Hypoxic Cell Marker Research Articles

Imaging hypoxia after oxygenation-modification: Comparing [ 18F]FMISO autoradiography with pimonidazole immunohistochemistry in human xenograft tumors

Purpose Hypoxia is one of the reasons for radiation therapy resistance. Positron emission tomography using 18F-labeled misonidazole ([ 18F]FMISO) is a non-invasive method of imaging tumor hypoxia. Aim of this study was to validate [ 18F]FMISO against the clinically most widely used hypoxic cell marker pimonidazole under different oxygenation conditions. Materials and methods One human head and neck squamous cell carcinoma (SCCNij3) and two human glioblastoma (E102 and E106) xenograft tumor lines were studied after injection of [ 18F]FMISO and pimonidazole. Control mice were compared with a second group breathing carbogen to reduce tumor hypoxia and with a third group with clamped tumors to increase hypoxia. Tumor sections were analyzed on a phosphor imaging system and consecutively stained immunohistochemically (IHC) for visualization of pimonidazole. Pixel-by-pixel analysis was performed and the hypoxic fraction, obtained after segmentation of the pimonidazole signal, was related to the mean optical density of [ 18F]FMISO and pimonidazole. Results A moderate pixel-by-pixel correlation between [ 18F]FMISO autoradiography and pimonidazole IHC was found for the control tumors, after carbogen breathing and after clamping for SCCNij3. For E102 and E106, mean signal intensities for pimonidazole significantly decreased after carbogen breathing and increased after clamping, mean [ 18F]FMISO signal intensities increased significantly after clamping and a significant correlation between the hypoxic fractions and the mean [ 18F]FMISO signal intensities was found. Conclusions [ 18F]FMISO autoradiography and pimonidazole immunohistochemistry can both be used to visualize treatment induced changes in tumor hypoxia. However, the response to these modifications differs widely between xenograft tumor lines.

Synthesis and Evaluation of Novel 8‐Oxo‐8H‐cyclopenta[a]acenaphthylene‐7‐carbonitriles as Longwavelength Fluorescent Markers for Hypoxic Cells in Solid Tumor.

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Novel fluorescent markers for hypoxic cells of naphthalimides with two heterocyclic side chains for bioreductive binding

Novel naphthalimides with two heterocyclic side chains of 2-nitroimidazole for bioreductive binding were designed, synthesized, and used as fluorescent markers for hypoxic cells. Their evaluation for imaging tumor hypoxia was carried out in V79 cells, CHO cells, and 95D cells in vitro by using fluorescence scan ascent. A 2 and A 4 showed a very large differential fluorescence between hypoxic and oxic cells (V79 cells) in vitro and are promising candidate markers for hypoxic cells.

Synthesis and evaluation of novel 8-oxo-8H-cyclopenta[a]acenaphthylene-7-carbonitriles as long-wavelength fluorescent markers for hypoxic cells in solid tumor

Novel bioreductive and long-wavelength fluorescent markers for hypoxic cells in solid tumor, 9-isocyano-8H-acenaphtho[l,2-b]pyrrol-8-one with the side chain of 2-nitroimidazole, were designed, synthesized, and evaluated in V79 379A Chinese hamster cells in vitro. Compounds A2 and A4 showed good hypoxic–oxic fluorescence differential in vitro (V79 cells) by using fluorescence scan ascent.

Does heterogeneity of pimonidazole labelling correspond to the heterogeneity of radiation-response of FaDu human squamous cell carcinoma?

Background and purpose Pimonidazole is a marker for hypoxic cells which are radioresistant and thereby important for the outcome of radiotherapy. The present study evaluates heterogeneity in pimonidazole binding within and between tumours and relates the results to the heterogeneity of radiation response in the same tumour cell line. Materials and methods FaDu, a poorly differentiated human squamous cell carcinoma line, was transplanted subcutaneously into the right hind-leg of NMRI nude mice. Tumours were irradiated with graded single doses either under ambient or clamped blood flow conditions and local tumour control was evaluated after 120 days. Complete dose–response curves for local tumour control were generated and the slope, a measure of heterogeneity of radiation response, was determined. In parallel, 12 unirradiated tumours were examined histologically. Seven serial 10 μm cross-sections per tumour were evaluated using fluorescence microscopy and computerised image analysis to determine the pimonidazole hypoxic fraction (pHF). Heterogeneity in pHF was quantified by its coefficient of variation (CV). Poisson-based model calculations considering the intertumoural heterogeneity of pHF were performed and the slopes of the predicted and the observed dose–response curves were compared. Results The mean pHF was 11% [CV 50%] when one central section per tumour was evaluated. Measurements of multiple sections per tumour resulted in a mean pHF of 12% [CV 46%] ( P=0.7). Intertumoural heterogeneity in pHF was more pronounced than heterogeneity in individual tumours by a factor of 2. Model calculations based on the variability in pHF resulted in similar slopes of the dose–response curve for local tumour control in comparison with the observed slope when the heterogeneity in an unknown and arbitrarily chosen additional radiobiologically relevant parameter, in this example clonogen density, was taken into account. Conclusions While the average pimonidazole hypoxic fraction in FaDu tumours corresponds well to the radiobiological hypoxic fraction, the variability of pHF in FaDu tumours was not sufficient to explain the heterogeneity of radiation response in the same tumour line. Information on at least one additional parameter is expected to substantially enhance the predictive power of histological markers of tumour hypoxia.

Comparison of different methods of CAIX quantification in relation to hypoxia in three human head and neck tumor lines

Purpose In head and neck cancer, it has been shown that hypoxic tumors respond poorly to therapy. Methods to identify hypoxic tumors are, therefore, of importance to select patients for oxygenation modifying or other intensified treatments. The aim of this study was to compare tumor cell hypoxia assessed by the hypoxic cell marker pimonidazole (PIMO) with expression of the endogenous hypoxia-related marker carbonic anhydrase IX (CAIX) in three human head and neck tumor lines. Material and methods Forty-five tumors of three human head and neck tumor lines, SCCNij3, SCCNij59 and MEC82, xenografted in athymic mice, were used. CAIX was quantified by biodistribution (% injected dose/g tumor) after injecting 3–5 μl 111In-labeled G250 mouse antibody 3 days prior to euthanizing. In a tissue section from the same tumor, fractions of tumor area positive for PIMO, CAIX and Hoechst 33342 (perfusion marker) were assessed after immunohistochemical staining, using a digital image analysis system. Results SCCNij3 and MEC82 were relatively hypoxic tumor lines with fractions of tumor area positive for pimonidazole of 0.16 and 0.15, respectively. SCCNij59 was a better-oxygenated tumor line with a PIMO-fraction of 0.03. The three tumor lines showed different levels and patterns of CAIX immunohistochemical staining, but only in MEC82 there was a good correlation between PIMO-fraction and CAIX-fraction ( r 2=0.92, P<0.0001). Correlations between 111In-G250 uptake and CAIX-fraction or PIMO-fraction within tumor lines were weak or absent. Conclusions Assessment of CAIX expression depends largely on the techniques and tumor lines used. Furthermore, the immunohistochemical staining pattern of CAIX relative to PIMO differs between human tumor lines of similar anatomical origin. Therefore, the use of CAIX as endogenous marker of tumor hypoxia remains questionable.

Enhanced response to radiotherapy in tumours deficient in the function of hypoxia-inducible factor-1

Background and purpose To test the hypothesis that deficiency in expression of the transcription factor, HIF-1, renders tumours more radioresponsive than HIF-1 proficient tumours. Patients and methods Tumours comprising mouse hepatoma cells lacking HIF-1β (and thereby HIF-1 function) were grown in nude mice and radiation-induced growth delay compared with that seen for wild-type tumours and tumours derived from HIF-1β negative cells where HIF-1 function had been restored. Results The xenografts that lack HIF-1 activity take longer to establish their growth and are more radioresponsive than both parental xenografts and those with restored HIF-1 function. Pre-treatment of the HIF-1 deficient xenografts with the hypoxic radiosensitizer misonidazole, had little effect on radioresponse. In contrast this treatment radiosensitized the parental xenografts. In spite of this, no difference in oxygenation status was found between the tumour types as measured by Eppendorf O 2-electrodes and by binding of the hypoxic cell marker NITP. Admixing wild type and HIF-1 deficient cells in the same tumour at ratios of 1 in 10 and 1 in 100 restores the growth of the mixed tumours to that of a 100% HIF-1 proficient cell population. However, when comparing the effects of radiation on the mixed tumours, radioresponsiveness is maintained in those tumours containing the high proportion of HIF-1 deficient cells. Conclusions The differences in radioresponse do not correlate with tumour oxygenation, suggesting that the hypoxic cells within the HIF-1 deficient tumours do not contribute to the outcome of radiotherapy. Thus, hypoxia impacts on tumour radioresponsiveness not simply because of the physio-chemical mechanism of oxygen with radiation-induced radicals causing damage ‘fixation’, but also because hypoxia/HIF-1 promotes expression of genes that allow tumour cells to survive under these adverse conditions. Further, the results from the cell mixing experiments uncouple the growth promoting effects of HIF-1 and the underlying mechanism by which HIF-1 may increase radiation resistance in solid tumours.

Automated preparation of hypoxic cell marker [ 18F]FRP-170 by on-column hydrolysis

An automated synthesis for the preparation of the novel hypoxic cell marker, [ 18F]FRP-170 3 , [ 18F]1-[2-fluoro-1-(hydroxymethyl)ethoxy]methyl-2- nitroimidazole, was developed using an on-column basic-hydrolysis step. The 18F-labeled protected intermediate 2 was retained on a Sep-Pak Plus C18 cartridge and, in the same cartridge at room temperature, hydrolyzed by NaOH for deacetylation to give [ 18F]FRP-170. The elution method from the cartridge was optimized for direct injection of the crude product into an HPLC column. Thus, [ 18F]FRP-170 was prepared in 20–30% decay-corrected radiochemical yield within 60 min.

Utility of 19F MRS detection of the hypoxic cell marker EF5 to assess cellular hypoxia in solid tumors

Background and purposeThe present studies were undertaken to determine whether 19F MRS could be used to quantify the binding of the pentafluorinated derivative of etanidazole (EF5) in hypoxic cells of solid tumors. Materials and methodsA 4.7T imaging magnet was used for the in situ and in vitro evaluation of EF5 signals. In order to develop a better understanding of these NMR measurements the characteristics of parent, reduced unbound, and reduced bound EF5 signals were examined in vitro using a 12T spectrometer. ResultsIn situ data acquired using a 4.7T imaging magnet, showed retention of EF5 signals in KHT sarcomas that was absent in muscles for 6h after EF5 injection. In vitro studies showed no difference in the NMR detectable signal of parent and reduced unbound EF5. T2 values determined using parent EF5 samples revealed a T2 time of 675ms. In contrast, EF5 bound to KHT tumor cells gave rise to signals of low intensity, broad line widths, and T2 relaxation times of less than 30ms. When the same samples were analyzed using the 4.7T imaging magnet, the CF3 and CF2 fluorine peaks were readily identifiable in the parent EF5 sample but no fluorine signal could be detected from EF5 bound to KHT tumor cells. ConclusionThe inability to resolve bound EF5 metabolites even at high field strengths (12T), coupled with the short T2 relaxation times of the bound EF5, and the limits of detection of the in situ applied imaging magnet (4.7T), meant that hypoxic cells could not be quantified in tumors using the 19F MRS technique. In situ 19F MRS measurements of EF5 signals (parent/reduced unbound) may reflect conditions of tumor physiology and thus indicate the extent of tumor hypoxia but they are not capable of resolving the cellular oxygenation status of the tumor cells.

Imaging the ischemic penumbra with 18F-fluoromisonidazole in a rat model of ischemic stroke.

The ischemic penumbra is a major focus of stroke research. 18F-fluoromisonidazole (18F-FMISO), a positron emission tomography (PET) marker of hypoxic cells, has shown promise as a technique to image the penumbra in humans. Our aim was to delineate the pattern of 18F-FMISO binding in a rat middle cerebral artery transient thread-occlusion model, and correlate this with tissue outcome at 24 hours. We hypothesized that the pattern of 18F-FMISO binding would mimic that seen in humans. Thirty-eight rats underwent 2 hours transient middle cerebral artery (MCA) occlusion, and then received 18F-FMISO at time points from 0.5 to 22 hours post-MCA occlusion and were killed 2 hours later. Autoradiographic assessment of 18F-FMISO binding and assessment (triphenyltetrazolium chloride) of the area of infarction were performed on tissue slices. Until 1 hour after MCA occlusion, 18F-FMISO binding was increased in the entire MCA territory, with little or no infarction visible. Over the next 5 hours, the pattern of binding evolved to a small rim of intensely binding tissue surrounding the infarct core, which itself showed reduced binding compared with the contralateral hemisphere. By 24 hours, there was minimal accumulation of 18F-FMISO binding and a large area of infarction. The pattern of 18F-FMISO binding rats reproduced the pattern seen in humans, consistent with this tracer being a marker of the ischemic penumbra in both species. This technique may have application in studying the ischemic penumbra in animal models, and correlating this with similar studies in humans.

The transcription factors hypoxia-inducible factor 1alpha and Ets-1 colocalize in the hypoxic synovium of inflamed joints in adjuvant-induced arthritis.

To determine the relationship between hypoxia and the expression of Ets-1 and hypoxia-inducible factor 1alpha (HIF-1alpha) in both normal and inflamed joints. Adjuvant-induced arthritis (AIA) was used as the model system, since it mirrors many aspects of the pathology of rheumatoid arthritis. Adjuvant arthritis was induced in a group of 10 female Lewis rats. A second group of 10 uninjected female Lewis rats served as naive controls. When a maximum clinical joint score was achieved in the AIA group, all 20 rats were injected with the specific hypoxic cell marker Hypoxyprobe-1 and subsequently killed. Hypoxyprobe-1 adducts, Ets-1, and HIF-1alpha were localized in the joints of the hind feet from these groups using immunohistochemistry. Compared with the joints from control rats, inflamed joints contained markedly more cells with Hypoxyprobe-1 adduct immunoreactivity, Ets-1-immunoreactive nuclei, and nuclear immunoreactivity for both Ets-1 and HIF-1alpha. Our results demonstrate the presence of hypoxia in inflamed joints in this experimental model of arthritis. The colocalization of Ets-1 and HIF-1alpha in these hypoxic areas suggests that hypoxia may induce Ets-1 and HIF-1alpha expression during joint inflammation.

Preparation of fluorine‐18‐labelled fluoromisonidazole using two different synthesis methods

Abstract18F‐labelled fluoromisonidazole [1H‐1‐(3‐[18F]fluoro‐2‐hydroxypropyl)‐2‐nitroimida‐zole; ([18F]FMISO)] is used as an in vivo marker of hypoxic cells in tumours and ischaemic areas of the heart and the brain. The compound plays an important role in evaluating the oxygenation status in tumours during radiotherapy. In this paper, we report experiments carried out in our laboratory in synthesizing [18F]FMISO using two different methods. The first method (I) for the [18F]FMISO synthesis was the fluorination of (2R)‐(−)‐glycidyl tosylate to [18F]epifluorohydrin. The subsequent nucleophilic ring opening, achieved with 2‐nitroimidazole, leads to labelled FMISO. The second method (II) was the fluorination of the protected precursor 1‐(2′‐nitro‐1′‐imidazolyl)‐2‐O‐tetrahydropyranyl‐3‐O‐toluenesulphonyl‐propanediol, followed by a rapid removal of the protecting group. With the first method, the radiochemical yield was about 10% at the end of the synthesis (EOS), and the radiochemical purity was over 99%. The radiochemical yield in the second method was 21% (EOS) on an average, and the radiochemical purity was over 97%. When an automated commercial synthesis module was used with method II, slightly better and more reproducible yields were achieved. The improvement in the synthesis yield with the automated apparatus will be valuable when working with high activities, and therefore it is under further development. Copyright © 2003 John Wiley &amp; Sons, Ltd.

Vascular architecture, hypoxia, and proliferation in first-generation xenografts of human head-and-neck squamous cell carcinomas

Purpose: To quantify the physiologic status of human tumor cells in relation to the tumor vasculature. Methods and Materials: Fourteen tumors of 11 first-generation xenograft lines of human head-and-neck squamous cell carcinoma were injected with the hypoxic cell marker pimonidazole, the proliferation marker BrdUrd, and the perfusion marker Hoechst 33342. Consecutive tissue sections were processed with immunohistochemical methods and analyzed with image-analysis techniques. Results: Three different hypoxic patterns were found: patchy, ribbon-like, and mixed. An image-analysis method was developed to quantify these, and an elongation index (length/width) was calculated for hypoxia. The mean elongation indices ranged from 2.0 to 28.3 and showed a good correlation with the visual scoring of hypoxic patterns. Comparative analysis of hypoxic and proliferating cells in zones around the tumor vasculature showed the presence of both hypoxic and proliferating cells in all zones up to 250 μm from the vessels. The largest coexistence of hypoxic and proliferating cells seemed to occur at 50–100 μm from the vessels. Conclusions: The three hypoxic patterns could be quantified by an elongation index, which is an additional parameter that allows distinction of tumors with similar fractions of hypoxic cells. The analysis of hypoxic and proliferating cells as a function of distance from the tumor vasculature indicates that proliferation does occur also at low oxygen tensions.

Changes in tumor hypoxia measured with a double hypoxic marker technique

Purpose: Development of a double hypoxic cell marker assay, using the bioreductive nitroimidazole derivatives CCI-103F and pimonidazole, to study changes in tumor hypoxia after treatments that modify tumor oxygenation. Methods and Materials: Both hypoxic markers were visualized by immunohistochemical techniques to detect changes in hypoxic fraction induced by carbogen breathing (95% O 2 and 5% CO 2) or hydralazine injection. The protocol was tested in a human laryngeal squamous cell carcinoma xenograft line. Quantitative measurements were derived from consecutive tissue sections that were analyzed by a semiautomatic image analysis system. Qualitative analysis was obtained by double staining of the two hypoxic markers on the same tissue section. Results: A significant correlation between the hypoxic fractions for the two markers, CCI-103F and pimonidazole, was found in air breathing animals. After carbogen breathing, the hypoxic fraction decreased significantly from 0.07 to 0.03, and after hydralazine treatment, the hypoxic fraction increased significantly. Reduction of hypoxia after carbogen breathing was most pronounced close to well-perfused tumor regions. Conclusions: With this method, employing two consecutively injected bioreductive markers, changes in tumor hypoxia can be studied. A significant reduction in hypoxia after carbogen breathing and a significant increase in hypoxia after hydralazine administration was demonstrated.

Optical sensor-based oxygen tension measurements correspond with hypoxia marker binding in three human tumor xenograft lines.

Hypoxia has a negative effect on the outcome of radiotherapy and surgery and is also related to an increased incidence of distant metastasis. In this study, tumor pO(2) measurements using a newly developed time-resolved luminescence-based optical sensor (OxyLitetrade mark) were compared with bioreductive hypoxia marker binding (pimonidazole). Single pO(2) measurements per tumor were compared to hypoxia marker binding in tissue sections using image analysis. Both assays were performed in the same tumors of three human tumor lines grown as xenografts. Both assays demonstrated statistically significant differences in the oxygenation status of the three tumor lines. There was also a good correlation between hypoxia marker binding and the pO(2) measurements with the OxyLitetrade mark device. A limitation of the OxyLitetrade mark system is that it is not yet suited for sampling multiple sites in one tumor. An important strength is that continuous measurements can be taken at the same position and dynamic information on the oxygenation status of tumors can be obtained. The high spatial resolution of the hypoxia marker binding method can complement the limitations of the OxyLitetrade mark system. In the future, a bioreductive hypoxic cell marker for global assessment of tumor hypoxia may be combined with analysis of temporal changes in pO(2) with the OxyLitetrade mark to study the effects of oxygenation-modifying treatment on an individual basis.

Effects of nicotinamide and carbogen on oxygenation in human tumor xenografts measured with luminescense based fiber-optic probes

Background and purpose: In head and neck cancer, addition of both carbogen breathing and nicotinamide to accelerated fractionated radiotherapy showed increased loco-regional control rates. An assay based on the measurement of changes in tumor pO 2 in response to oxygenation modification could be helpful for selecting patients for these new treatment approaches. Materials and methods: The fiber-optic oxygen-sensing device, OxyLite™, was used to measure changes in pO 2, at a single position in tumors, after treatment with nicotinamide and carbogen in three human xenograft tumor lines with different vascular architecture and hypoxic patterns. Pimonidazole was used as a marker of hypoxia and was analyzed with a digital image processing system. Results: At the position of pO 2 measurement, half of the tumors showed a local increase in pO 2 after nicotinamide administration. Steep increases in pO 2 were measured in most tumors during carbogen breathing although the increase was less pronounced in tumor areas with a low pre-treatment pO 2. A trend towards a faster local response to carbogen breathing for nicotinamide pre-treated tumors was found in all three lines. There were significant differences in hypoxic fractions, based on pimonidazole binding, between the three tumor lines. There was no correlation between hypoxic marker binding and the response to carbogen breathing. Conclusion: Temporal changes in local pO 2 can be measured with the OxyLite™. This system was used to quantitate the effects of oxygen modifying treatments. Rapid increases in pO 2 during carbogen breathing were observed in most tumor areas. The locally measured response to nicotinamide was smaller and more variable. Bio-reductive hypoxic cell marker binding in combination with OxyLite™ pO 2 determination gives spatial information about the distribution patterns of tumor hypoxia at the microscopic level together with the possibility to continuously measure changes in pO 2 in specific tumor areas.

A Novel Amine−Dioxime Chelator for Technetium-99m: Synthesis and Evaluation of 2-Nitroimidazole-Containing Analogues as Markers for Hypoxic Cells

A novel amine-dioxime chelator for (99m)Tc has been developed. It offers the advantages of ease of synthesis and flexibility in alteration of lipophilicity. Labeling by stannous reduction of pertechnetate takes place rapidly and efficiently at room temperature and is stable for 24 h. The (99m)Tc:ligand ratio is believed to be 1:2. Seven different alkyl moieties were used to achieve a range of lipophilicities. Three series of compounds were prepared: 2-nitroimidazoles as potential hypoxia-targeting agents, 4-nitroimidazoles as a less easily reduced isomer, and untargeted anilines. In an in vitro model of cellular hypoxia, the 2-nitroimidazole compounds all showed selective accumulation whereas 4-nitroimidazoles showed variable selectivity and aniline showed no selectivity. These experiments demonstrate the potential utility of the 2-nitroimidazole derivatives of the amine-dioxime class of chelator as hypoxia-targeting agents.

The constitutive level of vascular endothelial growth factor (VEGF) is more important than hypoxia-induced VEGF up-regulation in the angiogenesis of human melanoma xenografts.

Angiogenesis of tumours might develop as a result of environmental conditions, such as hypoxia, and/or as a result of genetic alterations specific for tumour cells. The relative contributions of these mechanisms were investigated by comparing the in vivo expression of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) to the hypoxic fraction, the angiogenic potential and the vascular density of four human melanoma lines (A-07, D-12, R-18, U-25) grown intradermally in Balb/c nu/nu mice. VEGF expression, bFGF expression and expression of pimonidazole, a marker of hypoxic cells, were investigated by immunohistochemistry. An association between high VEGF and bFGF expression and high angiogenic potential was detected, suggesting an important role for VEGF/bFGF in the angiogenesis of melanomas. High VEGF/bFGF expression was also related to low hypoxic fraction and high vascular density. Thus, the constitutive, genetically determined level of VEGF was probably more important than hypoxia-induced upregulation in the angiogenesis of the melanoma xenografts. © 2000 Cancer Research Campaign

Splanchnic tissues undergo hypoxic stress during whole body hyperthermia.

Exposure of conscious animals to environmental heat stress increases portal venous radical content. The nature of the observed heat stress-inducible radical molecules suggests that hyperthermia produces cellular hypoxic stress in liver and intestine. To investigate this hypothesis, conscious rats bearing in-dwelling portal venous and femoral artery catheters were exposed to normothermic or hyperthermic conditions. Blood gas levels were monitored during heat stress and for 24 h following heat exposure. Hyperthermia significantly increased arterial O2 saturation, splanchnic arterial-venous O2 difference, and venous PCO2, while decreasing venous O2 saturation and venous pH. One hour after heat exposure, liver glycogen levels were decreased approximately 20%. Two hours after heat exposure, the splanchnic arterial-venous O2 difference remained elevated in heat-stressed animals despite normal Tc. A second group of rats was exposed to similar conditions while receiving intra-arterial injections of the hypoxic cell marker [3H]misonidazole. Liver and intestine were biopsied, and [3H]misonidazole content was quantified. Heat stress increased tissue [3H]misonidazole retention 80% in the liver and 29% in the small intestine. Cellular [3H]misonidazole levels were significantly elevated in intestinal epithelial cells and liver zone 2 and 3 hepatocytes and Kupffer cells. This effect was most prominent in the proximal small intestine and small liver lobi. These data provide evidence that hyperthermia produces cellular hypoxia and metabolic stress in splanchnic tissues and suggest that cellular metabolic stress may contribute to radical generation during heat stress.

Comparison of two techniques for detecting tumour hypoxia: a fluorescent immunochemical method and an in vitro colony assay

The aim of this study was to compare the percentage of hypoxic cells obtained with two methods: an in vitro colony assay and a new method based on immunodetection of a marker for hypoxic cells (NITP) which could be used in patients. These studies have been carried out using one rodent tumour EMT6 (a mammary carcinoma) and one human tumour HRT18 (a rectal adenocarcinoma). The hypoxic cell fraction was assessed in control mice and in mice receiving two treatments: 250 mg/kg nicotinamide + carbogen, and 250 mg/kg nicotinamide + carbogen + 4 ml/kg perflubron emulsion. The two treatments increased the radiosensitivity of the two cell lines, nicotinamide plus carbogen plus perflubron emulsion having the greatest radiosensitising effect. For untreated and treated tumours, the percentage of hypoxic cells obtained with the in vitro colony assay were comparable to those obtained with immunodetection using NITP. Whatever the treatment, NITP detection was a convenient test to detect the hypoxic cell fraction in the two solid tumours we have studied.