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Purpose: In carcinomas of the uterine cervix, the tumor oxygenation status has been shown to be a prognostic indicator that is independent of treatment modality. In vitro studies suggest gene amplification and polyploidization to be among the major consequences of hypoxia (with or without consecutive reoxygenation) and to be associated with treatment resistance and tumor progression. This study analyzed whether hypoxia alters net DNA content in uterine cervix cancer cells to the extent that it is identifiable by DNA image cytometry., Materials and Methods: In 64 patients with primary cervical cancer, tumor oxygenation was assessed polarographically and correlated with cell DNA content (DNA image cytometry) in areas adjacent to the oxygen microsensor tracks in which oxygenation measurements were made., Results: No correlation between DNA content (stemline position, Auer classification, and 2c deviation index) and oxygenation status was observed. However, an association between DNA content and patient age and menopausal status was found., Conclusion: Using DNA cytometry, hypoxia-associated genomic changes in uterine cervix cancer cells could not be detected. The impact of tumor hypoxia on the genome may be masked by the effects of alternative mechanisms of genomic instability that can also influence DNA content.

Purpose: The effect of recombinant human erythropoietin (rhEPO) on the radiosensitivity of human tumor xenografts growing in anemic and nonanemic nude mice was studied., Methods and Materials: Anemia was induced by total body irradiation ([TBI], 2 x 4 Gy) of mice before tumor implantation into the subcutis of the hind leg. The development of anemia was prevented by rhEPO (750 U/kg s.c.) given 3 times weekly starting 2 weeks before TBI. Fourteen days after fractionated TBI (tumor volume of approx. 40 mm(3)), single-dose irradiation of the tumor with varying doses was performed so that in full dose-response relationship for the probability of tumor cure was obtained., Results: Radiation-induced anemia (hemoglobin concentration [cHb] = 9.9 g/dl) led to a reduced radiosensitivity compared to controls [49.4 vs. 40.1 Gy radiation dose to control 50% of the tumors (TCD50)]. Upon rhEPO treatment for anemia prevention (cHb = 13.3 g/dl), the TCD50 was 39.8 Gy, illustrating restored radiosensitivity compared to anemic mice., Conclusion: These data provide further experimental evidence for restored radiosensitivity upon prevention of anemia with rhEPO.

Purpose: Previous studies with rat rhabdomyosarcomas indicate that during fractionated irradiation profound alterations of the tumor microvasculature and the oxygenation status occur when the total dose exceeds 45 Gy. At this dose a destruction which included all structures of the vessels and a significant worsening in tumor oxygenation were found. The aim of the present study was to analyze whether these effects of fractionated irradiation on the microvasculature and on tumor oxygenation also induce changes in the bioenergetic and metabolic status in the tumors during radiation treatment., Methods and Materials: R1H rhabdomyosarcomas of the rat implanted into the flank were irradiated with 60Co-gamma-rays using 5 fractions of 3 Gy per week over 5 weeks. During this irradiation schedule, tumors were investigated each week for the microregional distributions of glucose, lactate, and ATP concentrations. For this, tumors were rapidly excised, shock-frozen and quantitative bioluminescence measurements were performed on tumor tissue sections., Results: ATP concentrations remained unchanged during fractionated irradiation up to a total dose of 45 Gy. Above this dose, a significant decrease in ATP levels was observed. Lactate concentrations changed only slightly during irradiation whereas glucose levels increased continuously over the whole irradiation period., Conclusions: During fractionated irradiation of R1H tumors with a total dose of 75 Gy, the bioenergetic and metabolic status of the tumors changed considerably. This became most obvious once a dose of 45 Gy had been achieved. The severe energy depletion and worsening of tumor oxygenation might be the result of destruction of tumor blood vessels as has been described previously in the same tumor model. The modification of the tumor micromilieu appears to be an important parameter in the responsiveness of tumor cells to radiation and for local tumor control.

Purpose: The use of methylxanthine derivatives has been postulated as a means of increasing tumor perfusion and thus ameliorating tumor hypoxia. The aim of this study was to quantify and compare the effects of three methylxanthine derivatives: pentoxifylline (PX), torbafylline (TB), and HWA 138 (HW) on tumor perfusion and oxygenation., Methods and Materials: Anesthetized Sprague Dawley rats with DS-sarcomas implanted subcutaneously onto the hind foot dorsum were used in this study. Mean arterial blood pressure (MABP) was measured throughout experiments. Regional red blood cell (RBC) flux was monitored using a multichannel laser Doppler device and tumor oxygenation on a more global level was assessed polarographically using an O2-sensitive catheter electrode. The methylxanthine derivatives were administered as a single dose intraperitoneally (for PX 50 mg/kg; for TB and HW 75 mg/kg)., Results: Following drug administration, initial decreases in MABP down to 75% of baseline values were observed for all three substances. PX, HW, and TB caused initial transient reductions in mean RBC flux followed by gradual increases to values of 137 +/- 27%, 139 +/- 14%, and 122 + 14% respectively at t = 60 min. Following a small initial decrease upon drug administration, O2 partial pressure (pO2) rose to 160 +/- 31%, 153 +/- 34%, and 121 +/- 11% for PX, HW, and TB, respectively at t = 60 min. At the end of the observation period (t = 90 min), increases in RBC flux and pO2 were still evident. When individual tumors were considered, a variety of patterns (including opposing effects) for changes in RBC flux were seen, not necessarily reflected in the mean values. Thus, while the methylxanthine derivatives caused an increased average tumor perfusion, there is evidence suggesting that a redistribution of tumor blood flow occurs which may amplify preexisting heterogeneity., Conclusions: Substantial improvements in tumor oxygenation and perfusion were observed after administration of the methylxanthine derivatives. These substances may therefore be of use during tumor therapies in which the outcome may be detrimentally affected by the presence of hypoxia.

Purpose: Several factors are known to influence the probability of tumor control after radiation. These include tumor oxygen tension distribution, glutathione content, intrinsic radiation sensitivity, rate of repopulation, tumor size, physician skill, etc. The relative impact of oxygen on human tumor response is unknown. The purpose of this analysis is to determine to what extent the observed shape of the radiation response curve for human tumors can be predicted by the tumor oxygenation status., Methods and Materials: The radiation dose response curve for patients treated with radiation alone for breast cancer was calculated based on pooled data. Tumor control rates as a function of radiation dose were fitted to a probit curve. Twenty-two women with breast cancer in Mainz (Germany) and at Stanford University had pO2 measurements made of their tumors. An average of 87 +/- 58 (range 21 to 300) measurements were made from each patient. Hypoxia was assumed to be a purely dose modifying factor with a maximum oxygen enhancement ratio of 2.5. Assuming patients are treated with daily radiation doses of 2 Gy, the breast cancer alpha/beta ratio is 10 Gy, tumors have a mean of 10(8) stem cells, and using the linear quadratic formula for modelling surviving fraction, it was possible to estimate tumor control probability., Results: Tumor oxygenation was an extremely important modifier of the shape of the dose response curve and alone was sufficient to account for the slope of the observed dose response curve for human breast carcinoma. Tumor size distribution had a smaller effect on the shape and the slope of the dose response curve. Two models of radiation induced reoxygenation were tested, one that allowed full reoxygenation to the baseline state between the daily radiation fractions and another with no reoxygenation between fractions. The clinical data fell between these two models in accordance with the expected incomplete reoxygenation between treatments., Conclusion: The results support the conclusion that in human breast carcinoma, oxygen tension distribution is a critical modifier of radiation treatment response.

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 (pO2) 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 pO2 readings less than or equal to 2.5 mmHg and also showed increased acidosis with a 0.17 unit decrease in pHNMR. When tumors growing in pre-irradiated tissue reached approximately 100 mm3 in volume, a high frequency of gross and microscopic necrosis and hemorrhage was already observed. Consistent with these observations, the phosphocreatine/inorganic phosphate (PCr/Pi) and nucleoside triphosphate/inorganic phosphate (NTP/Pi) ratios were significantly lower in the tumors in a pre-irradiated bed compared to tumors in a non-irradiated bed (PCr/Pi: 0.51 vs 0.79, p less than 0.05; and NTP/Pi: 0.64 vs 0.93, p less than 0.05). The longitudinal relaxation time (T1) of Pi 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).

A computerized pO2 measurement system with a novel electrode motion pattern (Sigma-pO2-histography) was evaluated in vitro and in vivo. The system was found to be reliable in 0.9% saline and 10% hydroxyethylene starch solution and in fresh donor blood. Marked deviations were found in lipid and hemoglobin solutions and in fluorocarbon emulsions. Histograms obtained in rat liver, mouse muscle, and subcutis were similar to previously reported distributions. Direct comparison between Sigma-Eppendorf and self-constructed Whalen-type electrodes in hypoxic tumors gave similar results. A large series of measurements indicated that hypoxic and anoxic tissue areas were frequently found both in isografted rodent and in xenografted human tumors. The extent of oxygen deprivation depended on the cell line studied, tumor size, implantation site, the vascularity, and the actual tissue perfusion. Pentobarbital anesthesia redistributed the tumor oxygenation without affecting the median pO2 value. Tumors growing in a pre-irradiated bed were less oxygenated than those at untreated sites. Hyperthermia at therapeutically relevant temperatures reduced pO2 levels in adequately oxygenated tumors whereas little change was detected in poorly oxygenated tumors. First measurements in tumors in patients revealed marked inter- and intratumor heterogeneity. It is concluded that this novel technique is suitable for routine measurements of tissue oxygenation of solid tumors in situ.

Energy metabolism of murine FSaII foot tumors was studied by in vivo 31P-MRS in C3Hf/Sed mice. Spectroscopy was performed following exposure to escalating doses of hydralazine (HYD) ip. At 0.25 mg/kg, HYD caused a 20% increase in PCr/Pi and had no significant effect on mean arterial blood pressure. HYD doses greater than or equal to 2 mg/kg lead to hypotension which was associated with a decrease in PCr, NTP, pH, and an increase in Pi (p less than 0.01 for control vs 10 mg/kg HYD). When mice were given ip injections of HYD (0.25, 1, 2 and 10 mg/kg) 10 min prior to whole body irradiation, spleen stem cell survival after 6 Gy was increased (2.19 colonies in control animals vs 6.74 colonies per spleen in animals treated with greater than or equal to 2 mg/kg HYD), as was the LD50/30 dose (6.49 Gy [control] vs 9.00 Gy [10 mg/kg HYD]). The data provide evidence that PCr/Pi is a useful indicator of perfusion efficiency (and indirectly of hypoxic cell fraction) in FSaII tumors. These observations suggest that HYD may be a useful adjuvant for hyperthermic treatment of tumors and for potentiation of agents specifically toxic to hypoxic or nutrient-deprived cancer cells. HYD should be used with care in patients receiving radiation treatments or other therapies for which hypoxia can unfavorably affect treatment outcome.

Culture medium that was recovered from tumor cell or fibroblast cultures during the plateau phase, and that was replenished by addition of glucose, glutamine, and serum and readjustment of pH had a distinct growth-inhibiting effect on monolayer cell cultures. The effect, which was not specific for a given cell strain, may be partially responsible for the "density inhibition" commonly observed in malignant cells grown in monolayer cultures. By modifying fresh growth media, it was shown that the growth inhibition observed can be partly attributed to the accumulation of lactate in the culture medium of plateau phase cells. This substance reduced the plating efficiency and the number of cells per petri dish in the plateau phase. It is concluded that this effect may be used for inducing growth inhibition in tumors in vivo by manipulating the cellular production of lactate and/or by impeding its removal from the cellular microenvironment.

Problems arising during application of a method proposed recently to predict tissue blood flow from measured thermal clearance curves have been discussed here. In this method, the treated tissue is divided into temperature equivalent zones with the aim to improve the mathematical description of experimental washout curves compared to descriptions based on monoexponential temperature decay. However, when applying the mathematical procedure suggested, any blood flow can be calculated by variation of the number of isothermal zones chosen. Unfortunately, to date no method for determination of the proper division of the treated tissue into temperature equivalent zones is available. Thus, any choice of the number of temperature equivalent zones is arbitrary. Therefore, we suggest that other investigators should not follow the analysis recently proposed for the determination of blood flow from thermal washout curves.

The benefit of Tetrachlorodecaoxide (TCDO) in improving tumor tissue oxygenation has been tested using multicellular tumor spheroids. Measurements of oxygen tension (PO2) values with O2-sensitive microelectrodes revealed a distinct enhancement of the spheroid oxygenation after both, bolus injection or continuous infusion of TCDO into the medium surrounding the spheroids. The effect of TCDO on local oxygenation appeared to be less pronounced in spheroid regions with low initial PO2 values compared to well-oxygenated areas of these cellular aggregates. By continuously infusing TCDO into the measuring system, a sustained increase of local PO2 was obtained which was proportional to the local steady state concentration of TCDO in the medium surrounding the tumor spheroids. During experiments with TCDO in vivo, this substance should, therefore, be tested for its potential of improving the oxygenation status of solid tumors, and hence, of sensitizing tumors to classical treatments, such as radiotherapy.

Oxygen diffusion distances in human breast cancer xenografts are computed considering cell line-specific in vivo data. By introducing variations into a set of experimental data characterizing a "standard" situation, the impacts of various tumor-specific or systemic mechanisms are studied. Evidence is given that radioresistance may develop at intercapillary distances above 100 micron. If intercapillary distances exceed 140 micron, hypoxia is present at the arterial end of microvessels already. Hypoxia evidenced under "standard" conditions is distinctly aggravated by reduction of microvascular blood flow, increased arterio-venous shunt perfusion, reduced red blood cell flux, elongation of tumor microvessels, anemia or arterial hypoxemia. Hypoxic tissue fractions calculated for these various conditions are found to be in accordance with the proportion of tumor tissue Po2-readings at 0-1 mmHg.

The effects of intraperitoneally administered glucose or mannitol (5 mg/g body weight, 25% solutions) on tumor energy metabolism and tumor red blood cell flux were studied using 31P-nuclear magnetic resonance spectroscopy and laser Doppler flowmetry. Isotransplants of a spontaneous murine fibrosarcoma growing in the hind foot dorsum were used. 31P-nuclear magnetic resonance and laser Doppler flowmetry studies in glucose treated animals were performed on small (congruent to 100 mm3) and large (congruent to 300 mm3) tumors. In mannitol treated animals, tumors with an average volume of congruent to 200 mm3 were used. Using this tumor model, intraperitoneally administration of the hypertonic sugar solutions caused similar declines in tumor microcirculation (mannitol, 60 +/- 8% flow reduction; glucose, 72 +/- 4% flow reduction; t = 60 min). These changes were not glucose-specific and can primarily be explained by a water shift into the abdominal cavity and an associated hypovolemic hemoconcentration. A stable (small tumors) or transiently increased (large tumors) tumor energy metabolism which occurred after glucose administration was probably caused by a transiently increased glucose availability. The decline in energy metabolism after mannitol, a non-metabolized sugar alcohol, and the earlier decline in tumor pH seen in the glucose treated animals, supports this conclusion. The differences in the high energy phosphate response to glucose seen in small compared with large tumors, suggests that the baseline metabolic state of larger tumors includes a glucose deficiency in addition to tumor hypoxia.