Tumor oxygenation and radiosensitization by pentoxifylline and a perflubron emulsion carbogen breathing

Oxygen tension measurements were made in three tumors: (i) the murine FSaII fibrosarcoma, (ii) the rat 9L gliosarcoma and (iii) the rat 13672 mammary adenocarcinoma using a pO2 histograph. Tumor oxygenation measurements were made while the animals breathed air or breathed carbogen (95% oxygen/5% carbon dioxide). Pentoxifylline or a perflubron emulsion was administered to the animals and tumor oxygen measurements were repeated under both breathing conditions. Both pentoxifylline and the perflubron emulsion improved the oxygenation of the FSaII fibrosarcoma under air breathing conditions but did not alter the oxygen profiles of either rat tumor compared with air breathing alone. Carbogen breathing increased the oxygenation of all tumors. Pentoxifylline administration did not change the oxygen profiles of the tumors under carbogen breathing conditions but administration of the perflubron emulsion increased the oxygenation of all three tumors under carbogen breathing conditions compared with carbogen breathing alone. Co-administration of pentoxifylline and the perflubron emulsion enhanced the radiation response of the Lewis lung tumor to daily fractionated radiation under air breathing conditions with a dose modifying factor of 1.65 and under carbogen breathing conditions with a dose modifying factor of 2.25. Over a range of perflubron emulsion doses, pentoxifylline increased the growth delay of the Lewis lung tumor in a constant manner. These results indicate that pentoxifylline and the perflubron emulsion have the largest impact on the oxygenation of more hypoxic tumors and that administration of the perflubron emulsion/carbogen breathing is the most effective means of increasing tumor oxygenation and radiation response.     

Tumor oxygenation after hyperthermia in the rat 13762 mammary carcinoma and the DU-145 human prostate carcinoma

Tumor oxygenation was measured in the rat 13762 mammary carcinoma over a time course from 30 min to 3 days after hyperthermia treatment of 43 degrees C, 30 min or 40 degrees C, 80 min, while the animals breathed air, carbogen or after administration of a perflubron emulsion with air or carbogen breathing. Shortly (30 min and 3 h) after treatment with 43 degrees C, 30 min, the 13762 tumors were more hypoxic, as determined by the percent of pO(2) readings <5 mmHg and median pO(2), than prior to treatment and by 24 h had returned to initial oxygenation. Administration of the perflubron emulsion and carbogen breathing abrogated the increase in hypoxia and improved tumor oxygenation. Athymic mice bearing the DU-145 human prostate carcinoma were treated with hyperthermia regimens 40 degrees C, 41 degrees C, 42 degrees C or 43 degrees C for 60 min and tumor oxygenation was then measured over a time course while the animals breathed air or carbogen. Shortly (30 min) after hyperthermia, the DU-145 tumors were more hypoxic than initially when the animals breathed air. Three days later, the DU-145 tumors treated with 41 degrees C: 42 degrees C or 43 degrees C remained hypoxic but the tumors treated with 40 degrees C were better oxygenated than initially. When the animals bearing the DU-145 tumor breathed carbogen during the tumor oxygenation measurements only tumors treated with 43 degrees C were more hypoxic shortly after hyperthermia treatment. Three days after hyperthermia, the DU-145 tumors treated with 40 degrees C and 43 degrees C were better oxygenated than initially while those treated with 41 degrees C and 42 degrees C were less oxygenated when the animals breathed carbogen during the oxygen measurements. Thus, the effect of hyperthermia on tumor physiology can persist for days post treatment and vary with the temperature of the treatment.     

Influence of an anti-angiogenic treatment on 9L gliosarcoma: Oxygenation and response to cytotoxic therapy

Tissue oxygen tensions were measured in subcutaneously growing rat 9L gliosarcoma under normal air and carbogen breathing conditions prior to and after i.v. administration of a perflubron emulsion. When these animals were treated with the anti-angiogenic agents TNP-470 and minocycline for 5 days prior to oxygen measurement, tumor hypoxia was decreased compared with untreated tumors. Hypoxia, defined as the percent of p^O₂ readings ≤5 mm Hg, was decreased from 71% in untreated air-breathing controls to 34% in animals treated with the anti-angiogenic agents, the perflubron emulsion and carbogen breathing. These effects were manifest in the increased response of the tumor to single-dose (10, 20 and 30 Gy) radiation therapy. Twenty-four hours after treatment with BCNU oxygenation of the tumors was not altered; however, 24 hr after administration of adriamycin oxygenation of the tumors was increased such that hypoxia in adriamycin-treated tumors in animals receiving the perflubron emulsion and carbogen was reduced to 21%. Tumor growth delay in the s.c. tumors was increased by the addition of treatment with the anti-angiogenic agents from day 4 through day 18 post-tumor cell implantation along with BCNU or adriamycin on days 7–11. Administration of the perflubron emulsion and carbogen breathing resulted in increased tumor growth delay with the chemotherapeutic agents alone and in combination with the anti-angiogenic agents. Life span in animals bearing intracranially implanted 9L gliosarcoma progressively increased with administration of the anti-angiogenic agents and then the anti-angiogenic agents and perflubron emulsion/carbogen compared to treatment with BCNU or adriamycin. © 1995 Wiley-Liss, Inc.     

Modification of photodynamic therapy-induced hypoxia by fluosol-DA (20%) and carbogen breathing in mice

The administration of a perfluorochemical emulsion and carbogen (95% O2, 5% CO2) breathing before photodynamic therapy (PDT) was studied to determine how increased levels of tumor oxygenation may affect PDT-induced tumor destruction. C3H/HeJ mice bearing the RIF tumor were given injections of 5 to 10 mg/kg of dihematoporphyrin ethers 24 h prior to treatment. Animals were given injections of 12 ml/kg of Fluosol-DA (20%) followed by carbogen breathing or 12 ml/kg of saline and air breathing (controls) 1 h before tumors were exposed to 135 J/cm2 of 630-nm light treatment. Changes in the hypoxic fraction of tumors, the time course for decreases in tumor cell clonogenicity, and tumor response were measured immediately and at various times after treatment. The administration of Fluosol-DA (20%) and carbogen breathing was found to delay the onset of PDT-induced hypoxia through the first hour posttreatment. Progressive tumor hypoxia was observed after 4 h posttreatment. The time period in which tumors remained well oxygenated coincided with observations of increased tumor cell survival. Decreases in tumor cell clonogenicity were observed only after tumor cells became hypoxic. These findings were consistent with the 24-h delay in complete tumor response in animals given Fluosol-DA (20%) and carbogen breathing before PDT. There were only minor variations in long-term tumor response and cure observed between the two groups tested. A second series of experiments was done to assess any treatment advantage of the adjuvant use of Fluosol-DA (20%) and carbogen breathing with PDT at high tumor photosensitizer levels. At an injected dose of 50 mg/kg of dihematoporphyrin ethers, no such advantage was observed. The administration of Fluosol-DA (20%) and carbogen breathing did not reduce the extent of PDT-induced microvascular damage, maintain high levels of tumor oxygenation through light treatment, or modify the extent of tumor cell kill following treatment.     

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.     

Increase in pO2 and radiosensitivity of tumors by Fluosol-DA (20%) and carbogen

The potential usefulness of i.v. injection of perfluorochemicals and breathing carbogen (95% O2 and 5% CO2) to improve the radiation-induced control of tumors was investigated. When C3H mice, bearing RIF-1 tumors in the legs, were given i.v. injections of Fluosol-DA (20%) at 12 ml/kg, and allowed to breathe carbogen for 1 h before and during a single dose of X-irradiation, the curability of tumors increased by a dose modification factor of 1.47 +/- 0.03 (SE). Such a treatment also increased the radiation-induced skin damage by a factor of 1.15 +/- 0.12, resulting in a therapeutic gain of 1.28 +/- 0.04. Measurement of intratumor pO2 by oxygen microelectrodes demonstrated small increases in pO2 when the animals breathed carbogen, and marked increases in pO2 when Fluosol-DA (20%) was injected into the animals and the animals breathed carbogen. It was concluded that i.v. injection of Fluosol-DA (20%) followed by carbogen breathing significantly improved the oxygen supply to hypoxic cells in the RIF-1 tumors and thus increased the control of tumors by radiation.     

Modulation of tumor oxygenation and radiosensitivity by a perfluorooctylbromide emulsion

The effect of a concentrated perfluorooctylbromide emulsion (Oxygent) on the radiosensitivity and oxygenation of solid tumors was examined using EMT6 mammary tumors in BALB/c mice and BA1112 rhabdomyosarcomas in WAG/rij rats. Treatment with Oxygent plus carbogen or oxygen breathing increased the radiosensitivity of both tumors. Analysis of tumor cell survival data and polarographic measurements of intratumoral pO2 indicated that this potentiation reflected an increase in the proportion of well-oxygenated tumor cells. Treatments with carbogen breathing alone, with Oxygent plus air-breathing, or with a vehicle emulsion containing all the components except the perfluorocarbon did not produce comparable improvements in tumor radiosensitivity. Concentrated perfluorooctylbromide emulsions appear to warrant further development and preclinical testing as adjuncts to cancer therapy.     

Effect of carbogen on tumor oxygenation: combined fluorine-19 and proton MRI measurements

PURPOSE: Blood oxygen level dependent (BOLD) contrast in magnetic resonance imaging (MRI) has been widely used for noninvasive evaluation of the effects of tumor-oxygenating agents. However, there have been few tests of the validity of this method. The goal of the present work was to use the T(1) of fluorine-19 in perfluorocarbon (PFC) emulsions as a "gold standard" for comparison with BOLD MRI. MATHODS AND MATERIALS: Rats bearing R3230AC tumors implanted in the hind limb were injected with an emulsion of perfluoro-15-crown-5-ether for 2-3 days before experiments, which ensured that the PFC emulsion concentrated in the tumors. We correlated changes in tumor oxygenation caused by carbogen inhalation measured by (1)H BOLD MRI with quantitative (19)F measurements. The (19)F spin-lattice relaxation rate R(1) (= 1/T(1)) was measured to determine initial oxygen tension (pO(2)) in each image pixel containing the PFC, and changes in pO(2) during carbogen (95% O(2), 5% CO(2)) breathing. In a second carbogen breathing period, changes in water signal linewidth were measured using high spectral and spatial resolution imaging. (19)F and (1)H measurements were used to classify pixels as responders to carbogen (pixels where oxygen increased significantly) or nonresponders (no significant change in tumor oxygenation). RESULTS: The (19)F and (1)H measurements agreed in 65% +/- 11% of pixels (n = 14). Agreement was even stronger among pixels where (1)H showed increased oxygenation; (19)F measurements agreed with (1)H measurements in over 79% +/- 11% of these pixels. Similarly, there was strong agreement between the two modalities in pixels where (19)F reported no change in pO(2); (1)H also showed no changes in 76% +/- 18% of these pixels. Quantitative correlation of changes T(2)* (DeltaT(2)*) in (1)H and changes R(1) (DeltaR(1)) in (19)F was weak during carbogen breathing, and averaged over the whole tumor was approximately 0.40 for 14 experiments. However, the spatial patterns of (1)H and (19)F changes were qualitatively very similar. In hypoxic regions that were identified based on long (19)F T(1) (>2.53 s), (19)F and (1)H MRI agreed that carbogen had relatively weak effects. CONCLUSIONS: These results suggest that (1)H BOLD MRI reliably identifies increases in tumor pO(2). In hypoxic regions where increases in pO(2) are most desirable, carbogen was ineffective. The data suggest that (19)F and (1)H MRI can be used individually or in combination to guide the design of improved tumor-oxygenating agents.     

Optimization of perfluorochemical levels with radiation therapy in mice

We have examined the effects of a wide range of levels of Therox, a perfluorochemical emulsion containing bis-perfluorobutyl ethylene (F44E) with carbogen breathing on the tumor growth delay of the Lewis lung carcinoma produced by single dose radiation and fractionated radiation. The enhancement in tumor growth delay with single dose radiation therapy increased as the dose of F44E was increased from 1.2 g/kg (0.03 ml) to 4 g/kg (0.1 ml). As the dose was increased further from 6 g/kg (0.15 ml) to 8 g/kg (0.2 ml) and then to 12 g/kg (0.3 ml), there was a progressive decrease in the tumor growth delay observed. The dose of 4 g/kg was the optimal F44E level with single dose radiation therapy, giving a dose modifying factor of 2.4 +/- 0.2. This was true whether administered as a 48% (v/v) emulsion in 0.1 ml or as a 16% (v/v) emulsion in 0.3 ml. When the injection volume was varied from 0.1 ml to 0.4 ml at the 4 g/kg or 6 g/kg dose, thereby varying the emulsion concentration from 48% (v/v) to 12% (v/v) or 18% (v/v), the results tended to indicate that the volume of injection may be more important than the emulsion concentration, i.e., an injection volume of 0.2 ml produced the greatest tumor growth delay for both doses, and the emulsion concentration of 0.2 ml and 4 g/kg of F44E is 24% (v/v) whereas the emulsion concentration of 0.2 ml and 6 g/kg of F44E is 36% (v/v). Administering any dose of the emulsion with carbogen for 1 h prior to and during the radiation fraction on Day 1 only of a daily fractionated radiation protocol (3 Gy/fraction x 5 days) had very little effect on tumor growth delay compared to radiation and daily carbogen breathing. When F44E was administered on treatment Days 1, 3, and 5 with carbogen breathing, there was an increased effect on tumor growth delay which reached a maximum at 4 g/kg (0.1 ml) of 10.0 +/- 1.2 days compared with 6.7 +/- 1.0 days for radiation with daily carbogen breathing. However, when the F44E emulsion was administered every day with fractionated radiation and carbogen breathing, there was a marked enhancement in tumor growth delay observed across the entire dosage range, from 1.2 g/kg to 12 g/kg.(ABSTRACT TRUNCATED AT 400 WORDS)     

Carbogen breathing with nicotinamide improves the oxygen status of tumours in patients.

Nicotinamide and carbogen breathing are both effective radiosensitisers in experimental tumour models and are even more effective in combination. This study was to investigate the feasibility of using the agents in combination in patients and to measure their effect on tumour oxygenation. Twelve patients with advanced malignant disease were treated with 4-6 g of oral nicotinamide (NCT) in tablet formulation. Ten of these 12 patients breathed carbogen (95% oxygen, 5% carbon dioxide) for up to 20 min at presumed peak plasma NCT concentration (Cpeak) and had tumour oxygen partial pressure (pO2) measured using the Eppendorf pO2) histograph. The mean Cpeak values were 82, 115 and 150 micrograms ml-1 for NCT doses of 4, 5 and 6 g respectively and were dose dependent. The time of Cpeak was independent of dose with an overall mean of 2.4 h (range 0.7-4 h). NCT toxicity occurred in 9 out of 12 patients and was mild in all but one; carbogen was well tolerated in all patients. Following NCT only two patients had significant rises (P < 0.05) in tumour median pO2. During carbogen breathing, eight out of ten patients had early highly significant rises in pO2 (P < 0.0001), of which six continued to rise or remained in plateau until completion of gas breathing. Six patients had hypoxic pretreatment values less than 5 mmHg, which were completely abolished in three and reduced in two during carbogen breathing. In conclusion, the combination of NCT and carbogen breathing was generally well tolerated and gave rise to substantial rises in tumour pO2 which were maintained throughout gas breathing. These results should encourage further study of this potentially useful combination of agents as radiosensitisers in the clinic.     

Potentiation of cytotoxic therapies by TNP-470 and minocycline in mice bearing EMT-6 mammary carcinoma

Summary The ability of the antiangiogenic agents TNP-470 and minocycline, singly or in combination, to potentiate the antitumor effects of several cytotoxic therapies was assessed in the murine EMT-6 mammary carcinoma as well as in two drug resistant sublines of that tumor designated EMT-6/CTX and EMT-6/CDDP.The antiangiogenic agents alone or in combination did not alter the growth of the tumors. However, their administration along with cyclophosphamide, CDDP, or thiotepa substantially increased the tumor growth delay produced by these cytotoxic therapies in tumors responsive to the drugs — the increase was about 2-fold for TNP-470 and minocycline together. In drug resistant tumors, treatment with the antiangiogenic agents did not reverse drug resistance but did increase the effect of the cytotoxic drugs.Treatment with TNP-470/minocycline also increased the oxygenation of each of the three tumors. Thus, TNP-470/minocycline administration increased the efficacy of fractionated radiation therapy, especially when used along with a perflubron emulsion oxygen delivery agent/carbogen.These results indicate that treatment regimens including therapies directed toward the proliferating normal cells within a tumor mass as well as therapies directed toward the malignant cells can produce improved outcomes.     

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% O₂ and 5% CO₂) 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.     

Changes in tumor oxygenation/perfusion induced by the no donor, isosorbide dinitrate, in comparison with carbogen: monitoring by EPR and MRI

PURPOSE: In an effort to improve radiotherapy treatments, methods aimed at increasing the quantity of oxygen delivered to tumors were investigated. The aim of this study was to evaluate the effect of one nitric oxide (NO) donor (isosorbide dinitrate) on pO(2) and blood flow in a murine tumor model. The effect was compared to carbogen, used as a reference treatment. METHODS AND MATERIALS: Thirty-six liver tumors implanted in mouse thighs were imaged using magnetic resonance imaging (MRI) at 4.7 Tesla with dynamic Gd-DTPA and blood oxygen level-dependent (BOLD) contrast-enhanced imaging after administration of isosorbide dinitrate or carbogen. The effect on the pO(2) was also tested by EPR oximetry (1.1 GHz) on 52 mice. RESULTS: A significant increase in MRI intensity was observed for both treatments in comparison with the control group. EPR oximetry showed a dose-dependant increase in tumor pO(2) for isosorbide dinitrate (by 5.9 mmHg at 0.2 mg/kg) and a substantially greater change for carbogen breathing (by 23 mmHg). CONCLUSION: Both tumor blood flow and pO(2) were increased by isosorbide dinitrate and carbogen. Carbogen is more efficient than isosorbide dinitrate in increasing the BOLD image intensity, as well as the tumor pO(2), but as efficient as isosorbide dinitrate in the Gd-DTPA contrast-enhanced imaging. We conclude that the effects of carbogen on improving tumor pO(2) involve both improved blood flow and improved hemoglobin oxygenation, whereas the effects of isosorbide dinitrate are predominantly mediated by improved blood flow alone.     

Oxygen tension measurements of tumors growing in mice.

PURPOSE: Clinical studies using the Eppendorf histograph have shown that patients whose tumors have a low pO2 have worse local control after radiotherapy, and have higher metastatic rates. Because preclinical studies of methods of overcoming, or exploiting, hypoxia generally use transplanted tumors in mice, we have compared the oxygenation of mouse tumors with human tumors to determine the appropriateness of the transplanted mouse model for such preclinical studies. METHODS AND MATERIALS: We evaluated the oxygenation status of subcutaneous (s.c.) tissue and of 12 intradermally (i.d.)- and 7 s.c.-growing mouse or human transplanted tumors in mice using the Eppendorf histograph, and compared the values obtained with measurements of human head and neck nodes. RESULTS: The normal tissue pO2 profile of air-breathing mice showed a nearly Gaussian distribution (38.2+/-14.9 mmHg). Breathing 10% O2 or carbogen resulted in dramatic changes in normal tissue oxygenation. Tumors growing intradermally in the back of air-breathing mice were extremely hypoxic and resistant to expected changes in oxygenation (carbogen breathing, size, and use of anesthetics). Tumors growing s.c. in the foot showed higher oxygen profiles with marked changes in oxygenation when exposing the animals to different levels of oxygen. However, the oxygenation of the mouse tumors transplanted in either site was only a fraction of that of the majority of human tumors. CONCLUSION: Experimental mouse tumors are markedly hypoxic, with median values of 10-20% of those of human tumors. Hence, mouse tumors are probably good models for the most hypoxic human tumors that respond poorly to radiotherapy; however, caution has to be exercised in extrapolating data from mouse to man.     

A pilot study comparing intratumoral oxygenation using the comet assay following 2.5% and 5% carbogen and 100% oxygen

PURPOSE: Tumor hypoxia has been purported to be an important biologic factor in the failure of radical radiotherapy to achieve local control in many tumor types. This study was designed to evaluate the effect of breathing high oxygen content gas mixtures (oxygen with 0%, 2.5%, or 5% carbon dioxide) on tumor oxygenation measured using the Eppendorf polarographic oxygen electrode and the comet assay in accessible, hypoxic human tumors. METHODS AND MATERIALS: Using Eppendorf pO2 histography to identify hypoxic tumors (median pO2 < or = 10 mmHg), eligible patients were systematically allocated either 100% oxygen (O2) or oxygen with 2.5% or 5% carbon dioxide (CO2). Tumors were treated with 6-10 Gy during which two fine needle aspirates (FNA) were obtained from different regions of the lesion, one at midway and the other at completion of the radiation exposure. Gas breathing was initiated 4 min before radiation was commenced. A 10-min interval was specified between the first and second halves of the radiation exposure to allow near maximal DNA repair prior to the second half of the radiation treatment. FNAs were performed within 2 min of cessation of radiation and the cells immediately suspended in buffered saline at 4 degrees C for analyses of hypoxic fraction using the comet assay. RESULTS: Fifteen evaluations were performed in 13 patients with hypoxic tumors (median O2 tension 2.75 mmHg) treated with a median dose of 8 Gy. The median hypoxic fraction determined using the comet assay fell from 0.36 to 0.13 (p = 0.001, Wilcoxon signed rank test) due to the addition of high oxygen content gases. CONCLUSIONS: In tumors defined as hypoxic using Eppendorf pO2 histography, a statistically significant reduction in the hypoxic fraction with the comet assay was found following administration of high oxygen content gases. These preliminary findings reveal a trend suggesting that 5% carbogen may reduce the hypoxic fraction by a greater margin than either 100% oxygen or 2.5% carbogen.     

Quantitative changes in tumor metabolism, partial pressure of oxygen, and radiobiological oxygenation status postradiation.

Hypoxia is considered to be a major cause of tumor radioresistance. Reoxygenation of previously hypoxic areas after a priming dose of radiation is associated with an increase in tumor radiosensitivity. In a study of a hypoxic mammary carcinoma, 31P nuclear magnetic resonance spectra showed statistically significant increases in metabolite ratios (phosphocreatine/Pi and nucleotide triphosphate/Pi) after 65 and 32 Gy. The maximum changes in metabolite ratios after 32 Gy occurred at 48 h, although significant changes were detected at 24 h. A corresponding increase in the mean tumor pO2 (polarographic microelectrode measurements) and a decrease in hypoxic cell fraction [changes in paired (clamped versus unclamped) tumor control dose for 50% of tumors] were also shown to occur 48 h after a priming dose of 32 Gy. A significant increase in the mean tumor pO2, phosphocreatine/Pi, and nucleotide triphosphate/Pi, compared to initial values, was noted at 24, 48, and 96 h post 65-Gy radiation. An increase in the downfield component of the phosphomonoester peak relative to the upfield component (phosphoethanolamine), is also noted after doses of 65 and 32 Gy. These are likely to be due to cell kill and/or decreased cell proliferation. In this tumor model, 31P nuclear magnetic resonance spectroscopic changes postradiation are temporally coincident with and may be indicative of tumor reoxygenation as measured by the tumor control dose for 50% of tumors and oxygen-sensitive microelectrodes.     

Increase in tumor pO2 by perfluorochemicals and carbogen

The effects of perfluorochemicals (Fluosol-DA) in combination with carbogen (95% O2 and 5% CO2) breathing on the tumor pO2 was investigated. The pO2 in RIF-1 tumors grown in the leg of C3H mice was determined by polarographic method using oxygen microelectrodes with diameters of 50-60 micron. The average and median pO2 in the control RIF-1 tumors was about 13 mm Hg and 6 mm Hg, respectively. Carbogen breathing alone could cause a significant increase in pO2 in some tumors and Fluosol-DA injection (i.v.) alone caused a slight change in tumor pO2. When the animals were treated with both Fluosol-DA injection and carbogen breathing, the pO2 markedly increased in most of the tumors resulting in an average and median tumor pO2 of 80 mm Hg and 60 mm Hg, respectively. Such an increase in tumor pO2 may account for the previous observations by us and others that the response of experimental tumors to radiation could be markedly increased by treating the tumor-bearing animals with carbogen and Fluosol-DA.     

The effect of hypoxia and hyperoxia on nucleoside triphosphate/inorganic phosphate, pO2 and radiation response in an experimental tumour model.

This study has evaluated the effect of breathing 100% oxygen, carbogen and carbon monoxide (at 660 p.p.m.) on the bioenergetic and oxygenation status and the radiation response of 200-mm3 C3H mammary carcinomas grown in the feet of CDF mice. Bioenergetic status was assessed by 31P magnetic resonance spectroscopy (MRS) using a 7-tesla spectrometer with both short (2 s) and long (6 s) pulse repetition times. Tumour partial pressure of oxygen (PO2) was measured with an Eppendorf polarographic electrode; the oxygenation parameters were the median pO2 and fraction of pO2 values < or = 2.5 mmHg. The radiation response was estimated using a tumour growth delay assay (time to grow three times treatment volume). Carbon monoxide breathing decreased tumour pO2 and compromised the radiation response, but the beta-nucleoside triphosphate (NTP)/Pi ratio was unchanged. Both carbogen and oxygen (100%) increased tumour pO2 and beta-NTP/Pi and enhanced the radiation response, the effects being similar under the two gassing conditions and dependent on the gas breathing time. Thus, in this tumour model, 31P-MRS can detect hyperoxic changes, but because cells can remain metabolically active even at low oxygen tensions the beta-NTP/Pi did not correlate with low tissue oxygenation. An analysis of variance showed that gas breathing time induced a significant systematic effect on beta-NTP/Pi, the MRS pulse repetition time had a significant effect on beta-NTP/Pi change under hypoxic but not under hyperoxic conditions and the type of gas that was inhaled had a significant effect on beta-NTP/Pi.     

Minimal effective dose of perfluorochemical emulsion as a radiosensitizer

Perfluorochemical emulsion can dissolve large amount of oxygen, carry it into tissues, and consequently enhance the response of hypoxic tumor cells to radiation. Previously we reported on the radiosensitizing effect of perfluorochemical emulsion. In the present study, the minimal effective dose of Fluosol-DA Saline 20% (FDAS), one of preparations of perfluorochemical emulsion, was determined. Mice bearing Lewis lung carcinoma in their thighs were injected in single i.v. of FDAS (1.25-20 ml/kg) and were allowed to breath carbogen (95% O2 + 5% CO2) for 30 min. before and during irradiation (15 Gy). The effect of FDAS was measured by the growth delay of the treated tumor. Administration of FDAS at over 5 ml/kg together with carbogen breathing significantly (p less than 0.01) enhanced the tumor growth delay as compared with treatment of carbogen breathing without FDAS. There was a significant difference (p less than 0.05) in the radiosensitizing effect between injection of FDAS at 5 ml/kg and 20 ml/kg, while there were not significant differences between injection of FDAS at 5 ml/kg and 10 ml/kg, or 10 ml/kg and 20 ml/kg, respectively. These results indicate that the minimal effective dose of FDAS is 5 ml/kg, which could be applied to clinical fractionated irradiation therapy as a standard FDAS-dose.     

Preferential action of arsenic trioxide in solid-tumor microenvironment enhances radiation therapy

PURPOSE: To investigate the effect of arsenic trioxide, Trisenox (TNX), on primary cultures of endothelial cells and tumor tissue under varying pH and pO(2) environments and the effects of combined TNX and radiation therapy on experimental tumors. METHODS AND MATERIALS: Human dermal microvascular endothelial cells were cultured in vitro and exposed to TNX under various combinations of aerobic, hypoxic, neutral, or acidic conditions, and levels of activated JNK MAP kinase were assessed by Western blotting. FSaII fibrosarcoma cells grown in the hind limb of female C3H mice were used to study the effect of TNX on tumor blood perfusion and oxygenation. The tumor-growth delay after a single or fractionated irradiation with or without TNX treatment was assessed. RESULTS: A single intraperitoneal injection of 8 mg/kg TNX reduced the blood perfusion in FSaII tumors by 53% at 2 hours after injection. To increase the oxygenation of the tumor vasculature during TNX treatment, some animals were allowed to breathe carbogen (95% O(2)/5% CO(2)). Carbogen breathing alone for 2 hours reduced tumor perfusion by 33%. When carbogen breathing was begun immediately after TNX injection, no further reduction occurred in tumor blood perfusion at 2 hours after injection. In vitro, TNX exposure increased activity JNK MAP kinase preferentially in endothelial cells cultured in an acidic or hypoxic environment. In vivo, the median oxygenation in FSaII tumors measured at 3 or 5 days after TNX injection was found to be significantly elevated compared with control tumors. Subsequently, radiation-induced tumor-growth delay was synergistically increased when radiation and TNX injection were fractionated at 3-day or 5-day intervals. CONCLUSIONS: Trisenox has novel vascular-damaging properties, preferentially against endothelium in regions of low pH or pO(2), which leads to tumor cell death and enhancement of the response of tumors to radiotherapy.