2-(2-nitro-1h-imidazol-1-yl)-n-(2-2-3-3-3-pentafluoropropyl)acetamide and pimonidazole

Atherosclerotic plaques with large lipid cores and inflammation contain regions of hypoxia. We examined the uptake of 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl) acetamide ([18F]EF5), a specific marker of hypoxia labeled for positron emission tomography, in mouse atherosclerotic plaques.. Atherosclerotic mice of 2 different genetic backgrounds (low-density lipoprotein receptor-/- apolipoprotein B100/100 and insulin-like growth factor II/low-density lipoprotein receptor-/- apolipoprotein B100/100) were first fed a Western diet to induce development of plaques with variable phenotypes and then injected with [18F]EF5. C57BL/6N mice served as controls. Aortas were dissected for biodistribution studies, autoradiography, histology, and immunohistochemistry. Uptake of [18F]EF5 was significantly higher in the aortas of mice with large atherosclerotic plaques than in the C57BL/6N controls. Furthermore, autoradiography demonstrated, on average, 2.0-fold higher [18F]EF5 uptake in atherosclerotic plaques than in the adjacent normal vessel wall. Hypoxia in plaques was verified by using an EF5 adduct-specific antibody and pimonidazole. The blood clearance of [18F]EF5 was slow, with blood radioactivity remaining relatively high up to 180 minutes after injection.. Large atherosclerotic plaques in mice contained hypoxic areas and showed uptake of [18F]EF5. Despite its slow blood clearance, the high uptake of [18F]EF5 in plaques suggested that plaque hypoxia is a potential target for identifying high-risk plaques noninvasively.

Topics: Analysis of Variance; Animals; Aorta; Apolipoprotein B-100; Atherosclerosis; Autoradiography; Disease Models, Animal; Etanidazole; Female; Fluorine Radioisotopes; Genotype; Hydrocarbons, Fluorinated; Hypoxia; Immunohistochemistry; Insulin-Like Growth Factor II; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Nitroimidazoles; Phenotype; Positron-Emission Tomography; Radiopharmaceuticals; Receptors, LDL; Tissue Distribution

Although hypoxia is a known prognostic factor, its effect will be modified by the rate of reoxygenation and the extent to which the cells are acutely hypoxic. We tested the ability of exogenous and endogenous markers to detect reoxygenation in a xenograft model. Our technique might be applicable to stored patient samples.. The human colorectal carcinoma line, HT29, was grown in nude mice. Changes in tumor hypoxia were examined by injection of pimonidazole, followed 24 hours later by EF5. Cryosections were stained for these markers and for carbonic anhydrase IX (CAIX) and hypoxia-inducible factor 1alpha (HIF1alpha). Tumor hypoxia was artificially manipulated by carbogen exposure.. In unstressed tumors, all four markers showed very similar spatial distributions. After carbogen treatment, pimonidazole and EF5 could detect decreased hypoxia. HIF1alpha staining was also decreased relative to CAIX, although the effect was less pronounced than for EF5. Control tumors displayed small regions that had undergone spontaneous changes in tumor hypoxia, as judged by pimonidazole relative to EF5; most of these changes were reflected by CAIX and HIF1alpha.. HIF1alpha can be compared with either CAIX or a previously administered nitroimidazole to provide an estimate of reoxygenation.

Topics: Animals; Antigens, Neoplasm; Biomarkers, Tumor; Carbon Dioxide; Carbonic Anhydrase IX; Carbonic Anhydrases; Cell Hypoxia; Etanidazole; Female; HT29 Cells; Humans; Hydrocarbons, Fluorinated; Hypoxia-Inducible Factor 1, alpha Subunit; Immunohistochemistry; Mice; Mice, Nude; Nitroimidazoles; Oxygen; Radiation-Sensitizing Agents

The endothelin-1 antagonist, Atrasentan (ABT-627) was used to modify perfusion in the human tumor xenograft model, HT29, growing in nude mice. Atrasentan produced a significant increase in perfusion, as measured in vivo by Gd-DTPA DCE-MRI. Changes in tumor hypoxia were assessed by comparing the binding of two hypoxia tracers, pimonidazole and EF5 given before and after Atrasentan administration. In vehicle-treated controls, the distribution of EF5 and pimonidazole was very similar. However, Atrasentan treatment was associated with decreased uptake of the second hypoxia tracer (EF5), relative to the first (pimonidazole). Although Atrasentan had no independent effect on the growth of HT29 tumors, Atrasentan combined with 20 Gy radiation led to a modest but significant increase in tumor growth delay compared to radiation alone.

Topics: Adenocarcinoma; Animals; Atrasentan; Colonic Neoplasms; Combined Modality Therapy; Etanidazole; Gadolinium DTPA; HT29 Cells; Humans; Hydrocarbons, Fluorinated; Hypoxia; Magnetic Resonance Imaging; Male; Mice; Mice, Nude; Nitroimidazoles; Perfusion; Pyrrolidines; Radiation-Sensitizing Agents; Radiotherapy; Treatment Outcome; Xenograft Model Antitumor Assays

To study the changes in hypoxia resulting from mild temperature hyperthermia (MTH) in a subcutaneous xenograft model using dual-tracer immunohistochemical techniques.. HT29 tumors were locally heated at 41 degrees C. Changes in tumor hypoxia were investigated by pimonidazole and EF5. Pimonidazole was given 1h preheating, EF5 at various times during or after treatment, 1h later the animals were sacrificed. Blood vessels were identified by CD31 staining, and perfusion by Hoechst 33342 injected 1 min pre-sacrifice.. The overall hypoxic fraction was significantly decreased by MTH during and immediately after heating. However, MTH induced both increases and decreases in tumor hypoxia in different parts of the tumor. Specifically, MTH decreased hypoxia in the regions with relatively well-perfused blood vessels, but increased hypoxia in regions that were poorly perfused. At 24-h post heating, newly formed hypoxic regions surrounded previously-hypoxic foci, which in turn surrounded pimonidazole-stained debris. Quantitative analysis did not evince changes in tumor oxygenation due to MTH at 24h post-treatment.. In this xenograft model, the effect of MTH on tumor oxygenation was variable, both spatially and kinetically. Overall tumor oxygenation was improved during and after heating, but the effect was short-lived.

Topics: Adenocarcinoma; Analysis of Variance; Animals; Cell Hypoxia; Colorectal Neoplasms; Etanidazole; Female; Hydrocarbons, Fluorinated; Hyperthermia, Induced; Immunohistochemistry; Mice; Nitroimidazoles; Oxygen; Radiation-Sensitizing Agents; Transplantation, Heterologous

Tumor hypoxia is important in the development and treatment of human cancers. We have developed a novel xenograft model for studying and imaging of hypoxia-induced gene expression. A hypoxia-inducible dual reporter herpes simplex virus type 1 thymidine kinase and enhanced green fluorescence protein (HSV1-TKeGFP), under the control of hypoxia response element (9HRE), was stably transfected into human colorectal HT29 cancer cells. Selected clones were further enriched by repeated live cell sorting gated for hypoxia-induced eGFP expression. Fluorescent microscopy, fluorescence-activated cell sorting, and radioactive substrate trapping assays showed strong hypoxia-induced expression of eGFP and HSV1-tk enzyme in the HT29-9HRE cells in vitro. Sequential micropositron emission tomography (PET) imaging of tumor-bearing animals, using the hypoxic cell tracer (18)F-FMISO and the reporter substrate (124)I-FIAU, yielded similar tumor hypoxia images for the HT29-9HRE xenograft but not in the parental HT29 tumor. Using autoradiography and IHC, detailed spatial distributions in tumor sections were obtained and compared for the following hypoxia-associated biomarkers in the HT29-9HRE xenograft: (124)I-FIAU, (18)F-FMISO, Hoechst (perfusion), lectin-TRITC (functional blood vessels), eGFP, pimonidazole, EF5, and CA9. Intratumoral distributions of (124)I-FIAU and (18)F-FMISO were similar, and eGFP, pimonidazole, EF5, and CA9 colocalized in the same areas but not in well-perfused regions that were positive for Hoechst and lectin-TRITC. In enabling the detection of hypoxia-induced molecular events and mapping their distribution in vivo with serial noninvasive positron emission tomography imaging, and multiple variable analysis with immunohistochemistry and fluorescence microscopy, this human xenograft model provides a valuable tool for studying tumor hypoxia and in validating existing and future exogenous markers for tumor hypoxia.

Topics: Animals; Antigens, Neoplasm; Arabinofuranosyluracil; Autoradiography; Biomarkers; Carbonic Anhydrase IX; Carbonic Anhydrases; Cell Hypoxia; Etanidazole; Female; Gene Expression; Green Fluorescent Proteins; Herpesvirus 1, Human; HT29 Cells; Humans; Hydrocarbons, Fluorinated; Immunohistochemistry; Mice; Mice, Nude; Misonidazole; Neoplasm Transplantation; Neoplasms; Nitroimidazoles; Positron-Emission Tomography; Thymidine Kinase; Tissue Distribution; Transplantation, Heterologous

The binding kinetics of the hypoxia marker EF5 can be quantified by uptake of (14)C-labeled drug or calibrated flow cytometry using antibodies specific for drug adducts. Maximum EF5 binding is cell-line dependent and varies directly with drug exposure (area under the curve; concentration integrated over time) but inversely with pO(2) from 0 to >100 mmHg. For pimonidazole, binding is reported to be independent of the cell line and drug AUC, being zero above 10 mmHg, with an easily discriminated increase at lower pO(2). The basis for these kinetic differences is unknown, but the main experimental variable distinguishing the two marker techniques is antibody concentration ([Ab] - pimonidazole << EF5). In this study, EF5 and pimonidazole binding kinetics were compared as a function of pO(2) and antibody concentration in cells of two rat (9L and R3230) and two human (HT1080 and SiHa) cancer cell lines. For both markers, binding varied directly with AUC at all pO(2). The dynamic range of observed binding (maximum change from 0 to 76 mmHg oxygen) decreased with antibody concentration. The pO(2) dependence of binding for pimonidazole, but not EF5, varied dramatically with antibody concentration. Thus the data presented herein do not support the reported binding kinetics of pimonidazole. In particular, it is shown that the common use of antibody concentrations much lower than antigen concentrations can lead to unreliable estimations of adduct level and hence pO(2).

Topics: Animals; Area Under Curve; Calibration; Cell Hypoxia; Cell Line, Tumor; Etanidazole; Flow Cytometry; Humans; Hydrocarbons, Fluorinated; Kinetics; Microscopy, Fluorescence; Nitroimidazoles; Oxygen; Radiation-Sensitizing Agents; Rats; Time Factors

Topics: Antigens, Neoplasm; Biomarkers; Carbonic Anhydrase IX; Carbonic Anhydrases; Carcinoma, Squamous Cell; Cell Hypoxia; Electrodes; Etanidazole; Head and Neck Neoplasms; Humans; Hydrocarbons, Fluorinated; Intracellular Signaling Peptides and Proteins; Mitochondrial Proteins; Neoplasm Proteins; Nitroimidazoles; Osteopontin; Oxygen; Partial Pressure; Radiation-Sensitizing Agents; Tirapazamine; Triazines