RBIO-13. Shift in Subcellular Expression and Increase in Immune Response Pathways After Radiation Suggests that HMGB2 is a Damage-Associated Molecular Pattern (DAMP) in Gliomas

Abstract While HMGB2 nuclear expression in gliomas is associated with enhanced cell proliferation, high HMGB2 expression in adult glioblastomas yielded better response to treatments with radiation+temozolomide and with viral vectors. Here we aimed to explore causes for this divergence and to generate a stable cell line with lentiviral-mediated HMGB2-GFP overexpression (HMGB2-GFP OE) for mechanistic studies on the role of HMGB2 in radiation response. METHODS AND RESULTS 1) Mass spectrometry label-free proteomic analysis of THP-1 and U87 cells’ secretomes identified HMGB2 as one of the top proteins with decreased abundance in conditioned media from co-culture, compared to that from each cell line individually. 2) Subcellular immunoblotting indicated HMGB2 nucleus-to-cytoplasm translation 1h after 3Gy radiation of U87, SF188, and KNS42 cell lines compared to un-irradiated cells. 3) HMGB2 cytoplasmic expression was associated with up-regulation of immune response pathways at microarray transcriptomic analysis from SF188 cells, compared to clones with shRNA-mediated HMGB2-knockdown 1h and 4h after 3Gy radiation. 4) Lentiviral vectors containing GFP (eGFP and HMGB2-GFP) were successfully transduced in KNS42 cells and HMGB2 expression and subcellular location in both clones were assessed with immunoblot and direct fluorescence visualization. CONCLUSION The differential secretion under mono- and co-culture suggested that HMGB2 may be an agent of intercellular communication between tumor (U87) and immune (THP-1) cells. HMGB2 nucleus-cytoplasmic translocation along with upregulation of immune pathways occurring after radiation in glioma cells in monoculture raises the hypothesis that HMGB2 is involved in immunogenic cell death and acts as a damage-associated molecular pattern (DAMP), like its paralog HMGB1, in pediatric and adult glioma cell lines. Our stable clones with HMGB2-GFP OE will allow us to explore the mechanistic aspects and the role of HMGB2 in glioma response to radiation, with improved direct visualization of the tumor-immune cells crosstalk in future in vitro and in vivo experiments.