Comparison between the binding of [3H]misonidazole and AF-2 in Chinese hamster V79 spheroids

Binding of two hypoxia probes, [3H]misonidazole and AF-2 (2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide), was compared in Chinese hamster V79 spheroids incubated under different oxygen concentrations. Fluorescence-activated cell sorting based on Hoechst 33342 penetration was used to obtain populations of cells from different depths within the spheroid, and sorted cells were analyzed by cytofluorometry for AF-2 content and by liquid scintillation counting for [3H]misonidazole content. The patterns of AF-2 and misonidazole binding were very similar, with about 20-fold more localization of both drugs in anoxic compared to aerobic cells. Similar results were obtained when cells were sorted on the basis of AF-2 rather than Hoechst 33342 fluorescence. When mean cellular fluorescence of AF-2 was plotted versus cpm misonidazole/cell for different oxygen tensions, it appeared that oxygen was equally effective in inhibiting AF-2 and misonidazole binding. Internal cells of anoxic spheroids bound about twice as much AF-2 and misonidazole as external cells, apparently due to an increased rate of nitroreduction by chronically hypoxic cells. AF-2 was found to enhance the retention of misonidazole in oxic and hypoxic spheroids when both drugs were present.     

The radiation response of hypoxic cells in EMT6 spheroids in suspension culture does model data from EMT6 tumors

Radiation survival curves of EMT6/Ed spheroids have been obtained under conditions which eliminate changes in oxygen concentration between growth and irradiation. These curves show a high-dose, resistant component which is nearly parallel to the curves obtained when spheroids were irradiated under nitrogen. Thus EMT6 spheroids appear to model accurately the radiation responses of EMT6 tumors. In contrast, when spheroids were grown to relatively high density (300-400 spheroids per 250-ml spinner flask), then separated into several flasks for irradiation, an increase in oxygen concentration in the medium occurred which fully oxygenated the previously hypoxic cells. The two causes for the oxygen depletion in sealed growth flasks were quantitated. Depletion of total oxygen in the flask occurred, and, more importantly, oxygen consumption kept the growth medium well below equilibrium with the oxygen in the gas phase. Smaller but similar effects on oxygen concentration were found in flasks containing V79 spheroids.     

Sub-Sets of Cancer Stem Cells Differ Intrinsically in Their Patterns of Oxygen Metabolism

The glycolytic response of hypoxic cells is primarily mediated by the hypoxia inducible factor alpha (HIF-1α) but even in the presence of abundant oxygen tumours typically show high rates of glycolysis. Higher levels of HIF-1α in tumours are associated with a poorer prognosis and up-regulation of markers of epithelial mesenchymal transition (EMT) due to HIF-1α actions. We have recently shown that EMT occurs within the CD44^high cancer stem cell (CSC) fraction and that epithelial and EMT CSCs are distinguished by high and low ESA expression, respectively. We here show that hypoxia induces a marked shift of the CSC fraction towards EMT leading to altered cell morphology, an increased proportion of CD44^high/ESA^low cells, patterns of gene expression typical of EMT, and enhanced sphere-forming ability. The size of EMT fractions returned to control levels in normoxia indicating a reversible process. Surprisingly, however, even under normoxic conditions a fraction of EMT CSCs was present and maintained high levels of HIF-1α, apparently due to actions of cytokines such as TNFα. Functionally, this EMT CSC fraction showed decreased mitochondrial mass and membrane potential, consumed far less oxygen per cell, and produced markedly reduced levels of reactive oxygen species (ROS). These differences in the patterns of oxygen metabolism of sub-fractions of tumour cells provide an explanation for the general therapeutic resistance of CSCs and for the even greater resistance of EMT CSCs. They also identify potential mechanisms for manipulation of CSCs.     

Further characterization of 4-bromomisonidazole as a potential detector of hypoxic cells

[14C]Bromomisonidazole was prepared by direct bromination of [ring-2] [14C]misonidazole in dioxane. The uptake and binding of the two labeled sensitizers were compared in vitro in 1-mm EMT-6 spheroids which contain a necrotic core. Using liquid scintillation counting it was shown that spheroids incubated with 50 microM [14C]bromomisonidazole concentrated drug above levels in the medium by 1 1/2 hr and achieved maximum concentration by 10 hr with no further increase at 23 hr. Spheroids incubated with 50 microM [14C]misonidazole may concentrate the sensitizer more slowly but ultimately reached the same fivefold increase over levels in the medium by 23 hr as was observed for bromomisonidazole. Autoradiographs prepared from spheroids after incubation with [14C]misonidazole or [14C]bromomisonidazole showed silver grains preferentially located over viable hypoxic cells in the inner half of the spheroid rim adjacent to the necrotic center, with lower grain density over nonviable necrotic areas and many fewer grains over oxic cells at the periphery of the spheroid. The results indicate that both severely and moderately hypoxic cells may preferentially bind [14C]bromomisondiazole. The data support the potential of radiolabeled bromomisonidazole for in vivo imaging pending additional studies of the metabolism of this agent.     

Characterization of radiolabeled fluoromisonidazole as a probe for hypoxic cells

Radiolabeled fluoromisonidazole has been characterized as a probe for hypoxic cells in vitro and in vivo. The uptake and retention of [3H]fluoromisonidazole and [3H]misonidazole were compared in V-79 cell monolayers and spheroids by varying incubation time and O2 levels in contact with the medium. The two labeled drugs were retained similarly in cell populations isolated from different depths in spheroids, and the amount of each drug bound in cells at the spheroid periphery increased with decreasing O2 level. The labeling patterns in autoradiographs were similar for spheroids incubated with the two labeled drugs, with most silver grains located over a zone of viable and presumed hypoxic cells intermediate between the necrotic center and the periphery of the spheroid. Biodistribution of the two tritiated drugs was compared in C3H mice bearing KHT tumors with 15% radiobiologically hypoxic cells. Tumor:blood and tumor:muscle ratios greater than 5.0 were achieved in mice sacrificed 4 h after the last of three injections of 5 or 20 mumol/kg of [3H]fluoromisonidazole. These ratios are compatible with imaging and are higher than those obtained with 50 mumol/kg misonidazole in a similar administration protocol. TLC analysis of plasma from mice injected with [3H]fluoromisonidazole indicated that the drug was stable in vivo for up to 2 h and that the metabolites formed were too polar to be dehalogenation products. Fluoromisonidazole labeled with 18F at the end of the alkyl side chain would retain the label on metabolites that bind in hypoxic cells in vivo. Fluoromisonidazole binds stably in the same populations of hypoxic cells as does misonidazole, and we conclude that [18F]fluromisonidazole has potential use as a hypoxia imaging agent in vivo.     

The Role of Glycolysis and Hexose Monophosphate Pathway in the Hypoxic Toxicity of Misonidazole

The metabolic activation of misonidazole (MISO) and its effects on the hexose monophosphate pathway (HMP) and clonogenicity were studied in hypoxic EMT6/R0, wildtype Chinese hamster ovary (CHO) and mutant CHO cells deficient in glucose-6-phosphate dehydrogenase. In all three cell lines metabolic activation of MISO, as indicated by the binding of ^l4C-MISO to the acid-insoluble fraction of these cells, was increased by the presence of glucose. In EMT6/R0 cells and wildtype CHO cells, MISO caused a significant stimulation of the activity of the HMP while in the mutant CHO cells no HMP activity was measurable, even in the presence of MISO. Loss of clonogenicity induced by MISO occurred markedly earlier in EMT6/R0 cells than in the CHO cells. In the latter cells, however, only a small difference was observed between the wildtype and mutant cell line. From these results it is concluded that not only the HMP but also glycolysis and other, glucose-independent, metabolic pathways are able to provide electrons for the reductive activation of MISO and hence contribute to the hypoxic toxicity of this compound.     

The in vitro sensitivity of chronically hypoxic EMT6/SF cells to X-radiation and hypoxic cell radiosensitizers

We have investigated the effect of extreme, prolonged hypoxia on the radiosensitivity of EMT6/SF cells in vitro. As cells were kept hypoxic for 1-24 h, their radiosensitivity increased, but no further change was noted for hypoxic incubation beyond 24 h. Chronically hypoxic (45 h) cells were more radiosensitive than acutely hypoxic (1 h) cells by a factor of 1.43. When chronically hypoxic cells were re-aerated, the increased radiosensitivity persisted, although it was reduced. Misonidazole (MISO) radiosensitization was equally effective under conditions of acute and chronic hypoxia. In contrast, MISO, SR2555 and SR2508 were more cytotoxic in chronically hypoxic cultures than in acutely hypoxic cells. Measurements suggested that intracellular thiols may play an important role in the effects observed.     

Determining hypoxic fraction in a rat glioma by uptake of radiolabeled fluoromisonidazole

The usefulness of radiolabeled nitroimidazoles for measuring hypoxia will be clarified by defining the relationship between tracer uptake and radiobiologically hypoxic fraction. We determined the radiobiologically hypoxic fraction from radiation response data in 36B10 rat gliomas using the paired cell survival curve technique and compared the values to the radiobiologically hypoxic fraction inferred from mathematical modeling of time-activity data acquired by PET imaging of [(18)F]FMISO uptake. Rats breathed either air or 10% oxygen during imaging, and timed blood samples were taken. The uptake of [(3)H]FMISO by 36B10 cells in vitro provided cellular binding characteristics of this radiopharmaceutical as a function of oxygen concentration. The radiobiologically hypoxic fraction determined for tumors in air-breathing rats using the paired survival curve technique was 6.1% (95% CL = 4.3- 8.6%), which agreed well with that determined by modeling FMISO time-activity data (7. 4%; 95% CL = 2.5-17.3%). These results are consistent with the agreement between the two techniques for measuring radiobiologically hypoxic fraction in Chinese hamster V79 cell spheroids. In contrast, the FMISO-derived radiobiologically hypoxic fraction in rats breathing 10% oxygen was 13.1% (95% CL 7.9-8.3%), much lower than the radiobiologically hypoxic fraction of 43% determined from the radiation response data. This discrepancy may be due to the failure of FMISO to identify hypoxic cells residing at or above an oxygen level of 2-3 mmHg that will still confer substantial protection against radiation. The presence of transiently hypoxic cells in rats breathing reduced oxygen may also be under-reported by nitroimidazole binding, which is strongly dependent on time and concentration.     

The Role of Glycolysis and Hexose Monophosphate Pathway in the Hypoxic Toxicity of Misonidazole

The metabolic activation of misonidazole (MISO) and its effects on the hexose monophosphate pathway (HMP) and clonogenicity were studied in hypoxic EMT6/R0, wildtype Chinese hamster ovary (CHO) and mutant CHO cells deficient in glucose-6-phosphate dehydrogenase. In all three cell lines metabolic activation of MISO, as indicated by the binding of ^l4C-MISO to the acid-insoluble fraction of these cells, was increased by the presence of glucose. In EMT6/R0 cells and wildtype CHO cells, MISO caused a significant stimulation of the activity of the HMP while in the mutant CHO cells no HMP activity was measurable, even in the presence of MISO. Loss of clonogenicity induced by MISO occurred markedly earlier in EMT6/R0 cells than in the CHO cells. In the latter cells, however, only a small difference was observed between the wildtype and mutant cell line. From these results it is concluded that not only the HMP but also glycolysis and other, glucose-independent, metabolic pathways are able to provide electrons for the reductive activation of MISO and hence contribute to the hypoxic toxicity of this compound.     

Evaluation of various types of new hypoxic cell sensitizers using the EMT6 single cell-spheroid-solid tumour system

Eleven new hypoxic cell sensitizers representative of those developed in Japan between 1980 and 1985 were evaluated in vitro and in vivo in comparison with misonidazole (MISO), SR-2508, Ro 03-8799, and ANT (2-amino-5-nitrothiazole). The new compounds included 2-nitroimidazole nucleoside analogues, nitrotriazoles and other nitroaromatics, non-nitro compounds, and electron-affinic compounds that readily intercalate DNA. The sensitizing activity in the EMT6 single cells correlated not only with the reduction potential but, for some compounds, also with the reactivity with non-protein sulphydryls. The sensitizers were also tested using the EMT6 spheroids and solid tumours. The patterns of changes in sensitizer enhancement ratios (SERs) for single cells, spheroids, and solid tumours were classified into two types: (1) SERs for the three testing systems were similar; and (2) SERs decreased in the order of: single cells, spheroids, and solid tumours. Only nitroimidazole and nitrotriazole derivatives belonged to the former type. RK-28 and RK-29, 2-nitroimidazoles with sugar analogue components, had in vivo effects almost equal to those of MISO. Also 3- and 4-nitrotriazole derivatives had definite in vivo effects.     

The combined use of 131I-labeled antibody and the hypoxic cytotoxin SR 4233 in vitro and in vivo.

Radioimmunotherapy is hindered by the slow penetration of antibody molecules into tumors. Cells that are poorly targeted by antibody, because of their distance from feeding blood vessels, receive the lowest radiation dose, and this problem is compounded if there are radioresistant hypoxic cells present. It would be desirable to combine radioimmunotherapy with an agent that is preferentially toxic to these cells. SR 4233 is a potent hypoxic cytotoxin, and it was combined with 131I-NR-LU-10 to treat LS174T human colon adenocarcinoma multicell spheroids and nude mouse xenografts for these studies. Under conditions of severe hypoxia (< 0.01% O2), 2 h of pretreatment or 18 h of simultaneous treatment with SR 4233 did not significantly enhance the effectiveness of 131I-NR-LU-10 in spheroids. However, under aerobic conditions with a 10% fraction of hypoxic cells, there was more toxicity than would be predicted from simple additivity. Xenografts treated with 131I-NR-LU-10 + SR 4233 had a growth delay that was significantly longer than that achieved with 131I-NR-LU-10 alone. In both spheroids and xenografts, combined treatment produced about 10 times more cell killing than 131I-NR-LU-10 alone. The lack of enhancement in spheroids under complete hypoxia suggests that SR 4233 does not sensitize hypoxic cells to radiation damage. The results with aerobic spheroids and in vivo, where a portion of the cells were hypoxic, could be explained by the targeting of different cell populations (hypoxic and aerobic) by each therapeutic modality. This effect should also be enhanced by reoxygenation and reestablishment of the hypoxic fraction during treatment, thus allowing more than the initially hypoxic fraction of cells to be killed by the SR 4233.     

Buoyant density of EMT6 fibrosarcoma cells: time course of the density changes after growth in hypoxia

EMT6 fibrosarcoma cells were grown to the exponential phase in tissue culture and incubated at 37 degrees C under hypoxic conditions. Buoyant density was determined as a function of the time in hypoxia. Hypoxia was produced in two ways. The first involved incubation of the cells in sealed aluminum chambers containing 95% N2, 5% CO2 gas, and < 10 ppm oxygen, resulting in the cells rapidly becoming exposed to the hypoxic environment. After incubation at 37 degrees C, they were centrifuged in linear Ficoll gradients to their isopycnic density. A significant decrease in density was found after 4 h, and prolonged incubation up to 24 h did not result in further change. This density change was reversible on transfer back to aerobic conditions, with the hypoxic cells reverting to their aerobic density after about 10 h reincubation in air. The second method of producing hypoxia involved growing about 8 X 10(6) cells in a medium-filled air-tight container. Hypoxia was produced gradually as the oxygen in the medium was consumed by cellular respiration. Similar results were obtained; that is, hypoxic cells became significantly less dense. However, when the level of hypoxia was varied between 4000 and < 10 ppm at 2-h intervals after the cells had depleted all of the original oxygen, no significant difference in density was found between hypoxic and aerobic cells.     

Radiosensitization, pharmacokinetics, and toxicity of a 2-nitroimidazole nucleoside (RA-263)

A 2-nitroimidazole nucleoside, 1-(2',3'-dideoxy-alpha-D-erythro-hex-2'-enopyranosyl)-2-nitroimida zole (RA-263), has been investigated for its radiosensitization, pharmacokinetics, and toxicity properties. The in vitro radiosensitization tests against hypoxic Chinese hamster (V-79) cells demonstrated that RA-263 was a more potent radiosensitizer than misonidazole and at 2 mM concentration approached the oxic curve. Significant in vitro radiosensitization activity was also observed in EMT6 mammary tumor cells. The in vitro cytotoxicity data suggested that RA-263 is considerably more toxic to hypoxic cells than misonidazole. The increased cytotoxicity may be related to its higher depletion of nonprotein thiols (NPSH) than misonidazole. The combined effects of radiosensitization and hypoxic cell toxicity were measured by preincubation of the V-79 cells for 4 h under hypoxic conditions before irradiation. The results demonstrated a synergistic response by causing a significant decrease in the extrapolation number with loss of shoulder of the radiation survival curves. The in vivo radiosensitization experiments conducted by the in vivo-in vitro cloning assay with the EMT6 mammary tumor indicate that RA-263 is an effective sensitizer. Pharmacokinetic data suggested that RA-263 was eliminated from plasma by a rapid alpha phase and a slower beta phase with T 1/2 of 36 and 72 min, respectively. The concentration in the brain was approximately one-sixth of tumor concentration, suggesting that RA-263 is excluded from the CNS. Moreover, RA-263 was two times less toxic than misonidazole on equimolar basis by acute LD50 tests. This agent was also significantly less mutagenic than misonidazole in a strain of Escherichia coli.     

The effect of hyperglycemia on the tumor response to irradiation given alone or in combination with hyperthermia

The effect of hyperglycemia (elevated blood glucose level) on the response of a murine tumor to irradiation given alone or in combination with hyperthermia was studied. Tumors were early generation isotransplants of a spontaneous C3H/Sed mouse fibrosarcoma, FSa-II. Single-cell suspensions were transplanted into the foot, and irradiation was given when each tumor reached an average diameter of 7 mm. Following irradiation, the tumor growth time to reach 1000 mm3 was studied and the dose-response curve between the tumor growth time and radiation dose was fitted. Preadministration of glucose increased the size of the hypoxic and chronically hypoxic cell fractions without altering the slope of the dose-response curve where the chronically hypoxic cell fraction is determined as the fraction of cells which were not oxygenated under hyperbaric oxygen conditions. Hyperthermia given prior to irradiation enhanced the tumor response to irradiation, but simultaneously increased the size of the hypoxic and chronically hypoxic cell fractions. Similar results were observed following hyperthermia given after irradiation. When hyperthermia at 43.5 degrees C was given 24 h before irradiation, the size of the hypoxic cell fraction increased with increasing treatment time, while a substantial decrease in the chronically hypoxic cell fraction was observed. Administration of glucose 60 min before hyperthermia further increased the size of the hypoxic cell fraction. Possible mechanisms explaining why glucose administration increases the hypoxic cell fractions are discussed.     

Detection of different hypoxic cell subpopulations in human melanoma xenografts by pimonidazole immunohistochemistry

This study aimed at developing immunohistochemical assays for different subpopulations of hypoxic cells in tumors. BALB/c-nu/nu mice bearing A-07 or R-18 tumors were given a single dose of 90 mg/kg body weight or three doses (3 h apart) of 30 mg/kg body weight of pimonidazole hydrochloride intravenously. The fraction of pimonidazole-labeled cells was assessed in paraffin-embedded and frozen tumor sections and compared with the fraction of radiobiologically hypoxic cells. The staining pattern in paraffin-embedded sections indicated selective staining of chronically hypoxic cells. Frozen sections showed a staining pattern consistent with staining of both chronically and acutely/repetitively hypoxic cells. Fraction of pimonidazole-labeled cells in paraffin-embedded sections was lower than the fraction of radiobiologically hypoxic cells (single-dose and triple-dose experiment). In frozen sections, fraction of pimonidazole-labeled cells was similar to (single-dose experiment) or higher than (triple-dose experiment) fraction of radiobiologically hypoxic cells. Three different subpopulations of hypoxic cells could be quantified by pimonidazole immunohistochemistry: the fraction of cells that are hypoxic because of limitations in oxygen diffusion, the fraction of cells that are hypoxic simultaneously because of fluctuations in blood perfusion, and the fraction of cells that are exposed to one or more periods of hypoxia during their lifetime because of fluctuations in blood perfusion.     

Reduced bone marrow pO2 following treatment with radioprotective drugs

The sensitizer adduct technique [( 3H]misonidazole binding) was used to assess the extent of murine bone marrow hypoxia following treatment with a variety of radioprotectors. The binding rates previously determined in vivo were compared to those obtained by incubating marrow cells in atmospheres of varying oxygen content. Parallel experiments demonstrated that the oxygen dependence of [3H]misonidazole binding (Km approximately 0.15% oxygen) was similar to the oxygen dependence of marrow radiosensitivity (Km approximately 0.2% oxygen). Maximally radioprotective doses of several drugs have been shown to increase the binding of [3H]misonidazole significantly in vivo. A comparison to the in vitro binding rates suggests that the average oxygen concentration in the marrow at times associated with radioprotection was on the order of 0.5 to 0.8% oxygen. The relative importance of marrow hypoxia to the overall radioprotective effects of different drugs may vary considerably. However, these results have demonstrated that certain radioprotective drugs can induce marrow hypoxia and this reduced pO2 may contribute to the efficacy of these agents.     

Comparisons among pimonidazole binding, oxygen electrode measurements, and radiation response in C3H mouse tumors.

Pimonidazole binding was compared with oxygen electrode measurements and with measurements of the radiobiologically hypoxic fraction in C3H mammary tumors in which oxygenation was manipulated by means of subjecting tumor-bearing CDF1 mice to air breathing, carbogen breathing, oxygen breathing, hydralazine injection or tumor clamping. Hypoxia measured by pimonidazole binding could be correlated with both pO2 (r2 = 0.81) and radiobiologically hypoxic fraction (r2 = 0.85) in this system. The scope and limitation of pimonidazole as an immunohistochemical marker for tumor hypoxia is discussed.     

Variations in tumor cell growth rates and metabolism with oxygen concentration, glucose concentration, and extracellular pH.

Tumors and multicellular tumor spheroids can develop gradients in oxygen concentration, glucose concentration, and extracellular pH as they grow. In order to calculate these gradients and assess their impact on tumor growth, it is necessary to quantify the effect of these variables on tumor cell metabolism and growth. In this work, the oxygen consumption rates, glucose consumption rates, and growth rates of EMT6/Ro mouse mammary tumor cells were measured at a variety of oxygen concentrations, glucose concentrations, and extracellular pH levels. At an extracellular pH of 7.25, the oxygen consumption rate of EMT6/Ro cells increased by nearly a factor of 2 as the glucose concentration was decreased from 5.5 mM to 0.4 mM. This effect of glucose concentration on oxygen consumption rate, however, was slight at an extracellular pH of 6.95 and disappeared completely at an extracellular pH of 6.60. The glucose consumption rate of EMT6/Ro cells increased by roughly 40% when the oxygen concentration was reduced from 0.21 mM to 0.023 mM and decreased by roughly 60% when the extracellular pH was decreased from 7.25 to 6.95. The growth rate of EMT6/Ro cells decreased with decreasing oxygen concentration and extracellular pH; however, severe conditions were required to stop cell growth (0.0082 mM oxygen and an extracellular pH of 6.60). Empirical correlations were developed from these data to express EMT6/Ro cell growth rates, oxygen consumption rates, and glucose consumption rates, as functions of oxygen concentration, glucose concentration, and extracellular pH. These empirical correlations make it possible to mathematically model the gradients in oxygen concentration, glucose concentration, and extracellular pH in EMT6/Ro multicellular spheroids by solution of the diffusion/reaction equations. Computations such as these, along with oxygen and pH microelectrode measurements in EMT6/Ro multicellular spheroids, indicated that nutrient concentration and pH levels in the inner regions of spheroids were low enough to cause significant changes in nutrient consumption rates and cell growth rates. However, pH and oxygen concentrations measured or calculated in EMT6/Ro spheroids where quiescent cells have been observed were not low enough to cause the cessation of cell growth, indicating that the observed quiescence must have been due to factors other than acidic pH, oxygen depletion, or glucose depletion.     

Effect of misonidazole on formation of thymine damage by gamma rays

The effect of the radiosensitizer misonidazole (Ro-07-0582) on the formation of thymine base damage of the 5,6-dihydroxydihydrothymine-type by gamma rays was measured under aerobic and hypoxic conditions. HeLa cells, prelabeled with [methyl-3H]thymidine, were suspended in phosphate-buffered saline in the presence and absence of misonidazole. Concentrations of misonidazole up to 15 mM were used. The cell suspensions were irradiated at ice temperature with 60Co gamma rays. Dose-response curves under aerobic and hypoxic conditions showed a much depressed base damage formation under hypoxia, which was created by blowing a stream of nitrogen across the cell suspensions for 30 min on ice. The presence of misonidazole had little or no detectable effect under hypoxia. It is concluded that an effect on the level of formation of thymine base damage is not primarily responsible for the radiosensitization by misonidazole under hypoxic conditions.