Jian Cao, Becky Jenkins, Uroob Haris, Jeni Gerberich
As complex organisms, animals and humans rely on a delicate interplay between many connected systems to maintain proper cellular and whole-body function. Oxygen regulation and homeostasis are vital for proper function in healthy cells, as are core components of aerobic metabolism and efficient ATP generation to supply our energy needs. Maintaining pH homeostasis is also of primary importance; cells maintain pH homeostasis through a host of active and passive transporters to maintain pH balance both inside and outside of the cell.
Events of uncontrolled cellular proliferation, also known as cancer, survive and thrive under conditions that perturb the delicate balance of both oxygen and pH homeostasis. Because tumors rapidly divide, they outgrow the vasculature that supplies them with the nutrients they require, including O2. This causes the hypoxia dependent expression of enzymes that shifts the tumor microenvironment into anaerobic metabolic states, resulting in the overproduction of lactic acid and concomitant decrease in extracellular pH within the tumor relative to that of healthy cells.
This thesis is focused on the development of chemical tools to image oxygen and pH (mis)regulation within the tumor microenvironment. To accomplish this, we use chemiluminescent 1,2-dioxetane compounds that emit light in response to analytes within their chemical environment. Specifically, we developed an activity-based hypoxia sensing chemiluminescence probe that emits light under low O2 conditions. HyCL-4-AM provides a selective 60,000-fold increase in luminescence emission in the presence of rat liver microsomes (RLM), and provides highly sensitive and reproduceable O2 dependent emission in cells. Whole animal imaging experiments in muscle tissue and tumor xenografts show that HyCL-4-AM can differentiate between well oxygenated muscle tissue and hypoxic tumors, demonstrating potential for monitoring tumor reoxygenation via hyperoxic treatment.
Furthermore, we report a chemiluminescence resonance energy transfer (CRET) probe Ratio-pHCL-1, that transfers energy from the dioxetane substrate to a pH responsive fluorophore. The probe provides an accurate measurement of pH between 6.8 and 8.4, making it a viable tool for measuring pH in biological systems. Using an IVIS Spectrum, pH can be measured through tissue with Ratio-pHCL-1, which is shown in vitro and calibrated in sacrificed mouse models. Intraperitoneal injections of Ratio-pHCL-1 into live mice show high photon outputs and consistent increases in flux ratio when measured at pH 6, 7, and 8. This design could ultimately be used to study pH heterogeneity within the tumor microenvironment.
Finally, we report UVC-454, UVA-454, and Spiro-CL as photoactivatable chemiluminescence compounds. UVC-454 and UVA-454 are protected with ortho-nitrobenzyl protecting groups that provide irreversible photochemical uncaging of the chemiluminophore species through UV light irradiation. UVC-454 and UVA-454 can be selectively activated based on uncaging wavelength, and demonstrate ability to be photoactivated in water. Spiro-CL is a novel chemiluminescent spiropyran that can reversibly interconvert from its stable spiropyran form to a metastable merocyanine form through UV or visible light irradiation, respectively. Further, this compound exhibits chemiluminescence in its open form upon irradiation with UV light in DMSO.
Biological Sciences, General, Chemistry, Life Sciences, Pharmacology
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RYAN, LUCAS, "Imaging the Tumor Microenvironment Using 1,2-Dioxetane Chemiluminescence Agents" (2020). Chemistry Theses and Dissertations. 18.