A novel in vivo tumor oxygen profiling assay: Combining functional and molecular imaging with multivariate mathematical modeling

Purpose: The objective of this study is to develop and test a novel high spatio-temporal in vivo assay to quantify tumor oxygenation and hypoxia. The assay implements a biophysical model of oxygen transport to fuse parameters acquired from in vivo functional and molecular imaging modalities. ^ Introduction: Tumor hypoxia plays an important role in carcinogenesis. It triggers pathological angiogenesis to supply more oxygen to the tumor cells and promotes cancer cell metastasis. Preclinical and clinical evidence show that anti-angiogenic treatment is capable of normalizing the tumor vasculature both structurally and functionally. The resulting normalized vasculature provides a more efficient and uniform microcirculation that enhances oxygen and drug delivery to the tumor cells and improves second-line treatments such as traditional radiation or chemotherapy. Early studies using the overall or average tumor hypoxia as a prognostic biomarker of anti-angiogenic therapy efficacy was ambivalent; however, recent studies have discovered that the etiology of hypoxia and its heterogeneity could be used as reliable prognostic biomarkers. The capability to longitudinally map tumor hypoxia with high spatial and temporal resolution has the potential to enhance fundamental cancer research and ultimately cancer patient care. ^ Method: A novel methodology to identify and characterize tumor hypoxia by fusing the physiological hemodynamic parametric maps obtained from functional and molecular imaging modalities and technique using a modified Krogh model of oxygen transport (MPO2) was developed. First, simulations studies were performed to validate this technique. Microscopy data of tumor and brain tissue (control) provided both the vasculature and rheology data. A Green\u27s function algorithm was used to solve the ordinary differential equation and calculate the oxygen profile at a microscopic scale (15 μm) (GPO2), which was used as a reference. From this data, simulated physiological maps (perfusion, fractional plasma volume, fractional interstitial volume) and hemoglobin status (oxygen saturation, hemoglobin concentration) was used as input to MPO2 and used to calculate pO2 levels as a function of scanner spatial resolution and noise. Second, MPO2 was compared to pO2 measurements in xenograft breast tumors using OxyLite oxygen sensor as a Gold Standard, where DCE-CT and PCT-S images were acquired to obtain hemodynamic images. Finally, the vascular physiology measurements obtained from an anti-angiogenic therapeutic study in pancreatic tumors was applied to MPO2 and compared to therapeutic response. ^ Results: The simulation results using Green\u27s function pO2 as standard showed that the MPO2 model performance was dependent on the spatial resolution (voxel size) of the images. Sensitivity and error analysis of this model were also investigated in this study. These oxygen transport simulations results suggest the oxygen saturation and hemoglobin concentration were two key factors in tissue oxygenation, and concomitant with blood perfusion and tumor metabolic rate. Comparisons of the pO2 profile obtained from MPO2 and OxyLite probe in MCF7 tumor model demonstrated a significant correlation and approached a slope of one (after accounting for a few outliers). Simulation studies implementing the physiological data obtained from the anti-angiogenic therapeutic study in pancreatic tumors using the MPO2 model agreed with the experimental findings that blood perfusion is a valuable prognostic biomarker in therapeutic efficacy. This model also predicted the oxygenation improvement difference from two vascular renormalization modes (topological normalization and geometrical normalization). ^ Conclusion: The results from the simulation and in vivo studies demonstrated the feasibility of this novel hypoxia assay. Simulation results of the pancreatic tumors provide an example of the impact the MPO2 model in conjunction with imaging can provide when evaluating the therapeutic significance of various normalization modes in anti-angiogenic therapy, and suggests potential approaches to further improve anti-angiogenic therapy efficacy

Development of a mathematical model to estimate intra-tumor oxygen concentrations through multi-parametric imaging

Background Tumor hypoxia is involved in every stage of solid tumor development: formation, progression, metastasis, and apoptosis. Two types of hypoxia exist in tumors—chronic hypoxia and acute hypoxia. Recent studies indicate that the regional hypoxia kinetics is closely linked to metastasis and therapeutic responses, but regional hypoxia kinetics is hard to measure. We propose a novel approach to determine the local pO2 by fusing the parameters obtained from in vivo functional imaging through the use of a modified multivariate Krogh model. Methods To test our idea and its potential to translate into an in vivo setting through the use of existing imaging techniques, simulation studies were performed comparing the local partial oxygen pressure (pO2) from the proposed multivariate image fusion model to the referenced pO2 derived by Green’s function, which considers the contribution from every vessel segment of an entire three-dimensional tumor vasculature to profile tumor oxygen with high spatial resolution. Results pO2 derived from our fusion approach were close to the referenced pO2 with regression slope near 1.0 and an r2 higher than 0.8 if the voxel size (or the spatial resolution set by functional imaging modality) was less than 200 μm. The simulation also showed that the metabolic rate, blood perfusion, and hemoglobin concentration were dominant factors in tissue oxygenation. The impact of the measurement error of functional imaging to the pO2 precision and accuracy was simulated. A Gaussian error function with FWHM equal to 20 % of blood perfusion or fractional vascular volume measurement contributed to average 7 % statistical error in pO2. Conclusion The simulation results indicate that the fusion of multiple parametric maps through the biophysically derived mathematical models can monitor the intra-tumor spatial variations of hypoxia in tumors with existing imaging methods, and the potential to further investigate different forms of hypoxia, such as chronic and acute hypoxia, in response to cancer therapies

Development of Follicle-Stimulating Hormone Receptor Binding Probes to Image Ovarian Xenografts

The Follicle-Stimulating Hormone Receptor (FSHR) is used as an imaging biomarker for the detection of ovarian cancer (OC). FSHR is highly expressed on ovarian tumors and involved with cancer development and metastatic signaling pathways. A decapeptide specific to the FSHR extracellular domain is synthesized and conjugated to fluorescent dyes to image OC cells in vitro and tumors xenograft model in vivo. The in vitro binding curve and the average number of FSHR per cell are obtained for OVCAR-3 cells by a high resolution flow cytometer. For the decapeptide, the measured EC50 was 160 μM and the average number of receptors per cell was 1.7 × 10(7). The decapeptide molecular imaging probe reached a maximum tumor to muscle ratio five hours after intravenous injection and a dose-dependent plateau after 24-48 hours. These results indicate the potential application of a small molecular weight imaging probe specific to ovarian cancer through binding to FSHR. Based on these results, multimeric constructs are being developed to optimize binding to ovarian cells and tumors

Circulating Markers Reflect Both Anti- and Pro-Atherogenic Drug Effects in ApoE-Deficient Mice

Background: Current drug therapy of atherosclerosis is focused on treatment of major risk factors, e.g. hypercholesterolemia while in the future direct disease modification might provide additional benefits. However, development of medicines targeting vascular wall disease is complicated by the lack of reliable biomarkers. In this study, we took a novel approach to identify circulating biomarkers indicative of drug efficacy by reducing the complexity of the in vivo system to the level where neither disease progression nor drug treatment was associated with the changes in plasma cholesterol.Results: ApoE-/- mice were treated with an ACE inhibitor ramipril and HMG-CoA reductase inhibitor simvastatin. Ramipril significantly reduced the size of atherosclerotic plaques in brachiocephalic arteries, however simvastatin paradoxically stimulated atherogenesis. Both effects occurred without changes in plasma cholesterol. Blood and vascular samples were obtained from the same animals. In the whole blood RNA samples, expression of MMP9, CD14 and IL-1RN reflected pro and anti-atherogenic drug effects. In the plasma, several proteins, e.g. IL-1β, IL-18 and MMP9 followed similar trends while protein readout was less sensitive than RNA analysis.Conclusion: In this study, we have identified inflammation-related whole blood RNA and plasma protein markers reflecting anti-atherogenic effects of ramipril and pro-atherogenic effects of simwastatin in a mouse model of atherosclerosis. This opens an opportunity for early, non-invasive detection of direct drug effects on atherosclerotic plaques in complex in vivo systems

A novel in vivo tumor oxygen profiling assay: Combining functional and molecular imaging with multivariate mathematical modeling

Purpose: The objective of this study is to develop and test a novel high spatio-temporal in vivo assay to quantify tumor oxygenation and hypoxia. The assay implements a biophysical model of oxygen transport to fuse parameters acquired from in vivo functional and molecular imaging modalities. Introduction: Tumor hypoxia plays an important role in carcinogenesis. It triggers pathological angiogenesis to supply more oxygen to the tumor cells and promotes cancer cell metastasis. Preclinical and clinical evidence show that anti-angiogenic treatment is capable of normalizing the tumor vasculature both structurally and functionally. The resulting normalized vasculature provides a more efficient and uniform microcirculation that enhances oxygen and drug delivery to the tumor cells and improves second-line treatments such as traditional radiation or chemotherapy. Early studies using the overall or average tumor hypoxia as a prognostic biomarker of anti-angiogenic therapy efficacy was ambivalent; however, recent studies have discovered that the etiology of hypoxia and its heterogeneity could be used as reliable prognostic biomarkers. The capability to longitudinally map tumor hypoxia with high spatial and temporal resolution has the potential to enhance fundamental cancer research and ultimately cancer patient care. Method: A novel methodology to identify and characterize tumor hypoxia by fusing the physiological hemodynamic parametric maps obtained from functional and molecular imaging modalities and technique using a modified Krogh model of oxygen transport (MPO2) was developed. First, simulations studies were performed to validate this technique. Microscopy data of tumor and brain tissue (control) provided both the vasculature and rheology data. A Green\u27s function algorithm was used to solve the ordinary differential equation and calculate the oxygen profile at a microscopic scale (15 μm) (GPO2), which was used as a reference. From this data, simulated physiological maps (perfusion, fractional plasma volume, fractional interstitial volume) and hemoglobin status (oxygen saturation, hemoglobin concentration) was used as input to MPO2 and used to calculate pO2 levels as a function of scanner spatial resolution and noise. Second, MPO2 was compared to pO2 measurements in xenograft breast tumors using OxyLite oxygen sensor as a Gold Standard, where DCE-CT and PCT-S images were acquired to obtain hemodynamic images. Finally, the vascular physiology measurements obtained from an anti-angiogenic therapeutic study in pancreatic tumors was applied to MPO2 and compared to therapeutic response. Results: The simulation results using Green\u27s function pO2 as standard showed that the MPO2 model performance was dependent on the spatial resolution (voxel size) of the images. Sensitivity and error analysis of this model were also investigated in this study. These oxygen transport simulations results suggest the oxygen saturation and hemoglobin concentration were two key factors in tissue oxygenation, and concomitant with blood perfusion and tumor metabolic rate. Comparisons of the pO2 profile obtained from MPO2 and OxyLite probe in MCF7 tumor model demonstrated a significant correlation and approached a slope of one (after accounting for a few outliers). Simulation studies implementing the physiological data obtained from the anti-angiogenic therapeutic study in pancreatic tumors using the MPO2 model agreed with the experimental findings that blood perfusion is a valuable prognostic biomarker in therapeutic efficacy. This model also predicted the oxygenation improvement difference from two vascular renormalization modes (topological normalization and geometrical normalization). Conclusion: The results from the simulation and in vivo studies demonstrated the feasibility of this novel hypoxia assay. Simulation results of the pancreatic tumors provide an example of the impact the MPO2 model in conjunction with imaging can provide when evaluating the therapeutic significance of various normalization modes in anti-angiogenic therapy, and suggests potential approaches to further improve anti-angiogenic therapy efficacy

Circulating Markers Reflect Both Anti- and Pro-Atherogenic Drug Effects in ApoE-Deficient Mice

Background Current drug therapy of atherosclerosis is focused on treatment of major risk factors, e.g. hypercholesterolemia while in the future direct disease modification might provide additional benefits. However, development of medicines targeting vascular wall disease is complicated by the lack of reliable biomarkers. In this study, we took a novel approach to identify circulating biomarkers indicative of drug efficacy by reducing the complexity of the in vivo system to the level where neither disease progression nor drug treatment was associated with the changes in plasma cholesterol. Results ApoE-/- mice were treated with an ACE inhibitor ramipril and HMG-CoA reductase inhibitor simvastatin. Ramipril significantly reduced the size of atherosclerotic plaques in brachiocephalic arteries, however simvastatin paradoxically stimulated atherogenesis. Both effects occurred without changes in plasma cholesterol. Blood and vascular samples were obtained from the same animals. In the whole blood RNA samples, expression of MMP9, CD14 and IL-1RN reflected pro-and anti-atherogenic drug effects. In the plasma, several proteins, e.g. IL-1β, IL-18 and MMP9 followed similar trends while protein readout was less sensitive than RNA analysis. Conclusion In this study, we have identified inflammation-related whole blood RNA and plasma protein markers reflecting anti-atherogenic effects of ramipril and pro-atherogenic effects of simwastatin in a mouse model of atherosclerosis. This opens an opportunity for early, non-invasive detection of direct drug effects on atherosclerotic plaques in complex in vivo systems