Kinan Alhallak

Optical redox ratio identifies metastatic potential-dependent changes in breast cancer cell metabolism.

The development of prognostic indicators of breast cancer metastatic risk could reduce the number of patients receiving chemotherapy for tumors with low metastatic potential. Recent evidence points to a critical role for cell metabolism in driving breast cancer metastasis. Endogenous fluorescence intensity of nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) can provide a label-free method for assessing cell metabolism. We report the optical redox ratio of FAD/(FAD + NADH) of four isogenic triple-negative breast cancer cell lines with varying metastatic potential. Under normoxic conditions, the redox ratio increases with increasing metastatic potential (168FARN>4T07>4T1), indicating a shift to more oxidative metabolism in cells capable of metastasis. Reoxygenation following acute hypoxia increased the redox ratio by 43 ± 9% and 33 ± 4% in the 4T1 and 4T07 cells, respectively; in contrast, the redox ratio decreased 14 ± 7% in the non-metastatic 67NR cell line. These results demonstrate that the optical redox ratio is sensitive to the metabolic adaptability of breast cancer cells with high metastatic potential and could potentially be used to measure dynamic functional changes that are indicative of invasive or metastatic potential.

Optical imaging of radiation-induced metabolic changes in radiation-sensitive and resistant cancer cells

Abstract. Radiation resistance remains a significant problem for cancer patients, especially due to the time required to definitively determine treatment outcome. For fractionated radiation therapy, nearly 7 to 8 weeks can elapse before a tumor is deemed to be radiation-resistant. We used the optical redox ratio of FAD/(FAD+NADH) to identify early metabolic changes in radiation-resistant lung cancer cells. These radiation-resistant human A549 lung cancer cells were developed by exposing the parental A549 cells to repeated doses of radiation (2 Gy). Although there were no significant differences in the optical redox ratio between the parental and resistant cell lines prior to radiation, there was a significant decrease in the optical redox ratio of the radiation-resistant cells 24 h after a single radiation exposure (p=0.01). This change in the redox ratio was indicative of increased catabolism of glucose in the resistant cells after radiation and was associated with significantly greater protein content of hypoxia-inducible factor 1 (HIF-1α), a key promoter of glycolytic metabolism. Our results demonstrate that the optical redox ratio could provide a rapid method of determining radiation resistance status based on early metabolic changes in cancer cells.

A Radiosensitizing Inhibitor of HIF-1 alters the Optical Redox State of Human Lung Cancer Cells In Vitro

Treatment failure caused by a radiation-resistant cell phenotype remains an impediment to the success of radiation therapy in cancer. We recently showed that a radiation-resistant isogenic line of human A549 lung cancer cells had significantly elevated expression of hypoxia-inducible factor (HIF-1α), and increased glucose catabolism compared with the parental, radiation-sensitive cell line. The objective of this study was to investigate the longitudinal metabolic changes in radiation-resistant and sensitive A549 lung cancer cells after treatment with a combination of radiation therapy and YC-1, a potent HIF-1 inhibitor. Using label-free two-photon excited fluorescence microscopy, we determined changes in the optical redox ratio of FAD/(NADH and FAD) over a period of 24 hours following treatment with YC-1, radiation, and both radiation and YC-1. To complement the optical redox ratio, we also evaluated changes in mitochondrial organization, glucose uptake, reactive oxygen species (ROS), and reduced glutathione. We observed significant differences in the optical redox ratio of radiation-resistant and sensitive A549 cells in response to radiation or YC-1 treatment alone; however, combined treatment eliminated these differences. Our results demonstrate that the optical redox ratio can elucidate radiosensitization of previously radiation-resistant A549 cancer cells, and provide a method for evaluating treatment response in patient-derived tumor biopsies.

Optical imaging and spectroscopy of microenvironmental changes associated with radiation resistance in tumors

We present our recent work using optical imaging and spectroscopy to investigate changes associated with oxygenation and metabolism in radiation-resistant and sensitive tumors.

Abstract 1673: Optical metabolic imaging of response to radiation in radiation-sensitive and resistant lung cancer cells

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.

Optical Imaging of Cancer Cell Metabolism in Murine Metastatic Breast Cancer

Results from imaging the optical redox ratio in murine breast cancer cells at normoxic and hypoxic conditions are presented. These data compare well with measurements of glycolysis and oxidative phosphorylation using a metabolic flux analyzer.