A new mouse tumor model system (RIF-1) for comparison of end-point studies.

A new tumor model system (RIF-1) was developed that is very suitable for studies in which clonogenic survival is compared with growth delay and control probability following various forms of treatment. The tumor was a radiation-induced sarcoma in the inbred female C3H/Km mouse. It had a low median tumor dose, had a satisfactory plating efficiency direct from in vivo to in vitro, was nonimmunogenic or minimally immunogenic, and metastasized only at a relatively advanced stage of growth. The cell line grew either as a monolayer on plastic dishes, as tumor spheroids in spinner culture, as lung nodules following injection of a single-cell suspension into the tail veins of syngeneic mice, or as a solid tumor. Both diploid and tetraploid clonogenic cells were found in monolayer cultures of the RIF-1 line.

Schedule-dependent therapeutic gain from the combination of fractionated irradiation and cis-diamminedichloroplatinum (II) in C3H/Km mouse model systems.

The interaction of cis-diamminedichloroplatinum(II) (c-DDP) with daily fractionated radiotherapy was studied in the SCCVII tumor, the duodenum, and the lungs of C3H/Km mice. The experimental end points were the time required for treated tumors to reach 3 times their treatment size, the survival of stem cells in the duodenal crypts, and the breathing rate measured early (19-23 weeks) and late (41-46 weeks) after treatment. In the 8 treatment schedules that were evaluated, radiation was delivered in 5 daily doses of 2-7 Gy, for total doses of 10-35 Gy; and c-DDP was administered either daily (2.4 or 1.6 mg/kg/day) or as a single bolus (8 or 12 mg/kg). Schedule 2, in which 2.4 mg/kg c-DDP was administered immediately before X-ray on 5 consecutive days produced the highest degree of enhancement of radiation effect (expressed as dose-effect factor); and the next greatest enhancement was produced by 12 mg/kg c-DDP administered 24 h before the start of fractionated daily radiotherapy. Those schedules also caused some enhancement in the normal tissues, but the dose-effect factors for those tissues were lower than for the tumor, which was reflected in the finding of maximal therapeutic gain factors for those same schedules. There was little or no enhancement nor were the therapeutic gain factors significantly greater than 1.00 when the 2 modalities were administered more than 24 h apart. Thus, for both normal tissue toxicity and antitumor effect there is striking schedule dependence with respect to both sequence and timing of these 2 modalities. This is of major relevance in clinical treatment planning.

Recruitment of noncycling tumor cells into proliferation by isoproterenol.

The resistance of tumors to conventional anticancer therapy is partially determined by the fraction of noncycling cells they contain. In several normal tissues, isoproterenol can stimulate cell proliferation. The effect of isoproterenol on the EMT6/St tumor grown both intradermally in BALB/c mice and in vitro as multicell spheroids was investigated. The incorporation of [3H]thymidine (5 microCi/g, 6.7 Ci/mmol) into DNA was determined as a function of time after i.p. injection of isoproterenol (0.15 mg/g). Tissue section autoradiographs of the EMT6 tumors were prepared, and labeling indices were determined. EMT6 spheroids were exposed to isoproterenol and labeled with [3H]thymidine in a manner designed to simulate drug exposure in vivo. In EMT6 tumors, the labeling index rose from a control level of 30% to a peak of 53% at 25 hr; it then declined gradually for the next 30 hr. This may be interpreted as evidence that cells were recruited into proliferation. In contrast, isoproterenol had minimal effects on EMT6 spheroids, implying that recruitment caused by this drug was not a direct effect on individual cells.

Interleukin 1 beta initially sensitizes and subsequently protects murine intestinal stem cells exposed to photon radiation.

Interleukin 1 (IL-1) has been shown to prevent early bone marrow-related death following total-body irradiation, by protecting hematopoietic stem cells and speeding marrow repopulation. This study assesses the effect of IL-1 on the radiation response of the intestinal mucosal stem cell, a nonhematopoietic normal cell relevant to clinical radiation therapy. As observed with bone marrow, administration of human recombinant IL-1 beta (4 micrograms/kg) to C3H/Km mice 20 h prior to total-body irradiation modestly protected duodenal crypt cells. In contrast to bone marrow, IL-1 given 4 or 8 h before radiation sensitized intestinal crypt cells. IL-1 exposure did not substantially alter the slope of the crypt cell survival curve but did affect the shoulder: the X-ray survival curve was offset to the right by 1.01 +/- 0.06 Gy when IL-1 was given 20 h earlier and by 1.28 +/- 0.08 Gy to the left at the 4-h interval. Protection was greatest when IL-1 was administered 20 h before irradiation, but minimal effects persisted as long as 7 days after a single injection. The magnitude of radioprotection at 20 h or of radiosensitization at 4 h increased rapidly as IL-1 dose increased from 0 to 4 micrograms/kg. However, doses ranging from 10 to 100 micrograms/kg produced no further difference in radiation response. Animals treated with saline or IL-1 had similar core temperatures from 4 to 24 h after administration, suggesting that thermal changes were not responsible for either sensitization or protection. Mice irradiated 20 h after IL-1 had significantly greater crypt cell survival than saline-treated irradiated controls at all assay times, which ranged from 54 to 126 h following irradiation. The intervals to maximum crypt depopulation and initiation of repopulation were identical in both saline- and IL-1-treated groups, suggesting that IL-1 altered absolute stem cell survival but not the kinetics of repopulation.

The importance of schedule and drug dose intensity in combinations of modalities.

PURPOSE: To review a series of laboratory experiments that have been conducted to investigate the degree to which therapeutic effectiveness is a function of the scheduling of two or more therapeutic agents. METHODS AND MATERIALS: The therapeutic effectiveness of a few combinations of modalities (fractionated irradiation + cis-DDP alone and with 5FU, cyclophosphamide, or etoposide) has been evaluated systematically for a large number of schedules. For every such schedule, therapeutic gain factors were calculated as the ratio of effectiveness for tumor growth inhibition to each of three normal tissue endpoints in laboratory mice: duodenal crypt cell survival, pneumonitis (breathing rate at 5 months after treatment), and pulmonary fibrosis (breathing rate at 10 months). RESULTS: For every combination tested, at least one schedule was distinctly therapeutically superior to the others. Most often, the greatest therapeutic gain was achieved with divided doses of cis-DDP administered simultaneously with five daily x-ray dose fractions. Even greater gain was found when cyclophosphamide was administered as a single bolus 1 day before fractionated cis-DDP/irradiation. Still greater therapeutic gain was achieved by protecting against normal tissue toxicity by administering the cytokine, interleukin 1, prior to chemotherapy-radiotherapy. CONCLUSION: Performing experiments of the type described in this paper can be of great value in the optimization of treatment with combinations of agents or modalities.

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.

Tumor oxygenation and reoxygenation during radiation therapy: 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.

Reoxygenation in the RIF-1 tumor after chemotherapy.

The proportion of hypoxic cells in the RIF-1 tumor was observed for 24 hr after treatments with bleomycin, cisplatin, cyclophosphamide, and mitomycin C. The assays were based upon the paired survival curve method for determining the hypoxic fraction using irradiation of aerobic and artificially hypoxic tumors. It was observed that at 1/2 hr after bleomycin, hypoxic fraction was elevated but returned to baseline levels by 2 hr. Following cisplatin, hypoxic fraction did not rise above baseline. However, after cyclophosphamide, hypoxic fraction was elevated and did not return to baseline over the 24-hr observation period of this study. At 1/2 hr after mitomycin treatment, the hypoxic fraction was raised but within 1 hr returned to baseline. These results indicate that tumor reoxygenation varies after different drug treatments and that the determination of drug specificity for aerobic versus hypoxic cells may be strongly biased by the choice of the time after treatment for making the determination.

Reoxygenation of the RIF-1 tumor after fractionated radiotherapy.

The regrowth delay assay was used to assess hypoxic fraction in the RIF-1 tumor. Results approximating those of earlier paired survival curve data were obtained for previously untreated tumors and tumors treated with a single dose of 15 Gy. Further studies showed that after 5 daily fractions of 5 Gy, the hypoxic fraction returned to approximate pretreatment values within 24 hr.

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.

Schedule-dependent therapeutic gain from the combination of fractionated irradiation plus c-DDP and 5-FU or plus c-DDP and cyclophosphamide in C3H/Km mouse model systems.

Cytotoxic drugs were administered either in single or fractionated doses before, during, or after a standard course of 5 daily X ray exposures. SCCVII and RIF-1 tumors were grown from cells implanted in the gastrocnemius muscles of syngeneic C3H/Km mice, and treatments were evaluated by regrowth delay (GD). Non-tumor-bearing mice were irradiated locally to the upper abdomen for analysis of intestinal crypt cell survival, an acute normal tissue effect; other non-tumor-bearing mice were irradiated locally to the thorax for analysis of early (pneumonitis) and late (fibrosis) effects on the lungs, as reflected in changes in breathing rates. In a series of experiments to test the combination of i.p. 5-FU, cis-DDP, and X ray, dose effect factors (DEF's) were compared so that therapeutic gain factors (TGF's) could be calculated from the ratio, DEF (tumor)/DEF (normal tissue). The highest TGF, 6.7 (tumor/duodenum), was obtained for the schedule in which 100 mg/kg 5-FU was given 24 hr before the simultaneous administration of 1.6 mg/kg cis-DDP and X ray for 5 consecutive days. The following summary refers only to tumor growth delay data. In confirmation of previous extensive experiments, the combination of cis-DDP + X ray showed supra-additivity, whether the drug was given in a single dose (abbreviated P) or simultaneously with X ray (abbreviated px), that is, P x x x x x or px px px px px. For CY + X ray, the greatest supra-additivity was obtained for either C x x x x x or x x x x x C. 5-FU alone did not act supra-additively with fractionated irradiation, but the addition of 5-FU to cis-DDP + X ray was supra-additive for certain schedules, maximally for F px px px px px. CY combined to give greater than additivity with either cis-DDP or X ray alone, and the combination of CY + cis-DDP + X ray appeared to be supra-additive for five different schedules, maximally for C x x x x x P. Normal tissue effects are being evaluated for these same schedules so that TGF's might soon be obtained.

Interleukin-1 modification of the effects of cyclophosphamide and fractionated irradiation.

Studies were performed to determine whether recombinant human interleukin-1 (IL-1) modifies the tumor cytotoxicity of cyclophosphamide (CY) combined with fractionated X-irradiation. RIF-1 tumors were implanted intradermally in C3H/Km mice and therapeutic effect was evaluated by the regrowth delay method, that is, the time for treated tumors to grow to 3 times their volume at the start of treatment relative to that for untreated tumors. A single intraperitoneal treatment of 15 micrograms/kg IL-1 given 24 hr after 100 or 200 mg/kg CY and immediately before the first of 5 daily fractionated treatments of 1-4 Gy increased tumor growth delay beyond that produced by CY and irradiation without the IL-1. However, the IL-1 given with either CY or fractionated irradiation did not extend the time for tumor regrowth beyond that produced by the agents themselves. Thus, while CY and fractionated irradiation together produce a greater than additive effect, IL-1 seems to extend this phenomenon. From these findings, it appears that IL-1 enhances the cytotoxic effects of CY and X ray against tumors, an effect that would have considerable practical significance in the light of the protective effects shown elsewhere for the same lymphokine on normal tissues.

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.

Interleukin 1 increases thymidine labeling index of normal tissues of mice but not the tumor.

PURPOSE: This study was conducted to investigate the action of human recombinant interleukin 1 as a radioprotector for different mouse normal cells other than bone marrow cells. METHODS AND MATERIALS: Semi-continuous injections of tritiated thymidine were administered every 6 h, over 24 h to determine thymidine labeling index. Mice were injected with recombinant human interleukin 1 24 h prior to tritiated thymidine and were compared to control animals that did not receive interleukin 1. Mice were killed 1 h after the last thymidine injection. The 24 h thymidine labeling index for normal tissues and RIF-1 tumor was determined. Labeling indices were also determined 1-14 days after a series of fractionated irradiations with or without pretreatment with a single dose of interleukin 1 administered 24 h prior to the first radiation. RESULTS: The thymidine labeling index of normal tissues was higher following the injection of recombinant human interleukin 1 24 h before radiolabeling. This was found in all normal tissues tested, including the lip and tongue mucosal basal cell layers, crypt cells of the duodenum, alveolar cells of the lung, hepatocytes, and basal skin cells. The thymidine labeling index of RIF-1 fibrosarcoma was not affected by interleukin 1 injection. A single interleukin 1 injection 24 h before the first radiation fraction also increased the thymidine labeling indices of normal tissues after localized fractionated irradiation. The thymidine labeling index of RIF-1 tumor was not increased by interleukin 1 administration except after relatively high radiation doses (20 Gy in five fractions). The ability of interleukin 1 to enhance the thymidine labeling index declined after the first day following the completion of fractionated irradiation. CONCLUSION: Recombinant human interleukin 1 increased the 24 h thymidine labeling index in normal tissues in mice, but not in RIF-1 tumor. Fractionated irradiation could maintain the effect of a single dose of interleukin 1, administered 24 h prior to the first fraction, up to 24 h after the end of radiation.

Interleukin-1 modulatory effect on the action of chemotherapeutic drugs and localized irradiation of the lip, duodenum, and tumor.

PURPOSE: This study was conducted to examine the radioprotective and radiochemoprotective capabilities of interleukin 1 beta (IL-1) on two acute-reacting normal tissues of the C3H mouse, the mucosa of the lip and the duodenum. Also assessed was the modulating effect of IL-1 on tumor growth in the same strain of mice. METHODS AND MATERIALS: IL-1 was administered to C3H/Km mice in combination with fractionated irradiation, or with cyclophosphamide, cisplatin, or 5-fluorouracil (5FU) followed by irradiation. Normal tissue damage was evaluated in the mouse lip, using a subjective scoring system for tissue reaction, and in the duodenum, using the crypt cell survival assay. RIF-1 fibrosarcoma tumor response was assayed with the regrowth delay method. RESULTS: IL-1 protected against the acute reaction produced by fractionated irradiation in the lip mucosa, shifting the dose-response curve by 3.8 Gy. IL-1 was protective when injected intraperitoneally 24 hr before CY or c-DDP, which were given immediately before the first of five daily radiation dose fractions. The dose-response curves for cyclophosphamide and cisplatin were shifted 4.0 Gy and 1.6 Gy, respectively. IL-1 did not protect against 5FU toxicity when treatments were administered in that same sequence; however, when 5FU was given 4 or 8 hr before IL-1 and the first radiation dose fraction followed 20 or 16 hr later, there was significant protection and the curves were separated by 1.5 Gy or 3.5 Gy. IL-1 also protected duodenal crypt cells against the cytocidal effect of fractionated irradiation, with a dose difference of 1.5 Gy and an improvement of crypt survival of 11.7%. It was even more protective for these cells against the enhanced drug toxicity when cyclophosphamide or 5FU were administered immediately before the first of five daily radiation doses, with the dose differences of 4.4 and 5.3 Gy, respectively, and improvements of crypt survival of 33.8 and 29.9%, respectively. There was no modification by IL-1 of the effect of irradiation alone on the RIF-1 tumor. CONCLUSION: This study demonstrates the potential for use of IL-1 as an auxiliary in combinations with chemotherapeutic agents and radiation. It also indicates that for some drugs, such as 5FU, IL-1 effects may be sequence dependent.

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.

Protection by interleukin 1 against lung toxicity caused by cyclophosphamide and irradiation.

Interleukin 1 has been shown to provide protection against the toxic effects of cyclophosphamide given in combination with localized irradiation of the lung. A single dose of 15 micrograms/kg interleukin 1 was given 24 h before cyclophosphamide (25-125 mg/kg) which was followed 1 day later by five daily exposures of 4.5 or 5.0 Gy, localized to the lung. Interleukin 1 significantly reduced early measurements of breathing rate for mice treated with high cyclophosphamide doses and irradiation, and at cyclophosphamide doses above 50 mg/kg, there was a significant reduction in the lethality from the combined treatment. At lower levels of cyclophosphamide, however, there seemed to be no effect.