Muneyasu Urano

Thermal enhancement of new chemotherapeutic agents at moderate hyperthermia.

AbstractBackground: Hyperthermia enhances the cytotoxicity of some chemotherapeutic agents. We have studied the effect of moderate hyperthermia (41.5°C) on the cytotoxicity of five new chemotherapeutic agents (docetaxel, paclitaxel, irinotecan, oxaliplatin, and gemcitabine) and melphalan against a spontaneous murine fibrosarcoma. Methods: The tumor was an early-generation isotransplant of a spontaneous C3Hf/Sed mouse fibrosarcoma, FSa-II. Hyperthermia was administered by immersing the tumor-bearing foot into a constant temperature water bath set at 41.5°C for 30 minutes when the tumor reached 34 mm3. Chemotherapy was administered intraperitoneally immediately before hyperthermia. Tumor response was studied by the mean tumor growth time and the mean tumor growth delay time. Results: Hyperthermia significantly increased the tumor growth times of the animals treated with docetaxel, irinotecan, and gemcitabine at low dose and these drugs plus oxaliplatin at high dose. Docetaxel at high dose showed the greatest control of tumor growth by hyperthermia, with a 26% reduction. Concerning the taxanes, paclitaxel cytotoxicity was not enhanced by hyperthermia, but docetaxel was enhanced by hyperthermia at both doses of drug. Conclusions:Moderate hyperthermia increases the cytotoxicity of docetaxel, irinotecan, and gemcitabine on mouse fibrosarcoma. Paclitaxel did not show heat enhancement. Oxaliplatin and docetaxel showed greater heat enhancement when the drug dose was high.

Relative biological effectiveness of modulated proton beams in various murine tissues

The relative biological effectiveness (RBE) of proton beams produced by Harvard University 160 MeV synchrocyclotron was studied in various murine tissues. Reference radiation was Cobalt-60 gamma-rays from a teletherapy unit at the Massachusetts General Hospital. Animals were C3Hf/Sed mice derived from our defined flora mouse colony. Test tissues are: lens, lung, testes and tail vertebrae. The RBE of the third generation isotransplants of a spontaneous mouse mammary carcinoma was also investigated. The proton and Cobalt-60 irradiations were carried out simultaneously by 2 teams. The dose response curves obtained for testes weight loss and growth stunting of tail vertebrae indicated that the RBE for our protons was independent of radiation dose in the range of 0.4 to 16 Gy. This finding was identical to our previous studies of the murine fibrosarcoma, skin and small intestine. The RBE values for lens and lung tissues were obtained by determining radiation dose to result in a complete cataract in half the irradiated eyes in 210 days and a 50% mortality in 180 days respectively. We have studied proton RBE in 7 normal tissues and 2 tumors including previously reported results. The RBE values for these tissues were found to fall between 1.09 and 1.32. No significant differences in the proton RBE were found between the several normal and tumor tissues studied.

Tumor size dependent changes in a murine fibrosarcoma: Use of in vivo31P NMR for non-invasive evaluation of tumor metabolic status☆

Tumor tissue contains viable hypoxic regions that are radioresistant and often chemoresistant and may therefore be responsible for some treatment failures. A subject of general interest has been the development of non-invasive means of monitoring tissue oxygen. Pulse Fourier transform 31P NMR spectroscopy can be used to estimate intracellular nucleotide triphosphates (NTP), phosphocreatinine (PCr), inorganic phosphate (Pi) and pH. We have obtained 31P NMR spectra as an indirect estimate of tissue oxygen and metabolic status in a C3H mouse fibrosarcoma FSaII. Sequential spectra were studied during tumor growth in a cohort of animals and peak area ratios for several metabolites were computed digitally by computer. During growth, tumors showed a progressive loss of PCr with increasing Pi, and most tumors greater than 250 mm3 in volume had little or no measurable PCr. The smallest tumors (38 mm3 average volume) had PCr/Pi ratios of 1.03 +/- .24, whereas tumors 250 mm3 or more had an average PCr/Pi ratio of 0.15 +/- .04. Similarly derived NTP/Pi ratios decreased with tumor size, but this change was not significant (p = .17). Radiobiologic hypoxic cell fractions were estimated using the radiation dose required to control tumor in 50% of animals (TCD50) or by the lung colony technique. Tumors less than 100 mm3 had a hypoxic cell fraction of 4% (TCD50) while tumors 250 mm3 had a 40% hypoxic cell fraction (lung colony assay). These hypoxic fraction determinations correlated well with the depletion of PCr and decline in NTP/Pi ratios seen at 250 mm3 tumor volumes. Tumor spectral changes with acute ischemia were studied after ligation of the tumor bearing limb and were similar to changes seen with tumor growth. PCr was lost within 7 minutes, with concurrent increase in Pi and loss of NTP. Complete loss of all high energy phosphates occurred by 40 minutes of occlusion. In vivo tumor 31P NMR spectroscopy can be used to estimate tissue metabolic status and may be useful in non-invasive prediction of hypoxic cell fraction, reoxygenation, and radiation treatment response.

Visualization of hypoxia in microscopic tumors by immunofluorescent microscopy

Tumor hypoxia is commonly observed in primary solid malignancies but the hypoxic status of subclinical micrometastatic disease is largely unknown. The distribution of hypoxia in microscopic tumors was studied in animal models of disseminated peritoneal disease and intradermal (i.d.) growing tumors. Tumors derived from human colorectal adenocarcinoma cell lines HT29 and HCT-8 ranged in size from a few hundred microns to several millimeters in diameter. Hypoxia was detected by immunofluorescent visualization of pimonidazole and the hypoxia-regulated protein carbonic anhydrase 9. Tumor blood perfusion, cellular proliferation, and vascularity were visualized using Hoechst 33342, bromodeoxyuridine, and CD31 staining, respectively. In general, tumors of <1 mm diameter were intensely hypoxic, poorly perfused, and possessed little to no vasculature. Larger tumors (approximately 1-4 mm diameter) were well perfused with widespread vasculature and were not significantly hypoxic. Patterns of hypoxia in disseminated peritoneal tumors and i.d. tumors were similar. Levels of hypoxia in microscopic peritoneal tumors were reduced by carbogen breathing. Peritoneal and i.d. tumor models are suitable for studying hypoxia in microscopic tumors. If the patterns of tumor hypoxia in human patients are similar to those observed in these animal experiments, then the efficacy of systemic treatments of micrometastatic disease may be compromised by hypoxic resistance.

Repair of potentially lethal radiation damage in acute and chronically hypoxic tumor cells in vivo

The ability of animal tumor cells to repair potentially lethat damage was studied in vivo. Fifth-generation isotransplants of a spontaneous mouse squamous-cell carcinoma were irradiated under tourniquet-induced hypoxia or in air. Tumors were removed either immediately or 6 hours after irradiation and dose-response curves were determined by TD50 assays. Repair was attributed to cells in the hypoxic cell component for animals irradiated in air. Extensive repair was also noted for those irradiated under typoxic conditions. Implications of these results are discussed.

Thermal enhancement of melphalan and oxaliplatin cytotoxicity in vitro

It has been established that hyperthermia can enhance cytotoxicity of some chemotherapeutic agents. This has led to various clinical trials of thermochemotherapy, although many questions remain unanswered. The effects of various agents have been studied on animal tumours with different histopathology at elevated temperatures. These studies indicated that alkylating agents were most effective to all tumours at a moderately elevated temperature. Cisplatin was also effective to all tumours, but its effectiveness at 41.5°C was less than that of alkylating agents. To quantitatively study these findings, the magnitude of thermal enhancement of melphalan, an alkylating agent, and that of oxaliplatin, a new platinum compound, were studied at 37-44.5°C by the colony formation assay. The dose of each agent was kept constant, and cell survival was determined as a function of treatment time. The cell survival curve was exponentially related with treatment time at all test temperatures, and the T 0 (the time to reduce survival from 1 to 0.37) decreased with an increasing temperature. These results suggested that the cytotoxic effect of these agents occurred with a constant rate at 37°C, and the rate was facilitated with an increasing temperature. This suggests that heat can accelerate the cytotoxic chemical reaction, leading to substantial thermal enhancement. The thermal enhancement ratio (TER, the ratio of the T 0 at 37°C to the T 0 at an elevated temperature) increased with an increase in the temperature. The activation energy for melphalan at moderately elevated temperatures was largest among the agents tested in the laboratory and that for oxaliplatin was approximately half of the melphalan activation energy. This suggests that the thermal enhancement for the cytotoxicity of melphalan or alkylating agents might be the greatest. Potential mechanisms of thermal enhancement of cytotoxicity were discussed.

The effect of various chemotherapeutic agents given with mild hyperthermia on different types of tumours

It has been shown that hyperthermia can enhance the cytotoxicity of some chemotherapeutics. However, the most effective agent(s) at elevated temperatures have yet to be determined. A previous study suggests that the drug of choice at elevated temperatures may be different from that at the physiological temperature, and that the alkylating agents may be most effective at elevated temperatures. To further investigate these possibilities, the effect of chemotherapeutic agents were compared. These agents were cyclophosphamide, ifosfamide, melphalan, cis-diamminedichloroplatinum (II), 5-fluorouracil, mitomycin C and bleomycin. Three tumours (mammary carcinoma, osteosarcoma and squamous cell carcinoma) were used. They were transplanted into the feet of C3H/He mice. When tumours reached 65 mm 3 , a test agent was injected intraperitoneally. Tumours were immediately heated at 41.5°C for 30 min, and the tumour growth (TG) time was studied for each tumour. Using the TG times, the TG-50 (the time required for one-half of the total number of the treated tumours to reach the volume of 800 mm 3 from 65 mm 3 ) was calculated. Subsequently, the tumour growth delay time (GDT) and the thermal enhancement ratio (TER) were obtained. The GDT was the difference between the TG-50 of treated tumours and that of non-treated control tumours. The TER was the ratio of the GDT of a group treated with an agent at 41.5°C to that of a group treated with the agent at room temperature. Results showed that the top three effective agents tested at 41.5°C were solely alkylating agents--CY, IFO and L-PAM--for each kind of tumour. A GDT of cisplatin was smaller than those of the alkylating agents. The smallest TER, 1.1, was observed for 5-fluorouracil, which was given for mammary carcinoma, and for mitomycin C, which was given for squamous cell carcinoma. It could be concluded that the alkylating agents at elevated temperatures might be the drugs of choice for many types of tumours. The possible mechanisms of thermal enhancement associated with these agents are discussed.

Relative biological effectiveness of a high energy modulated proton beam using a spontaneous murine tumor In vivo

Abstract The relative biological effectiveness (RBE) for proton beams produced by 160 MeV Harvard University synchrocyclotron was studied in C3Hf/Sed mouse fibrosarcoma (FSa-II). Reference radiation was Cobalt-60 γ-ray. Cell survival was determined by 2 types of lung colony assays. Type A assays involved irradiation of solid tumors followed by immediate preparation of single cell suspension and the intravenous injection of the suspension. In type B assays, intravenous injection of single cell suspension, prepared from non-treated tumors, was followed by mouse thorax irradiation. The type B assay was used to determine survival of aerobic cells while the type A assay was used to analyze the survival of acutely hypoxic cells. Tumors were irradiated with 1 to 10 doses in the center of spread-out Bragg peak (SOBP) or in an incident plateau area. Survival curves were analyzed by alpha-beta (linear quadratic) model and RBE was determined at various survival levels. The RBE in SOBP was found to be independent of fraction size although 2 % of the proton beam was reported to be a result of high linear energy transfer (LET) events of greater than 100 KeV/μ. The RBEs at survival levels of 0.5, 0.1, and 0.01 were 1.16 ± 0.12, 1.16 ± 0.06 and 1.17 ± 0.05 respectively. The RBE in the incident plateau was not significantly different from that in SOBP.

Studies on fractionated hyperthermia in experimental animal systems II. Response of murine tumors to two or more doses

The effect of multiple hyperthermia and the kinetics of thermal resistance were studied in experimental murine tumors. A spontaneous C3Hf mouse mammary carcinoma, MCa had a chemically-induced fibrosarcoma, FAa-1, were used. Assay methods included determination of the TCD50, i.e., the treatment time to yield a tumor control in half the treated animals and TG (tumor growth) time analysis, i.e., the time required for a tumor to reach a given size after first treatment. After equal dose fractions the TCD50 of MCa increased with increasing overall treatment time. This increase was most predominant when treatments were given with a time interval of one day. Isoeffect curves for the MCa were comparable to those for normal tissue damage (foot reaction), which was reported in the first part of this series of communications. The kinetics of thermal resistance in the MCa was compared with that in FSa-I since the fractionated hyperthermia for the FSa-I was reported to have resulted in an appreciable therapeutic gain. The magnitude of thermal resistance was far greater in the MCa than in the FSa-I, although the kinetics of thermal resistance was similar in both tumors; i.e., (a) the resistance reached a maximum in 24 hours after treatment and then decayed slowly, and (b) the development of thermal resistance increased with increasing initial dose. The thermal resistance in these tumors appeared to be greater than that in animal feet.

Evaluation of the clinical applicability of proton beams in definitive fractionated radiation therapy.

We report on the treatment of 317 patients treated either wholly or in part with proton beams at the Harvard Cyclotron Laboratory. These include: 130 patients treated for definitive radiation therapy of choroidal melanoma; 17 patients treated for tumors of the base of skull, cervical spine and cranium, which abut structures of the central nervous system (CNS); 23 patients treated for sarcomas of soft tissue and bone; 65 patients treated for carcinoma of the prostate; 14 patients treated for carcinoma of the rectum and anus; and 23 patients treated for squamous carcinoma of the oral cavity and oro-pharynx. Data on causes of failure and morbidity of treatment are presented. Overall the results are judged to be extremely encouraging. In particular, the treatment of the choroidal melanomas and sarcomas abutting CNS structures have clear clinical value, and the treatment of prostatic tumors and tumors of the head and neck are thought to be promising.