Ionizing radiation and glioblastoma exosomes: implications in tumor biology and cell migration

Abstract

Exosomes are nanometer-sized lipid vesicles released ubiquitously by cells, which have been shown to have a normal physiological role, as well as influence the tumor microenvironment and aid metastasis. Recent studies highlight the ability of exosomes to convey tumor-suppressive and oncogenic mRNAs, microRNAs, and proteins to a receiving cell, subsequently activating downstream signaling pathways and influencing cellular phenotype. Here, we show that radiation increases the abundance of exosomes released by glioblastoma cells and normal astrocytes. Exosomes derived from irradiated cells enhanced the migration of recipient cells, and their molecular profiling revealed an abundance of molecules related to signaling pathways important for cell migration. In particular, connective tissue growth factor (CTGF) mRNA and insulin-like growth factor binding protein 2 (IGFBP2) protein levels were elevated, and coculture of nonirradiated cells with exosomes isolated from irradiated cells increased CTGF protein expression in the recipient cells. Additionally, these exosomes enhanced the activation of neurotrophic tyrosine kinase receptor type 1 (TrkA), focal adhesion kinase, Paxillin, and proto-oncogene tyrosine-protein kinase Src (Src) in recipient cells, molecules involved in cell migration. Collectively, our data suggest that radiation influences exosome abundance, specifically alters their molecular composition, and on uptake, promotes a migratory phenotype.

Figure 1

Confirming isolation of exosomes by ultracentrifugation. (A) Scanning electron micrograph of U87MG cell surface with vesicular protrusions (arrows; scale bar, 250 nm) and (B) transmission electron micrograph of purified U87MG exosomes (scale bar, 100 nm). (C) U87MG-GFP cells with PKH26-labeled exosomes in the cytoplasm and on the cell surface (white arrows). (D) Immunoblot analysis shows presence of exosome markers in U87MG exosomes and parent cell line.

Figure 2

Cellular irradiation increases exosome release. (A) Exosome abundance was measured by NTA at 48 hours after 4-Gy treatment of GBM cells (LN18, U251, and U87MG; n = 3), GBM stem-like cells (GBAM1 and GBMJ1; n = 2), and normal astrocytes (n = 3). (B) U87MG exosomes isolated at 24 hours posttreatment with 2 to 8 Gy were measured by NTA (n = 3). (C) Measurement of exosomes released by irradiated (4 Gy) and control cells from 12 to 48 hours after treatment, relative to 0-Gy 12-hour condition (n = 3). All graphs represent averaged values relative to 0-Gy condition + SEM; values normalized to number of cells present at time of exosome isolation. *P < .05; #P < .01.

Figure 3

Radiation-derived exosomes are more readily taken up by recipient cells. (A) Confocal microscopic visualization of exosomes within cells. Exosomes are stained red with PKH26, and nuclei are stained blue with DAPI. (B) Flow cytometric quantitation of exosome uptake by cells after 1 to 24 hours in coculture. (C and D) Radiation-derived exosome uptake (4-Gy exosomes) in recipient cells at 24 hours compared with exosomes from nonirradiated cells (0-Gy exosomes) incubated for the same time; (D) is the averaged value + SEM of three independent experiments, relative to the 0-Gy condition. *P = .03.

Figure 4

Radiation-derived exosomes enhance cell migration. (A) Schematic of cell migration experiments. (B-E and G) Exosome effect on the migration of U87MG cells across transwell membranes when used as a chemoattractant (B, D, E, and G) or by preincubating exosomes with cells before assay setup (C); n = 3. (D) Cell migration in response to increasing exosome concentrations, quantified in E as the averaged values + SEM; n = 3. (F) Uptake of PKH26-labeled exosomes before and after Tx-100 lysis. (G) Influence of Tx-100-lysed exosomes and Tx-100/dFBS on cell migration; n = 3. Values represent the averaged values + SEM of three independent experiments. Radiation-derived exosomes, 4-Gy exosomes; exosomes from nonirradiated cells, 0-Gy exosomes. *P < .05.

Figure 5

Radiation alters the molecular composition of exosomes. (A) Two-way hierarchical clustering representation of exosome mRNA content. Values shown compare radiation-derived exosomes to those derived from nonirradiated cells at 24 and 48 hours, using a cutoff P value of <.05. Up-regulation, red; down-regulation, blue. (B) Venn diagram of the number of significant transcript changes with ≥1.33-fold increased/decreased expression in radiation-derived exosomes compared to exosomes from nonirradiated cells. (C) Protein array analysis of differentially expressed proteins in radiation-derived exosomes compared to exosomes from nonirradiated cells. Values represent those with a ≥1.33-fold increased/decreased expression cutoff comparing radiation-derived exosomes to exosomes from nonirradiated cells (n = 2).

Figure 6

Molecular profile of radiation-derived exosomes relates to pathways involved in cell movement. IPA of top networks from mRNA and protein targets with greater abundance in radiation-derived exosomes. (A) Upper table: Associated Network Functions, lower table: Molecular and Cellular Functions. (B) Cell movement network is overlaid with values from protein (outlined in blue) and mRNA (outlined in green) arrays using targets with increased abundance in radiation-derived exosomes (red-filled molecules).

Figure 7

Radiation-derived exosomes augment signaling pathways associated with cell motility. (A) CTGF gene expression levels in U87MG exosomes and cells by quantitative reverse transcription-PCR. Data represent averaged values + SEM, n = 3; radiation-derived exosomes/cells were compared to nonirradiated controls. *P = .0184. (B) CTGF and (C) IGFBP2 protein levels on immunoblot analysis exosome and whole-cell lysates. (D) Immunoblot analysis of CTGF on lysates from cells incubated for 24 hours in serum-free DMEM or in coculture with exosomes (radiation-derived, 4-Gy exosomes; nonirradiated controls, 0-Gy exosomes). (E) Transfer of BrUTP-labeled mRNA in exosomes to recipient cells (BrUTP exosomes, middle panel). Distribution of PKH26-labeled exosomes incubated for the same time for comparison (right panel). (F) Influence of radiation-derived exosomes and control exosomes on activation of signaling molecules involved in cell migration after coculture with cells for 24 hours.