TMIC-64. CHARACTERIZATION OF THE TUMOR MICROENVIRONMENT IN DIFFUSE MIDLINE GLIOMAS (DMG) FOR 3D IN VITRO MODEL BUILDING

Abstract

Abstract BACKGROUND Diffuse midline glioma (DMG) is an aggressive pediatric brain cancer. Few comprehensive tumor characterization studies have been conducted due to the relative scarcity of tissue samples. However, it is well known that the surrounding microenvironment, and particularly the cells of the normal brain, influence tumor outcomes. Here, we investigate the cell type distribution among patient autopsy samples, comparing the primary tumor, metastatic tumors, and normal brain tissue. METHODS Patient samples (n=10) were obtained at Children’s National through a postmortem autopsy program. All patients were diagnosed with DMG between the ages of four and thirteen. Brain sections were embedded in paraffin, sectioned into 5 μm thick slices, and stained using fluorescent immunohistochemistry. Targets included astrocytes (ALDH1L1), microglia (IBA1), neurons (NeuN), blood vessels (CD31), and tumor cells (H3K27M). Images were captured using the VS200 whole slide scanner. Automatic cell detection was conducted using QuPath, and statistical analysis was performed in GraphPad Prism. RESULTS From initial primary tumor IHC analysis, microglia appear to be present in slightly higher ratios than astrocytes (0.203 vs. 0.148, fraction of total cells, p=0.062). Furthermore, neurons appear in lower numbers compared to microglia and astrocytes (0.036 vs. 0.203 and 0.148, fraction of total cells, p< 0.0001). There are relatively few blood vessels when adjusted for total area (0.084% of total area). In addition, within a single patient, we anticipate fewer microglia and astrocytes in normal tissue and metastatic tissue compared to the primary site. We also anticipate more neurons in normal tissue and metastatic tissue compared to the primary site. FUTURE DIRECTIONS Using this tumor composition data, we plan to create a 3D gel in vitro cell culture model of DMG. In collaboration with individuals at Children’s National and Columbia, we will use this model in conjunction with focused ultrasound and drug testing.