P01.07 * IMPACT OF EXTRACELLULAR VESICLES RELEASED BY GLIOBLASTOMA CELLS AFTER IRRADIATION ON TUMOR MICROENVIRONMENT

Abstract

AIMS: Glioblastoma (GBM) is the most lethal of all human tumors. Ionizing radiation (IR), as a major therapeutic modality, induces multiple types of DNA lesions in cells, therefore causes cell death. However, IR could also affect neighboring unirradiated cells, inducing Bystander Effects as chromosomal aberrations, increased proliferation, etc. Intercellular communication through the release of different components is involved in the mechanism. GBM cells release different soluble factors as well as tumor microvesicles (TMVs) to modify the phenotype of neighboring cells, thus participating in the tumor progression. The present study was designed to investigate in vitro the impact of IR on the communication between tumor cells and endothelial cells in the tumor microenvironment via soluble factors and TMVs. MATERIALS AND METHODS: Two GBM cell lines (T98G, U87) were grown and sham-irradiated (0Gy) or irradiated (2 or 10Gy) using a Clinac iX linear accelerator. Cell culture media (CM) were collected. Filtrate (containing only soluble factors) and TMVs were separated with successive centrifugations and Pierce concentrator. Cell viability was assessed by cell counting using trypan blue. TMVs quantifications were performed by flow cytometry. The effect of CM/Filtrate/TMVs on the global behavior (proliferation, adhesion) of bystander tumor cells or HUVEC was investigated using the xCELLigence system (ACEA). RESULTS: As expected, irradiation caused a loss of cell number in U87 and T98G: 20% at 2Gy and 60% at 10Gy 48h post-IR as compared to untreated cells. Both CM and Filtrate collected from sham-irradiated tumor cells induced a 50% reduction of bystander tumor cells proliferation, while CM and Filtrate recovered from 10Gy-irradiated cells had different influence on the proliferation: herein, the inhibitory properties of CM were less marked than those of Filtrate. The discordant effects between CM and Filtrate led us to investigate the role of TMVs. When quantified using flow cytometry, two-fold more TMVs were found in 10Gy-irradiated T98G as compared to untreated cells 48h post-IR, while in U87 this stimulation of release was observed at 2h. TMVs were shown to induce a 2-fold increase of HUVEC cell index. Interestingly after 10Gy-irradiation, TMVs from both cell lines slightly also favored both bystander tumor cells and HUVEC proliferation. CONCLUSION: IR inhibits GBM cells proliferation. IR stimulates TMVs release by GBM cells in a time- and line-dependent manner. IR facilitates tumor progression by stimulating the proliferation of bystander tumor cells as well as favoring tumor angiogenesis via TMVs.