Robert F. Kallman

The effect of treatment in fractionated schedules with the combination of x-irradiation and six cytotoxic drugs on the RIF-1 tumor and normal mouse skin

RIF-1 tumors, implanted syngeneically in the gastrocnemius muscles of the right hind legs of C3H/Km mice, were treated either with X ray alone, drug alone, or drug and X ray combined. The drugs tested were bleomycin, BCNU, cis-diamminedichloro platinum, adriamycin, cyclophosphamide, and actinomycin-D. All drugs were administered either in the maximum tolerated dose or a dose that causes minimal tumor growth delay. Both drugs and X rays were administered either as a single dose or in five daily fractions. In addition to the single modality controls, seven different schedules of combined modalities were tested. Tumors were measured periodically after treatment in order that the day at which each tumor reached 4 times its initial cross-sectional area, i.e., its size at the time of treatment, could be determined. The effect of treatment on tumors was based upon excess growth delay (GD), i.e., T400% (treated)-T400% (untreated control). Treatment effects for the same combined modality schedules were also determined for normal skin, using the early skin reaction as an endpoint. Dose effect factors (DEF) were computed for all combined modality schedules and were based upon calculated radiation dose equivalents. We also calculated supra-additivity ratios, SRI and SRII, therapeutic gain factors and adjusted therapeutic gain factors. The only drugs to produce significant supra-additivity with X rays were cis-Pt and cyclo. Maximum supra-additivity for cis-Pt was afforded by divided doses of the drug (5 X 2.4 mg/kg/day) given immediately before X ray (5 X 1000 rad/day) on 5 consecutive days, although 3 other schedules also produced significant supra-additivity. Maximum supra-additivity for cyclo was seen for a single dose of 100 mg/kg followed 1 day later by a course of 5 daily X ray doses (5 X 1000 rad/day), and at least one other schedule produced almost as great an effect.

Therapeutic enhancement in mice by clinically relevant dose and fractionation schedules of cis-diamminedichloroplatinum(II) and irradiation

The interaction of cis-diamminedichloroplatinum(II) (c-DDP) and irradiation was investigated in the RIF-1 tumor, skin and duodenum in C3H/Km mice. The c-DDP enhanced the effects of fractionated irradiation in the tumor, as measured by both growth delay and cell survival determined by excision and colony formation in vitro. This enhancement was at least additive and possibly supra-additive. The combined treatment was effective in clinically relevant treatment regimens. The same doses of c-DDP failed to enhance acute radiation skin reactions and caused only a moderate enhancement of the radiation-induced cell killing in duodenal crypt cells. The latter was found when c-DDP was administered immediately before each irradiation dose in a fractionated treatment schedule, but not when it was given in a single larger dose 24 h before the start of fractionated radiotherapy. Comparing the effects on tumor with those on normal tissues, the optimum treatment appeared to be c-DDP given once a week 24 h before daily fractionated irradiation.

Tumor oxygenation and reoxygenation during radiation theraphy: Their importance in predicting tumor response

The degree of oxygenation of a tumor is a major determinant of the effectiveness of radiotherapy. From animal experiments, it is known that hypoxia is common in tumors, that there can be marked heterogeneity in cellular oxygenation within a given tumor, and that the hypoxic fraction is influenced by tumor size and site. The three methods used in obtaining such information are discussed. They are not applicable to tumors in man, but other kinds of evidence suggest qualitatively that the patterns of oxygenation in human tumors are not dissimilar to those in animals. Reoxygenation of animal tumors appears to proceed rapidly after single conditioning radiation doses, but its rate and extent depend upon the size and scheduling of the conditioning dose(s). The importance of reoxygenation in determining the outcome of fractionated radiotherapy may be inferred from the results of recent tumor growth delay experiments wherein we have found that reoxygenation in the RIF-1 sarcoma is nearly complete by 24 hr. after the completion of 5 daily 5 Gy fractional doses.

The effect of time between x-irradiation and chemotherapy on the growth of three solid mouse tumors—II. cyclophosphamidet

Abstract Experiments have been carried out to determine the effect of different time intervals between the administration of X-radiation (1200 red) and cyclophosphamide (100 mg/kg) on the growth delay produced in 3 mouse tumors. The tumors used were the EMT6 tumor in BALB/c mice and the KHT and RIF-1 sarcomas in C3H mice. All tumors were grown intramuscularly in the gastrocnemius muscle and treatment was carried out at a mean tumor weight of 450 mg. Time to reach 2X (for KNIT) or 4X (for EMT6 and RIF-1) treatment volume was used as the endpoint of response. The drug was administered intraperitoneally either 24, 6, or 2 hr before radiation, immediately before the start of radiation, or 3, 6, or 24 hr after radiation. All irradiations were carried out in unanesthetized mice. For the RIF-1, EMT6, and KHT tumors, the growth delays due to the drug alone were 11, 4.5, and 12 days, respectively. In the RIF-1 system, the growth delays following combination treatment tended to be longer than predicted by the addition of the single agent delays. For the KHT tumor, the opposite trend was seen, whereas in EMT6, there was no significant trend in either direction. No consistent dependence upon the timing between irradiation and drug administration was seen from system to system.

Migration and internalization of cells and polystyrene microspheres in tumor cell spheroids

Abstract The movement and internalization of 3H-labelled cells and of inert polystyrene microspheres within multicellular spheroids has been examined through histological sectioning and autoradiography. EMT6 and RIF-1 spheroids were cultured in spinner flasks for approx. 2.5 weeks. At this time, 3H-labelled cells and/or microspheres were allowed to adhere to the spheroid surface. Microspheres, 3H-labelled RIF-1 monolayer cells and 3H-labelled EMT6 monolayer cells were observed to move centripetally as a wave into EMT6 spheroids. In contrast, 3H-labelled trypsinized RIF-1 and EMT6 spheroid cells became mixed with the other non-labelled spheroid cells in homotypic RIF-1 and EMT6 spheroids, respectively. Reduction of spheroid growth by maintaining the spheroids at room temperature and by treatment with 2500 rads irradiation did not prohibit the internalization of 3H-labelled EMT6 cells and microspheres in EMT6 spheroids.

Effects of fractionated schedules of irradiation combined with cis-diamminedichloroplatinum II on the SCCVII/St tumor and normal tissues of the C3H/km mouse

The interaction of cis-diamminedichloroplatinum II (c-DDP) and a course of 5 daily irradiations was investigated in the SCCVII/St tumor and normal tissues (duodenal crypt cells and lung) of the C3H mouse. Two schedules with daily doses of 2.4 mg/kg c-DDP given immediately before 4 Gy X ray on 5 consecutive days and a single 12 mg/kg c-DDP dose followed 24 hr later by the first of 5 daily 4 Gy X ray doses produced the most consistent and significant supra-additive effects on the SCCVII tumor. This supra-additive effect was also achieved with lower and much less toxic drug doses. These schedules produced high enhancement ratios (dose effect factors DEF) for mouse duodenal crypt cells, but the degree of enhancement was less than that for the SCCVII tumor. Schedules with a 72-hr interval between drug and radiation treatments, which produced low enhancement ratios for the SCCVII tumor and duodenal crypt cells, gave high enhancement ratios for the lung. It is concluded that c-DDP has the potential of enhancing the radiation effect on normal tissues, and the degree of enhancement depends upon the interval between X ray and c-DDP. The enhancement ratios for the SCCVII tumors are greater than for normal tissues, and this results in high therapeutic gain factors (TGF). Comparing the effects on tumor with those on normal tissues, it may be seen that there is clinical usefulness in simultaneous combination treatments and perhaps moreso in the administration of a single drug dose 24 hr before the first of 5 daily X ray fractions.

In vitro and in vivo labelling of animal tumours with tritiated thymidine.

The labelling indices obtained by incubating tumour specimens with tritiated thymidine in vitro under hyperbaric oxygen have been compared with those obtained by labelling a matched tumour in vivo. The correspondence between these individual labelling indices is sometimes very poor; however, the average labelling index derived from groups of tumours labelled in the two ways does not differ significantly. There was a large variation from field to field within any tumour, and considerable variation from one tumour to another within each tumour type. The mitotic index was also compared in the matched tumour preparations; the mitotic index in vitro was almost always considerably lower than the values observed in vivo.

Reoxygenation in the RIF-1 tumor

The proportion of hypoxic cells in the RIF-1 tumor was examined for 13 days following a 15 Gy conditioning dose. The paired survival curve technique indicated that 100% of the surviving cells were hypoxic immediately following this treatment. However, within 1 hour, only about 50% remained hypoxic; this proportion continued to drop to about 10% but did not reach the pretreatment level of 1.1% for the duration of the study.

The effect of time between X-irradiation and chemotherapy on the growth of three solid mouse tumors. III. Cis-diamminedichloroplatinum.

Abstract Experiments have been carried out to detemine the effect of different time intervals between the administration of X-irradiation (1200 rad) and cis -diamminedichloroplatinum ( cis -DDP) (7 mg/kg) on the growth delay produced in three mouse tumors. The tumors used were the EMT6 tumor in BALB/c mice and the KHT and RIF-1 sarcomas in C3H mice. All tumors were grown intramuscularly in the gastrocnemius muscle and treatment was carried out at a mean tumor weight of 450 mg. Time to reach 2X (for KHT) or 4X (for EMT6 and RIF-1) treatment volume was used as the endpoint of response. The drug was administered by the intraperitoneal route either 24, 6, or 2 hr before radiation, immediately before the start of radiation, or 3, 6, or 24 hr after radiation. All irradiations were carried out in unanesthetized mice. The growth delays due to the drug alone were 2, 10, and 2 days for the EMT6, RIF-1, and KHT tumors, respectively. In the RIF-1 and KHT tumors, the combined modality groups tend to show longer growth delays than predicted by the addition of the growth delays for the single agents. For the EMT6 tumor, however, the trend is in the opposite direction. There is no particular timing between irradiation and drug administration which appears to produce consistently longer or shorter growth delays from system to system.

The effect of time between X-irradiation and chemotherapy on the growth of three solid mouse tumors—I. Adriamycin☆

Experiments have been carried out to determine the effect of different time intervals between X-irradiation (120Orad) and the administration of adrfamycin (ADR) (6mg/kg) on the growth deiay produced in 3 mouse tumors. The tumors used were the EMTo/St/i.d. tumor in BALB/c mice and the KIIT and RIF-1 sarcomas in C3H mice. Ail tumors were grown intramuscularly in the gastrocnemius muscie and treatment was carried out at a mean tumor weight of 450 mg. Time to reach 2X (for KHT) or 4X (for EMT6/St/i.d. and RIF-1) treatment volume was used as the endpoint of response. The drug was administered by the intraperitoneal route either 24, 6, or 2 hr before radiation, immediately before the start of radiation, or 3, 6, or 24 hr after radiation. Ail irradiations were carried out in unanesthetixed mice. For a single administration at this dose leve1 (close to the maximum tolerated dose), ADR alone produced oniy minima1 growth delay in any of the tumors. Furthermore, the growth delays produced by drug/radiation combinations were not significantly different from the additfon of the growth delays produced by the single modalities.