D Campos

Radiation Promptly Alters Cancer Live Cell Metabolic Fluxes: An In Vitro Demonstration

Quantitative data is presented that shows significant changes in cellular metabolism in a head and neck cancer cell line 30 min after irradiation. A head and neck cancer cell line (UM-SCC-22B) and a comparable normal cell line, normal oral keratinocyte (NOK) were each separately exposed to 10 Gy and treated with a control drug for disrupting metabolism (potassium cyanide; KCN). The metabolic changes were measured live by fluorescence lifetime imaging of the intrinsically fluorescent intermediate metabolite nicotinamide adenosine dinucleotide (NADH) fluorescence; this method is sensitive to the ratio of bound to free NADH. The results indicated a prompt shift in metabolic signature in the cancer cell line, but not in the normal cell line. Control KCN treatment demonstrated expected metabolic fluxes due to mitochondrial disruption. The detected radiation shift in the cancer cells was blunted in the presence of both a radical scavenger and a HIF-1α inhibitor. The HIF-1α abundance as detected by immunohistochemical staining also increased substantially for these cancer cells, but not for the normal cells. This type of live-cell metabolic monitoring could be helpful for future real-time studies and in designing adaptive radiotherapy approaches.

Potential role of the glycolytic oscillator in acute hypoxia in tumors

Tumor acute hypoxia has a dynamic component that is also, at least partially, coherent. Using blood oxygen level dependent magnetic resonance imaging, we observed coherent oscillations in hemoglobin saturation dynamics in cell line xenograft models of head and neck squamous cell carcinoma. We posit a well-established biochemical nonlinear oscillatory mechanism called the glycolytic oscillator as a potential cause of the coherent oscillations in tumors. These data suggest that metabolic changes within individual tumor cells may affect the local tumor microenvironment including oxygen availability and therefore radiosensitivity. These individual cells can synchronize the oscillations in patches of similar intermediate glucose levels. These alterations have potentially important implications for radiation therapy and are a potential target for optimizing the cancer response to radiation.

On the importance of prompt oxygen changes for hypofractionated radiation treatments

This discussion is motivated by observations of prompt oxygen changes occurring prior to a significant number of cancer cells dying (permanently stopping their metabolic activity) from therapeutic agents like large doses of ionizing radiation. Such changes must be from changes in the vasculature that supplies the tissue or from the metabolic changes in the tissue itself. An adapted linear-quadratic treatment is used to estimate the cell survival variation magnitudes from repair and reoxygenation from a two-fraction treatment in which the second fraction would happen prior to significant cell death from the first fraction, in the large fraction limit. It is clear the effects of oxygen changes are likely to be the most significant factor for hypofractionation because of large radiation doses. It is a larger effect than repair. Optimal dose timing should be determined by the peak oxygen timing. A call is made to prioritize near real time measurements of oxygen dynamics in tumors undergoing hypofractionated treatments in order to make these treatments adaptable and patient-specific.

Corrigendum: Potential role of the glycolytic oscillator in acute hypoxia in tumors (2015 Phys. Med. Biol. 60 9215).

At the time of publication, our group had performed short tandem repeat (STR) testing on the SCC22B cell line and believed that had been correctly identified. As part of a recent comprehensive process to confirm the identity of cell lines in use in our lab, we repeated STR testing on all cell lines. These results were compared to the ExPASy Cellosaurus database (http://web.expasy.org/cellosaurus/). One cell line used in this manuscript was a near perfect match for T24 (CVCL_0554), a bladder carcinoma cell line commonly found as a cellular contaminant. Although we are unable to test the exact cells used in this manuscript, we believe that the cells labeled as SCC22B are most likely to actually be T24. The authors believe that neither the results nor the conclusions have been significantly changed on the basis of the specific cell line utilized.

SU-G-TeP3-10: Radiation Induces Prompt Live-Cell Metabolic Fluxes

PURPOSE To compare metabolic dynamics and HIF-1α expression following radiation between a cancerous cell line (UM-SCC-22B) and a normal, immortalized cell line, NOK (Normal Oral Keratinocyte). HIF-1 is a key factor in metabolism and radiosensitivity. A better understanding of how radiation affects the interplay of metabolism and HIF-1 might give a better understanding of the mechanisms responsible for radiosensitivity. METHODS Changes in cellular metabolism in response to radiation are tracked by fluorescence lifetime of NADH. Expression of HIF-1α was measured by immunofluorescence for both cell lines with and without irradiation. Radiation response is also monitored with additional treatment of a HIF-1α inhibitor (chrysin) as well as a radical scavenger (glutathione). Changes in oxygen consumption and respiratory capacity are also monitored using the Seahorse XF analyzer. RESULTS An increase in HIF-1α was found to be in response to radiation for the cancer cell line, but not the normal cell line. Radiation was found to shift metabolism toward glycolytic pathways in cancer cells as measured by oxygen consumption and respiratory capacity. Radiation response was found to be muted by addition of glutathione to cell media. HIF-1α inhibition similarly muted radiation response in cancer. CONCLUSION The HIF-1 protein complex is a key regulator cellular metabolism through the regulation of glycolysis and glucose transport enzymes. Moreover, HIF-1 has shown radio-protective effects in tumor vascular endothelia, and has been implicated in metastatic aggression. Monitoring interplay between metabolism and the HIF-1 protein complex can give a more fundamental understanding of radiotherapy response.

SU-C-303-02: Correlating Metabolic Response to Radiation Therapy with HIF-1alpha Expression

Purpose: To understand radiation induced alterations in cellular metabolism which could be used to assess treatment or normal tissue response to aid in patient-specific adaptive radiotherapy. This work aims to compare the metabolic response of two head and neck cell lines, one malignant (UM-SCC-22B) and one benign (Normal Oral Keratinocyte), to ionizing radiation. Responses are compared to alterations in HIF-1alpha expression. These dynamics can potentially serve as biomarkers in assessing treatment response allowing for patient-specific adaptive radiotherapy. Methods: Measurements of metabolism and HIF-1alpha expression were taken before and X minutes after a 10 Gy dose of radiation delivered via an orthovoltage x-ray source. In vitro changes in metabolic activity were measured via fluorescence lifetime imaging (FLIM) to assess the mean lifetime of NADH autofluorescence following a dose of 10 Gy. HIF-1alpha expression was measured via immunohistochemical staining of in vitro treated cells and expression was quantified using the FIJI software package. Results: FLIM demonstrated a decrease in the mean fluorescence lifetime of NADH by 100 ps following 10 Gy indicating a shift towards glycolytic pathways for malignant cells; whereas this benign cell line showed little change in metabolic signature. Immunohistochemical analysis showed significant changes in HIF-1alpha expression in response to 10 Gy of radiation that correlate to metabolic profiles. Conclusion: Radiation induces significant changes in metabolic activity and HIF-1alpha expression. These alterations occur on time scales approximating the duration of common radiation treatments (approximately tens of minutes). Further understanding these dynamics has important implications with regard to improvement of therapy and biomarkers of treatment response.

WE-E-BRE-12: Tumor Microenvironment Dynamics Following Radiation

PURPOSE This work aims to understand the radiation-induced interplay between tumor oxygenation and metabolic activity. These dynamics can potentially serve as biomarkers in assessing treatment response allowing for patient-specific adaptive radiotherapy. METHODS Using patient-derived xenografts of head and neck cancer we assessed tumor oxygenation via fiber-optic probe monitored hemoglobin saturation and Blood Oxygen Level Dependent (BOLD) MRI. Measurements were taken before and after a 10 Gy dose of radiation. Changes in metabolic activity were measured via Fluorescence Lifetime IMaging (FLIM) with the appropriate controls following a 10 Gy dose of radiation. FLIM can non-invasively monitor changes in fluorescence in response to the microenvironment including being able to detect free and bound states of the intrinsically fluorescent metabolite NADH (Nicotinamide Adenine Dinucleotide). With this information FLIM can accurately quantify the metabolic state of cells that have been radiated. To model the observed changes, a two-compartment, source-sink simulation relating hemoglobin saturation and metabolic activity was performed using MATLAB. RESULTS Hemoglobin saturation as measured by interstitial probe and BOLD-MRI decreased by 30% within 15 minutes following radiation. FLIM demonstrated a decrease in the mean fluorescence lifetime of NADH by 100 ps following 10 Gy indicating a shift towards glycolytic pathways. Simulation of radiation-induced alterations in tumor oxygenation demonstrated that these changes can be the result of changes in either vasculature or metabolic activity. CONCLUSION Radiation induces significant changes in hemoglobin saturation and metabolic activity. These alterations occur on time scales approximately the duration of common radiation treatments. Further understanding these dynamics has important implications with regard to improvement of therapy and biomarkers of treatment response.

MO-G-BRF-06: Radiotherapy and Prompt Oxygen Dynamics

PURPOSE Adaptive radiotherapy requires a knowledge of the changing local tumor oxygen concentrations for times on the order of the treatment time, a time scale far shorter than cell death and proliferation. This knowledge will be needed to guide hypofractionated radiotherapy. METHODS A diffuse optical probe system was developed to spatially average over the whole interior of athymic Sprague Dawley nude mouse xenografts of human head and neck cancers. The blood volume and hemoglobin saturation was measured in real time. The quantities were measured with spectral fitting before and after 10 Gy of radiation is applied. An MRI BOLD scan is acquired before and after 10 Gy that measures regional changes in R2* which is inversely proportional to oxygen availability. Simulations were performed to fit the blood oxygen dynamics and infer changes in hypoxia within the tumor. RESULTS The optical probe measured nearly constant blood volume and a significant drop in hemoglobin saturation of about 30% after 10 Gy over the time scale of less than 30 minutes. The averaged R2* within the tumor volume increased by 15% after the 10 Gy dose, which is consistent with the optical results. The simulations and experiments support likely dynamic metabolic changes and/or fast vasoconstrictive signals are occurring that change the oxygen concentrations significantly, but not cell death or proliferation. CONCLUSION Significant oxygen changes were observed to occur within 30 minutes, coinciding with the treatment time scale. This dynamic is very important for patient specific adaptive therapy. For hypofractionated therapy, the local instantaneous oxygen content is likely the most important variable to control. The invention of a bedside device for the purpose of measuring the instantaneous response to large radiation doses would be an important step to future improvements in outcome.

SU‐E‐T‐304: On Dose Error Sensitivities for Hypofractionated Helical Tomotherapy Treatments

PURPOSE Tour purpose is to explore the efficacy of optimizing and adapting hypofractionated treatments for oxygen dynamics, and with Helical Tomotherapy (HT) in particular. Hypofractionated treatments such as SBRT induce a higher sensitivity to any dose error that would have been averaged away with a typical number of fractions. The greatest uncertainty relates to the effectiveness of the dose and that aspect is dominated by the short term oxygen dynamics induced by the radiation. METHODS This work builds on a suite of HT studies related to 1. the interplay or lack thereof for respiratory motion, 2. Ill-posed optimizations that can lead to cold spots, 3. Proper choices for pitch, but the largest issue is 4. Reoxygenation dynamics for which we have started to use a new optical device. RESULTS For each of the first three items mentioned above, simple conditions to enhance robustness can be specified as follows: 1. Control motion on the order of the couch speed relative to the beam width, and use probabilistic optimization, 2. Do not prescribe gradients sharper than the beam profile can accomplish, 3. Use a pitch value of 0.86/integer, and 4. Find a way to robustly track the tumor oxygen dynamics in real-time per patient and find robust adaptations for these dynamics. CONCLUSION For hypofractionated treatments, there will be an increased sensitivity to critical physics based dose error issues. However, the more severe issues are biological, and in particular, reoxygenation which is more complex for fewer fractions because the dynamics prior to cell death make the of the linear-quadratic equation difficult. Physicists need to extend their work into new directions for these new types of treatments.

SU-E-T-292: In-Vivo Blood Oxygen Measurements Via Interstitial Fiber Optic Probe and Photoacoustic Imaging

PURPOSE Better understanding of hemodynamics and oxygen transport in response to radiation will enable clinicians to optimize radiotherapy treatment regimens. It is well-known that oxygenated tissue is significantly more responsive to damage caused by irradiation. We sought to explore the real-time hemodynamics and blood oxygenation dynamics in response to radiation using a novel interstitial fiber-optic probe and a photoacoustic imaging system. METHODS Mice bearing xenografted tumors composed of a head and neck cancer cell line (UM-SCC47) and a patient-derived xenograft (UW-SCC36) were irradiated (10 Gy). Blood oxygena saturation (S) and blood volume fraction (B) were monitored in real time using an interstitial fiber optic probe as well as a photoacoustic imaging system (using a 21 MHz transducer, and infra red wavelengths of 750 and 850nm on the Vevo LAZR (R)). RESULTS A significant drop in blood oxygen saturation was found within 15 minutes following irradiation using the interstitial fiber optic probe. Meanwhile, Blood volume fraction remained constant. Preliminary photoacoustic imaging provided evaluation of the spatial characteristics of blood-oxygen dynamics and showed qualitative agreement with probe measurements. The measurements were compared to gain a better understanding of the fiber optic probes spatial averaging, also. CONCLUSION While development of the interstitial fiber optic probe continues, it appears to provide real-time assessment of blood-oxygen dynamics. Correlation of optimal reoxygenation with split dose radiation response is ongoing and may provide insight that could lead to improvements in the delivery of radiation therapy. This is particularly valuable for hypofractionated treatments as this information can guide the timing, size, and number of fractions for radiotherapy treatments. Minalini Lakshman is an employee of the VisualSonics company; however, the company did not provide any financial support for this work.