Bicarbonate Receptor SLC4A4 Sensitizes Infiltrating Glioblastoma Cells to Changes in Extracellular Ph

Introduction: Glioblastoma cells can survive in acidic microenvironments, but the pH regulation strategies of glioblastoma are poorly understood. Cortical astrocytes regulate pH through the bidirectional Na+/HCO3- transporter SLC4A4.

Methods: Contrast-enhancing solid tumor and non-enhancing infiltrating tumor were sampled from patients undergoing surgery for newly diagnosed IDH-wildtype glioblastoma (n=30). SLC4A4 was characterized in both regions using single-cell sequencing (n=17), spatial transcriptomics (n=2) and immunohistochemistry (IHC, n=20). Lentiviral knockdown and overexpression models were developed, and SLC4A4 pharmacologic inhibitor (DIDS) was acquired. Extracellular pH (pHe) was quantitated using microelectrode recordings. Intracellular pH (pHi) was quantitated using fluorescent BCECF. An acid challenge assay exposed cells to pHe changes. Tumor growth was evaluated using a tumor-cortical organoid model.

Results: Solid tumor gliomaspheres had lower pH than infiltrating tumor spheres (p=0.001). SLC4A4 molecular characterization showed higher expression in infiltrating tumor relative to solid tumor (FDR p< 0.0001) and confirmed with IHC (p=0.039). SLC4A4 knockdown showed minimal changes in solid tumor but infiltrating tumor had significantly increased pHi, decreased pHe, and decreased cell death with pHe challenge (p=0.0105). Similarly, pHe challenge in SLC4A4 overexpression revealed minimal changes in infiltrating tumor but decreased pHi, increased pHe, and increased cell death in solid tumor (p=0.0201). SLC4A4 knockdown showed increased proliferation (p=0.0041) and overexpression demonstrated decreased proliferation (p=0.0016) in a cortical organoid model. IHC showed increased Ki67 and decreased Cas9 with decreased SLC4A4 (p=0.0001).

Conclusion : Na+/HCO3- transporter SLC4A4 contributes to pH buffering in infiltrating glioblastoma. We propose that reduced SLC4A4 expression in the contrast-enhancing region desensitizes these tumor cells to decreased pHe, permitting continued growth despite an unfavorable acidic environment. This differs from the non-enhancing infiltrating tumor cells which strongly express SLC4A4 and are sensitive to pHe changes. Therapeutic approaches to target infiltrating glioblastoma cells through pH regulation may help to reduce tumor recurrence by eliminating glioblastoma cells likely to persist after contrast-enhancing tumor resection.