Application of autobioluminescence toward continuous and real-time in vitro/in vivo pre-clinical brain imaging for disease therapeutics

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Project Summary: This project proposes to develop autonomously bioluminescent (autobioluminescent) patient-derived glioblastoma (GBM) cellular and rodent models capable of substrate-free, continuous, and noninvasive assessment of therapeutic efficacy to enable effective and efficient translational GBM research. GBM is one of the most lethal of human cancers, with less than 3% of patients surviving beyond a five-year period. To assist in the battle against GBM, bioluminescent imaging technologies that facilitate the noninvasive and longitudinal visualization of tumor dynamics have served as valuable tools in translational efforts to better understand the molecular mechanisms of GBM progression and the evaluation of novel therapeutics in pre- clinical small animal models. However, existing bioluminescent imaging approaches that rely upon conventional luciferase reporter systems (firefly, Gaussia, Renilla, etc.) are handicapped for brain imaging studies because they require that the animal subject be injected with a light-activating substrate prior to each and every measurement. For brain imaging in particular, the extraneous addition of this chemical substrate confounds imaging endpoints because its biodistribution and bioavailability is interfered with by the blood-brain barrier and the brain’s efflux pumping mechanisms. Although research is being dedicated toward the synthesis of novel bioluminescent reaction substrates with improved access to the brain environment, we have engaged in an entirely different approach by eliminating the need to add substrate altogether. Our technology leverages the development of a synthetic luciferase (lux) cassette that efficiently expresses bioluminescence in mammalian cells independent of any extraneous addition of a light-activating substrate. These cells are able to self-synthesize all of the requisite substrates from intracellular endogenous metabolites and are therefore capable of self- generating ‘autobioluminescent’ signals under both constitutive and inducible genetic controls. Within the brain environment, such cells go beyond conventional bioluminescent imaging to ultimately enable continuous, noninvasive, and authentic real-time visualization of neurobiological processes. In this research effort, we propose to express autobioluminescence in patient-derived GBM cell lines and validate their application potential in high-throughput in vitro drug discovery assays and in in vivo rodent models. We will specifically develop and characterize 2D, 3D, and 3D astrocyte co-culture assays, create signaling pathway-specific autobioluminescent cellular models for targeted small molecule screening, and establish an orthotopic autobioluminescent xenograft mouse model for in vivo evaluation of chemotherapeutics that we will test in both conscious and anesthetized subjects. The innovative autobioluminescent cellular and animal models developed in this project will improve the status quo of glioblastoma drug screening and testing and facilitate the development of novel glioblastoma therapeutics within a research environment designed to intellectually stimulate and challenge undergraduate student researchers.