CAREER: A multimodal imaging platform to investigate spatiotemporal changes in tumor bioenergetics that drive treatment resistance.

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Nearly 50% of all cancer patients each year are treated with radiation therapy, either alone or in combination with chemotherapy. In the case of radiation therapy, which is the focus of this project, hypoxia (very low levels of oxygen) is an important cause of radiation resistance or treatment failure. Recent evidence suggests that tumors experiencing brief episodes of hypoxia can also harbor radiation-resistant cancer cells. Of specific interest are cancer cells that are situated close to the oxygen diffusion limit in tumors, a region that experiences fluctuations in oxygen availability due to poorly developed vasculature. It is not known if metabolic adaptations (adaptations to decrease energy expenditure) of these cancer cells to fluctuations in oxygen delivery can help promote radiation resistance. The research goal of this project is to develop a multimodal imaging platform to investigate the relationship between microvascular oxygenation and cellular metabolism, and how this relationship helps promote treatment resistance. Knowledge of these relationships in response to radiation therapy can lead to the development of targeted therapeutics to reverse resistance. These studies will provide opportunities for students to gain valuable research experience in design and development of optical imaging technologies and basic science investigations of cancer biology. The educational and outreach goal of this proposal is to develop year-long learning kits based on light and optics, which is part of the middle school curriculum. The purpose of these kits is to equip rural middle school teachers with active teaching modules to better communicate optics and light-based concepts to students.The principal investigator's long-term career goal is to develop optical imaging technologies that can visualize and answer basic biological questions about the tumor micro-environment that would optimally lead to the development of new biomarkers that can spur the development of cost-effective tools to help advance public health. Towards this goal, this project will build and validate a label-free multimodal imaging platform (a two-photon microscope integrated with a hyperspectral darkfield microscope (TP-HDMI)) to quantitatively visualize the relationship between microvascular oxygenation and cellular metabolism and determine how the relationship between these two hallmarks promotes radiation resistance. Hyperspectral microscopy will be used to assess oxygenation levels based on light absorption by hemoglobin and the differences in absorption profiles of oxygenated (HbO2) and deoxygenated (dHb) hemoglobin. The oxygenation level metric for spatial comparison will be [HbO2]/([HbO2]+[dHb]). Two-photon microscopy will be used to assess cellular metabolism based on the system's ability to image natural fluorescing NADH and FAD, molecules that are critical for the energy producing processes. The cellular metabolism metric for spatial comparison will be the optical redox ratio FAD/(FAD + NADH). The transformative nature of the project lies in the ability to investigate dynamic changes in the spatiotemporal relationship between tumor oxygenation and cellular metabolism in vivo in response to radiation therapy, and how these changes might be distinct in radiation-resistant and sensitive tumors. The central hypothesis of this work is that metabolic reprogramming under these intermittently hypoxic conditions can cause cells to develop a radiation-resistant phenotype. The Research Plan is organized under two objectives. The FIRST OBJECTIVE is to build the multimodal system and to validate its ability to investigate spatiotemporal relationships between vascular oxygenation and cellular metabolism. Once assembled, the integrated imaging platform will be validated in normal tissue within a window chamber model established in athymic nude mice. Image analysis algorithms will be used to separate vascular and non-vascular tissue. Dynamic metabolic responses to changes in oxygen supply will be imaged while the mice are subjected to varying levels of hypoxia. The optical redox ratio as a function of distance from the nearest blood vessel will be determined. The SECOND OBJECTIVE is to investigate the spatiotemporal changes in tumor bioenergetics in radiation-resistant and sensitive tumors in response to radiation therapy. The integrated imaging platform will be used to investigate dynamic spatiotemporal changes in cellular metabolism in response to different doses of radiation therapy in human head and neck tumors of known radiation resistance and sensitivity implanted in the mouse window chambers. All of the imaging-related endpoints will be further validated using immunohistochemical assays. The studies will establish a model for metabolic changes within cancer cells as a function of microvascular oxygenation, distance from the nearest microvessel, and time after radiation therapy. Results obtained are expected to significantly expand our understanding of how the spatial location of cells with respect to the oxygen diffusion limit influences their resistance to treatment.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.