Tissue-Oxygen Quantification in Preclinical Oncological Models

Abstract

Hypoxia, a prevalent characteristic of most solid malignant tumors, contributes to diminished therapeutic responses and more aggressive phenotypes. The impact of hypoxia on cancer therapeutics is significant: hypoxic tissue is 3x less radiosensitive than normoxic tissue, the impaired blood flow found in hypoxic tumor regions influences chemotherapy delivery, and the immune system is dependent on oxygen for functionality. Despite the clinical implications of hypoxia, there is not a clinically-accepted, universal, ideal method for quantifying hypoxia, particularly cycling hypoxia because of its complexity and heterogeneity across tumor types and individuals. This dissertation includes four studies describing oxygen quantification and its role in cancer, beginning with a comprehensive overview introducing the preclinical and clinical methods for quantifying hypoxia with the advantages and disadvantages of each. The first two studies utilized a dual-emissive boron nanoparticle that - through ratiometric sensing and optical imaging techniques - quantifies oxygen. These boron nanoparticles were found to be an appropriate alternative for quantifying the oxygen consumption rate (versus the gold-standard Seahorse Assay) in two breast-cancer in vitro models. In addition, we confirmed that radiation plays a significant role in altering the oxygen consumption rate of cells. The second study used an in vivo dorsal skinfold window chamber model to quantify the effects of both anesthesia and hypoxic environments on tissue pO2. Using a multivariable linear regression model, we found that tissue pO2 can be reasonably predicted by the interaction term between respiratory rate and anesthesia state (p=0.0107, overall R2 = 0.8224). Boron nanoparticles, while a valuable preclinical tool, are inherently limited by their penetration depth and are most useful in a murine dorsal window chamber model. However, optical imaging remains clinically useful, so we investigated quantifying passive Cherenkov radiation produced during megavoltage radiation therapy in client-owned canines as a metric for characterizing tumors. Six dogs receiving fractionated radiation therapy for subcutaneous cancerous tumor were recruited into this trial. By recording naturally-occurring Cherenkov emissions during radiation exposure, we documented significant intra-subfraction differences between the Cherenkov emission intensities for normal tissue, whole-tumor tissue, tissue at the edge of the tumor and tissue at the center of the tumor (p<0.05). Additionally, intra-subfraction changes suggest that Cherenkov emissions may have captured fluctuating absorption properties within the tumor. Optical imaging techniques for measuring oxygen characteristics are severely limited by the penetration depth of optical wavelengths. The characteristics of the ideal oxygen-imaging modality include: 1) 3D volumetric tumor images, 2) high spatial, temporal and oxygen resolution, and 3) directly quantify tissue oxygen in a native environment (i.e.: not a window chamber) at any depth. Electron paramagnetic resonance (EPR) oxygen imaging fulfills those requirements. A precommercial EPR oxygen-imager was used to quantify tumor hypoxia and investigate the hypoxia-modifying effects of the FDA-approved vasodilator papaverine (PPV). We aimed to absolutely quantify the change in tumor hypoxia induced by papaverine in two murine tumor models: E0771 and primary p53/MCA sarcomas. We hypothesized that 1) there is a PPV dose-related change in tissue pO2, 2) papaverine radiosensitizes tumors, increasing tumor control and survival probability, and 3) pre-screening tumors for baseline tumor hypoxia predicts radiation + PPV treatment response. We confirmed that hypoxic tumors are more radioresistant than normoxic tumors in the primary sarcoma model (p=0.0057) via oxygen quantification with EPR. Additionally, in a Cox Hazard Regression analysis, baseline hypoxic fractions proved to be a significant (p=0.01450) hazard in survivability. Additionally, we found papaverine alters tumor hypoxia in both tumor models; however, the radiosensitizing effect was not apparent. Papaverine’s effect on tumor vasculature (in combination with its oxygen consumption rate decrease) requires further study before concluding it is a radiosensitizer.