Radiobiological hypoxia, oxygen tension, interstitial fluid pressure and relative viable tumour area in two human squamous cell carcinomas in nude mice during fractionated radiotherapy

Very little is known about the correlation between the radiobiological hypoxic fraction (rHF) and other measures of tumour oxygenation during fractionated irradiation. In the present study the rHF is determined in untreated human FaDu and GL squamous cell carcinoma in nude mice and in tumours irradiated with 10 fractions in 2 weeks and 20 fractions in 4 weeks, using tumour control as the experimental endpoint. The results were compared with measurements of the pO2, the interstitial fluid pressure (IFP) and the relative viable tumour area. In FaDu tumours the radiobiological hypoxic fractions (rHFs) before and during irradiation were not statistically different from 100%. Depending on the assumptions made for D0, the rHFs of GL tumours were between 0.2 and 4% or 30 and 53%. The median pO2 values were 2.8 mmHg for untreated FaDu tumours and 0.2 mmHg for GL tumours (p < 0.001). The median IFP values were 2.6 mmHg in FaDu and 5.3 mmHg in GL tumours (p = 0.01). No important changes in the pO2 and IFP values were observed during fractionated irradiation. The relative viable tumour area during irradiation decreased by 83% in FaDu tumours (p = 0.002) and by 54% in GL tumours (p = 0.003). It is concluded that differences in rHF exist between FaDu and GL tumours before and during fractionated irradiation and that these differences are not reflected by pO2 and IFP values and the relative viable tumour area.     

Detection of hypoxic cells in a C3H mouse mammary carcinoma using the comet assay.

The comet assay was used to estimate radiobiological hypoxic fraction across a full range of tumour oxygenations in C3H mammary tumours implanted into the feet of female CDF1 mice. Tumours were either clamped before irradiation or mice were allowed to breath air, 100% oxygen, carbogen or carbon monoxide for 5-35 min before and during exposure to 15 Gy. For the alkaline comet assay, tumours were excised after irradiation and individual tumour cells were analysed for DNA single-strand breaks. Hypoxic cells were defined as those cells with approximately three times fewer single-strand breaks than aerobic cells. Radiobiological hypoxic fraction was calculated by fitting DNA damage histograms to two normal distributions, representing the response of the aerobic and hypoxic populations. The percentage of hypoxic cells estimated using the comet assay was then compared with hypoxic fraction measured using a clamped tumour control assay. Carbogen and oxygen breathing reduced the normal hypoxic fraction from 14% to 2-3% in this tumour, whereas 75-660 p.p.m. carbon monoxide progressively increased the hypoxic fraction from 18% to 82%. The slope of the line comparing the two methods was 1.23 with 95% confidence limits of 1.12-1.33 (r2 = 0.994). In the SCCVII squamous cell carcinoma growing subcutaneously in C3H mice, a similar correlation was observed between hypoxic fraction measured using the comet assay and hypoxic fraction measured in the same tumour cells using the paired survival curve assay (slope = 1.20 with 95% confidence limits of 1.03-1.37). These results confirm the ability of the comet assay to provide an accurate estimate of radiobiological hypoxic fraction over a wide range of tumour oxygenations and between two tumour types.     

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 microm 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.     

Relationship Between Tumour Oxygenation, Bioenergetic Status and Radiobiological Hypoxia in an Experimental Model

Tumour oxygenation and bioenergetic status were measured in the same tumour and these results related to radiobiological hypoxia. A C3H mouse mammary carcinoma grown in the feet of CDF1 mice was used. Bioenergetic status was assessed by ³¹P MRS using a SISCO 7 Tesla magnet, oxygen measurements were done by a polarographic electrode and the hypoxic fraction was determined from direct analysis of the radiation dose-response data. During all examinations restrained, non-anaesthetized mice were allowed to breathe either 100% oxygen, carbogen, normal air, carbon monoxide (CO) at 75, 220, or 660 ppm or had blood flow occluded by clamping. Results showed a significant correlation between the radiobiological hypoxic fraction and % pO₂ ≤5 mmHg under the different treatment conditions, whereas no correlation was found between beta nucleosidetriphosphate/inorganic phosphate (β-NTP/Pi) ratio and either the hypoxic fraction or the % of pO₂ values ≤5 mmHg under the different treatment conditions. In conclusion, oxygen electrode measurements were sensitive to changes in tumour hypoxia whereas the bioenergetic status alone seemed to be a less precise measure of hypoxia in this tumour model. Furthermore, the present study demonstrated that tumour cells in vivo can actually maintain the bioenergetic status during a period of severe hypoxia.     

Preclinical assessment of hypoxic marker specificity and sensitivity

Purpose: In the search for a sensitive, accurate, and noninvasive technique for quantifying human tumor hypoxia, our laboratory has synthesized several potential radiodiagnostic agents. The purpose of this study was to assess and compare the hypoxic marking properties of both radioiodinated and Tc-99m labeled markers in appropriate test systems which can predict for in vivo activity.Materials and Methods: Preclinical assessment of hypoxic marker specificity and sensitivity employed three laboratory assays with tumor cells in vitro and in vivo. Radiolabeled marker uptake and/or binding to whole EMT-6 tumor cells under extremely hypoxic and aerobic conditions was measured and their ratio defined hypoxia-specific factor (HSF). Marker specificity to hypoxic tumor tissue was estimated from its selective avidity to two rodent tumors in vivo, whose radiobiologic hypoxic fractions (HF) had been measured. The ratios of % injected dose/gram (%ID/g) of marker at various times in EMT-6 tumor tissue relative to that in the blood and muscle of scid mice were used to quantify hypoxia-specific activity. This tumor in this host exhibited an average radiobiologic HF of ∼35%. As well, nuclear medicine images were acquired from R3327-AT (HF ≃15%) and R3327-H (no measurable HF) prostate carcinomas growing in rats to distinguish between marker avidity due to hypoxia versus perfusion.Results: The HSF for FC-103 and other iodinated markers were higher (5–40) than those for FC-306 and other Tc-99m labeled markers. The latter did not show hypoxia-specific uptake into cells in vitro. Qualitative differences were observed in the biodistribution and clearance kinetics of the iodinated azomycin nucleosides relative to the technetium chelates. The largest tumor/blood (T/B) and tumor/muscle (T/M) ratios were observed for compounds of the azomycin nucleoside class in EMT-6 tumor-bearing scid mice. These markers also showed a 3–4 × higher uptake into R3327-AT tumors relative to the well-perfused R3327-H tumors. While both FC-306 and CERETEC® rapidly distributed at unique concentrations to different tissues, their avidity to EMT-6 and R3327-AT tumors did not correlate with tumor HF.Conclusions: The halogenated azomycin nucleosides with the lowest lipid/water partition coefficient values were found to yield the optimal hypoxia-specific signal in these animal tumors. Our Tc-99m-labeled azomycin chelates showed little or no hypoxia-specific uptake and had in vivo biodistribution and clearance kinetics similar to those of CERETEC®, a perfusion agent with no known hypoxic binding activity.     

Conversion of xanthine dehydrogenase to xanthine oxidase as a possible marker for hypoxia in tumours and normal tissues

The enzyme activities of endogenous xanthine dehydrogenase (XDH) and xanthine oxidase (XO) have been measured in 10 different types of mouse tumour and seven normal tissues. The conversion of XDH to XO has been observed in two tumour types upon the prolonged clamping off of the blood supply to the tumours. It is proposed that a similar conversion might also occur naturally in chronically hypoxic cells and that the ratio of the XO activity to the combined XO + XDH activities (%XO activity) could well serve as a marker for tissue hypoxia. A qualitative relationship exists between the %XO activity and literature values of the hypoxic fraction for some tumours measured by radiobiological assays. The influence of tumour size (about 0.2-1.8 g) on %XO activity is presented for all 10 tumours as well as %XO activity determinations for four of the normal tissues.     

Hypoxia in larynx carcinomas assessed by pimonidazole binding and the value of CA-IX and vascularity as surrogate markers of hypoxia

Tumour hypoxia as driving force in tumour progression and treatment resistance has been well established. Assessment of oxygenation status of tumours may provide important prognostic information and improve selection of patients for treatment. In this study, a large homogenous group of 103 laryngeal carcinomas has been investigated in the presence of hypoxia by pimonidazole binding and the usefulness of Carbonic anhydrase IX (CA-IX) and vascular parameters as surrogate markers of hypoxia. These parameters are further related to clinical and biological characteristics.One hundred and three patients with T2–T4 larynx carcinoma were included. They were given the hypoxia marker pimonidazole intravenously (i.v.) 2 h prior to taking a biopsy. Expression of all the parameters was examined by immunohistochemistry, excluding large necrotic areas. Among tumours a large variation in pimonidazole positivity (hypoxic fraction based on pimonidazole, HFpimo) (range 0–19%) and CA-IX expression (hypoxic fraction based on CA-IX staining, HFCA-IX) (range 0–34%) was observed. In 67% of the tumours, hypoxia involved 1% of the viable tumour area. HFpimo and HFCA-IX correlated significantly albeit weak (p = 0.04). Both parameters showed weak inverse correlations with the relative vascular area (RVA) (p = 0.01). HFpimo was further associated with histopathological grade, with poorly differentiated tumours being more hypoxic. The fraction of the tumour area positive for both pimonidazole and CA-IX correlated significantly with N stage.From these results, it was concluded that CA-IX and RVA have only limited value for measuring hypoxia and are not as robust as pimonidazole, probably due to the influence of other factors in the microenvironment. A combination of staining patterns of exogenous and endogenous markers might give important additive information about tumour biology and behaviour.     

Effect of induced host anaemia on the viability and radiosensitivity of murine malignant cells in vivo

Within 48 hours of the institution of severe phenylhydrazineinduced anaemia in mice bearing ascites tumours or generalised leukaemia, a substantial proportion of the malignant cells disappeared respectively from the peritoneal cavity or infiltrated liver. The results of radiobiological experiments permitting determination of the proportion of viable leukaemia cells which were severely hypoxic and relatively radioresistant in the livers of leukaemic mice, showed that induction of anaemia was associated with a several hundredfold increase in the proportion of such cells. The proportion of hypoxic cells was greatly reduced when the anaemic leukaemic mice were transfused with packed erythrocytes or allowed to breathe oxygen under high pressure. Similar experi - ments with solid sarcomas indicated that a high proportion of the tumour cells were hypoxic in non-anaemic mice breathing air. The hypoxic fraction was not significantly reduced when tumour-bearing mice were made severely anaemic during growth of the tumour and were later transfused. Thus, the hypoxic cells in leukaemic livers and those in solid tumours are markedly different in their capacity for oxygenation following the induction of relative hyperoxaemia.     

Fraction of radiobiologically hypoxic cells in human melanoma xenografts measured by using single-cell survival, tumour growth delay and local tumour control as end points.

Four human melanoma xenograft lines (A-07, D-12, R-18, U-25) grown orthotopically in Balb/c nu/nu mice were characterized with respect to the fraction of radiobiologically hypoxic cells. The purpose of the study was to establish a firm radiobiological basis for future use of the lines in the development and evaluation of non-invasive assays of tumour hypoxia. The hypoxic fractions were assessed using three different assays, the single cell survival assay, the tumour growth delay assay and the local tumour control assay, and the means +/- s.e. were found to be 6 +/- 3%, 3 +/- 1% and 5 +/- 2% respectively (A-07), 26 +/- 5%, 25 +/- 6% and 22 +/- 6% respectively (D-12), 55 +/- 9%, 65 +/- 8% and 48 +/- 7% respectively (R-18) and 52 +/- 8%, 59 +/- 7% and 47 +/- 7% respectively (U-25). The three assays gave numerical values for the hypoxic fraction that were not significantly different for any of the lines. The hypoxic fraction differed significantly among the lines; the R-18 and U-25 lines showed higher hypoxic fractions than the D-12 line (P < 0.05), which in turn showed a higher hypoxic fraction than the A-07 line (P < 0.05), regardless of the assay. The wide range of the hypoxic fractions and the significant differences among the lines suggest that A-07, D-12. R-18 and U-25 tumours should be useful models in future studies attempting to develop non-invasive assays of tumour hypoxia.     

Non-invasive assessment of human tumour hypoxia with 123I-iodoazomycin arabinoside: preliminary report of a clinical study.

Non-invasive predictive assays which can confirm the presence or absence of hypoxic cells in human tumours show promise for understanding the natural history of tumour oxygenation, and improving the selection of patient subsets for novel radiotherapeutic strategies. Sensitiser adducts have been proposed as markers for hypoxic cells. Misonidazole analogues radiolabelled with iodine-123 have been developed for the detection of tumour hypoxia using conventional nuclear medicine techniques. In this pilot study, we have investigated one such potential marker, 123I-iodoazomycin arabinoside (123I-IAZA). Patients with advanced malignancies have undergone planar and single-photon emission computed tomographic (SPECT) imaging after intravenous administration of 123I-IAZA. We have observed radiotracer avidity in three out of ten tumours studied to date. Normal tissue activity of variable extent was also seen in the thyroid and salivary glands, upper aerodigestive tract, liver, intestine, and urinary bladder. Quantitative analysis of those images showing radiotracer avidity revealed tumour/normal tissue (T/N) ratios of 2.3 (primary small cell lung carcinoma), 1.9 (primary malignant fibrous histiocytoma) and 3.2 (brain metastasis from small cell lung carcinoma) at 18-24 h post injection. These preliminary data suggest that the use of gamma-emitter labelled 2-nitroimidazoles as diagnostic radiopharmaceuticals is feasible and safe, and that metabolic binding of 123I-IAZA is observed in some, but not all tumours. The inference that tumour 123I-IAZA avidity could be a non-invasive measure of tumour hypoxia deserves independent confirmation with needle oximetry.     

Intraoperative detection of somatostatin-receptor-positive neuroendocrine tumours using indium-111-labelled DTPA-D-Phe1-octreotide.

After injection of 111In-labelled DTPA-D-Phe1-octreotide, intraoperative tumour localisation was performed using a scintillation detector in 23 patients with neuroendocrine tumours. Count rates from suspect tumour lesions and adjacent normal tissue were expressed as a ratio before (Rin situ) and after (Rex vivo) excision. 111In activity concentration ratios of tumour tissue to blood (T/B) were determined in a gamma counter. In patients with midgut carcinoids, (all scintigraphy positive), false Rin situ recordings were found in 4/29 macroscopically identified tumours. T/B ratios were all high (27-650). In patients with medullary thyroid carcinomas (eight out of ten scintigraphy positive), misleading Rin situ results were found in 4/37 macroscopically identified tumours. T/B ratios were lower (3-39) than those seen in midgut carcinoids. Two out of four patients with endocrine pancreatic tumours had positive scintigraphy, reliable intraoperative measurements and very high T/B ratios (910-1500). One patient with a gastric carcinoid had correct measurements in situ and ex vivo with high T/B ratios (71-210). In situ measurements added little information to preoperative scintigraphy and surgical findings using the present detection system. Rex vivo measurements were more reliable. The very high T/B ratios seen in midgut carcinoids and some endocrine pancreatic tumours would be favourable for future radiation therapy via somatostatin receptors.     

Measuring hypoxia in solid tumours--is there a gold standard?

Tumour hypoxia is known to be associated with aggressiveness and poor response to treatment, which has stimulated the development of several methods able to detect hypoxic tumours. To date, only one method, the oxygen microelectrode, has been used to provide pretreatment measures of tumour oxygenation that correlate with local control and disease-free survival. In an effort to validate new methods, comparisons have been made between the Eppendorf oxygen microelectrode, the comet assay, and hypoxia marker binding in tumours of patients undergoing curative treatment or palliative radiotherapy. These comparisons suggest that tumours with median oxygen tensions below 10 mmHg have relatively high hypoxic fractions as measured by the comet assay (> 0.20). The fraction of cells that binds pimonidazole, detected in cells obtained by fine-needle aspiration biopsy, correlates well with the hypoxic fraction measured using the comet assay. However, in general, hypoxic fractions measured by the comet assay and pimonidazole binding correlate only poorly with Eppendorf measurements performed for the same tumour. Factors that might be responsible for these differences, and problems associated with measuring the 'relevant' hypoxic population are discussed.     

Is tumour radiosensitization by misonidazole a general phenomenon?

The response of 14 mouse tumour sub-lines to the radiosensitizing action of a large single dose of misonidazole (MISO) has been assessed by regrowth delay. In 13 of these, significant enhancement of radiation effect occurred under ambient conditions, indicating sensitization of naturally hypoxic cells. The enhancement observed (SER'') varied with the radiation dose, as would be predicted for a mixed oxic/hypoxic cell population. The maximum SER'' in these 13 tumours did not depend on histology or regrowth rate. The 14th tumour, a slow-growing sarcoma, was not sensitized under ambient conditions, but showed marked sensitization when clamped to produce acutely hypoxic cells. This is consistent with no hypoxic cells occurring naturally in a sarcoma with a slow rate of growth. Faster-growing variants of this tumour showed radiosensitization under ambient conditions. The slow-growing carcinoma, RH, however, appears to contain hypoxic cells and did show sensitization. The cytotoxic action of MISO was compared with the radiosensitization by administering it after irradiation in 8 of the tumour lines. In 2 tumours no cytotoxicity was observed. In the rest cytotoxicity was significant, but much smaller than the sensitization observed when MISO was administered before irradiation. These regrowth-delay data have been used to calculate hypoxic fractions in 3 ways. Estimates of hypoxic fraction ranged from less than 0.1% in the slow sarcoma to greater than or equal to 30% in several tumours. There is considerable variation in the estimate, according to the technique used.     

Correlations between in vivo tumor weight,oxygen pressure, 31P NMR spectroscopy,hypoxic microenvironment marking by beta-D-iodinated azomycin galactopyranoside (beta-D-IAZGP), and radiation sensitivity

PURPOSE: The purpose of this study is to evaluate the amount of hypoxic fraction in a rodent tumor by means of polarographic oxygen electrode, phosphorus-31 magnetic resonance spectroscopy (31P-MRS), and a newly synthesized hypoxic marker, beta-D-iodinated azomycin galactopyranoside (beta-D-IAZGP). We also investigated the radiosensitivity for tumors of different weights. METHODS AND MATERIALS: Murine mammary carcinoma cells, FM3A, were subcutaneously implanted into the back of 5-week-old male C3H/He mice. beta-D-IAZGP radiolabeled with 123I or with 125I was injected intravenously into tumor-bearing mice, and marker distribution was measured by nuclear medicine procedures. Radiosensitivity of the tumor was measured by the in vivo/in vitro clonogenic assay. Tumor oxygenation status was also measured directly by polarographic oxygen electrodes and indirectly estimated from 31P-MR spectra. RESULTS: Higher accumulation of 123I-beta-D-IAZGP was observed in the tumors than in normal tissues at 24 h after administration. As to biodistribution of 125I-beta-D-IAZGP, the tumor/blood ratio varied widely, but correlated significantly with tumor weight. Mean oxygen pressure (pO2) values and ratios of nucleoside triphosphate beta to inorganic phosphate (beta-ATP/Pi) and of phosphocreatine to inorganic phosphate (PCr/Pi) decreased significantly with the increase in tumor volume. As tumor volume increased, the surviving fraction of cells from tumors irradiated in vivo increased significantly. CONCLUSIONS: The increase in tumor volume was significantly correlated with a reduction in mean pO2, a decrease in the ratios of beta-ATP/Pi or PCr/Pi, an increase in uptake of beta-D-IAZGP, and an increase in radioresistance. Because the uptake of beta-D-IAZGP can be measured noninvasively by nuclear medicine techniques, it could be clinically useful for monitoring hypoxia in human tumors.     

In vivo evaluation of [18F]fluoroetanidazole as a new marker for imaging tumour hypoxia with positron emission tomography

Development of hypoxia-targeted therapies has stimulated the search for clinically applicable noninvasive markers of tumour hypoxia. Here, we describe the validation of [(18)F]fluoroetanidazole ([(18)F]FETA) as a tumour hypoxia marker by positron emission tomography (PET). Cellular transport and retention of [(18)F]FETA were determined in vitro under air vs nitrogen. Biodistribution and metabolism of the radiotracer were determined in mice bearing MCF-7, RIF-1, EMT6, HT1080/26.6, and HT1080/1-3C xenografts. Dynamic PET imaging was performed on a dedicated small animal scanner. [(18)F]FETA, with an octanol-water partition coefficient of 0.16+/-0.01, was selectively retained by RIF-1 cells under hypoxia compared to air (3.4- to 4.3-fold at 60-120 min). The radiotracer was stable in the plasma and distributed well to all the tissues studied. The 60-min tumour/muscle ratios positively correlated with the percentage of pO(2) values <5 mmHg (r=0.805, P=0.027) and carbogen breathing decreased [(18)F]FETA-derived radioactivity levels (P=0.028). In contrast, nitroreductase activity did not influence accumulation. Tumours were sufficiently visualised by PET imaging within 30-60 min. Higher fractional retention of [(18)F]FETA in HT1080/1-3C vs HT1080/26.6 tumours determined by dynamic PET imaging (P=0.05) reflected higher percentage of pO(2) values <1 mmHg (P=0.023), lower vessel density (P=0.026), and higher radiobiological hypoxic fraction (P=0.008) of the HT1080/1-3C tumours. In conclusion, [(18)F]FETA shows hypoxia-dependent tumour retention and is, thus, a promising PET marker that warrants clinical evaluation.     

Radiobiological Hypoxia in the KHT Sarcoma: Predictions Using the Eppendorf Histograph

Purpose: To investigate whether electrode measurements of tumor oxygenation obtained under a range of different treatment conditions designed to alter the degree of tumor hypoxia could be correlated with estimates of radiobiological hypoxia measured under the same conditions.

Methods and Materials: Experiments were performed in restrained, nonanesthetized, female C3H/He mice, which had 0.5 g KHT sarcomas growing intramuscularly in the hind limbs. The treatments used to modify tumor oxygenation status included breathing gas mixtures of varying oxygen content, altering tumor blood flow, and shifting the hemoglobin oxygen dissociation curve. Radiobiological hypoxic fraction was estimated using the paired survival curve assay, while electrode measurements of tumor oxygenation were obtained with an Eppendorf histograph.

Results: With the selected manipulations it was possible to vary the radiobiological hypoxic fraction in the tumors from 1 to 100% of the total viable cell population. Furthermore, these changes in radiation response were directly reflected in the changes in tumor oxygenation measurements made with the Eppendorf histograph.

Conclusion: These findings suggest that in the KHT tumor model the Eppendorf electrode measurements could predict the response of the tumors to radiation as determined by the proportion of hypoxic cells.     

Enhancement of the cytotoxicity of SR 4233 to normal and malignant tissues by hypoxic breathing.

The bioreductive cytotoxic agent SR 4233 (1,2,4-benzotriazine 3-amine 1,4-dioxide) has been shown to markedly potentiate the cell killing of mouse tumours when combined with fractionated radiation therapy. Differential metabolism under oxic compared to hypoxic conditions results in SR 4233 exhibiting selective cytotoxicity to hypoxic cells. This is thought to result from the production of a cytotoxic free radical which is generated predominantly in the absence of oxygen. We have examined a way of enhancing the effectiveness of this antitumour agent in vivo by artificially increasing the hypoxic fraction of tumours by hypoxic breathing. Mice are placed in a chamber containing 10% Oxygen 90% Nitrogen for 1 h after each administration of SR 4233. Our results in the SCCVII tumour model indicate that this manoeuvre results in a 10-fold increase in antitumour effectiveness of SR 4233 when administered in a fractionated regime with radiotherapy (8 x 2.5 Gy and 0.08 mmol kg-1), but not when a single treatment regime (1 x 20 Gy and 0.3 mmol kg-1) is used. Mathematical modelling of this effect is used to illustrate this phenomenon and can be used to predict the dependence of this type of therapy on the modification of tumour oxygenation.     

Bioreductive drugs and the selective induction of tumour hypoxia

In this work tumour hypoxia is induced by physically occluding the tumour vascular supply by clamping, or by giving mice 5 mg kg-1 hydralazine. These methods have previously been shown to increase the radiobiological hypoxic fraction in tumours close to 100%. Their effectiveness in potentiating the bioreductive toxicity of: misonidazole (800 mg kg-1), RSU1069 (80 mg kg-1), mitomycin C (5 mg kg-1) and SR4233 (50 mg kg-1) is assessed in the RIF-1 and KHT tumours using regrowth delay as an assay. Clamping alone for 120 min gives little or no response, but when RSU1069 is administered 15 min before clamping, large growth delays result. RIF-1 tumours clamped for 90 or 120 min with RSU1069 give cure rates of 12.5% and 37.5% respectively. Less effect with clamping is seen for the other bioreductive agents. The effect of hydralazine with RSU1069 although significant in the RIF-1 tumour, is modest compared to that for clamping. Small enhancements of toxicity are seen with hydralazine in combination with misonidazole in the RIF-1 tumour and mitomycin C in both tumours. The varying effectiveness of these treatments is attributed to several factors which include the level and duration of hypoxia, concentration and contact time of the bioreductive drugs, the microenvironment of the tumour and the nature of the reductive metabolic pathways available in the different tumour cell types.     

Comparison between the comet assay and pimonidazole binding for measuring tumour hypoxia

Pimonidazole is finding increasing use in histochemical analyses of hypoxia in tumours. Whether it can identify every hypoxic cell in a tumour, and whether the usual subjective criteria used to define 'positive' cells are optimal, are less certain. Therefore, our aim was to develop an objective flow cytometry procedure for quantifying pimonidazole binding in tumours, and to validate this method by using a more direct indicator of radiobiologic hypoxia, the comet assay. SCCVII tumours in C3H mice were analysed for pimonidazole binding using flow cytometry and an iterative curve-fitting procedure, and the results were compared to the comet assay for the same cell suspensions. On average, cells defined as anoxic by flow analysis (n = 43 tumours) bound 10.8 +/- 0.95 times more antibody than aerobic cells. In samples containing known mixtures of aerobic and anoxic cells, hypoxic fractions as low as 0.5% could easily be detected. To assess the flow cytometry assay under a wider range of tumour oxygen contents, mice were injected with hydralazine to reduce tumour blood flow, or allowed to breathe various gas mixtures during the 90 min exposure to pimonidazole. Hypoxic fraction estimated by the pimonidazole binding method agreed well with the hypoxic fraction measured using the comet assay in SCCVII tumours (r2 = 0.87, slope = 0.98), with similar results in human U87 glioma cells and SiHa cervical carcinoma xenografts. We therefore conclude that this objective analysis of pimonidazole labelling by flow cytometry gives a convenient and accurate estimate of radiobiological hypoxia. Preliminary analyses of biopsies from 3 patients given 0.5 g m-2 pimonidazole also suggest the suitability of this approach for human tumours.