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Glioblastoma Extracellular Vesicles Modulate Immune PD-L1 Expression in Accessory Macrophages upon Radiotherapy

ABSTRACT: The aim of the study was to investigate the effects of ionizing radiation on CT-2A cells at the protein level, proteomics analyses were performed on CT-2A cells irradiated with 5 Gy. To investigate the effect of glioma-secreted extracellular vesicles (EVs) on bone-marrow-derived macrophages (BMDM) at the protein level, BMDM were exposed to EVs for 48 hours. Multiplexed mass spectrometry-based proteomics was performed using TMTpro barcoding reagents and the SPS-MS3 method on an Orbitrap Lumos mass spectrometer. Samples were labeled as follows: CT-2A CTRL samples: CT2A_Cells_CTRL_1 (126), CT2A_Cells_CTRL_2 (127n) CT-2A IR samples: CT2A_Cells_IR_1 (127c), CT2A_Cells_IR_2 (128n) BMDM samples: Macrophage_CTRL_1 (128c), Macrophage_CTRL_2 (129n) BMDM+EV samples: Macrophage_Plus_EV_1 (130c), Macrophage_Plus_EV_2 (131n) BMDM+irEV samples: Macrophage_Plus_irEV_1 (131c), Macrophage_Plus_irEV_2 (132n)

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Mus Musculus (ncbitaxon:10090)

SUBMITTER: Wilhelm Haas Koen Breyne Bakhos Tannous

PROVIDER: MSV000093696 | MassIVE | Tue Dec 19 12:49:00 GMT 2023

REPOSITORIES: MassIVE

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Project description:Acute radiation syndrome (ARS) is a severe condition that develops after exposure to high doses of ionizing radiation that features immune suppression and organ failure. Currently, there are no diagnostics to identify the occurrence or severity of radiation exposure. There are also no current treatments or preventative strategies to mitigate ARS. Extracellular vesicles (EVs) are mediators of intercellular communication that contribute to immune dysfunction across diseases. We investigated if EV cargo would be able to identify whole body irradiation (WBIR) exposure and if EVs promote ARS immune dysfunction. We hypothesized that beneficial EVs derived from mesenchymal stem cells (MSC-EVs) would blunt ARS immune dysfunction and might serve as prophylactic radioprotectants. Mice received WBIR (2 or 9 Gy) with assessment of EVs at 3 and 7 days after exposure. LC-MS/MS proteomic analysis of WBIR-EVs found dose-related changes as well as candidate proteins that were increased with both doses and timepoints (34 total) such as Thromboxane-A Synthase and lymphocyte cytosolic protein 2. Suprabasin and Sarcalumenin were increased only after 9 Gy suggesting these proteins may indicate high dose/lethal exposure. Analysis of EV miRNAs identified miR-376 and miR-136, which were increased up to 200- and 60-fold respectively by both levels of WBIR and select miRNAs such as miR-1839 and miR-664 that were increased only with 9 Gy. WBIR-EVs (9 Gy) were biologically active and blunted immune responses to LPS in RAW264.7 macrophages, inhibiting canonical signaling pathways associated with wound healing and phagosome formation. Accordingly, WBIR-EVs also blunted RAW macrophage phagocytosis. When given 3 days after exposure, MSC-EVs slightly modified immune gene expression changes in the spleens of mice in response to WBIR and a combined radiation plus burn injury exposure (RCI). MSC-EVs normalized the expression of certain key immune genes such as NFκBia and Cxcr4 (WBIR), Map4k1, Ccr9 and Cxcl12 (RCI) lowered plasma TNFα cytokine levels after RCI, though most gene changes were not influenced. When given prophylactically (24 and 3 hours before exposure), MSC-EVs prolonged survival to the 9 Gy lethal exposure. Thus, EVs are important participants in ARS. EV cargo might be used to diagnose WBIR exposure, and MSC-EVs might serve as radioprotectants to blunt the impact of toxic radiation exposure.

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Glioblastoma Extracellular Vesicles Modulate Immune PD-L1 Expression in Accessory Macrophages upon Radiotherapy

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