Eppendorf pO2 Histograph Research Articles

Oxygen Tension and Vascular Density in Adenocarcinoma and Squamous Cell Carcinoma of the Uterine Cervix

The prognosis of patients with carcinoma of the uterine cervix has been shown to depend on the oxygenation and vascularization status of the tumors. The purpose of the study reported here was to search for possible differences in oxygen tension and vascular density between adenocarcinomas and squamous cell carcinomas. Ten patients with adenocarcinoma and forty patients with squamous cell carcinoma were included in the study. Oxygen tension was measured polarographically using the Eppendorf pO2 Histograph 6650. Vascular density was determined by histological examination of tumor biopsies. The adenocarcinomas were significantly better oxygenated than the squamous cell carcinomas. The squamous cell carcinomas and the adenocarcinomas did not differ significantly in vascular density. The difference in prognosis between patients with adenocarcinoma and patients with squamous cell carcinoma is probably not attributable to differences in tumor oxygenation or vascularization.

Measurement of Tumor Oxygenation: A Comparison between Polarographic Needle Electrodes and a Time-Resolved Luminescence-Based Optical Sensor

A novel oxygen sensor which does not rely on electrochemical reduction has been used to measure the oxygenation of the murine sarcoma F in a comparative study with an existing polarographic electrode that is available commercially. The prototype luminescence sensor yielded an oxygen distribution comparable with readings made using a pO2 histograph. The percentage of regions detected that had a pO2 less than 5 mm Hg was 79 and 75 using the Eppendorf pO2 histograph and the luminescence fiber optic sensor, respectively. These values were compatible with a measured radiobiologically hypoxic fraction of 67% in this tumor. The polarographic method detected more regions with a pO2 of 2.5 mm Hg or less (69%) compared with the optical sensor (50%) (P < 0.05). This could reflect differences in the oxygen use of the sensing devices. This initial assessment indicates the potential of a fiber-optic-based oxygen-monitoring system. Such a system should have several advantages including monitoring temporal oxygen changes in a given microregion and use with NMR procedures.

Oxygen tensions in rodent tumors after irradiation with neutrons.

We started investigations on intratumoral oxygen tension after irradiations with reactor fission neutrons using the Eppendorf-pO2 Histograph. Isotransplanted AT17-mammary carcinomas on C3H-mice and osteosarcomas OTS-64 on balb C-mice received 2 or 6 Gy neutrons single dose. Before and at certain points of time after treatment the pO2 values were evaluated. Some tumors with initially low median pO2 values showed a short-lasting increase between 2 and 24 h after irradiation. In those tumors with relatively high pretherapeutic pO2 values the pO2 decreased to the range of hypoxia. A third group of tumors showed no marked changes after irradiation. No tumor stopped growth during the observation period.

Comparing Techniques of Measuring Tumor Hypoxia in Different Murine Tumors: Eppendorf pO 2 Histograph, [ 3 H]Misonidazole Binding and Paired Survival Assay

Using five transplantable murine tumors (SCC-VII, B16F1, KHT-C, KHT-LP1, RIF-1), measurements of tumor hypoxia have been made with two techniques which have the potential to be used for assessing oxygenation in human tumors (the Eppendorf pO2 Histograph and binding of [3H]misonidazole) and have been compared with an established radiobiological technique, the paired survival assay. There were significant differences in the pO2 measurements made in individual tumors both within and between the five different tumor types. Significant differences between the tumor types were also found for the [3H]misonidazole binding. A correlation was observed between the mean values of the hypoxic proportion as measured by the paired survival assay and the mean binding of [3H]misonidazole as measured by both tumor activity in dpm/100 mg tissue (r = 0.94, P = 0.02) and the tumor-to-muscle activity ratio (r = 0.87, P = 0.06). No biologically significant correlation was seen between the mean values of the hypoxic proportion from anesthetized mice as measured by the paired survival assay (range 20-58%) and the pooled Eppendorf pO2 Histograph measurements made on groups of tumors. These results with the Eppendorf pO2 Histograph are similar to those reported by others. When both Eppendorf pO2 Histograph measurements and paired survival measurements were made on the same individual KHT-C tumors, it was again found that there was no correlation between the two measurements of hypoxia.

PEG-Hemoglobin:Effects on tumor oxygenation and radiosensitization

Derivatization with polyethylene glycol (PEG) is a biocompatible method to improve the therapeutic benefit of administered proteins by increasing their biological half-life. The ability of a PEG-bovine hemoglobin preparation (2–8 ml/kg) to increase the oxygen content of hypoxic tumor regions was determined in rats bearing the 13762 mammary carcinoma using an Eppendorf pO2 histograph. Administration of PEG-hemoglobin decreased the percent of pO2 readings ≤ 5 mmHg in animals breathing air or breathing carbogen, respectively. In rats, PEG-hemoglobin has a circulating half-life of 20 hr. A single administration of PEG-hemoglobin was able to provide increased tumor oxygenation on the following at least 3 days if atmospheres of 28% oxygen or carbogen were breathed. PEG-hemoglobin administration along with fractionated radiation therapy (2, 3 or 4 Gy × 5) resulted in radiation dose modifying factors of 1.2 ± 0.2, 1.4 ± 0.2 and 1.5 ± 0.2 when the animals breathed air, 28% oxygen or carbogen prior to and during radiation delivery, respectively. Using the EMT-6 murine mammary carcinoma tumor cell survival assay, administration of PEG-hemoglobin (6 ml/kg) prior to single dose radiation therapy resulted in dose modifying factors of 1.4 ± 0.2 1.8 ± 0.2 and 1.9 ± 0.2 when the animals breathed air, 28% oxygen or carbogen prior to and during radiation delivery, respectively. In EMT-6 tumor growth delay, administration of PEG-hemoglobin (6 ml/kg) along with fractionated radiation therapy (2, 3 or 4 Gy × 5) resulted in dose modifying factors of 1.2 ± 0.2, 1.5 ± 0.2 and 1.7 ± 0.2 when the animals breathed air, 28% oxygen or carbogen prior to and during radiation delivery. Administration of PEG-hemoglobin only on the first treatment day of a 5-day fractionated radiation therapy regimen resulted in dose modifying factors of 1.3 ± 0.2 and 1.4 ± 0.2 if the animals breathed 28% oxygen or carbogen prior to and during each radiation fraction delivery. Further investigation of this potentially clinically important oxygen delivery agent is warranted. Radiat. Oncol. Invest. 4:200–210, 1996. © 1997 Wiley-Liss, Inc.

Tumor reoxygenation as a mechanism of taxol-induced enhancement of tumor radioresponse.

Paclitaxel is a novel chemotherapeutic agent that arrests cells in the radiosensitive G2 and M phases of the cell cycle and as such may act as a specific cell cycle radiosensitizer. We recently reported that paclitexel induces mitotic arrest in the MCA-4 murine mammary carcinoma and enhances radio-response of this tumor. However, the greatest enhancement was observed not when radiation was given at the time of peak mitotic arrest, which was 9 h after paclitaxel administration, but when it was given 24 h after paclitaxel. This implied the involvement of other mechanisms in radiosensitization; we hypothesized that tumor reoxygenation was a likely mechanism based on the observed massive loss of mitotically arrested cells at 24 h. The present study shows that paclitaxel greatly enhanced MCA-4 tumor radioresponse when radiation was given under air-breathing conditions (DMF = 1.74), but not when it was performed under hypoxic conditions. This observation supports the hypothesis of tumor reoxygenation as a mechanism of enhancement of tumor radioresponse. That reoxygenation occurred in tumors treated with paclitaxel 24 h earlier was confirmed by direct measurements of pO2 values, using the Eppendorf pO2 histograph. Median pO2 values increased from 6.2 mmHg in untreated tumors to 10.0 mmHg in tumors treated with paclitaxel. These observations emphasize the importance of timing of paclitaxel administration in relation to radiation treatment.

Cytotoxic Effect of Tumour Necrosis Factor-Alpha on Sarcoma F Cells at Tumour Relevant Oxygen Tensions

We have investigated the response of a murine tumour cell line, the sarcoma F (SaF), to tumour necrosis factor-alpha (TNF) at oxygen tensions known to occur in vivo. Using the Eppendorf pO2 histograph, the oxygen status of SaF tumours grown in situ was assessed. The median pO2 of the SaF is less than 1% oxygen with over 90% of values at or below 15 mmHg (< 2% O2). SaF cells primed in vitro for 24 h at tumour relevant oxygen tensions required at least four times more TNF to reduce cell number to 50% of controls following a 24 h incubation period in 21% oxygen. Chronic exposure of SaF cells to hypoxia during several passages increased resistance to TNF more than 60-fold. The oxygen sensitizing effect is transient as the resistance of hypoxic cells to TNF was reversed after 24 h incubation in air. These data clearly show that oxygen tension is a key modulator of the cytotoxic action of this important cytokine.

Increased Tumor Oxygenation and Radiation Sensitivity in two Rat Tumors by A Hemoglobin-Based, Oxygen-Carrying Preparation

The rat 13762 mammary carcinoma and the rat 9L gliosarcoma were grown subcutaneously in a hind limb of female, Fisher 344 rats. The oxygen content of the tumors was determined using an Eppendorf pO2 histograph. Fifty-to-sixty oxygen measurements were made per tumor and there were 8-to-10 animals per group. The percent of pO2 readings < or = 5 mmHg in the mammary carcinoma was 49%, this was decreased to 34% by administration of the hemoglobin preparation (8 ml/kg) and further decreased to 29% when carbogen (95% O2/5% CO2) breathing was added to administration of the hemoglobin preparation. The percent of pO2 readings < or = 5 mmHg in the gliosarcoma was 49%, this was decreased to 24% by administration of the hemoglobin preparation and further decreased to 0% when carbogen breathing was added to administration of the hemoglobin preparation. Therapeutic response was assessed over a single-dose range of radiation therapy (10, 20 and 30 Gray). The dose modifying factor produced by the hemoglobin preparation/air was 1.6 and by the hemoglobin preparation/carbogen was 2.7 in the rat 13762 mammary carcinoma. The dose modifying factor produced by the hemoglobin preparation/air was 1.9 and by the hemoglobin preparation/carbogen was 2.9 in the rat 9L gliosarcoma. Administration of a hemoglobin-based oxygen carrier reduced tumor hypoxia and increased tumor response to radiation therapy.

Reduced oxygenation in a rat mammary carcinoma after chemo- or radiation therapy and reoxygenation with perflubron emulsion/carbogen breathing.

Rat 13672 mammary carcinoma tumors were grown subcutaneously in the hind legs of female Fischer 344 rats to a volume of about 1 cm3. Tumor oxygenation was measured using an Eppendorf PO2 histograph. Tumor oxygen measurements were made under four conditions: (a) normal air breathing, (b) carbogen breathing, (c) after intravenous administration of a perflubron emulsion (8 ml/kg) with air breathing and (d) after intravenous administration of a perflubron emulsion (8 ml/kg) with carbogen breathing. Tumor oxygenation was examined without treatment or 24 h and 48 h after treatment with cyclophosphamide (300 mg/kg, i.p.) or cisplatin (8 mg/kg, i.p.] or after the fifth dose of a daily regimen of 3-Gy irradiation (5 x 3 Gy). Under normal air-breathing conditions 49% of the tumor had a PO2 < or = 670 Pa (5 mm Hg). The degree of hypoxia in the tumors increased after each treatment such that 24 h after treatment 65%-85% of the oxygen readings were < or = 670 Pa and 48 h after treatment 60%-74% of the oxygen readings were < or = 670 Pa. Administration of the perflubron emulsion/carbogen atmosphere increased the oxygen content of the tumors both without treatment and after each of the treatments. A knowledge of tumor oxygen content over the course of treatment and the ability to increase tumor oxygen should allow for the development of more rational treatment combinations and better treatment outcomes.

Oxygenation of Human Tumor Xenografts in Nude Mice by a Perfluorochemical Emulsion and Carbogen Breathing

Human solid tumors (prostate carcinomas PC-3 and DU-145, breast carcinoma MX-1, cervical carcinoma ME-180, small cell lung carcinoma SW2, and glioblastoma T98G) were grown as xenografts in nude mice. Using the Eppendorf pO2 histograph microelectrode system, the oxygen profiles of the tumors were determined while the animals breathed air or carbogen (95% O2/5% CO2), and after administration of the perfluorochemical emulsion Oxygent-CA (8 ml/kg) under air breathing and carbogen breathing conditions. Under normal air breathing with or without Oxygent-CA administration the mean oxygen tensions were between 4.9 and 9.3 mmHg and each tumor had severely hypoxic regions where the pO2 was less than 5 mmHg. The severely hypoxic regions comprised 41-71% of the oxygen tension measurements under normal air breathing conditions. Carbogen breathing alone increased the mean oxygen tensions to 10.9-23.9 mmHg. Administration of Oxygent-CA and carbogen breathing increased the mean oxygen tensions over the levels of carbogen breathing alone to varying degrees. The highest mean oxygen tensions were 40.8 mmHg in the T98G glioblastoma and 24.5 mmHg in the ME-180 cervical carcinoma. Investigation of the use of Oxygent-CA/carbogen to increase the oxygenation of clinical tumors is warranted.