Douglas F. Martin

ENHANCEMENT OF TUMOR RADIATION RESPONSE BY THE COMBINATION OF A PERFLUOROCHEMICAL EMULSION AND HYPERBARIC OXYGEN

The presence of radioresistant hypoxic cells in tumors is believed to be one of the limiting factors in achieving local tumor control by radiotherapy. Treatment with hyperbaric oxygen during irradiation has been shown to improve the radiation response of many solid tumors in rodents and of some tumors in patients. Intravenous administration of perfluorochemical emulsions combined with oxygen breathing at atmospheric pressure has also been shown to improve the radiation response of several rodent tumors. Theoretical considerations suggest that the combination of a perfluorochemical emulsion and hyperbaric oxygen should be significantly more effective than either agent alone. This hypothesis was tested by examining the radiation response of BA1112 rhabdomyosarcomas growing in WAG/rij-Y rats. Treatment with a perfluorochemical emulsion, Fluosol-DA, plus hyperbaric oxygen (3 Atmospheres O2) significantly increased the radiation response of the malignant cells in these solid tumors. The observed changes in the tumor cell survival curve suggest that the combination of Fluosol-DA and HBO decreases the proportion of severely hypoxic cells in the tumor to less than 1.5% of the original value. The effect of the Fluosol-DA dose and the duration of pretreatment with HBO are described.

Effect of a perfluorochemical emulsion on the radiation response of BA1112 rhabdomyosarcomas.

The effect of treatment with a perfluorochemical emulsion (Fluosol DA, 20%), carbogen, or the combination of these two agents on the radiation response of BA1112 tumors in WAG/rij rats was examined. Fluosol and carbogen as single agents had only small effects on the tumor cell survival curve. The combination of Fluosol plus carbogen had a larger effect on tumor cell survival, reducing the hypoxic fraction of the tumor from 23 to 1.6%. The amount of sensitization was a function of the Fluosol dose, with maximal augmentation of the radiation response obtained at doses of 7.5-15 ml/kg. Carbogen pretreatments ranging from 5 to 60 min in duration all had similar effects on tumor radiosensitivity. The effect of the perfluorochemical emulsion plus carbogen on the survival of irradiated tumor cells appears to reflect changes in tumor oxygenation, rather than cytotoxic or immunological effects, since the perfluorochemical emulsion (with or without carbogen) had no effect on the viability of cells in unirradiated tumors. These experiments extend previous studies by ourselves and others using mouse tumors to show that the combination of a perfluorochemical emulsion and carbogen breathing can also increase the radiation response of a nonimmunogenic rat tumor.

Radiation sensitivity of cultured rabbit aortic endothelial cells

The radiation response of cultured rabbit aortic endothelial cells was measured using colony formation to determine survival. The dose survival curve was qualitatively similar to those reported for other cell types. At doses of 400 rad and greater, the curve was an exponential function of dose with a D0 of 120 rad and an n of 7. Exponentially growing endothelial cell cultures recovered from sublethal damage between two doses of radiation. Plateau phase cultures recovered from potentially lethal damage when incubated for 6 hr between irradiation and assay.

Hypoxic Fraction Determinations with the BA1112 Rat Sarcoma: Variation within and among Assay Techniques

Measurement of the hypoxic fraction in the same tumor system by different assay techniques often gives incompatible results. The hypoxic fraction of the BA1112 sarcoma has been measured by three assay techniques: tumor control and growth delay assays in which the tumors remain in situ after irradiation, and paired survival curve assays in which the tumors are excised after irradiation. The assays were done with and without anesthesia on tumors growing in two different subcutaneous sites. Anesthesia of the hosts produced a statistically significant decrease in the calculated hypoxic fraction in a tumor control assay, but not in a paired survival assay. The excision assays gave consistently higher hypoxic fraction estimates than either the tumor control or growth delay assays. Some of the factors which could produce higher hypoxic fractions in excision assays than in in situ assays are discussed. Interpretation of the results was complicated by disagreements between the growth delay and tumor control assays, and by disparities between replicate determinations. The discrepancies among assays are caused almost entirely by variations in the response of the artificially hypoxic tumors, rather than variations in the response of aerobic tumors. These results indicate that assumptions commonly made when measuring hypoxic fractions may not be valid.

Tumor-to-tumor variability in the hypoxic fractions of experimental rodent tumors

Paired determinations of the radiation responses of normally-aerated and artificially hypoxic rodent tumors, performed to measure the hypoxic fractions of the tumors, were obtained from our own laboratories and from the literature. The data were reanalyzed to assess whether the variabilities in the radiation responses of the normally-aerated and artificially hypoxic tumors were similar. If there were large differences in the hypoxic fractions of individual tumors within the experiments, the variability in the data from aerobic tumors would be expected to be greater than the variability in the data from artificially hypoxic tumors (which should all be brought to uniform hypoxia and therefore uniform radioresistance). The analyses revealed the variability to be as great or greater for hypoxic tumors as for normally-aerated tumors. This finding suggests that factors other than tumor-to-tumor differences in oxygenation produce most of the variability in the radiation responses of individual tumors from an experimental tumor line.

Development of an in vitro assay for the survival of cells suspended from BA1112 rat sarcomas

A method has been developed for the in vitro assay of the survival of BA1112 rat sarcoma cells following treatment in vivo. The BA1112 tumor was not adapted for growth in culture and was maintained by serial transplantation in vivo. The assay used a feeder layer of heavily irradiated cells to eliminate any changes in plating efficiency caused by the large number of dying cells which must be plated when assaying cell survival after intensive treatments. The plating efficiency of the tumor cells was not affected significantly by the age of the feeder layer (between 1 and 4 days), the density at which the experimental cells were plated or the interval between preparation of the cell suspension and plating (up to 4 hr). Using the in vitro assay a survival curve was determined for cells from tumors irradiated in air-breathing animals. This curve was similar to that determined previously for BA1112 tumor cells using an in vivo assay to measure cell survival.

Identification of a X174 coded protein involved in the shut-off of host dna replication

Summary When Escherichia coli is infected with X174, the host cell DNA replication ceases 10 – 12 minutes after infection. This shut-off is prevented by the addition of 30μg/ml chloramphenicol indicating a requirement for post-infection protein synthesis. Some amber mutants in X174 cistron A, which codes for two proteins, do not shut off the host DNA replication. Amber mutants in all other X174 genes shut off host replication. Mutants near the amino-terminal end of cistron A, which still produce the small 35, 000 molecular weight cistron A polypeptide, shut off the host DNA synthesis whereas mutants near the carboxy-terminal end, which do not produce the small A polypeptide do not shut off the host.

Effectiveness and biological effects of techniques used to induce hypoxia in solid tumors

Acute hypoxia is often induced in rodent tumors during studies of the oxygenation or the therapeutic responses of the tumors, either by clamping the blood supply to the tumors or by asphyxiating the hosts with nitrogen. Analyses of data from such experiments generally assume that these processes have no effects other than the induction of hypoxia and that uniform, severe hypoxia is produced throughout the tumors. The studies described in this report test several aspects of these assumptions using BA1112 rat rhabdomyosarcomas and EMT6 mouse mammary tumors. Both clamping and asphyxiation appear to be effective in producing hypoxia in the tumors. Induction of artificial hypoxia for the times necessary for irradiation was not toxic to the tumor cells and generally did not alter the growth of unirradiated tumors. However, the techniques are not without significant effects. Prolonged clamping produced extensive cytotoxicity. Clamping BA1112 tumors for 30 min and removing the clamp just before irradiation altered the tumor cell survival curve and the TCD50. Furthermore, anesthesia and/or restraint (necessary during clamping) have significant effects on tumors and hosts. The data show that the assumptions underlying the use of clamping and N2-asphyxiation to produce hypoxia for short periods in vivo are generally reasonable for BA1112 and EMT6 tumors, but illustrate the necessity for carefully examining the effects and efficacies of the procedures in each tumor/host system.

Development of an 241Am applicator for continuous low-dose-rate irradiation of the rat sarcoma BA1112

An 241Am applicator for continuous low-dose-rate irradiation of the rat sarcoma BA1112 has been developed. The irradiator consists of two disc sources, each containing 800 mCi of 241Am, an isotope which emits primarily 60 keV photons. The disc sources are held in a specially-designed light-weight helmet which surrounds the tumor on the head of the rat. Dose distributions produced by these sources have been measured using an ionization chamber, thermoluminescent dosimeters and Fricke dosimeter. A computerized treatment planning system has been modified to compute dose distributions from 241Am sources, to optimize the design of this applicator. Computed and measured dose distributions for several values of separation between the 241Am discs are presented. A survival curve for cells from tumors irradiated in vivo with this applicator has been determined by an in vitro colony formation technique. The mean lethal dose DO was found to be 720 cGy for an average tumor dose rate of 95 +/- 7 cGy/hr. The major advantages of the 241Am applicator in comparison with the 192Ir applicator used previously for continuous low-dose-rate studies are: a considerably smaller half value layer thickness and the longer half life of the radionuclide. These features make it more suitable for long-term tumor cure studies because of the lower whole body dose to the animal, the availability of relatively constant dose-rate irradiators for many years, the decreased shielding requirements for the animal care facility and the diminished exposure to laboratory personnel involved with the implants on the animals.