PLoS One. 2014 Jan 27;9(1):e87031.

Evaluation of hyperpolarized [1-¹³C]-pyruvate by magnetic resonance to detect ionizing radiation effects in real time.

Sandulache VC1, Chen Y2, Lee J3, Rubinstein A4, Ramirez MS3, Skinner HD5, Walker CM3, Williams MD6, Tailor R4, Court LE4, Bankson JA3, Lai SY7.

1Bobby R. Alford Department of Otolaryngology, Head and Neck Surgery, Baylor College of Medicine, Houston, Texas, United States of America ; Department of Head and Neck Surgery, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, United States of America.
2Department of Head and Neck Surgery, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, United States of America.
3Department of Imaging Physics, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, United States of America.
4Department of Radiation Physics, The University of Texas MD Anderson Cancer Center, Houston, Texas, United States of America.
5Department of Radiation Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, United States of America.
6Department of Pathology, The University of Texas M.D. Anderson Cancer Center, Houston, Texas, United States of America.
7Department of Head and Neck Surgery, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, United States of America ; Department of Molecular and Cellular Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, Texas, United States of America.

Abstract

Ionizing radiation (IR) cytotoxicity is primarily mediated through reactive oxygen species (ROS). Since tumor cells neutralize ROS by utilizing reducing equivalents, we hypothesized that measurements of reducing potential using real-time hyperpolarized (HP) magnetic resonance spectroscopy (MRS) and spectroscopic imaging (MRSI) can serve as a surrogate marker of IR induced ROS. This hypothesis was tested in a pre-clinical model of anaplastic thyroid carcinoma (ATC), an aggressive head and neck malignancy. Human ATC cell lines were utilized to test IR effects on ROS and reducing potential in vitro and [1-¹³C] pyruvate HP-MRS/MRSI imaging of ATC orthotopic xenografts was used to study in vivo effects of IR. IR increased ATC intra-cellular ROS levels resulting in a corresponding decrease in reducing equivalent levels. Exogenous manipulation of cellular ROS and reducing equivalent levels altered ATC radiosensitivity in a predictable manner. Irradiation of ATC xenografts resulted in an acute drop in reducing potential measured using HP-MRS, reflecting the shunting of reducing equivalents towards ROS neutralization. Residual tumor tissue post irradiation demonstrated heterogeneous viability. We have adapted HP-MRS/MRSI to non-invasively measure IR mediated changes in tumor reducing potential in real time. Continued development of this technology could facilitate the development of an adaptive clinical algorithm based on real-time adjustments in IR dose and dose mapping.

PMID: 24475215

 

Mol Cancer Ther. 2012 Jun;11(6):1373-80.

Glycolytic inhibition alters anaplastic thyroid carcinoma tumor metabolism and improves response to conventional chemotherapy and radiation.

Sandulache VC, Skinner HD, Wang Y, Chen Y, Dodge CT, Ow TJ, Bankson JA, Myers JN, Lai SY.

Bobby R. Alford Department of Otolaryngology-Head and Neck Surgery, Baylor College of Medicine, Houston, Texas, USA.

Abstract

Anaplastic thyroid carcinoma (ATC) accounts for more than 50% of thyroid cancer mortality and is generally refractory to conventional treatment. On the basis of recent studies, we hypothesized that ATC metabolism can be targeted to improve response to chemoradiotherapy. Eight established and authenticated ATC cell lines were sequenced at 140 sites contained within 26 commonly mutated genes to identify novel potential therapeutic targets. Cellular proliferation, energy, and reducing potential stores were measured under conditions of specific nutrient deprivation. Tumor metabolism was evaluated using hyperpolarized (13)C MRI in a murine orthotopic xenograft model of ATC. Sensitivity to chemotherapeutic agents and radiation (XRT) was assayed using cytotoxicity assays. We identified mutations in BRAF, NRAS, and KIT but failed to identify generalized novel targets for therapeutic intervention. ATC cell lines exhibited a mesenchymal phenotype and generalized dependence on glucose for energy, reducing potential and survival. Glycolytic inhibition using 2-deoxyglucose (2-DG) sensitized ATC cells to conventional chemotherapy and external beam radiation. In vivo, 2-DG induced a transient, but significant reduction in ATC metabolic activity. Generalized dependence of ATC cells on glucose catabolism makes them susceptible to the sensitizing effects of 2-DG for radiation therapy and chemotherapy. Under in vivo conditions, 2-DG can inhibit ATC metabolism. However, the modest magnitude and transient nature of this effect suggest the need for antimetabolic agents with more favorable pharmacodynamics to achieve therapeutic effects. ©2012 AACR

PMID: 22572813

 

SUPPLEMENT

More than 200,000 new head and neck solid tumors are diagnosed each year in the United States. Ionizing radiation (IR) is commonly employed in the treatment of these tumors as definitive therapy or in the adjuvant setting. The effectiveness of IR has been well established in head and neck solid tumors such as squamous cell carcinoma and anaplastic thyroid carcinoma. Unfortunately, radiation toxicity, manifested as mucositis, xerostomia, dysphagia and dysphonia, limits the maximum tolerated dose. Successful IR treatment is therefore driven by its intrinsic therapeutic index or the ratio of tumoricidal effects to normal tissue toxicity. Since neither parameter can measured in real-time, total dose, dose fractionation and dose painting decisions are largely based on empirical data derived from previous prospective and retrospective trials.

Generation of reactive oxygen species (ROS) is crucial to radiation cytotoxicity by inducing DNA damage and triggering tumor cell death. Our group has demonstrated that ROS-generated metabolically in response to IR play an important role in radiation cytotoxicity. Levels of metabolically-derived ROS correlate with relative radiosensitivity/radioresistance, and exogenous manipulation of post-IR ROS levels can dramatically alter tumor cell response to IR. Although we can measure IR-driven ROS changes in cells without much difficulty, clinical applicability is not possible without the ability to measure ROS in solid tumors in a non-invasive manner.

To overcome this technological limitation, we focused our attention on tumor reducing potential (an aggregate measure of various reducing equivalents). Tumor cells maintain substantial levels of glutathione and other thiol-containing proteins, which can scavenge IR-generated ROS. Regeneration of these thiol moieties requires utilization of secondary reducing equivalents such as NAD(P)H, which are regenerated metabolically (Fig. 1). Exposure to a burst of IR-induced ROS therefore triggers detectable changes in tumor metabolism. By analogy, IR-induced ROS can be compared to a fast traveling ship which generates a wake. The magnitude of the wave is related to the energy of the ship; measurements of wave size can provide an indirect measure of the energy of the passing ship. We hypothesized that exogenous perturbations in cellular and tumor ROS levels should trigger compensatory changes in reducing equivalent levels. Our hypothesis was proven correct. Exogenous perturbation of cellular ROS using metabolic inhibition (2-deoxyglucose), exogenous ROS sources (H2O2) or IR triggered a compensatory decrease in cellular reducing potential. This decrease was transient, dose-dependent and reversible using exogenous ROS scavengers such as N-acetyl cysteine (NAC). This demonstrated link between cellular reducing potential and exogenous ROS perturbations presents an intriguing potential means of directing solid tumor treatment.

Although biochemical measurements of cellular reducing potential are routinely employed in the laboratory setting, they cannot be translated to patients for two reasons: 1) they are invasive and 2) they require a very rapid time frame which is difficult to achieve in a clinical setting. To allow for clinical translation of our biochemical findings, a non-invasive means of measuring reducing potential changes in real time is required. We hypothesized that HP-MRS/I using 13C pyruvate can provide indirect measurements of perturbations of tumor reducing potential. We tested this hypothesis in our model of anaplastic thyroid carcinoma (ATC), an aggressive solid tumor with rapid local growth and a high rate of locoregional invasion and distant metastasis. Within this model, we were able to demonstrate that increased ROS is reflected in altered conversion of pyruvate into lactate, as a function of decreased cellular reducing potential (Fig. 1). This altered conversion can be measured biochemically in cells and tumors, and more importantly is detectable using 13C pyruvate HP-MRS/I. Within 1 hour of either metabolic stress (2-DG) or irradiation of ATC orthotopic mouse tumors, we were able to detect a reproducible, measurable and transient decrease in tumor reducing potential. For the first time, we were able to demonstrate a real-time capability of measuring acute treatment effects in solid tumors in a non-invasive manner. For aggressive tumors such as ATC, the ability to monitor treatment effects in real time and react accordingly has the potential to radically alter clinical paradigms and improve outcomes. Not only could we use HP-MRS to guide IR dosing, fractionation and dose painting, but also to develop effective synergistic treatment strategies using other ROS-perturbing agents (metabolic or conventional).

With the use of high-throughput genomic and epigenetic analyses, we are obtaining a more comprehensive understanding of tumor growth and response to treatment. Development of HP-MRS/MRSI into a viable biomarker of radiation response, combined with a better understanding of molecular processes which drive radioresistance in tumor cell subpopulations can lead to more effective treatment strategies for aggressive solid tumors that may fundamentally change the practice of medicine.

fig1Figure 1. Integration of HP-MRS into a therapeutic framework that combines metabolic inhibition with IR and conventional chemotherapeutic agents. IR or cisplatin trigger ROS generation which is scavenged by primary reducing equivalents (i.e. glutathione). Primary reducing equivalents are regenerated by secondary reducing equivalents (i.e. NAD(P)H). Metabolic inhibitors such as 2-DG can block regeneration by interfering with generation of secondary reducing equivalents. Perturbations in levels of secondary reducing equivalents (i.e. NADH) are detected by altered LDH activity. This can be monitored non-invasively by measuring the conversion of 13C-labeled pyruvate into 13C-labeled lactate. This conversion rate is an indirect measure of cellular reducing potential. Real time HP-MRS measurements can allow for precise manipulation of drug dose and timing of administration in order to maximize synergy between metabolic inhibition and conventional treatment strategies.

 

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