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Children undergoing cranial radiation therapy (CRT) for pediatric central nervous system malignancies are at increased risk for neurological deficits later in life. We have developed a model of neurotoxic damage in adult Drosophila following irradiation during the juvenile stages with the goal of elucidating underlying neuropathological mechanisms and of ultimately identifying potential therapeutic targets. Wild-type third-instar larvae were irradiated with single doses of γ-radiation, and the percentage that survived to adulthood was determined. Motor function of surviving adults was examined with a climbing assay, and longevity was assessed by measuring lifespan. Neuronal cell death was assayed by using immunohistochemistry in adult brains. We also tested the sensitivity at different developmental stages by irradiating larvae at various time points. Irradiating late third-instar larvae at a dose of 20 Gy or higher impaired the motor activity of surviving adults. A dose of 40 Gy or higher resulted in a precipitous reduction in the percentage of larvae that survive to adulthood. A dose-dependent decrease in adult longevity was paralleled by a dose-dependent increase in activated Death caspase-1 (Dcp1) in adult brains. Survival to adulthood and adult lifespan were more severely impaired with decreasing larval age at the time of irradiation. Our initial survey of the Drosophila Genetic Reference Panel demonstrated that differences in genotype can confer phenotypic differences in radio-sensitivity for developmental survival and motor function. This work demonstrates the usefulness of Drosophila to model the toxic effects of radiation during development, and has the potential to unravel underlying mechanisms and to facilitate the discovery of novel therapeutic interventions.

Metastatic disease in the brain is difficult to control and predicts poor prognosis. Here, we analyze human brain metastases and demonstrate their robust infiltration by CD8+T cell subsets with distinct antigen specificities, phenotypic states, and spatial localization within the tumor microenvironment. Brain metastases are densely infiltrated by T cells; the majority of infiltrating CD8+T cells express PD-1. Single-cell RNA sequencing shows significant clonal overlap between proliferating and exhausted CD8+T cells, but these subsets have minimal clonal overlap with circulating and other tumor-infiltrating CD8+T cells, including bystander CD8+T cells specific for microbial antigens. Using spatial transcriptomics and spatial T cell receptor (TCR) sequencing, we show these clonally unrelated, phenotypically distinct CD8+T cell populations occupy discrete niches within the brain metastasis tumor microenvironment. Together, our work identifies signaling pathways within CD8+T cells and in their surrounding environment that may be targeted for immunotherapy of brain metastases.