Search

Supplemental Figure 2. IR triggers a temporary, reversible perturbation in cellular reducing potential.

Supplemental figure legends - detailed text

Ionizing radiation is the primary nonsurgical treatment modality for solid tumors. Its effectiveness is impacted by temporal constraints such as fractionation, hypoxia, and development of radioresistant clones. Biomarkers of acute radiation response are essential to developing more effective clinical algorithms. We hypothesized that acute perturbations in tumor lactate levels act as a surrogate marker of radiation response. In vitro experiments were carried out using validated human-derived cell lines from three histologies: anaplastic thyroid carcinoma (ATC), head and neck squamous cell carcinoma (HNSCC), and papillary thyroid carcinoma (PTC). Cellular metabolic activity was measured using standard biochemical assays. In vivo validation was performed using both an orthotopic and a flank derivative of a previously established ATC xenograft murine model. Irradiation of cells and tumors triggered a rapid, dose-dependent, transient decrease in lactate levels that was reversed by free radical scavengers. Acute lactate perturbations following irradiation could identify hypoxic conditions and correlated with hypoxia-induced radioresistance. Mutant TP53 cells and cells in which p53 activity was abrogated (shRNA) demonstrated a blunted lactate response to irradiation, consistent with a radioresistant phenotype. Lactate measurements therefore rapidly detected both induced (i.e., hypoxia) and intrinsic (i.e., mutTP53-driven) radioresistance. We conclude that lactate is a quantitative biomarker of acute genotoxic stress, with a temporal resolution that can inform clinical decision making. Combined with the spatial resolution of newly developed metabolic imaging platforms, this biomarker could lead to the development of truly individualized treatment strategies. Mol Cancer Ther; 14(12); 2901–8. ©2015 AACR.

Supplemental Figure 6. Loss of p53 function results in decreased radiosensitivity in HNSCC cell lines.

Supplemental Figure 1. Metabolic inhibition and exogenous ROS perturb cellular reducing potential.

Supplemental Figure 5. Acute IR effects on reducing potential and lactate can be used to detect intrinsic radioresistance driven by loss of p53 activity.

Supplemental Figure 4. Lactate accumulates in extra-cellular media as a function of cellular metabolism over time.

Supplemental Figure 3. IR effects on cellular lactate and clonogenic survival are driven by ROS.

Ionizing radiation is the primary nonsurgical treatment modality for solid tumors. Its effectiveness is impacted by temporal constraints such as fractionation, hypoxia, and development of radioresistant clones. Biomarkers of acute radiation response are essential to developing more effective clinical algorithms. We hypothesized that acute perturbations in tumor lactate levels act as a surrogate marker of radiation response. In vitro experiments were carried out using validated human-derived cell lines from three histologies: anaplastic thyroid carcinoma (ATC), head and neck squamous cell carcinoma (HNSCC), and papillary thyroid carcinoma (PTC). Cellular metabolic activity was measured using standard biochemical assays. In vivo validation was performed using both an orthotopic and a flank derivative of a previously established ATC xenograft murine model. Irradiation of cells and tumors triggered a rapid, dose-dependent, transient decrease in lactate levels that was reversed by free radical scavengers. Acute lactate perturbations following irradiation could identify hypoxic conditions and correlated with hypoxia-induced radioresistance. Mutant TP53 cells and cells in which p53 activity was abrogated (shRNA) demonstrated a blunted lactate response to irradiation, consistent with a radioresistant phenotype. Lactate measurements therefore rapidly detected both induced (i.e., hypoxia) and intrinsic (i.e., mutTP53-driven) radioresistance. We conclude that lactate is a quantitative biomarker of acute genotoxic stress, with a temporal resolution that can inform clinical decision making. Combined with the spatial resolution of newly developed metabolic imaging platforms, this biomarker could lead to the development of truly individualized treatment strategies. Mol Cancer Ther; 14(12); 2901–8. ©2015 AACR.

At present, there are over 100 clinical trials examining the role of metformin in malignancy, either alone or in combination with chemotherapy. However, despite significant preclinical data, trials examining the role of metformin as a radiosensitizer are few. Moreover, the effects of endogenous tumor mutations on response to metformin are unknown, as some data suggests that certain mutations (eg. TP53, STK11) may confer sensitivity to this agent. Finally, the effects of metformin on FDG-uptake in solid tumors is far from clear, as this agent can actually lead to an increased uptake of FDG in normal bowel. Because of these questions, we have designed an imagining-guided trial of 70 patients, randomized 6:1 in favor of metformin treatment in patients with non-small cell lung cancer to be treated with stereotactic body radiotherapy (SBRT). Patients receive either placebo or metformin at a loading dose of 1,000 mg per day for one week followed by 1-2 weeks of metformin at 2,000 mg per day. Prior to study entry, an FDG-PET scan is performed which is then repeated after 2-3 weeks of metformin treatment. Metformin or placebo is maintained throughout SBRT and the standard daily cone-beam CTs are taken. Metformin or placebo stops at the end of SBRT followed by an additional FDG-PET scan 6 months after completion of therapy. All tumors with available tissue will be subjected to targeted sequencing. The primary outcome is FDG-PET response following initial metformin or placebo monotherapy as well as at 6 months. Secondary outcomes include the effect of common mutations (TP53, STK11, Kras) on metformin FDG-PET response. Currently, 14 patients have been registered for this trial. We anticipate that this study will provide a unique insight on how metformin affects FDG-PET response, if this response is affected by tumor mutation status, and provide preliminary data on the clinical efficacy of metformin in the setting of SBRT. Citation Format: Heath D. Skinner, Thomas Guerrero, Brian Hobbs, Laurence Court, Paige L. Nitsch, John Heymach. Tumor mutation status will predict metabolic response to metformin in lung cancer treated with stereotactic body radiation therapy (SBRT). [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 CT075.

Purpose: To optimize translational fidelity of a murine model of anaplastic thyroid carcinoma (ATC) using real-time metabolic imaging. Background: Anaplastic thyroid carcinoma (ATC) is an aggressive solid malignancy with rapid growth, invasion of critical cervical structures and a high rate of pulmonary metastasis. Although rare in overall incidence (2-3% of thyroid cancer), ATC accounts for 50% of thyroid cancer related mortality. The rapid rate of growth and metastasis makes optimization of treatment algorithms challenging and development of novel therapeutic strategies difficult. Our group has developed an integrated preclinical model of ATC using validated human cell lines and a xenograft mouse model which recapitulates disease characteristics and closely mimics response to treatment including chemotherapy and ionizing radiation (IR). Experimental procedures: Validated human ATC cell lines were either injected into the right thyroid lobe of athymic nude mice in an orthotopic tumor model or intravenously (tail-vein) to generate pulmonary metastases. Tumors were exposed to varying doses of ionizing radiation (IR) and monitored using both anatomic magnetic resonance imaging (MRI) sequences as well as hyperpolarized (HP) [1-13C]-pyruvate (PYR). HP-MR data was acquired using either temporal or spatial acquisition sequences depending on the experimental aims. At the completion of intended experiments, primary thyroid tumors and pulmonary metastases were harvested and analyzed using standard histologic and immunohistochemical techniques. Results: We have employed HP-MR using labeled [1-13C]-pyruvate to measure tumor metabolic activity in real-time. We have translated this capability into precise measurements of tumor viability at the primary site, which when combined with anatomic MRI sequences provides enhanced visualization and identification of critical cervical structure invasion. HP-MR has allowed us to identify sub-centimeter pulmonary metastases and differentiate tumor from normal lung parenchyma based on the inherently higher rate of conversion of labeled pyruvate (PYR) into lactate (LAC). We had previously demonstrated that pharmacologic perturbations in tumor reducing potential can be detected in real time using HP-MR. In these studies we extended these initial experiments to demonstrate that HP-MR can detect parallel and opposing changes in tumor reactive oxygen species (ROS) levels following irradiation. Using the ATC xenograft mouse model we were able to measure acute (2hr) changes in tumor reducing potential following irradiation compared to control tumor (p=0.004). Furthermore, HP-MR allowed us to identify areas of ATC tumors which display enhanced reducing potential, potentially driving ATC radio-resistance. Conclusion: Utilization of HP-MR metabolic imaging has allowed us to further develop our preclinical model of ATC and improve our ability to develop novel translational applications. Specifically, we believe HP-MR can improve the ability to monitor loco-regional tumor growth and invasion, as well as the development of pulmonary metastases. Furthermore, the development of a quantifiable imaging-based biomarker of radiation effect may permit adaptive IR plans based upon tumor response that would optimize disease treatment and improve clinical outcomes. Citation Format: Vlad Sandulache, Yunyun Chen, Jaehyuk Lee, Marc Ramirez, Heath Skinner, Laurence Court, Jim Bankson, Stephen Lai. Using magnetic resonance and hyperpolarized tracers to monitor solid tumor progression, metastasis, and response to treatment. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr B07.