Abstract B45: Optical spectroscopy of tumor oxygenation and metabolism in preclinical head and neck tumors that fail radiation therapy
Authors: Rajaram Narasimhan , Murphy Helen A. , Ramanujam Nimmi 
Published: May 2016

Abstract
The purpose of this study was to determine the role of tumor metabolism and its relationship with tumor oxygenation in driving radiation resistance in head and neck cancer. The use of anatomical endpoints for therapy planning and the evaluation of response to radiation therapy in head and neck squamous cell carcinomas (HNSCC) is problematic because patients who do not respond favorably are losing critical time when alternate interventions could be attempted earlier. The use of a radiolabeled isotope severely limits the frequency of positron emission tomography (PET) of glycolytic demand and therefore the availability of immediate response to therapy. Although radiation-induced reoxygenation is a biomarker of tumor cell kill, radiation-induced reoxygenation can also lead to increased production of reactive oxygen species (ROS) that stabilize hypoxia-inducible factor (HIF-1). The transcription factor, hypoxia-inducible factor (HIF-1) and its downstream targets play an important role in the switch to aerobic glycolysis or metabolism of glucose to lactate under well-oxygenated conditions. Aerobic glycolysis confers tumors with a growth advantage, reduces the production of toxic free radicals by diverting energy production away from the mitochondria, and creates an acidic environment conducive for invasion and metastasis. Thus, it is important to measure both tumor glycolytic demand and oxygenation to determine whether re-oxygenation after radiation is accompanied by decreased glycolytic demand signaling cell death, or increased glycolytic demand signaling potential treatment failure. The use of non-ionizing radiation and the low cost of optical imaging allow measurements of these functional biomarkers several times after commencement of therapy and have the potential to significantly improve treatment outcome.
We have developed fiber-based diffuse optical spectroscopy for simultaneously quantifying vascular oxygenation (SO2) and glycolytic demand in solid tumors in vivo. Glycolytic demand was measured using a fluorescent glucose analog, 2-[N-(7-nitrobenz-2-oxa-1,3-diaxol-4-yl)amino]-2-deoxyglucose (2-NBDG). Quantification of label-free SO2 and 2-NBDG-fluorescence-based glycolytic demand 60 minutes after administration of the tracer (2-NBDG60) was performed using computational models of light-tissue interaction. In a pilot radiation study, a group of 10 mice was injected subcutaneously with FaDu head and neck cancer cells. Once tumor size reached 200 mm3, they were exposed to a 5-day dose of radiotherapy (1 dose/day; 7.5Gy/day) using a biological small animal X-ray irradiator (X-Rad320). In addition to pre-radiation baseline measurements of SO2 and 2-NBDG60, we also measured both parameters every other day during radiation (Days 1, 3, and 5) and for 10 days following the end of radiation. Total of 8 mice showed tumor recurrence after elimination of the primary tumor. Wide variability was observed in both recurrence and location after radiation. Mice that failed therapy showed improved oxygenation as well as increased glycolytic demand relative to local control mice as early as day 4 after commencing radiation. Mice that responded completely to radiation (no recurrence up to 90 days post treatment) showed similar trends in improved SO2 as the tumor was cured coupled with decreasing glycolytic demand. Our results illustrate the potential of optical measurements to provide valuable information about the metabolic status of tumors in vivo, with important implications for early cancer prognosis. Our future efforts will focus on a larger mouse cohort, identifying the specific role of HIF-1 in mediating treatment failure, and expanding the technology to measure larger areas of the tumor.
Citation Format: Narasimhan Rajaram, Helen A. Murphy, Nimmi Ramanujam. Optical spectroscopy of tumor oxygenation and metabolism in preclinical head and neck tumors that fail radiation therapy. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr B45.