Fraction Of Hypoxic Cells Research Articles

Activation of AP-1 and of a nuclear redox factor, Ref-1, in the response of HT29 colon cancer cells to hypoxia.

Many solid tumors contain substantial fractions of hypoxic cells which are relatively resistant to both radiation therapy and certain cytotoxic drugs. We have previously shown that exposure of human HT29 cells to hypoxic conditions results in the overexpression of certain enzymes involved in the detoxication of xenobiotics, including NAD(P)H:(quinone acceptor) oxidoreductase (DT)-diaphorase, and gamma-glutamylcysteine synthetase, the rate-limiting enzyme in glutathione synthesis. This hypoxic effect on DT-diaphorase was shown to involve both transcriptional induction and altered message stability. We have investigated the effects of hypoxia on elements in the promoter region of DT-diaphorase. Electrophoretic mobility shift assays demonstrate the induction of a binding activity to the AP-1 response element of DT-diaphorase. Supershift assays suggest that this binding is due to AP-1 nuclear factors and that members of the jun family are induced to a greater degree than fos by hypoxia. Analysis of the kinetics of transcription factor expression indicates that the expression of c-jun and junD is induced during hypoxic exposure; mRNA levels fall during reoxygenation. Induction of fos on the other hand is not as florid during hypoxia (5-fold) and is most pronounced (17-fold) 24 h after the restoration of an oxic environment. Thus, the hypoxic response of DT-diaphorase expression is mediated in part through AP-1, initially by a jun-related mechanism and then by the involvement of fos. The affinity of transcription factors for the AP-1 binding site depends on the redox state of a cysteine residue located close to the DNA-binding region of both Fos and Jun. A nuclear protein, Ref-1, maintains the reduced state of Fos and Jun and promotes binding to AP-1. Nuclear extracts of HT29 cells exposed to hypoxia show markedly increased Ref-1 protein content. Elevation of ref-1 steady-state mRNA levels occurs as an early event following induction of hypoxia and persists when cells are restored to a normally oxygenated environment. Nuclear run-on analysis demonstrates that induction of transcription is the mechanism of ref-1 mRNA elevation. Electrophoretic mobility shift assays and immunodepletion assays were used to further define the interaction of Ref-1 with specific AP-1-binding proteins under hypoxic conditions. These data demonstrate that the induction of detoxicating enzyme expression in HT29 cells exposed to hypoxia results from the induction of both transactivating factors that bind to the AP-1 element and of redox proteins that enhance their affinity for this element.

Discrepancies between measured changes of radiobiological hypoxic fraction and oxygen tension monitoring using two assay systems

Purpose : This study was conducted to assess the ability of computerized pO 2 histography to measure changes in tumor oxygenation produced by low oxygen breathing. Methods and Materials : Female syngeneic C3H/Km mice bearing SCC VII/St carcinomas were used in these experiments. Changes in tumor oxygenation produced by the mice breathing 10% oxygen were assessed with computerized pO2 histography, 3H-misonidazole binding, and the paired survival curve assay of radiosensitivity. Results : The hypoxic cell fraction of the tumors in mice breathing 10% oxygen was 3.1 times higher than that of tumors in mice breathing normal air determined by an in vivo-in vitro clonogenic assay. Binding of radiolabeled misonidazole to the tumors in mice breathing 10% oxygen was also significantly higher than that to tumors in mice breathing normal air ( p < 0.05). In addition, oxygen tension histograms for normal tissue showed a dramatic shift to a lower oxygen tension when the mice were breathing 10% oxygen. However, under identical conditions, there was only a minimal shift in the oxygen tension of tumor tissue. Although the number of oxygen tension readings in the relatively oxic class in tumor tissue was lower when the mice were breathing 10% oxygen than when breathing normal air, there was not a significant decrease in the median pO 2 value for the tumor. The number of pO 2 readings lower than 5 mmHg in the tumor was not affected by the 10% oxygen breathing. Conclusion : These findings indicate that increases in radiobiological hypoxic fraction produced by lower blood oxygen levels may not correlate well with the results of polarographic measurements of tumor pO 2 levels.

Impact of overall treatment time of fractionated irradiation on local control of human FaDu squamous cell carcinoma in nude mice

A series of experiments were performed to determine the impact of overall treatment time on local control of human FaDu squamous cell carcinoma irradiated with 30 fractions under ambient conditions in nude mice. The TCD 50 increased with treatment time between 15 days and 10 weeks from 43 Gy to 102 Gy. The data can be well described by a single linear function. The dose recovered per day is 1.0 Gy. However, the data can also be adequately fitted by two components with an initial delay of about 30 days followed by a steep increase at a rate of 1.5 Gy per day. Assuming that the increase of TCD 50 is solely caused by repopulation of clonogenic tumor cells, and that the cellular radiation sensitivity in vitro reflects the radiation sensitivity of FaDu cells in vivo, the doubling time of clonogenic tumor cells during treatment is estimated to be ∼ 1.8 days for the one-component model and, after an initial delay, ∼ 1.2 days for the two-component model. Both values are shorter than the doubling time of clonogenic cells in untreated FaDu tumors and similar to the potential doubling time determined by flow cytometry after BrdUrd labelling. It is concluded that the dose necessary to control FaDu squamous cell carcinoma increases considerably with increasing time of a fractionated radiation treatment. It appears most likely that this increase is caused by repopulation of clonogenic tumor cells; however, other mechanisms such as an increasing fraction of hypoxic tumor cells can not be ruled out at present.

Reoxygenation and rehypoxiation in the SCCVII mouse tumor

Purpose : To test the hypothesis that, following preferential killing of tumor hypoxic cells, the fraction of hypoxic cells in the tumor will reestablish itself to pretreatment levels (rehypoxiation) with the same kinetics as for reoxygenation. Methods and Materials : Mouse squamous cell carcinoma VII (SCCVII) tumors were treated with a single dose of 10 Gy or a single dose of the bioreductive hypoxic cell cytotoxin, tirapazamine (SR 4233, 0.2 mmol/kg), which preferentially kills hypoxic cells within the tumor. Hypoxic fractions were determined by the paired survival curve technique using the in vivo-in vitro clonogenic assay 0–24 h after treatment. Results : Immediately after irradiation with 10 Gy, the hypoxic fraction of the tumors increased to 80% and rapidly returned to pretreatment levels 3–6 h later. Within 1 h of injecting tirapazamine, the hypoxic fraction fell to 0.57% (about 7% of pretreatment levels) and returned to pretreatment levels 3–5 h later. Conclusion : The return to pretreatment levels of hypoxia among tumor cells surviving a single dose of radiation (reoxygenation) and of the hypoxic cell toxin tirapazamine (rehypoxiation) was rapid and occurred with similar kinetics for the two processes. These data support the hypothesis that reoxygenation and rehypoxiation are different manifestations of the same phenomenon and result from fluctuating tumor blood flow which creates acute hypoxia.

Effect of carbon monoxide breathing on hypoxia and radiation response in the SCCVII tumor in vivo

Purpose : To study the influence of a clinically relevant concentration of carbon monoxide (CO) on tumor oxygenation and response to irradiation. Methods and Materials : The murine tumor model was the SCCVII squamous cell carcinoma transplanted to the feet of C3H/Km mice. Results : Sixty minutes of breathing CO at 200 ppm resulted in a carboxyhemoglobin level of 15%. This resulted in a reduction in p50 (the oxygen partial pressure at which hemoglobin is 50% saturated) to 78% of the control value, and a decrease in tumor blood perfusion to 73% of the control value. The combined effect of a decrease in effective hemoglobin and blood perfusion resulted in a reduction in tumor oxygen supply to 62% of the control value. In agreement with this, intratumoral pO 2 measurements showed a significant increase in tumor hypoxia, such that the percentage of measurements with low PO 2 (≤ 5 mmHg) increased from 33% to 62%. The fraction of clonogenic hypoxic cells, measured radiobiologically by paired cell survival curves, similarly increased from 0.2% to 3.8%. Radiation sensitivity, evaluated from in vivo- in vitro excision assay, was significantly decreased by CO breathing with both single dose and fractionated irradiation. The observed enhancement ratios for radiation given in 1, 4, 8, and 12 fractions were 0.71, 0.77, 0.83, and 0.71, respectively. Conclusion : The present SCCVII tumor data confirm the general experimental observation that CO breathing significantly increases tumor hypoxia and reduces the effectiveness of ionizing irradiation.

Hypoxic fractions measured in murine tumors and normal tissues using the comet assay

Purpose : To apply the alkaline comet assay to the detection of radiobiologically hypoxic cells in solid tumors and normal tissues of mice, and to examine the influence of strand break repair on the oxygen enhancement ratio measured using the alkaline comet assay. Methods and Materials : In previous studies, we found that hypoxic fraction in squamous cell carcinomas growing in C3H mice could be reliably and easily measured using the alkaline comet assay. The comet assay applies fluorescence microscopy and image analysis to examine patterns of migration of deoxyribonucleic acid from individual cells embedded in agarose and exposed to an electric field. This method has sufficient resolution to detect subpopulations of hypoxic cells which show about 3 × fewer strand breaks than aerobic cells after irradiation. Results : Fast rejoining kinetics in vitro are comparable to those measured in vivo, and rejoining of strand breaks in hypoxic tumor cells occurs at a similar rate as rejoining in aerobic cells. Little residual damage was detectable using the comet assay in tumors 4–24 h following 15 Gy, allowing repeat measurements to be performed. Bone marrow and testis, but not liver, spleen, or jejunum contained a small fraction of hypoxic cells when mice breathed 10% oxygen during irradiation. Conclusion : The comet assay confirms that some normal tissues may border on hypoxia. Rejoining of strand breaks occurs rapidly in both oxic and hypoxic cells so that the oxygen enhancement ratio remains relatively constant with time after irradiation. Interestingly, a smaller oxygen enhancement ratio was observed in tumors than was expected, probably as a result of the presence of acutely hypoxic cells.

The effect of hyperthermia on reoxygenation during the fractionated radiotherapy of two murine tumors, FSa-II and MCa

Purpose : To investigate the effect of hyperthermia on the tumor reoxygenation during fractionated irradiations. It has been shown that hyperthermia increases the size of hypoxic cell fraction in some murine tumors and reoxygenation is critical for successful radiotherapy. Methods and Materials : Tumors were early generation isotransplants of spontaneous murine fibrosarcoma (FSaII) and mammary carcinoma (MCa) in C3Hf/Sed mice. Treatments were initiated when they reached an average diameter of 4 mm. A local heat treatment at 43.5°C for 45 min was given in a constant temperature water bath 24 h before irradiation(s). This interval was selected to avoid heat-radiation interaction and to simply investigate the heat effect on the reoxygenation process. Tumors were irradiated under hypoxic conditions or in air and observed for recurrences for 120 days. The foot reaction of animals with controlled-tumors was scored on the last day of experiments. The TCD 50 (50% tumor control dose) and RD 50 (dose to induce partial foot atrophy in 50% of treated animals) were calculated. Results The TCD 50s following a various number of fractions were obtained for FSa-II and MCa with or without hyperthermia. The difference between the TCD 50 (hypoxia) and TCD 50 (in air) without hyperthermia increased with an increasing number of fractions, suggesting that significant reoxygenation occured during the fractionated irradiation. The TCD 50s (with heat, in air) were smaller than the TCD 50s (radiation alone, in air) following fractionated irradiations, indicating that hyperthermia did not affect tumor reoxygenation. The difference between these TCD 50 values was greater for heat-sensitive MCa than for heat-resistant FSa-II, suggesting that this difference was due to additive heat cytotoxicity. An unexpected observation was that heat significantly enhanced the foot reaction with no resultant therapeutic gain for both MCa and FSa-II tumors. Conclusion : Hyperthermia given independently prior to fractionated irradiation did not affect tumor reoxygenation, nor was there a therapeutic gain for the two murine tumors. These results suggest that selective tumor heating is essential in clinical thermoradiotherapy.

The relationship between carbon monoxide breathing, tumour oxygenation and local tumour control in the C3H mammary carcinoma in vivo.

The effect of acute carbon monoxide (CO) breathing on blood oxygenation and tumour hypoxia was related to the radiation response of the C3H/Tif mammary carcinoma. Blood gas analysis showed that CO breathing caused a time- and dose-dependent formation of carboxyhaemoglobin (HbCO), a significant left shift of the oxygen dissociation curve and a reduction in tumour blood perfusion. These factors all contributed to a marked drop in tumour oxygen supply. In agreement with this, tumour hypoxia was found to be significantly increased: Microelectrode PO2 measurements showed a clear relationship between CO concentration and the proportion of low PO2 measurements (< or = 5 mmHg). The fraction of clonogenic hypoxic cells increased from 8% in air-breathing animals to 13%, 18% and 54% with 75,220 and 660 p.p.m. CO respectively. The tumour hypoxia resulted in significant radiation modification. The local tumour control after single-dose and fractionated irradiation gave TCD50 enhancement ratios (relative to air-breathing controls) of 0.90, 0.85 and 0.89 for single dose and five or ten fractions given in 5 days (P < 0.005 for all values). For 15 fractions in 5 days with 6- 6- and 12 h intervals, the TCD50 was similar in CO- and air-breathing mice, presumably as a consequence of insufficient reoxygenation during the short inter-fraction intervals. It is concluded that elevated HbCO levels to increased tumour hypoxia and that the induced hypoxia has a significant impact on the local tumour control also after fractionated irradiation.

Relative perfusion of tumours in two sites for up to 6 hours after the induction of anaemia.

Anaemia in the cancer patient is widely recognised as an important factor in determining the outcome of various forms of treatment. Animal studies have shown in several tumour lines that acute reductions in haematocrit produce a significant increase in the hypoxic fraction of tumour cells, thus rendering the tumour cells more resistant to radiotherapy (Hirst et al., 1984; Siemann et al., 1975). Where inspired air of low oxygen content has been used to induce anaemia, the reduced radiosensitivity has been attributed to a reduction in O2 availability resulting from the low pO2 in the arterial blood, whilst induction by phlebotomy, with subsequent plasma replacement, directly reduces the Hb concentration of the blood, thus reducing the O2 carrying capacity. It may be expected that compensatory mechanisms act to increase the cardiac output to maintain the supply of oxygen to tissues. The way in which any such increase affects the relative distribution of blood between tumours and normal tissues has not been closely investigated as yet. This study examines how the cardiac output is distributed between 5 minutes and 6 hours after the onset of anaemia.

The detection and modification of the hypoxic fraction in quiescent cell populations in murine solid tumours

Mice bearing SCC VII or EMT6/KU tumours were irradiated after receiving 10 injections of 5-bromo-2'-deoxyuridine (BUdR) to label all proliferating tumour cells, and the tumours were then excised and trypsinized. The tumour cell suspensions thus obtained were incubated with cytochalasin-B (a cytokinesis blocker), and the micronucleus (MN) frequency in cells without BUdR labelling was determined using immunofluorescence staining to BUdR. This MN frequency was then used to calculate the surviving fraction of unlabelled cells from the regression line for the relation between MN frequency and the surviving fraction of all tumour cells. Thus a cell survival curve could be determined for cells not labelled by BUdR, which can be regarded as quiescent tumour cells for all practical purposes. Assays performed immediately after irradiation of both normally aerated and hypoxic tumours showed that quiescent cells contained higher hypoxic fractions than the tumour cells as a whole. Furthermore, administration of nicotinamide before irradiation or the placement of mice in a circulating carbogen (95% O2, 5% CO2) chamber for 30 min before and during irradiation altered the acutely and chronically hypoxic fractions of the proliferating and quiescent tumour cell populations in a way which depended on the tumour system. Combined nicotinamide and carbogen therapy was shown to have a large potential to sensitize cells to low-dose radiation in vivo. In addition, this assay method appears to be useful for determining the size of the hypoxic fraction of quiescent tumour cells in murine solid tumours.

Timing and sequence of hyperthermia in fractionated radiotherapy of a murine fibrosarcoma

Purpose : This study investigated the effect of timing and sequence of hyperthermia on fractionated radiotherapy, since it has been shown that the heat increases the size of hypoxic cell fraction which could affect the effect of subsequent radiation doses. Methods and Materials : Animal-tumors were early generation isotransplants of a spontaneous fibrosarcoma, FSa-II, in C3Hf/Sed mice. Tumor response was studied by tumor growth time and TCD 50 (50% tumor control dose) assays. The tumor growth time is the time required for one-half of the treated tumors to reach 500 mm 3 from the first treatment day. The TCD 50 is a radiation dose to control one-half of the treated tumors for 120 days following treatments. One heat treatment at 43.5°C for 45 min was given in a water bath in combination with fractionated doses independently (24 hr interval) or simultaneously (2 min interval). For the normal tissue study, the mouse foot was treated, and the acute foot reaction was scored daily and averaged. The late foot reaction was scored in animals used in the TCD 50 assay that developed no recurrence for 120 days. The RD 50(2.0) and RD 50(5.0), or total radiation doses to induce an average score of 2.0 (complete epilation) and 5.0 (partial foot atrophy) in 50% of treated animals, were calculated. Results : Thermal radiosensitization was most prominent when heat was combined simultaneously with the first or last radiation dose in both the tumor growth time and TCD5o assays. However, the thermal enhancement was greatest when heat was given either with the first or last radiation dose in the TCD 50 assay; whereas it was greatest when heat was administered with the last radiation dose in the tumor growth time assay. Both acute and late skin reactions were significantly potentiated by heat administered 24 hr before the first radiation dose. Conclusion : A significant observation in this study was that, in both the tumor growth time and TCD 50 assays, heat given independently or simultaneously did not result in any therapeutic gain compared to the radiation alone treatment.

Influence of the Timing of a Concomitant Boost During Fractionated Irradiation of Rat Rhabdomyosarcoma R1H

Rhabdomyosarcomas R1H of the rat (WAG/Rij) were treated using fractionation schedules including a boost. The total dose was 60 Gy. Overall treatment time was 6 weeks. Four different boost schedules were applied: a single dose boost (12.15 Gy) at the last day of treatment, a single dose boost (12.15 Gy) at the first day of treatment, a schedule including the boost in 7 fractions during the first week, and a schedule including the boost in 10 fractions during the first week of treatment. A standard schedule with 30 fractions of 2 Gy without a boost was used for comparison. Initially accelerated schedules, i.e. those with a boost at start of treatment, revealed higher effect on tumour parenchyma as monitored by local control rate and net growth delay. This could be due to a decrease of radio-sensitivity, that is, an increase of the hypoxic fraction of clonogenic tumour cells during fractionated irradiation.

The Combined Use of 131 I-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.

Implications of a pre-existing tumor hypoxic fraction on photodynamic therapy

The presence of oxygen in tissue is a requirement for photodynamic therapy (PDT)-induced destruction of solid tumors, otherwise no cell death occurs. Since many tumors have been shown to have significant populations of hypoxic cells, it is of clinical interest to determine if pre-existing tumor hypoxia limits phototherapy. This question was examined using RIF tumors where tumor response to PDT of completely oxygenated tumors was compared to tumors with an induced hypoxic fraction. Tumor hypoxia was induced by using vasoactive drugs (epinephrine, chlorpromazine, or isoproterenol), given 30 min prior to PDT, or by a surgical method. PDT consisted of 5 mg/kg Photofrin II ip 24 hr prior to treatment and 135 J/cm 2 630-nm light. The administration of the various vasoactive agents induced hypoxic fractions of 2.2 to 10%. The surgical method induced hypoxic fractions of 35%. Tumor response and cure in animals given vasoactive agents did not differ from controls, suggesting that low levels of pre-existing tumor hypoxia do not limit photodynamic therapy in this tumor model. Animals with tumors made hypoxic by a surgical method showed significantly reduced tumor response to PDT. Only 14% of these animals had tumors which became flat and necrotic by the day following PDT, compared to nearly 100% for animals given vasoactive drugs or controls. Furthermore, no tumor cure was observed in animals treated by this method. The higher level of tumor hypoxia in these animals likely represents one point where large proportions of PDT-resistant cells can survive after treatment. These data suggest that tumors with very high fractions of hypoxic cells may have a poor prognosis to PDT, and that low numbers of hypoxic cells do not necessarily limit tumor response and cure following PDT.

Energy status in the murine FSaII and MCaIV tumors under aerobic and hypoxic conditions: An in-vivo and in-vitro analysis

The relationship between energy status and hypoxia was examined in two murine tumors with substantially different hypoxic cell fractions in situ and in cells derived from these tumors in vitro. Parameters of tumor energy status were NTP/P i; and PCr/P i; obtained by 31P-NMR spectroscopy and adenylate energy charge and energy status obtained by high-pressure liquid chromatographic analysis of tumor extracts. Adenylate energy charge and rates of high-energy phosphate degradation were determined on cells obtained from both tumor types (MCaIV and FSaII) under identical nutrient and oxygen conditions, that is, air and nitrogen for various durations (0–6 hr). No consistent or substantial differences were noted in the various parameters of tumor energy status obtained by nuclear magnetic resonance analysis or analysis of tumor extracts, even though the MCaIV contains a substantially larger hypoxic fraction (49% vs 12%). Under in vitro conditions, the two cell lines exhibited different responses to oxygen deprivation, the MCaIV being substantially more refractory to energy changes secondary to hypoxia. Noting with caution that this study is based on only two tumor types, our results suggest that differences in cellular capacity for energy maintenance preclude quantitative inferences regarding tumor oxygen status from energy status between tumor types.

Influence of carboxyhemoglobin level on tumor growth, blood flow, and radiation response in an experimental model

Carboxyhemoglobin (HbCO) is formed when carbon monoxide is bound to hemoglobin. High levels of HbCO are known to reduce the amount of oxygen that can be carried to the tissues. This experimental study focuses on the influence of HbCO on the growth, blood flow, and radiation response of an experimental mouse tumor. The study was designed to mimic the clinical situation where heavy smokers are undergoing radiotherapy while having a high HbCO level. The tumor was a C3H mammary carcinoma grown in the feet of CDF 1 mice. Chronic exposure to carbon monoxide, resulting in 10% HbCO, increased the tumor volume doubling time from 2.5 to 3.5 days ( p < 0.05). The acute exposure to carbon monoxide prior to and during irradiation significantly raised the radiation dose required to control the tumor locally. The TCD 50 increased from 54 Gy in air breathing mice (HbCO 0–2%) to 57 Gy (HbCO 7–9%) and 61 Gy (HbCO 20–23%). This increase corresponded to an increase in the fraction of clonogenic hypoxic tumor cells from 0.12 in air breathing animals to 0.21 and 0.41, respectively. The tumor blood flow, determined by the 86RbCl extraction technique, decreased to 63% (n.s.) and 50% ( p < 0.05) for low and high HbCO levels, respectively.

Improving the radiation response in a c3h mouse mammary carcinoma by normobaric oxygen or carbogen breathing

The limited therapeutic benefit from nitroimidazoles has renewed the interest in normobaric oxygen as a hypoxic cell radiosensitizer. In this experimental study we have tried to modify the oxygenation of a C3H mammary carcinoma by flushing tumor-bearing mice with oxygen or carbogen for 5 min before and during treatment. The response to these treatments was evaluated by the changes in radiation-induced tumor conntrol (TCD 50) and by the changes in tumor hypoxic fraction (HF). Irradiation was given either as a large, single dose or as rive equal, daily fractions. High levels of oxygen in the inspired air were found to decrease the TCD 50 significantly. The enhancement ratios were in the range of 1.2-1.4 ( p < 0.05) for both single dose and fractionated irradiation, which suggests that hypoxic cells may be important even when reoxygenation is believed to be complete between fractions. The change in TCD 50 corresponded to a decrease in the fraction of clonogenic hypoxic cells from 12% to 3–4% ( p < 0.05). Tumor blood flow was not significantly influenced by the gas treatment. This study thus shows that normobaric oxygen/carbogen inhalation may significantly improve the local tumor control by reducing the diffusion related hypoxia within tumors.

Increases in tumor response by pentoxifylline alone or in combination with nicotinamide

Pentoxifylline (PENTO), a derivative of methylxanthine, has been reported to improve fluidity of red blood cells (RBC), and thus improve the flux of RBC through narrow capillaries. Additionally, PENTO increases 2,3-DPG levels in RBC, thereby increasing the O 2 release from RBC. Nicotinamide (NA) has been known to increase tumor blood flow, reducing the hypoxic cell fractions in the tumors. The purpose of this study was to examine the effects of PENTO alone or in combination with NA (PENTO + NA) on the oxygenation and radioresponse of FSaII murine fibrosarcomas of mice. We observed a significantly enhanced, radiation-induced growth delay of the FSaII tumors by the treatment of either single or multiple injections of PENTO. The combination of PENTO and NA further delayed the growth of tumors. The TCD 50 of control tumors was about 56.6 Gy, whereas that of PENTO+NA treated tumors was about 31.9 Gy. Thus, TCD 50 was modified by a factor of 1.8. PENTO+NA exerted no effect on the acute skin damage of OH mice after local irradiation and the gastrointestinal death after whole body irradiation. However, PENTO+NA slightly increased the bone marrow death as demonstrated by the decrease in LD 50(30) from 5.5 Gy to 5.2 Gy. The average p0 2 in the saline-treated control group of FSaII tumors was 8 mmHg and it significantly increased to 19 mmHg in the PENTO+NA treated group ( p < 0.001). We concluded that the PENTO+NA treatment increased the radioresponse of tumors by improving tumor oxygenation.

Tumors growing in irradiated tissue: Oxygenation, metabolic state, and pH

Experimental tumors growing in irradiated tissue have been used to study the biological differences characteristic of locally recurrent tumors. Animal tumors were early generation isotransplants of a spontaneous fibrosarcoma in a C3Hf/Sed mouse, designated FSa-II. Since the hypoxic cell fraction of tumors growing in irradiated tissue is increased, these tumors are assumed to be metabolically deprived with hypoperfusion and acidosis. In this study we directly measured the oxygen partial pressure (pO 2) distribution, metabolic state, and pH of tumors growing in an irradiated tumor bed using oxygen sensitive electrodes and 31P-NMR. The results confirmed a three-fold increase in the number of pO 2 readings ≤ 2.5 mmHg and also showed increased acidosis with a 0.17 unit decrease in pH NMR. When tumors growing in pre-irradiated tissue reached ≈100 mm 3 in volume, a high frequency of gross and microscopic necrosis and hemorrhage was already observed. Consistent with these observations, the phosphocreatine/inorganic phosphate (PCr/P i) and nucleoside triphosphate/inorganic phosphate (NTP/P i) ratios were significantly lower in the tumors in a pre-irradiated bed compared to tumors in a non-irradiated bed (PCr/P i: 0.51 vs 0.79, p < 0.05; and NTP/P i,: 0.64 vs 0.93, p < 0.05). The longitudinal relaxation time (T 1) of P i was numerically shorter in control tumors (consistent with the better tissue oxygenation), but this did not reach statistical significance (2.09 ± .11 sec vs 2.25 ± .16 sec).

Reoxygenation in a rat rhabdomyosarcoma tumor following X-irradiation

The paired survival curve technique was used to characterize the rate at which the fraction of hypoxic cells in rat rhabdomyosarcoma R-1 tumors returns to the preirradiation value of 37% following a single dose of 225-kVp X rays. Tumors were administered a conditioning x-ray dose of 15-Gy, followed at 0, 3, 6, 12, 24, or 48 hr by a 5Gy, 10-Gy, or 15-Gy dose of X rays under air-breathing conditions or under hypoxic conditions produced by nitrogen-gas asphyxiation 5 min prior to irradiation. Cellular surviving fractions were determined by the tumor excision assay following in vivo irradiation. From the ratio of the survival fractions measured for tumor cells from air-breathing and hypoxic animals, the fraction of hypoxic cells was determined as a function of time postirradiation. These results indicated that immediately following a 15-Gy dose of X rays, essentially 100% of the viable cells remaining were hypoxic. The tumors reoxygenated rapidly, returning to the preirradiation level of 37% during the first 6 hr postirradiation.