Keywords

1 Introduction

In vivo oxygen concentration (pO2) has been found to be crucially important in determining normal tissue health as well as the aggressiveness of tumors and their response to various forms of treatment [1–4]. Due to the ubiquitous influence of tissue pO2 various methods for measuring and/or imaging pO2 have been developed [5–8].

One such method is electron paramagnetic resonance (EPR) oxygen imaging (EPROI). EPROI is a particularly robust method of imaging in vivo tissue pO2 distributions for several reasons. EPROI provides full 3D images of pO2. These images have good spatial resolution (~1 mm3 voxels) as well as pO2 resolution (1–3 torr). The low electromagnetic wave excitation frequencies (e.g., 250 MHz) currently used in EPRI are comparable to those used in 6T whole body MRI and can penetrate deep in tissue (>7 cm) in animals as large as humans. EPROI images are obtained non-invasively, which means they can be used to study in vivo pO2 distributions without perturbing the system. EPROI requires an intravenously injected, non-toxic spin probe, which distributes in the extracellular compartment of tumors, to report local pO2 [9]. The accuracy of EPROI oxygen measurements has been established by correlating with well-established optical fiber based oxygen measurement techniques [10].The information provided by EPROI can be applied to help study myriad interesting oxygen related biologic and physiologic topics. A number of studies demonstrating the array of interesting applications of 3D EPR oxygen images of normal tissue and/or tumor tissue will be presented here.

2 Methods

EPROI is used to non-invasively determine the effect of tumor pO2 on success of tumor treatment with radiation therapy. Two cancer models are used: fibrosarcoma (FSa) and murine mammary MCa4 carcinoma. Fraction of EPROI voxels with less than 10 torr pO2 (HF10) is used as a measure of tumor hypoxia. The variable HF10 is then correlated to success or failure of radiotherapy to see what role, if any, hypoxia as determined by EPROI plays in tumor resistance to treatment.

EPROI has been used to provide insight into the effect of fraction of inspired oxygen (FiO2) changes on the distribution of pO2 in various organs of mice. The result of cyclic FiO2 variation can also be observed in various organs using EPROI. This directly assesses levels of tissue pO2 in models of important disorders such as sleep apnea to understand their biologic effects.

To examine the role of BNIP3 in tumor progression and regulation of oxygenation, changes in pO2 levels and distributions in mouse mammary tumors are compared for BNIP3 null mice that have been crossed to the Mouse Mammary Tumor Viral (MMTV) promoted-polyoma middle T antigen (PyMT) mouse model of breast cancer and wild type mice. EPROI images are registered with anatomic CT images to analyse differences in oxygenation within these tumors.

3 Results

Using two cancer models (FSa and MCa4), we found that EPROI corroborates the theory that tumors exhibiting a higher degree of hypoxia tend to be more resistant to radiation therapy. A cohort of animals with either of the two tumor lines were treated to the previously determined 50 % tumor control dose (TCD50) for each tumor type. The HF10 as determined by EPROI for each tumor was correlated with radiation therapy treatment outcome. For the FSa tumors, hypoxic tumors (HF10 > 10 %) 37 % were successfully controlled while for tumors with HF10 < 10 %, 90 % were successfully controlled (p = 0.0138) [11, 12]. For the MCa4 tumors, hypoxic tumors (HF10 > 10 %) 23 % were successfully controlled while for tumors with HF10 < 10 %, 90 % were successfully controlled (p = 0.0072) [12]. An example of the dramatic oxygenation difference observed in animals whose tumors were successfully controlled with radiotherapy versus those for which radiotherapy failed is shown in Fig. 16.1.

Fig. 16.1
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Sample slices from representative EPROI of mouse legs bearing tumors (black outline) demonstrating the difference in tumor oxygenation observed in animals where treatment with radiation therapy eventually (a) successfully controlled the tumor or (b) failed to control the tumor

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Significant differences in the overall pO2 in three tissue types of a mouse breathing air versus a mouse breathing 12 % O2 are seen in whole-body EPROI (Fig. 16.2). It is seen that significant variations in tissue pO2 are observed with oscillating FiO2, however the response becomes damped over time (Fig. 16.3). The direct assessment of tissue pO2 responses in vivo provides data concerning the extent of hypoxia induced in these tissues and allows the development of models explaining the deleterious end organ effects.

Fig. 16.2
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Demonstration of the overall effect on the tissue pO2 of a mouse when the breathing gas oxygen content is changed from 21 % O2 to 12 % O2 as measured with EPROI. Whole body EPROI of a mouse breathing (a) 21 % O2 and (b) 12 % O2, with labeled regions of interest. The pO2 distributions for the whole body EPROI when the mouse is breathing (c) 21 % O2 and (d) 12 % O2

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Fig. 16.3
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Slices from an EPROI of a mouse are shown with regions of interest outlined in magenta. The breathing gas oxygenation for the mouse alternates from 21 % O2 to 12 % O2 (breathing gas O2 changes shown in red line). Next to each image with a particular region of interest (lung, liver, or leg muscle) the change in mean oxygenation within that region of interest in response to the breathing gas change is shown

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Preliminary studies of BNIP3 knock out (KO) mice show extremely hypoxic breast tumors (Fig. 16.4). This is consistent with the fact that BNIP3 null tumor cells show increased invasive properties, suggesting that BNIP3 is a metastasis suppressor required to maintain mitochondrial integrity and mitigate against the metastasis promoting activities of reactive oxygen and hypoxia [13].

Fig. 16.4
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(a) Photograph of MMTV-PyMT, BNIP3 null breast cancer tumors grown in the breast of a mouse. (b) Registered EPROI overlaid on an anatomical CT of the mouse, shown with the tumor outlined in white. (c) Distribution of pO2 found within the BNIP3 null tumor demonstrating that the tumor is extremely hypoxic

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4 Discussion and Conclusions

With EPROI we can begin to develop quantitative assays of hypoxia induced in tumors and normal tissues. These, in turn will allow the development of quantitative models of the response of tumors and normal tissues to anti-cancer therapies and models of end-organ damage from disease processes such as the intermittent hypoxia induced by sleep apnea.

The hypoxic fraction of a tumor, as determined by EPROI, has been found to be quite a powerful determinant in the eventual outcome of treatment with radiation. This validates EPROI as a tool to analyze spatial distributions of pO2 in vivo.

In preliminary studies EPROI has also proven to be a valuable tool for tracking tissue pO2 response to changes in FiO2. EPROI therefore has the potential to enhance studies investigating the biologic consequences of temporally fluctuating tissue oxygenations (e.g., sleep apnea conditions) by allowing noninvasive tracking of the response to FiO2 changes of whole body pO2 as well as pO2 of individual organs and how this response changes over time.

EPR oxygen images can also help investigate the complicated effects of hypoxia on several aspects of tumor and tissue development. This is evidenced by preliminary results from a study investigating the effect of the BNIP3 protein, which limits production of reactive oxygen species by promoting mitochondrial degradation at the autophagosome, on tumor oxygenation. Initial results show that tumors in BNIP3 KO mice are extremely hypoxic, which may be due to dysfunctional mitochondria. EPROI will help in further studies to investigate the effect of BNIP3 on oxygenation as well as radiation resistance.

In general, oxygen images from EPROI provide an important tool in understanding the relationship between microenvironment oxygenation and a wide variety of crucial physiologic functions.