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W T Jenkins

Publications and source records attributed to W T Jenkins.

At least 19 recordsLinked to original sources
PubMed2002

Noninvasive imaging of tumor hypoxia in rats using the 2-nitroimidazole 18F-EF5.

Tumor hypoxia is an important prognostic indicator for cancer therapy outcome. EF5 [2-(2-nitro-1[ H]-imidazol-1-yl)- N-(2,2,3,3,3-pentafluoropropyl)-acetamide] has been employed to measure tumor hypoxia in animals and humans using immunohistochemical methods. EF5 is a lipophilic molecule designed to have a very uniform biodistribution, a feature of obvious benefit for use in PET imaging. The present study represents the first demonstration of noninvasive PET imaging of rat tumors using fluorine-18 labeled EF5. Because of the small tumor size, partial volume effects may result in underestimation of concentration of the compound. Therefore, validation of the PET data was performed by gamma counting of the imaged tissue. The tumor models studied were the Morris 7777 (Q7) hepatoma (n=5) and the 9L glioma (n=2) grown subcutaneously in rats. Our previous studies have demonstrated that early passage 9L tumors are not severely hypoxic and that Q7 tumors are characterized by heterogeneous regions of tumor hypoxia (i.e., Q7 tumors are usually more hypoxic than early passage 9L tumors). The seven rats were imaged in the HEAD Penn-PET scanner at various time points after administration of 50-100 micro Ci (18)F-EF5 in 30 mg/kg carrier nonradioactive EF5. The carrier was used to ensure drug biodistribution comparable to prior studies using immunohistochemical methods. (18)F-EF5 was excreted primarily via the urinary system. Images obtained 10 min following drug administration demonstrated that the EF5 distributed evenly to all organ systems, including brain. Later images showed increased uptake in most Q7 tumors compared with muscle. Liver uptake remained relatively constant over the same time periods. Tumor to muscle ratios ranged from 0.82 to 1.73 (based on PET images at 120 min post injection) and 1.47 to 2.95 (based on gamma counts at approximately 180 min post injection). Tumors were easily visible by 60 min post injection when the final tumor to muscle ratios (based on gamma counts) were greater than 2. Neither of the 9L tumors nor the smallest Q7 tumor met this criterion, and these tumors were not seen on the PET images. These preliminary results suggest that (18)F-EF5 is a promising agent for noninvasive assessment of tumor hypoxia. Plans are underway to initiate a research project to determine the safety and preliminary evidence for the efficacy of this preparation in patients with brain tumors.

Animals↗
PubMed2002

Effects of hyperglycemia on oxygenation, radiosensitivity and bioenergetic status of subcutaneous RIF-1 tumors.

Since tissue oxygen tension is a balance between delivery and consumption of oxygen, considerable effort has been directed at increasing the former and/or decreasing the latter. Techniques to decrease the rate of cellular oxygen consumption (increasing the distance oxygen can diffuse into tissues) include increasing glycolysis by administering supra-physiologic levels of glucose. We have examined the effect of hyperglycemia produced by intravenous glucose infusion on the tissue oxygenation and radiation response of subcutaneously implanted murine radiation induced fibrosarcomas (RIF-1). A 0.3 M glucose solution was delivered via tail vein injection according to a protocol that maintained glucose at a plasma concentration of 17+/-1 mM. The effect of this treatment on radiation response (clonogenic and growth delay studies), tumor oxygenation (needle electrode pO2 and 2-[2-nitro-1H-imidazol-1-yl]-N-(2,2,3,3,3-pentafluoropropyl) acetamide (EF5) binding), and tumor bioenergetics and pH (31P NMR spectroscopy) was examined. Systemic measurements included hematocrit and blood glucose and lactate concentrations. The results of these studies suggest that these subcutaneously implanted RIF-1 tumors are both radiobiologically and metabolically hypoxic and that intravenous glucose infusion is not an effective method of modifying this metabolic state.

Animals↗
PubMed2001

Doppler ultrasound imaging detects changes in tumor perfusion during antivascular therapy associated with vascular anatomic alterations.

Noninvasive monitoring of antiangiogenic therapy was performed by serial power Doppler ultrasound imaging of murine tumors treated with recombinant interleukin 12, the results of which were correlated with assessments of tumor vascularity by microscopy. Growth of established K1735 tumors, but not of IFN-gamma-unresponsive K1735.N23 variants, was suppressed by treatment. Serial Doppler imaging of K1735 tumor vascularity during treatment revealed a progressive change from a diffuse perfusion pattern to a more punctate distribution. Quantitative analysis of the images revealed that color-weighted fractional average, representing overall tumor perfusion, consistently decreased in these tumors, primarily because of a decrease in fractional tumor cross-sectional area carrying blood flow. In contrast, these parameters increased in nonresponsive tumors during treatment. Confocal microscopy of thick tumor sections revealed a reduction in the density and arborization of vessels labeled in vivo by fluorochrome-conjugated lectin with effective treatment. Immunohistological examination of thin tumor sections confirmed the preferential loss of small vessels with successful therapy. Similar changes in tumor vascular anatomy and perfusion were also observed during recombinant interleukin 12 treatment of two other responsive murine tumor types. These results indicate that power Doppler ultrasound is a sensitive, noninvasive method for reporting functional consequences of therapy-induced vascular anatomical changes that can be used to serially monitor tumor perfusion and efficacy of antivascular therapy in clinical trials.

Animals↗
PubMed2001

Hypoxia in human intraperitoneal and extremity sarcomas.

PURPOSE: The presence of hypoxia, measured by needle electrodes, has been shown to be associated with poor patient outcome in several human tumor types, including soft tissue sarcomas. The present report emphasizes the evaluation of hypoxia in soft tissue sarcomas based upon the binding of the 2-nitroimidazole drug EF5 (2-[2-nitro-1H-imidazol-1-yl]-N-(2,2,3,3,3-pentafluoropropyl) acetamide). EF5 has previously been shown to be predictive of radiation response in animal tumors and in in vitro studies. We have also previously reported studies of EF5 binding in human squamous cell tumors. Using fluorescent immunohistochemical techniques, we provide data on the presence and distribution of EF5 binding, as a surrogate for hypoxia, in human spindle cell tumors. METHODS AND MATERIALS: Patients with spindle cell tumors who were scheduled for tumor surgery were asked to participate in the Phase I trial of EF5. Approximately 48 h preoperatively, EF5 was administered i.v. at doses between 9 and 21 mg/kg. Binding in frozen sections of biopsied tissues was determined using monoclonal antibodies labeled with the green-excited, orange-emitting fluorescent dye, Cy3. Calibration studies were performed in vitro by incubating fresh tumor tissue cubes obtained from each patient with EF3 (an analog of EF5) under hypoxic conditions ("reference binding"). The goal of these calibration studies was to quantify the maximal binding levels possible in individual patient's tissues. The relationship between binding (in situ based on EF5 binding) and reference binding (in vitro based on EF3 binding) was determined. RESULTS: Eight patients were studied; 3 of these patients had gastrointestinal stromal tumors (GIST). The incubation of tumor tissue cubes in EF3 under hypoxic conditions demonstrated that all tumors bound drug to a similar extent. Reference binding showed a 3.2-fold variation in median fluorescence (113-356) on an absolute fluorescence scale, calibrated by a Cy3 dye standard. In situ binding in the brightest tumor section varied by a factor of 25.4 between the lowest and highest binding tumor (7.5-190.2). Heterogeneity of highest binding was greater between tumors than within individual tumors. A correspondence between EF5 binding and Eppendorf needle electrode studies was seen in the 5 patients with non-GISTs. CONCLUSION: Inter- and intratumoral heterogeneity of EF5 binding in spindle cell tumors has been documented. Patterns of binding consistent with diffusion limited hypoxia are present in human spindle cell neoplasms.

Adult↗
PubMed2000

Detection of hypoxia in human squamous cell carcinoma by EF5 binding.

Localization and quantitation of 2-nitroimidazole drug binding in low pO2 tumors is a technique that can allow the assessment of hypoxia as a predictive assay. EF5 [2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl) acetamide] is such a drug, and it has been shown to be predictive of radiation response in rodent tumors. Using fluorescence immunohistochemical techniques, we provide data on the presence, distribution, and levels of EF5 binding as a surrogate for hypoxia in human head and neck and uterine cervix squamous cell cancers (SCCs). Six patients with SCC were studied. Four patients had head and neck tumors, and two had uterine cervix cancers. The incubation of fresh tissue cubes in EF3 under hypoxic conditions ("reference binding") demonstrated that all tumors were capable of binding drug, and that this binding varied by a factor of 2.9-fold (174.5-516.1) on an absolute fluorescence scale. In the five patients treated at the lowest drug doses (9 mg/kg), in situ binding was quantitatable. For all six patients, the maximum rate of in situ binding varied by a factor of 6.7 between the lowest and highest binding tumor (24.8-160.3) on an absolute fluorescence scale. In tumors with high binding regions, intratumoral heterogeneity was large, extending from minimal fluorescence (<1%) up to 88.6% of reference binding. In tumors with minimal binding, there was little intratumoral heterogeneity. These studies demonstrate substantial heterogeneity of in situ binding between and within individual squamous cell tumors.

Adult↗
PubMed2000

Hypoxia and necrosis in rat 9L glioma and Morris 7777 hepatoma tumors: comparative measurements using EF5 binding and the Eppendorf needle electrode.

PURPOSE: The purpose of this study was to assess the presence of tumor hypoxia using two independent techniques: binding of the 2-nitroimidazole EF5 and Eppendorf needle electrode measurements. The distribution of tumor hypoxia was assessed with respect to tumor necrosis in corresponding histological studies. METHODS AND MATERIALS: Each of several rats bearing a subcutaneous 9L glioma or Morris 7777 hepatoma tumor was given EF5 i.v. to a final, whole-body concentration of 100 microM. About 2.5 h later, each rat was anesthetized, and needle electrode measurements were made in the tumor along 1-5 tracks (30-200 individual measurements). At 3 h post-EF5 injection, the tumor was excised and frozen. Frozen sections were analyzed for the presence and distribution of binding of EF5 and necrosis using immunohistochemical techniques followed by staining with hematoxylin and eosin (H&E). The histochemical analysis and electrode readings in similar regions of the tumor were compared. RESULTS: Electrode measurements were taken at 0.4-mm intervals along one-dimensional tracks, whereas EF5 binding measurements from tissue sections contained two-dimensional information at high spatial resolution ( approximately 2.5 micro). The EF5 measurements showed greater spatial heterogeneity than did the electrode measurements. In tumor regions with minimal necrosis, needle tracks with relatively high pO(2) readings were usually found to contain relatively low EF5 binding, and vice versa. Because EF5 binding is inversely related to tissue pO(2), this result was expected. The expected inverse correlation of the two techniques was most disparate in necrotic tumor regions (confirmed by H&E staining), where needle electrode measurements showed low to zero pO(2) values, but little or no EF5 binding was found. CONCLUSION: The two methods compared in this study operate in fundamentally different ways and provide substantially different information. EF5 binding provided detailed spatial information on the distribution of hypoxia in viable tumor tissue. There was no EF5 binding in necrotic tumor tissue because cells in such tissue were unable to metabolize the drug. In contrast, output from the needle electrode method appeared to represent a "track-average" tissue pO(2) and did not distinguish between extreme hypoxia and either macroscopic or microscopic necrosis. At the present time, the importance of tumor necrosis in determining treatment response is unknown. However, our data suggest that the Eppendorf needle electrode technique will overestimate the presence of hypoxia. Both techniques are potentially limited by sampling errors in tumors with heterogeneous distributions of hypoxia.

Animals↗
PubMed2000

Noninvasive detection of tumor hypoxia using the 2-nitroimidazole [18F]EF1.

UNLABELLED: The noninvasive assessment of tumor hypoxia in vivo is under active investigation because hypoxia has been shown to be an important prognostic factor for therapy resistance. Various nuclear medicine imaging modalities are being used, including PET imaging of 18F-containing compounds. In this study, we report the development of 18F-labeled EF1 for noninvasive imaging of hypoxia. EF1 is a 3-monofluoro analog of the well-characterized hypoxia marker EF5, 2(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetami de, which has been used to detect hypoxia in tumor and nontumor systems using immunohistochemical methods. METHODS: We have studied 2 rat tumor types: the hypoxic Morris 7777 (Q7) hepatoma and the oxic 9LF glioma tumor, each grown in subcutaneous sites. PET studies were performed using a pharmacological dose of nonradioactive carrier in addition to [18F]EF1 to optimize and assess drug biodistribution. After PET imaging of the tumor-bearing rats, tissues were obtained for gamma-counting of the 18F in various tissues and immunohistochemical detection of intracellular drug adducts in tumors. In one pair of tumors, Eppendorf needle electrode studies were performed. RESULTS: [18F]EF1 was excreted dominantly through the urinary tract. The tumor-to-muscle (T/M) ratio of [18F]EF1 in the Q7 tumors was 2.7 and 2.4 based on PET studies and 2.1, 2.5, and 3.0 based on gamma-counting of the tissues (n = 3). In contrast, the T/M ratio of [18F]EF1 in the 9LF glioma tumor was 0.8 and 0.5 based on PET studies and 1.0, 1.2, and 1.4 based on gamma-counting of the tissues (n = 3). Immunohistochemical analysis of drug adducts for the two tumor types agreed with the radioactivity analysis. In the Q7 tumor, substantial heterogeneous binding was observed throughout the tumor, whereas in the 9LF tumor minimal binding was found. CONCLUSION: [18F]EF1 is an excellent radiotracer for noninvasive imaging of tumor hypoxia.

Animals↗
PubMed1999

Hypoxia-mediated apoptosis from angiogenesis inhibition underlies tumor control by recombinant interleukin 12.

The role of angiogenesis inhibition in the antitumor activity of recombinant murine interleukin 12 (rmIL-12) was studied in K1735 murine melanomas, the growth of which is rapidly and markedly suppressed by rmIL-12 treatment. On the basis of the prediction that tumor ischemia should result from therapeutic angiogenesis inhibition, tumor cell hypoxia was evaluated as a marker of ischemia using the EF5 [2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)aceta mide] approach. This method measures intracellular binding of the nitroimidazole EF5, which covalently binds to cellular macromolecules selectively under hypoxic conditions. Whereas 1 week of rmIL-12 treatment effectively inhibited K1735 cell-induced angiogenesis in Matrigel neovascularization assays, 2 weeks of treatment were needed before severe tumor cell hypoxia was detected in K1735 tumors. The hypoxia that developed was regional and localized to tumor areas distant from blood vessels. The great majority of severely hypoxic tumor cells were apoptotic, and in vitro studies indicated that the degree of hypoxia present within treated tumors was sufficient to trigger K1735 apoptosis. Tumor cell apoptosis was also prevalent in the first week of rmIL-12 treatment when few cells were hypoxic. In vitro studies indicated that this non-hypoxia-related apoptosis was induced directly by IFN-gamma produced in response to rmIL-12 administration. These studies reveal that rmIL-12 controls K1735 tumors initially by IFN-gamma-induced apoptosis and later by hypoxia-induced apoptosis. They also establish hypoxia as an expected result of tumor angiogenesis inhibition and a mediator of its therapeutic effect.

Animals↗
PubMed1998

MIBG inhibits respiration: potential for radio- and hyperthermic sensitization.

INTRODUCTION: Meta-iodobenzylguanidine (MIBG) in its 131I-labeled form is clinically used as a tumor-targeted radiopharmaceutical in the diagnosis and treatment of adrenergic tumors. This well established drug may have additional clinical applications as a radiosensitizer or hyperthermic agent, ie., MIBG reportedly inhibits mitochondrial respiration in vitro. The mechanism for MIBG inhibition of cellular oxygen consumption is uncertain. Moreover, MIBG reportedly stimulates glycolysis both in vitro and in vivo. Our studies show the effect of MIBG on 9L glioma oxygen consumption and redox status with tumors cells in vitro and in vivo. MATERIALS AND METHODS: The effects on electron transfer were determined by following oxygen consumption with a Clark oxygen electrode. Fluorescence measurements were used to determine effects of MIBG on intracellular electron acceptors, NADPH and flavoproteins, in vitro and in vivo. 31P-NMR was used to determine alterations in tumor cell pH in vivo. RESULTS: Our results show the inhibition of oxygen utilization with MIBG for cell suspensions in vitro. The same results were demonstrated for tumor cell suspensions rapidly isolated from tumors grown in rats. Moreover, NAD(P)H and flavoprotein (Fp) fluorescence changes were observed to rapidly occur following MIBG addition in vitro. Changes in intracellular pH measured with 31P-NMR, in vivo, precede the changes in fluorescence of NAD(P)H and Fp obtained with frozen sections of tumor. CONCLUSIONS: We conclude that 31P-NMR measurements and fluorescence changes, following MIBG injection, can be used as criterion for selecting the proper time to treat tumors with ionizing radiation or hyperthermia.

3-Iodobenzylguanidine↗
PubMed1998

Three solutions of the protein solubility problem.

Three simple equations are presented, which describe the variation of protein solubility (S) with changes in salt concentration, in terms of either the salt molality (M), the salt activity (ax), or the water activity (aw). Each equation yields, essentially independent, estimates of the numbers of salt ions (delta vx) and water molecules (delta vw) involved in the dissolution of a mol of the protein. The equations can be used to elucidate the physical significance of the parameters in other empirical equations for protein solubility.

Models, Chemical↗
PubMed1998

Oxygen distributions within R3230Ac tumors growing in dorsal flap window chambers in rats.

R3230Ac mammary tumors were grown in transparent window chambers implanted into the dorsal skin flap of 250 g Fischer 344 rats (see Dewhirst et al, 1992). The oxygen pressure distributions in the tumor and host tissue were measured by the oxygen dependent quenching of phosphorescence (see Vinogradov et al, 1996) after injection of Oxyphor R2 (7 mg, 0.3 ml) into the tail vein. The oxygen pressure maps show the R3230Ac tumors to be hypoxic relative to the surrounding tissue. The excitation spectrum for the phosphor has peaks at 419 nm (blue light) and at 524 nm (green light), and the emitted phosphorescence spectrum and lifetime are independent of the wavelength at which the phosphor is excited. The absorption by tissue is much greater for blue light than green light, due to intrinsic chromophores such as cytochromes, hemoglobin, myoglobin etc. Thus, blue excitation measures the oxygen pressures in a much thinner, superficial, surface layer (< 50 microns) than does green excitation, allowing "optical sectioning" of tissue oxygenation. The tissue can be further optically sectioned by making measurements from both sides of the window. Viewed from the tumor side, the superficial layers (blue excitation) of these tumors were hypoxic whereas the host tissue was well oxygenated. The oxygen pressures in the growing edge of the tumors are lower than those in the central core of the tumor, and much lower than those of the host tissue. This result is in agreement with the micro-oxygen electrode measurements of perivascular oxygen pressures reported by Dewhirst and coworkers (1992).

Animals↗
PubMed1998

Co-localization of hypoxia and apoptosis in irradiated and untreated HCT116 human colon carcinoma xenografts.

HCT116 human colon carcinoma xenografts were grown in nude mice. Frozen sections of control and irradiated tumors were stained and analysed for the distribution and extent of hypoxia and apoptosis. Tissue oxygen partial pressure was measured by immunohistochemical staining of hypoxia-dependent metabolites of the 2-nitroimidazole EF5. Apoptosis was assessed using a commercial kit which stains damaged DNA. Although the apoptosis stain was unlikely to exclude other forms of cell death (necrosis, pyknosis) all staining was found to associate with regions of near anoxia.

Animals↗
PubMed1997

Exchange of subunit interfaces between recombinant adult and fetal hemoglobins. Evidence for a functional inter-relationship among regions of the tetramer.

The inter-relationship between the interior subunit interfaces and the exterior diphosphoglycerate (DPG) binding region of the hemoglobin tetramer and the effects of a specific N-terminal acetylation on tetramer assembly have been evaluated. Tetrameric fetal hemoglobin F in the liganded state was found to dissociate to dimers much less than previously appreciated, i.e. about 70 times less than adult hemoglobin A (Kd = 0.01 microM and 0.68 microM, for HbF and HbA, at pH 7.5, respectively) over the pH range 6.2-7.5, whereas HbF1, in which the N termini of the gamma-chains are acetylated, dissociates like HbA. To determine whether this feature of HbF could be transferred to hemoglobin A, the single amino acid difference in their alpha1beta2/alpha1gamma2 interfaces and the 4 amino acid differences in their alpha1beta1/alpha1gamma1 interfaces have been substituted in HbA to those in HbF. This pentasubstituted recombinant HbA/F had the correct molecular weight as determined by mass spectrometry, the expected mobility on isoelectric focusing, the calculated amino acid composition, and normal circular dichroism properties, oxygen binding, and cooperativity. Although HbA/F has the same amino acid side chains that bind DPG as HbA, its diminished response to 2,3-DPG resembled that of HbF. However, its tetramer-dimer dissociation constant (Kd = 0.14 microM) was between that of HbA and HbF despite the fact that it was composed entirely of HbF subunit interfaces. The results indicate that regions of the tetramer distant from the tetramer-dimer interface influence its dissociation and, reciprocally, that the interfaces affect regions involved in the binding of allosteric regulators, suggesting flexible long range inter-relationships in hemoglobin.

2,3-Diphosphoglycerate↗
PubMed1997

Tamoxifen induces hypoxia in MCF-7 xenografts.

Tamoxifen is widely used as an adjunct therapy for breast cancer. We hypothesized that hypoxia develops in tumors as a result of tamoxifen treatment because tamoxifen has been reported to be antiangiogenic and thrombogenic. MCF-7 breast tumors were grown under estrogenic stimulation in 4-6-week-old CD-1 nu/nu female mice. When the tumors were approximately 5 mm in diameter, 17beta-estradiol pellets were replaced with either placebo or tamoxifen-containing pellets. Two days later, tissue oxygenation was measured using immunohistochemical detection of binding of the 2-nitroimidazole EF5. Intravascular oxygen partial pressures were measured noninvasively by oxygen-dependent quenching of phosphorescence of an injected dye that is excited by light pulses. Tamoxifen treatment increased hypoxia in the tumors, as measured by EF5 binding (P = 0.01 by Mann-Whitney test). This observation was not dependent on the presence of tamoxifen-induced necrosis. Intravascular oxygen partial pressures were lower in tumors relative to surrounding normal tissue in tamoxifen-treated tumors as compared to placebo-treated tumors. In vitro, tamoxifen did not modify the oxygen-dependent metabolism of EF5, indicating that the increased EF5 binding in tamoxifen-treated tumors reflects a physiological decrease in tissue oxygenation. The clinical significance of these observations is discussed in the context of the sequencing of tamoxifen with other therapies, and in light of recent data suggesting that hypoxia may be associated with genetic changes resulting in a more aggressive tumor phenotype.

Animals↗
PubMed1997

Evaluation of the concept of "hypoxic fraction" as a descriptor of tumor oxygenation status.

The presence and significance of tumor hypoxia has been recognized since the 1950's. Hypoxic cells in vitro and in animal tumors in vivo are documented to be three times more resistant to radiation-induced killing compared to aerobic cells. There is now evidence that tumor hypoxia is treatment-limiting in many human cancers. One common way to describe the extent of hypoxia in individual and groups of tumors is the "hypoxic fraction." This measurement infers that cells are present in only two radiobiologically significant states: oxygenated and hypoxic. In this paper, we demonstrate the qualitative and quantitative presence of hypoxic tumor cells using the oxygen dependent metabolism of the 2-nitroimidazole, EF5. Two assumptions concerning the calculation and interpretation of the hypoxic fraction are considered. The first is the use of multiple animals to describe the radiation response at a given radiation dose. We hypothesize that the presence of intertumor variability in radiation response due to hypoxia could negatively influenced the characterization of the change in slope required to calculate the hypoxic fraction. The studies presented herein demonstrate heterogeneity of radioresponse due to hypoxic fraction within and between tumor lines. The 9L subcutaneous tumor studied in air-breathing rats demonstrates a 2 log variation in surviving fraction at 17 Gy. The Morris 7777 hepatoma, in contrast, showed little variability of radiation response. Our second question addresses the limitations of using the "hypoxic fraction" to describe the radiation response of a tumor. This calculated value infers that radiobiological hypoxia is a binary measurement: that a tumor contains two cell populations, aerobic cells with maximal radiosensitivity and hypoxic cells with maximal radioresistance. The classic work of Thomlinson and Gray, however, implies the presence of an oxygen gradient from tumors vessel through the tissues. In both the 9L and Q7 tumors, flow cytometric analysis of EF5 binding demonstrates a continuous range of cellular pO2 levels. These studies suggest that: 1) there is extensive intertumor variability of radiation response in certain tumor lines; 2) the variability in radiation response between individual tumors in a group may affect the ability to describe a particular tumor type's "hypoxic fraction" and 3) The oxygen status of tumor cells is a continuum. This realization affects the ability to apply a binary concept such as the "hypoxic fraction" effectively in radiobiology.

Animals↗
PubMed1997

Oxygen distribution in the vasculature of mouse tissue in vivo measured using a near infra red phosphor.

Oxygen dependent quenching of phosphorescence has been used to measure the oxygenation of tissue in mice, including the differences between normal tissue and that of a murine tumor. Approximately 0.3 mg of the phosphorescence oxygen probe, Green 2W, was injected into the tail vein of tumor bearing mice. The mice were immobilized using an anesthetic cocktail and illuminated with flashes (< 4 microseconds t1/2) of light of 636 +/- 15 nm. The emitted phosphorescence (790 nm max.) was measured using an imaging phosphorimeter with an intensified CCD camera, an instrument which provides two dimensional digital maps of oxygen pressure. Both the illumination light and the phosphorescence were in the near infra red region of the spectrum, where skin and tissue have little absorption. The light can therefore readily pass through the skin and centimeter thickness of tissue. Mice are sufficiently small that the oxygen pressure maps could be obtained by illuminating from either the same or the opposite side as the camera (and tumor). The tumors were observed as regions with oxygen pressures substantially below those of the surrounding normal tissue. Thus, it is possible to non-invasively detect these tumors and to monitor their internal oxygen pressure in real time and through cm of tissue.

Animals↗
PubMed1996

2-Nitroimidazole (EF5) binding predicts radiation resistance in individual 9L s.c. tumors.

The presence of hypoxic tumor cells is known to be an important cause of radiation treatment resistance in vivo. The ability to predict the presence and extent of hypoxic cells in individual tumors would allow the addition of specific "antihypoxia"-based treatment regimes. Hypoxia can be monitored by measuring the binding of 2-nitroimidazoles. We have tested the hypothesis that binding of EF5, a fluorinated derivative of the 2-nitroimidazole, Etanidazole, can predict radioresistance in individual tumors. Fischer rats bearing 9L s.c. tumors were given injections i.v. with EF5 3 h before irradiation and tumor harvest. Tumor cells were dissociated for flow cytometric analysis and plating efficiency studies. EF5 binding was detected via monoclonal antibodies conjugated to the orange emitting dye, Cy3. In air breathing rats, for a given radiation dose, a large amount of variation in plating efficiency was seen. However, there was minimal variability of the plating efficiency for tumors irradiated in euthanized animals (hypoxic tumors; correlation coefficient for the fitted curve = 0.93) and in cells dissociated from tumors and irradiated in suspension (correlation coefficient for the fitted curve = 0.99), suggesting that varying sensitivity to the cell disaggregation technique was not responsible. In contrast, a good correlation between the relative radiation resistance or hypoxic survival and EF5 binding of "moderately" hypoxic cells in air breathing rats was identified using these techniques. In these 9L s.c. tumors, intertumor variation in oxygenation accounted for most of the range in individual tumor radiation response, and this was found to be independent of tumor size. This study provides evidence for the application of EF5 binding with monoclonal antibody detection as an in vivo predictive assay of individual tumor hypoxia and resultant therapy resistance.

Animals↗