Friedrich Kallinowski

Tumor tissue oxygenation as evaluated by computerized-po2-histography

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.

Correlations between 31P-NMR spectroscopy and tissue O2 tension measurements in a murine fibrosarcoma.

Size-dependent changes in therapeutically relevant and interrelated metabolic parameters of a murine fibrosarcoma (FSaII) were investigated in vivo using conscious (unanesthetized) animals and tumor sizes less than or equal to 2% of body weight. Tumor pH and bioenergetics were evaluated by 31P nuclear magnetic resonance spectroscopy (31P-MRS), and tumor tissue oxygen tension (pO2) distribution was examined using O2-sensitive needle electrodes. During growth FSaII tumors showed a progressive loss of phosphocreatine (PCr) and nucleoside triphosphate (NTP) with increasing inorganic phosphate (Pi) and phosphomonoester (PME) signals. Ratios for PCr/Pi, PME/Pi, NTP/Pi, and phosphodiester/inorganic phosphate (PDE/Pi) as well as pH determined by 31P-NMR (pHNMR) and the mean tissue pO2 progressively declined as the tumors increased in size. The only relevant ratio increasing with tumor growth was PME/NTP. When the mean tissue pO2 value was plotted against pHNMR, NTP/Pi, PCr/Pi, PME/Pi, and PDE/Pi for tumor groups of similar mean volumes, a highly significant positive correlation was observed. There was a negative correlation between mean tumor tissue pO2 values and PME/NTP. From these results we concluded that 31P-MRS can detect changes in tumor bioenergetics brought about by changes in tumor oxygenation. Furthermore, the close correlation between oxygenation and energy status suggests that the microcirculation in FSaII tumors yields an O2-limited energy metabolism. Finally, a correlation between the proportion of pO2 readings between 0 and 2.5 mmHg and the radiobiologically hypoxic cell fraction in FSaII tumors was observed. The latter finding might be of particular importance for radiation therapy.

Effects of hydralazine on in vivo tumor energy metabolism, hematopoietic radiation sensitivity, and cardiovascular parameters☆

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.

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 (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).

Oxygenation of Tumors Derived from ras Transformed Cells

In order to gain insight into mechanisms governing the development of tumor hypoxia, malignancies derived from spontaneously tumorigenic or ras-transformed cell lines were grown in nude mice. As a rule, tumors with ras oncogenes exhibited rapid growth rates and large areas with low pO2 readings even at small tumor sizes. The slow proliferation rate of a spontaneously tumorigenic cell line was consistent with more adequate tissue oxygen levels. In all lines, hypoxia was accentuated at larger tumor sizes. These results demonstrate that ras transformation can lead to accelerated proliferation rates and is then concomitant with the development of pronounced tumor hypoxia.