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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.

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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.

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The purpose of this study was to determine whether power Doppler ultrasound techniques could be used to direct biopsies into tumour regions with relatively low red blood cell flux, and therefore preferentially sample regions that were relatively hypoxic. Subcutaneous 9L glioma rat tumours were biopsied using power Doppler ultrasound guidance. Immunohistochemical detection of the 2-nitroimidazole EF5 was performed to determine the presence and level of hypoxia in the biopsy samples. Comparisons between the power Doppler-determined red blood cell flux and EF5 binding were made. In seven out of eight tumours studied, power Doppler ultrasound allowed differentiation of a relatively hypoxic region from a relatively oxic region by localizing relatively low vs high red blood cell flux areas respectively. In one of these seven tumours, RBC flux was high in both biopsied sites and hypoxia was not present in either. In two of these seven tumours, hypoxia was present in each biopsy and both of the red blood cell flux measurements were low. In the eighth tumour, both the EF5 binding and the red blood cell flux measurements were low. In this tumour, low EF5 binding was due to the dominance of necrotic cells, which will not reduce or bind EF5 in the biopsy specimen. Using EF5-binding techniques, we have confirmed that regions of relatively low red blood cell flux are more hypoxic than those with relatively high red blood cell flux. Counterstaining specimens with haematoxylin and eosin allows differentiation of low EF5-binding regions due to oxia vs necrosis. These methods have clinical implications for the expanded use of power Doppler ultrasound as a means to direct tissue sampling when it is important to identify the presence of hypoxia.

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The characteristic reduction and binding of nitroimidazoles to cellular macromolecules in the absence of oxygen allows their use for detection and characterization of hypoxia. The biodistribution of a new nitroimidazole, EF5 (2-[2-nitro-1H-imidazol-1-yl]-N-(2,2,3,3,3-pentafluoropropyl) acetamide), in mice bearing EMT6 tumors is described. Detection methods based on radioactivity and monoclonal antibody techniques are compared for liver and tumor. All nonexcretory tissues demonstrated similar levels of radioactivity at 0.5 hr postinjection of drug, demonstrating equivalent access of EF5 to all tissues. At 24 hr, when unbound drug has been cleared, the tissues with the highest binding are the liver, esophagus, bladder and tumor. Typically, liver tissue contains the highest level of radioactivity at this time. Examination of tumor and liver tissue by use of fluorescence microscopy and Cy3-bound monoclonal antibodies specific for EF5 adducts showed that the patterns of binding in tumor are considerably more heterogeneous than those of liver. Histograms of fluorescence intensity, with use of these antibodies, demonstrate average and maximal binding higher in tumors than in the liver. This divergence from the radioactivity data was determined to be unrelated to sampling error, differential antibody access or staining efficiency of liver vs. tumor tissue. A possible cause is the scavenging of radioactive drug metabolites by liver. The data presented herein suggest that EF5 is useful as a hypoxia detector and that monoclonal antibody detection methods can give detailed information on the distribution of EF5 binding. This technology may allow an accurate estimation of the oxygenation and/or nitroreductase levels in both tumor and normal tissues.

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One of the most sensitive hypoxia detection methods is based on the observation that binding of nitroimidazoles to cellular macromolecules occurs as a result of hypoxia-dependent bioreduction by cellular nitroreductases. Nitroimidazole-binding techniques provide measurements of hypoxia to virtually any degree of spatial resolution and with a multiplicity of techniques. This paper demonstrates hypoxia imaging using in vivo EF5 binding with detection by a fluorochrome-conjugated monoclonal antibody. We investigated these techniques in the 9L glioma tumour, in part because the exact nature of the hypoxia in this tumour system is controversial. Our results demonstrate that following intravenous injection of EF5, binding and detection using a monoclonal antibody in 9L gliomas is specific and oxygen dependent. Detection of binding using fluorescence microscopy can be performed on frozen tissues; tissue sections can be counterstained with haematoxylin and eosin for light microscopic analysis. Alternatively, the distribution of hypoxia in a tumour can be inferred by examining individual tumour cells using flow cytometric techniques. Based upon the results presented herein, the radiation-resistant phenotype of 9L epigastric tumours grown in our laboratories can be associated with the presence of hypoxic cells.

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Radical hysterectomy, a procedure used in the treatment of women with cervical cancer, is frequently a cause of either temporary or permanent bladder dysfunction in the postoperative period. Four major types of dysfunction are seen: hypertonus of the bladder muscle, loss of sensation of bladder fullness, difficulty initiating micturition, and bladder hypotonia. Nursing interventions can affect adaptation to some of these problems and prevention of others. A review of rehabilitative literature is used to develop a rationale for nursing care using Levine's Conservation Principles as a conceptual framework.

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