Studying LNCGRS-1 role in human gliomas treatment using 2D culture and a novel mature brain organoid model
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BACKGROUND: Malignant gliomas are a fatal disease that despite extensive research in the field, are still poorly understood. The primary form of treatment has been tumor reduction using a combination of radiotherapy, chemotherapy and surgery, but median survival of adult and pediatric patients is about 2 years. Thus, it is imperative to understand this disease and develop therapies that can eradicate these infiltrative tumor cells without having significant toxicity to the normal functioning of the brain. CRISPR-interference (Clustered Regularly Interspaced Short Palindromic Repeats; CRISPRi) screens have identified long noncoding RNAs (lncRNAs) that may potentiate the effects of radiation in gliomas. lncRNAs exhibit highly cell type-specific expression and function, making these attractive targets for potential cancer therapies. This class of transcript is poorly studied thus far as a potential therapeutic target. Current models of human glioma fall short of recapitulating the tumor microenvironment, and therefore, make testing potential therapies difficult. For example, because CTC338M12.4 (lncGRS-1), a potential glioma therapeutic target, is only expressed in primates, it would be time-consuming and expensive to perform studies on genetically-engineered mouse models. OBJECTIVES: 1) To explore the role of lncGRS-1 as a glioma-specific therapeutic target and in the p53-mediated DNA damage response, 2) To build upon a new experimental model system that can better represent the characteristics of human glioma. METHODS: Project One: I performed knockdown assays of lncGRS-1 and analyzed levels p53 and p21 levels by Western Blot analysis. I also identified presence of phospho-53 binding protein (P53BP1) and γ H2AX by immunohistochemistry. Finally, I performed double knockdowns of lncGRS-1 and p53 to study the effect on cell proliferation. Project Two: I generated mature brain organoids (MBOs) of 10,000 cells comprised of astrocytes, neurons, and pediatric glioma cells. Tumor burden of glioma cells growing in the MBOs was assessed over a period of 10 days and quantified with ImageJ. I generated MBOs of 60,000 cells comprised of astrocytes, neurons, pediatric glioma cells, and two different types of endothelial cells labeled with calcein. Organoids were placed in three different media compositions and combinations of epithelial growth factor and basic fibroblast growth factor and followed for 8 days. RESULTS: Project One: lncGRS-1 knockdown reduced both p53 and p21 levels. P53BP1 levels increased in the presence of radiation and lncGRS-1 knockdown, as well as with knockdown alone. γ-H2AX levels were not impacted significantly by lncGRS-1 knockdown. U87 glioma cells had decreased proliferation with p53 and lncGRS-1 double knockdown than with lnGRS-1 knockdown alone. Project Two: Introducing EGF/FGF-2 at 10 ng/mL concentration produced MBOs with tumors that did not overtake the organoid within 10 days. Cortical organoid media appeared to be the best media composition to support MBOs comprised of endothelial cells, astrocytes, neurons, and glioma cells. CONCLUSIONS: Project One: Our results suggest that lncGRS-1 plays a role in the DNA damage response by modifying a cell’s ability to pass cell cycle checkpoints. Project Two: I generated mature brain organoids comprised of 4 different cell types – astrocytes, neurons, endothelial cells, and glioma cells. Furthermore, I identified a media composition (cortical organoid media) that allowed for all cell types to survive for at least a one week.
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