Investigating Pediatric Diffuse Midline Glioma Invasion: Chloride Modulation and Tumor Co-Culture Dynamics

Abstract

This thesis investigates the invasive nature of diffuse midline glioma (DMG), a rare yet lethal brain tumor most common in children, by exploring chloride ion modulation and tumor-tumor interactions. The primary objectives were to examine the regulation of potassium-chloride cotransporter 2 (KCC2) expression in DMG cells through transcription factor knockouts, modulate intracellular chloride levels through genetic engineering for KCC2 and sodium-potassium-chloride cotransporter 1, and assess tumor-tumor spheroid interactions under various conditions. The study used molecular biology techniques, including short guide RNA plasmid cloning and CRISPR-Cas9 gene editing, to manipulate chloride cotransporter expression in DMG XIII cells. Immunofluorescence imaging and analysis were used to evaluate KCC2 expression following transcription factor knockouts. Additionally, tumor spheroid co-culture experiments were conducted to investigate infiltration patterns between different DMG cell lines under varying media conditions. Results showed that knockout of the transcription factor STAT5A significantly increased KCC2 expression in DMG XIII cells. Genetic engineering efforts to overexpress KCC2 and sodium-potassium-chloride cotransporter 1 (NKCC1) were successful, though further selection and growth were required for comprehensive analysis. Tumor spheroid co-culture experiments revealed distinct infiltration patterns between different DMG cell lines, with DMG XIII and DMG XXIV showing the highest level of interaction. However, media conditions did not significantly affect infiltration levels. This work establishes a foundation for understanding chloride ion homeostasis in DMG progression and invasion. The findings suggest potential therapeutic targets for managing DMGs and provide insights into tumor-tumor interactions that may guide future strategies to limit tumor invasion into healthy brain tissue.