Optogenetic engineering of tumor topography in native tissue environments

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Project Summary: In nearly every cancer, tumors demonstrate a predilection for certain locations within a tissue and this is associated with unique clinical phenotypes. For example, in pancreatic cancer, lesions that arise in the head of the pancreas have a better prognosis and favorable histological subtypes compared to those developing in the tail. Thus, the anatomic position of a tumor within an organ can provide the context on which mutations act to define clinical and molecular phenotypes. Despite the clinical significance, the mechanisms that determine regional differences in tumor phenotypes remains enigmatic. The overall goal of this proposal is to develop novel approaches to gain mechanistic insights into how anatomic position directs tumor phenotypes. A major technical gap in our ability to interrogate tumor topography in disease progression is the lack of relevant genetic models that recapitulate spatial patterns and behaviors of human cancers. For example, pancreatic tumors can be readily generated in multiple murine models. However, localizing tumor formation to the head or tail of the pancreas is not possible. This stems from current tumor models that use Cre-recombinase technology to induce oncogenic transformation by activating oncogenes or deleting tumor suppressors in tissues. While tissue specificity can be achieved in some cases, targeting Cre activity and by extension oncogenic mutations to specific anatomic locations in a tissue is not possible and limits spatial control of tumor formation. To overcome these limitations, we propose to add a layer of spatial/regional control to tumor induction in genetically engineered murine models (GEMMs). We accomplish this by developing and leveraging optogenetic technologies for cancer research. Optogenetics involves introducing genetically encoded light sensitive proteins to a cell that can be activated by light and enables spatially defined protein regulation in tissues. Here we propose to engineer an optogenetic recombinase platform that allows for control of Cre activity with targeted light beams to enable precise spatial and temporal control of tumor formation. Though risky, we employ a systematic strategy to accomplish this. First, we will combine protein engineering with in vivo barcoded screening methods to develop a novel photoactivable Cre capable of eliminating background recombinase activity while maintaining robust recombination with light. Second, we will develop this construct into an optogenetic-Cre GEMM and optimize parameters to enable precise gene activation at a regional and single-cell level. Finally, we will integrate this system into Cre inducible oncogene models to generate tumors within defined anatomic locations and characterize their phenotypes and molecular features with single-cell sequencing technologies. The potential to control spatiotemporal patterns of tumor development in vivo would be a major achievement in cancer research. This will transform our ability to model spatial patterns of human cancers and study the impact on tumor biology. Furthermore, our technology could advance research beyond cancer, as the biology of many diseases are influenced by anatomic location but cannot be interrogated with existing models.