Project Summary Radiopharmaceuticals have shown tremendous clinical promise, resulting in regulatory approvals of several agents. Such targeted radiotherapy exposes tumor cells to continuous, exponentially decaying radiation, resulting in biological effects distinct from those of external beam radiotherapy. Despite this fundamental difference, there is limited understanding of the impact of radiopharmaceuticals on tumor cell populations and other components of the tumor microenvironment such as stromal cells, immune compartments, and endothelial cells. The proposed work will provide novel insights into tumor cell-intrinsic and -extrinsic mechanisms of radiation response that can inform the study of radiopharmaceuticals across oncology. Against this backdrop, Project 2 will deeply characterize samples, imaging, and data from a cohort of children with high-risk neuroblastoma treated with the targeted radiopharmaceutical 131I-MIBG. In Aim 1, we will evaluate pre-treatment tumor cell-intrinsic and tumor cell-extrinsic factors, including somatic and host genomic variants, single cell and bulk transcriptomics, and DNA damage repair profiling. In Aim 2, we will assess dynamic changes in these factors in response to 131I-MIBG therapy, including evaluation of tumor materials resected after 131I-MIBG as well as serial circulating tumor DNA samples. In Aim 3, we will leverage two large national studies to assess the late effects of 131I-MIBG in this vulnerable pediatric population. By the conclusion of Project 2, we will understand predictors of response and toxicity following 131I-MIBG therapy and, more fundamentally, will understand the changes in a comprehensive set of molecular markers reflective of radiation biology following treatment with this radiopharmaceutical. We have embedded our work within the only randomized phase 3 trial of 131I-MIBG ever conducted, COG ANBL1531 (NCT03126916). However, beyond our utilization of goal-oriented clinical trials to enable a question-oriented, hypothesis-driven study plan in close collaboration with our Data Science and AI Cores, Project 2 advances several transformative innovations. A major barrier limiting our understanding of the biological effects of radiopharmaceuticals is a lack of paired tumor samples obtained before and after exposure to radiation. Our work overcomes this barrier by exploiting our unique access to detailed, cross-sectional data on long-term medical, psychological, and educational outcomes, coupled with paired, longitudinal biologic specimens, dosimetry, serial images, data science, mechanistic computational modeling, and radiomics to identify the critical changes in our markers of tumor heterogeneity that are associated with 131I-MIBG response and late effects. Importantly, biospecimens are available from patients treated with or without 131I-MIBG therapy on this trial, enabling us to validate specific markers as truly predictive rather than simply prognostic. Finally, our proposal leve...