TMIC-24. BICARBONATE RECEPTOR SLC4A4 SENSITIZES INFILTRATING GLIOBLASTOMA CELLS TO CHANGES IN EXTRACELLULAR PH

Abstract BACKGROUND Glioblastoma progression involves changes in extracellular pH (pHe) related to rapid cell turnover and growth. There exist differences in pH between contrast-enhancing solid tumor and non-enhancing infiltrating tumor. SLC4A4 is a two-way bicarbonate transporter used in cortical astrocytes to buffer intracellular pH. METHODS In patients undergoing surgery for newly diagnosed IDH wild-type glioblastoma, we obtained tissue from solid tumor and infiltrating tumor. Single-nucleus RNA sequencing (n=15), immunohistochemistry (n=20) were carried out to identify SLC4A4 in infiltrating tumor beyond the contrast enhancing edge. SLC4A4 knock-down models were used to evaluate functional biology of this bicarbonate receptor using our tumor – cortical organoid model of infiltrating glioma and gliomaspheres. pH measurements were recorded using pH sensitive microelectrodes to evaluate changes on pH buffering ability of tumor cells during acid titration assays. RESULTS Single-nucleus RNA sequencing (n=15) and spatial RNA sequencing (n=3) showed SLC4A4 to be the highest expressed transmembrane receptor on infiltrating glioblastoma cells but not solid tumor. IHC (n=20) confirmed higher protein expression in infiltrating tumor (p=0.0390). Infiltrating cells were specifically sensitive to decreasing pHe (p=0.0105). In the infiltrating glioma cortical-organoid model, the slc4a4- had increased proliferation (GFP+ cell quantitation; n=10, p=0.0410). There was also increased proliferation in wild-type cells treated with SLC4A4 inhibitor DIDS (n=10, p=0.0016). Immunostaining of these models showed that downmodulation of SLC4A4 was associated with increased Ki67 and decreased caspase staining. CONCLUSIONS SLC4A4 plays a role in pH buffering in glioblastoma. We hypothesize that loss of SLC4A4 in solid tumor allows for desensitization of these cells to an acidic extracellular environment and allows for increased tumor growth and density while infiltrating tumor remains sensitivity to changes in pHe. An understand of pH regulation may lead to therapeutic efficacy through pH modulation of tumor.