Julia A. O’Hara

Spin traps: in vitro toxicity and stability of radical adducts.

We have evaluated the effects of DMPO, CMPO, EMPO, BMPO, and DEPMPO on functioning CHO cells and the stability of the radical adducts in the presence of cells. The potential toxic effects of the spin traps were measured by two estimates of cell viability (trypan blue exclusion and colony formation) and one of cell function (rate of oxygen consumption). We also studied the effects of the spin traps on colony formation in a second cell line, 9L tumor cells. Toxicity varied with the type of cell line and the parameter that was measured. In aqueous solutions the order of stability for all spin adducts was SO(3) > OH > CH(3), while in cell suspensions it was SO(3) > OH approximately CH(3). The radical adducts of the new spin traps have significantly increased stability as compared to DMPO. These results indicate that the new spin traps potentially offer increased stability of spin adducts in functioning cells. It also is clear that it is necessary to carry out appropriate studies of the stability and toxicity in the system that is to be studied for any particular use of these spin traps. It then should be feasible to select the spin trap(s) best suited for the proposed study.

Quantitative imaging of scattering changes associated with epithelial proliferation, necrosis, and fibrosis in tumors using microsampling reflectance spectroscopy

Highly localized reflectance measurements can be used to directly quantify scatter changes in tissues. We present a microsampling approach that is used to raster scan tumors to extract parameters believed to be related to the tissue ultrastructure. A confocal reflectance imager was developed to examine scatter changes across pathologically distinct regions within tumor tissues. Tissue sections from two murine tumors, AsPC-1 pancreas tumor and the Mat-LyLu Dunning prostate tumor, were imaged. After imaging, histopathology-guided region-of-interest studies of the images allowed analysis of the variations in scattering resulting from differences in tissue ultra-structure. On average, the median scatter power of tumor cells with high proliferation index (HPI) was about 26% less compared to tumor cells with low proliferation index (LPI). Necrosis exhibited the lowest scatter power signature across all the tissue types considered, with about 55% lower median scatter power than LPI tumor cells. Additionally, the level and maturity of the tumors fibroplastic response was found to influence the scatter signal. This approach to scatter visualization of tissue ultrastructure in situ could provide a unique tool for guiding surgical resection, but this kind of interpretation into what the signal means relative to the pathology is required before proceeding to clinical studies.

Deferoxamine Iron Chelation Increases δ-Aminolevulinic Acid Induced Protoporphyrin IX in Xenograft Glioma Model

Exogenous administration of δ‐aminolevulinic acid (δ‐ALA) leads to selective accumulation of protoporphyrin IX (PpIX) in brain tumors, and has shown promising results in increasing extent of resection in fluorescence‐guided resection (FGR) of brain tumors. However, this approach still suffers from heterogeneous staining and so some tumor margins may go undetected because of this variation in PpIX production. The aim of this study was to test the hypothesis that iron chelation therapy could increase the level of fluorescence in malignant glioma tumors. Mice implanted with xenograft U251‐GFP glioma tumor cells were given a 200 mg kg−1 dose of deferoxamine (DFO), once a day for 3 days prior to δ‐ALA administration. The PpIX fluorescence observed in the tumor regions was 1.9 times the background in animal group without DFO, and 2.9 times the background on average, in the DFO pre‐treated group. A 50% increase in PpIX fluorescence contrast in the DFO group was observed relative to the control group (t‐test P‐value = 0.0020). These results indicate that iron chelation therapy could significantly increase δ‐ALA‐induced PpIX fluorescence in malignant gliomas, pointing to a potential role of iron chelation therapy for more effective FGR of brain tumors.

What Does EPR Oximetry with Solid Particles Measure—and How Does this Relate to Other Measures of PO2?

The technique of in vivo EPR oximetry has been more widely introduced only recently (Ferrari et al., 1994, Halpern et al., 1994, Swartz et al., 1994). Our laboratory has concentrated on spectroscopy at 1.2GHz, using particulate oxygen-sensitive paramagnetic materials placed at the site (or sites) of interest (Dunn et al., 1995, Gallez et al., 1996, Goda et al., 1996, Goda et al, 1995, Jiang et al., 1995, Liu et al., 1995, Liu et al., 1994a, Liu et al., 1994b, O’Hara et al., 1995a, O’Hara et al., 1995b, Swartz et al., 1992). This form of EPR oximetry has a number of features that seem potentially advantageous for making biologically useful measurements. These features include 1) the capability of making repeated non-invasive measurements from the same site without the need for anesthesia (after the placement of oxygen-sensitive paramagnetic materials at the site (or sites) of interest); 2) high accuracy and sensitivity, especially for lower levels of oxygen; 3) rapid response (seconds); 4) measurements which are not perturbed by factors such as pH, temperature, osmotic strength; 5) a high degree of stability and inertness in biological systems; 6) the availability of the oxygen-sensitive paramagnetic materials in a variety of forms ranging from a slurry of very small particles to a single macroscopic crystal; 7) the calibration is very stable; and 8) there is a very high degree of specificity of the measurements because there usually are no other EPR responsive materials present in sufficient concentrations to affect the measurements. EPR oximetry, of course, also has some potential limitations and uncertainties. These include 1) a certain level of invasiveness; for many uses the particulate oxygen-sensitive paramagnetic materials need to be physically placed at the site(s) of interest 2) availability; at the present time the instrumentation required for these measurements is not yet widely available 3) depth of penetration; the most sensitive approach, using an exciting frequency of I GHz, limits non-invasive measurements to a depth of about 10 mm; and 4) the nature of the parameter that is measured by the technique is not fully understood by many scientists. The purpose of this paper is to address the last question, doing so in a context that relates EPR oximetry to other methods.

Measurements of pO2 in Vivo, Including Human Subjects, by Electron Paramagnetic Resonance

The purpose of this paper is to provide an illustrative description of the current state of development of the use of electron paramagnetic resonance (EPR, or completely equivalently, electron spin resonance or ESR) to measure the partial pressure of oxygen (pO2) in tissues in vivo under physiological conditions. This summary is based on published and unpublished results from our laboratory (1–7) and does not attempt to describe the results of other laboratories which also are working along related lines (8–10). The pertinent features of our technique are illustrated. We also consider the current limitations of the technique and likely developments in the near future. Our evaluation is that: this technique now is suitable for immediate use in small animals; within a short period of time instruments will be available facilitating its use in larger animals; and preliminary studies are imminent in human subjects (7).

Practical conditions and limitations for high-spatial-resolution multisite EPR oximetry

We have previously reported a high-spatial-resolution multisite electron paramagnetic resonance (EPR) oximetry method that is based on consecutive applications of magnetic field gradients with the same direction but different magnitudes. This method that could be called also two-gradient convolution EPR oximetry has no restrictions for the shape of solid paramagnetic materials implanted in tissue and is applicable for any particulate EPR oxygen-sensitive matieral with a Lorentzian line shape. To enhance the utilization of this method, a previously described algorithm was used to develop user-friendly Windows-based software. Practical conditions of application of the method were established using several different model systems. It has been shown that the spectral overlap from the adjacent sites can be neglected if the splitting between the corresponding lines exceeds the largest line width by at least a factor of 1.3. An additional requirement of the method is that the second field gradient should exceed by at least 30% the value of the first gradient. It was confirmed that the error in line width determination at L-band is proportional to the noise-to-signal ratio, and does not exceed 1% a noise-to-signal ratio of 0.1 in a typical in vivo experiment. We demonstrate that the line widths of up to 10 different sites can be determined.

Protoporphyrin IX fluorescence contrast in invasive glioblastomas is linearly correlated with Gd enhanced magnetic resonance image contrast but has higher diagnostic accuracy

The sensitivity and specificity of in vivo magnetic resonance (MR) imaging is compared with production of protoporphyrin IX (PpIX), determined ex vivo, in a diffusely infiltrating glioma. A human glioma transfected with green fluorescent protein, displaying diffuse, infiltrative growth, was implanted intracranially in athymic nude mice. Image contrast from corresponding regions of interest (ROIs) in in vivo MR and ex vivo fluorescence images was quantified. It was found that all tumor groups had statistically significant PpIX fluorescence contrast and that PpIX contrast demonstrated the best predictive power for tumor presence. Contrast from gadolinium enhanced T1-weighted (T1W+Gd) and absolute T2 images positively predicted the presence of a tumor, confirmed by the GFP positive (GFP+) and hematoxylin and eosin positive (H&E+) ROIs. However, only the absolute T2 images had predictive power from controls in ROIs that were GFP+ but H&E negative. Additionally, PpIX fluorescence and T1W+Gd image contrast were linearly correlated in both the GFP+ (r = 0.79, p<1×10(-8)) and H&E+ (r = 0.74, p<0.003) ROIs. The trace diffusion images did not have predictive power or significance from controls. This study indicates that gadolinium contrast enhanced MR images can predict the presence of diffuse tumors, but PpIX fluorescence is a better predictor regardless of tumor vascularity.

In vivo EPR spectroscopy

This chapter is intended to provide a brief overview of the principles of electron paramagnetic resonance (EPR, or completely equivalently electron spin resonance, ESR) spectroscopy applied to living animals. It attempts to indicate especially those areas in which this approach is likely to be of value because it can provide useful information that cannot be provided as well by other approaches. As a matter of convenience the descriptions are drawn principally from the authors’ laboratory but it should be noted that there are a number of laboratories around the world, especially in Japan, which are also actively pursuing these developments. Because of the need for brevity in this volume, the coverage is illustrative rather than comprehensive but this fits well with the aim of the book which is to provide a review that will be useful for the longer term rather than only a review of the current state of development.

Intratumoral pO2 Measured Using a New Oxygen Sensitive Paramagnetic Material, Gloxy

There is increasing evidence that tumor oxygenation is clinically important in pre-dicting tumor response to radiation, chemotherapy and/or overall prognosis (Vaupel, et al., 1989; Hall, 1988). More recently, tumor hypoxia has been implicated in promoting sur-vival of tumor cell phenotypes that are more resistant and have lost their apoptotic (self- killing) ability (Giaccia, 1996; Shrieve and Begg, 1985). A valid method for obtaining measurements of intratumor oxygen tensions repeatedly and non-invasively is therefore very desirable. Electron Paramagnetic Resonance (EPR) oximetry is a recently developed technique which has the potential to provide such data. Its full use will be facilitated by the development of oxygen-sensitive materials which can be used under the various cir-cumstances in which repeated measurements of pO2 are desired.

Tumor pO2 assessments in human xenograft tumors measured by EPR oximetry: location of paramagnetic materials.

Radioantibody immunotherapy (RAIT) is a promising treatment modality but the effectiveness of this targeted low dose radiation varies from tumor to tumor. Since RAIT is an oxygen dependent treatment, baseline pO2 or growth-induced changes in the microenvironment may alter treatment response. In this pilot work we monitored tumor pO2 in untreated human xenograft tumors growing s.c. in nude mice. These data will be used to plan a study of the relationship between the effectiveness of RAIT and tumor pO2. Growth or treatment-induced changes in the microenvironment may alter the tumor pO2 and thus affect the response to therapy but may also affect location and microenvironment of the particulate oxygen sensor. We monitored tumor pO2 during growth and also examined the tumor histological structure overall and in the region of the paramagnetic material in the tumor at the time of necropsy.