Abstract 1673: Optical metabolic imaging of response to radiation in radiation-sensitive and resistant lung cancer cells
Authors: Alhallak Kinan , Dings Ruud P.M. , Rajaram Narasimhan 
Published: July 2016

Tumor resistance to radiation therapy is a significant obstacle that patients and doctors face while treating tumors. Meaningful changes in therapy are contingent upon the ability to identify an unfavorable response very early (first week) in the course of treatment. Optical imaging can perform noninvasive, frequent and quantitative measurements of tumor biology at the point of care. The goal of this study was to identify optically measurable metabolic endpoints in a matched model of radiation resistance before and after treatment. A human lung cancer cell line was induced to develop radiation resistance through repeated exposure to a clinically relevant dose of X-ray radiation (2 Gy). We used a fluorescent glucose analog called 2-NBDG to quantify glucose uptake. We measured the endogenous fluorescence of nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD), metabolic coenzymes found primarily in the cytoplasm and mitochondria, respectively. We determined the redox ratio [FAD/(NADH + FAD)] in both cell lines. We evaluated the behavior of the cellular antioxidant system by comparing the levels of reactive oxygen species (ROS) and ROS scavengers, such as glutathione (GSH) for each cell group. Finally, we analyzed the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) using a metabolic flux analyzer.

Radiation-resistant A549 lung cancer cells were induced by exposure to 25 fractions of X-ray radiation (2.2 Gy/fraction). Clonogenic assays were performed to confirm radiation resistance. All fluorescence images were obtained using a multiphoton microscope. 2-NBDG, ROS, GSH, NADH, and FAD fluorescence were excited at 960, 930, 780, 755 and 860 nanometers, respectively. The cells were exposed to 2 Gy 60 minutes prior to the Seahorse assay. All experiments were performed in both the parental A549 cell line (A549C) and the radiation-resistant A549 cell line (A549R) at baseline (prior to radiation) and after radiation.

At baseline, the A549R cells revealed a significantly lower level of oxidative stress and a higher level of GSH. Interestingly, glucose uptake, quantified using 2-NBDG fluorescence, was significantly lower in the A549R cells compared to the A549C cells at baseline. The redox ratio was significantly higher in the A549R cells prior to radiation, indicating a preference for mitochondrial respiration. However, after radiation, the OCR of the A549R cells was significantly lower than the parental cell line, indicating a lower use of mitochondrial respiration. There were no significant differences in ECAR levels between both cell lines after radiation. Our results indicate that the radiation-resistant A549 lung cancer cells present very distinguishable metabolic properties at baseline and after exposure to radiation relative to the parental cell line that could potentially be exploited in vivo.

Citation Format: Kinan Alhallak, Ruud P.M. Dings, Narasimhan Rajaram. Optical metabolic imaging of response to radiation in radiation-sensitive and resistant lung cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1673.