused ultrasound (FUS) hyperthermia is a technique for non-invasive, non-ionizing sublethal heating of cells in a near-instantaneous manner; while it has been shown to improve drug delivery and immunological recognition of tumors, its impact on EVs has not been explored to da ...[more]

PMID: 32642004

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Project description:Introduction: Glioma cells exert influence over the tumor-microenvironment in part through the release of extracellular vesicles (EVs), membrane-enclosed structures containing proteins, lipids, and RNAs. In this study, we evaluated the function of Ras-associated protein 27a (Rab27a) in glioma and evaluated the feasibility of assessing its role in EV release in glioma cells in vitro and in vivo. Methods: Rab27a was knocked down via a short hairpin RNA (shRNA) stably expressed in mouse glioma cell line GL261, with a scrambled shRNA as control. EVs were isolated by ultracentrifugation and quantified with Nanoparticle Tracking Analysis (NTA) and Tunable Resistive Pulse Sensing (TRPS). CellTiter-Glo viability assays and cytokine arrays were used to evaluate the impact of Rab27a knockdown. GL261.shRab27a cells and GL261.shControl were implanted into the left striatum of eight mice to assess tumor growth and changes in the tumor microenvironment. Results: Knockdown of Rab27a in GL261 glioma cells decreased the release of small EVs isolated at 100,000 × g in vitro (p = 0.005), but not the release of larger EVs, isolated at 10,000 × g. GL261.shRab27a cells were less viable compared to the scramble control in vitro (p < 0.005). A significant increase in CCL2 expression in shRab27a GL261 cells was also observed (p < 0.001). However, in vivo there was no difference in tumor growth or overall survival between the two groups, while shRab27a tumors showed lower proliferation at the tumor borders. Decreased infiltration of IBA1 positive macrophages and microglia, but not FoxP3 positive regulatory T cells was observed. Conclusion: Rab27a plays an important role in the release of small EVs from glioma cells, and also in their viability and expression of CCL2 in vitro. As interference in Rab27a expression influences glioma cell viability and expression profiles, future studies should be cautious in using the knockdown of Rab27a as a means of studying the role of small EVs in glioma growth.

Dataset Information

Focused Ultrasound Hyperthermia Augments Release of Glioma-derived Extracellular Vesicles with Differential Immunomodulatory Capacity.

Publications

Focused Ultrasound Hyperthermia Augments Release of Glioma-derived Extracellular Vesicles with Differential Immunomodulatory Capacity.

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