Determining mechanisms of heterogeneous treatment response in glioblastoma using multi-omic and multi-organ approaches - Glioblastoma (GBM) is an aggressive brain tumor that recurs in treatment-refractory form within months of initial standard-of-care (SOC) treatment. Extensive study of primary (pre-treatment) GBM has revealed numerous characteristics that impede treatment, including marked intratumoral genetic, transcriptional, and spatial heterogeneity; rapid infiltration throughout the brain parenchyma; a uniquely compromised immune microenvironment; and immunosuppression beyond the central nervous system. However, the mechanisms by which these factors enable disease progression are not understood, and the critical disease entity -- recurrent GBM -- is comparatively understudied. The central hypothesis of this study is that identifying the unique characteristics of recurrent GBM and the mechanisms driving its evolution from primary GBM will reveal new therapeutic targets. Our overall objective is to identify transcriptional, genetic, neuronal, and immunological factors that influence GBM formation, progression, and treatment response, with an emphasis on therapeutically targetable ligand-receptor interactions. We utilize unique human tissue repositories, novel experimental designs, and spatially-resolved single-cell approaches with the rationale that reversible interactions between tumor cells and diverse nonmalignant cells drive tumor formation, progression, and drug resistance. We pursue our objective through three Specific Aims: (1) Identify unique molecular, cellular, and spatial features of recurrent human GBM; (2) Identify mechanisms underlying distal pathological effects of GBM, including distant brain infiltration and systemic immunosuppression, in whole-brain and whole-body autopsy settings; and (3) Elucidate the dynamic reconfiguration of the GBM ecosystem during tumor formation and treatment response using mouse models, single-cell lineage tracing, and spatial transcriptomics. In Aim 1, we will integrate spatial transcriptomics (ST) with novel long-read single-nucleus RNA-sequencing (snRNA-seq) and exome sequencing to identify cell states, single-cell clonal architecture, multicellular niches, and heterotypic ligand-receptor interactions specific to recurrent GBM. In Aim 2, we will leverage unique resources at MGH that enable the analysis of diverse brain regions and peripheral immune organs in post-mortem GBM patients. We will combine snRNA-seq, ST, and single-cell T-cell receptor sequencing to study brain invasion and immunosuppression within the brain and peripheral immune system. In Aim 3, we will perform time-resolved single-cell RNA-seq (scRNA-seq) and ST in a mouse model of GBM to map the temporal and spatial co-evolution of tumor and immune cells during tumor development and response to SOC. Critically, we will integrate single-cell lineage tracing with scRNA-seq and ST to distinguish among possible mechanisms by which treatment reconfigures the tumor ecosystem. This study addresses innovative questions using unique resources, novel study designs, and new combinations of genomic and computational approaches. The work is significant because it will enable the discovery of therapeutic vulnerabilities associated with GBM development, progression, and recurrence.
