A comparison between the effect of step-down heating in a tumour and a normal tissue in vivo

A comparison between the effect of step-down heating (SDH) obtained in a C3H mammary carcinoma grown in the feet of CDF1 mice and the skin of normal CDF1 feet is presented. Water-bath heating was used, and SDH was obtained by giving a 44.7 degrees C/10 min treatment followed by heating at 42.2 degrees C for variable times. Single heating at 42.2 degrees C and step-up heating (SUH), i.e. 42.2 degrees C followed by 44.7 degrees C/10 min, were used as controls. The endpoint was the heating time at 42.2 degrees C to obtain either a definite tumour growth time (TGT50) or a specific skin score level (RD50) in 50% of the animals. The effect of SDH and SUH was quantified by the step-down ratio (SDR), calculated as the ratio of the heating times at 42.2 degrees C to obtain the specific endpoint. In both assays the effect of SDH was seen as a significant left shift of the SDH dose-response curve compared to the curve for single heating and SUH. For the comparison of the tumour and the normal tissue response, damage levels with comparable heating times for single heating were used. The therapeutic effect was then investigated by calculating the therapeutic gain factor (TGF), where TGF = SDR(tumour)/SDR(normal tissue). Neither SUH nor SDH gave a TGF significantly different from 1. The results suggest that SDH may be used clinically to shorten the heating time without decreasing the therapeutic effect.     

A comparison between the effect of step-down heating in a tumour and a normal tissue in vivo

A comparison between the effect of step-down heating (SDH) obtained in a C3H mammary carcinoma grown in the feet of CDFl mice and the skin of normal CDFl feet is presented. Water-bath heating was used, and SDH was obtained by giving a 44.7°C/10 min treatment followed by heating at 42.2°C for variable times. Single heating at 42.2°C and step-up heating (SUH), i.e. 42.2°C followed by 44.7°C/10 min, were used as controls. The endpoint was the heating time at 42.2°C to obtain either a definite tumour growth time (TGT50) or a specific skin score level (RD50) in 50% of the animals. The effect of SDH and SUH was quantified by the step-down ratio (SDR), calculated as the ratio of the heating times at 42–2°C to obtain the specific endpoint. In both assays the effect of SDH was seen as a significant left shift of the SDH dose-response curve compared to the curve for single heating and SUH. For the comparison of the tumour and the normal tissue response, damage levels with comparable heating times for single heating were used. The therapeutic effect was then investigated by calculating the therapeutic gain factor (TGF), where TGF = SDR(tumour)/SDR(normal tissue). Neither SUH nor SDH gave a TGF significantly different from 1. The results suggest that SDH may be used clinically to shorten the heating time without decreasing the therapeutic effect.     

Effect of chronic thermotolerance on thermosensitization in Chinese hamster ovary cells studied at various temperatures

The effect of chronic thermotolerance on the thermal responses of Chinese hamster ovary (CHO) cells to single and step-down heating was studied. Thermotolerance was induced by pre-heating exponentially growing cells at 39 degrees C for 9 h, followed by test treatments for variable times at temperatures ranging from 39 to 43 degrees C. In the temperature range studied, the heat sensitivity of thermotolerant CHO cells was characterized by an Arrhenius activation energy of Ea = 1175 +/- 40 kJ/mol. This value agreed well with Ea = 1180 +/- 45 kJ/mol measured after single heating, indicating that the induction of chronic thermotolerance did not affect the activation energy for cell killing by heat. Thermosensitization was studied after a priming treatment at 43 degrees C for 50 min followed by step-down heating at temperatures ranging from 39 to 43 degrees C. The temperature dependence of the thermal response after step-down heating was characterized by an activation energy of Ea = 490 +/- 17 kJ/mol. When the cells were pre-treated for 1-16 h at 39 degrees C prior to step-down heating (43 degrees C, 50 min, followed by graded exposure to 39-43 degrees C), the activation energy was gradually enhanced and approached Ea = 825 +/- 42 kJ/mol for 39 degrees C, 16 h. This change in Ea reflects the effect of thermotolerance on the priming treatment at 43 degrees C for 50 min, whereas the effect on the final test treatment resulted in a parallel shift of the Arrhenius curve without changing the slope, indicating that the effect of thermotolerance on the priming and the test treatment is expressed in the Arrhenius diagram in different ways.     

Factors of importance for the development of the step-down heating effect in a C3H mammary carcinoma in vivo

The effect of step-down heating (SDH) was investigated in a C3H mammary carcinoma inoculated into the feet of CDF1 mice. The SDH effect was evaluated by comparing slopes of time versus growth delay curves of SDH-heated with the curve for single-heated controls. The effect was quantified by a ratio: 'step-down ratio' (SDR), defined as slope (SDH-heated)/slope (single-heated). Step-down heating resulted in thermosensitization in contrast to step-up heating which did not affect the heat sensitivity. The kinetics of the step-down heating effect was investigated by inserting an interval between a 44.5 degrees C/10 min sensitizing treatment (ST) and a 42.0 degrees C test treatment (TT). The effect of SDH was maximal with no interval between ST and TT (SDR = 2.3), decayed within 2 h and turned into thermotolerance. This thermotolerance was maximal after 12 h and decayed within 120 h. The effect of varying the TT temperature was investigated in the range 39.0-44.5 degrees C (ST = 44.5 degrees C/10 min). Below 42.5 degrees C the SDR value increased exponentially, and even a 39 degrees C TT produced a significant heat damage. An Arrhenius analysis was made showing a straight line in the whole temperature range with an activation energy of 526 kJ/mol and an increased activation entropy. These data show that thermosensitization can be induced by SDH in C3H mammary carcinomas in vivo. The effect seems to decay within 2 h, and by decreasing the heat activation energies the effect of low temperature heating is increased.     

Effect of step-down heating on hyperthermic radiosensitization in an experimental tumor and a normal tissue in vivo

The effect of step-down heating (SDH) on the radiosensitization induced by simultaneous hyperthermia and radiation was investigated in a C3H mammary carcinoma inoculated into the feet of CDF1 mice and the skin of normal CDF1 feet. SDH consisted of a sensitizing treatment (ST) of 44.5 degrees C/10 min followed by a test treatment (TT) of 41.5 degrees C for 30, 60 or 120 min. Simultaneous administration of radiation and hyperthermia was achieved by delivering radiation in the middle of the TT. The endpoint selected was the radiation dose needed to achieve either tumor control or moist desquamation in 50% of the animals. The results were evaluated by the thermal enhancement ratio (TER), defined as dose of radiation needed to achieve endpoint in relation to dose of combined radiation and hyperthermia needed to achieve the endpoint. SDH of tumors increased the TER significantly compared with step-up heating (SUH). The ratios between TCD50 values for corresponding SDH and SUH increased with TT heating time and at 120 min a 2.5-fold increase in the radiosensitizing effect was achieved. It has previously been shown that SDH alone causes thermosensitization in tumors by decreasing the activation energy. However, the effect was too small to explain the increased radiosensitization observed with SDH. In the normal tissue studies SDH combined with radiation treatment gave a lower TER compared to the SDH tumor results, suggesting a possible therapeutic gain.     

Skin responses to step-up and step-down heating in C3H mice

The effects of step-up (42 leads to 44 degrees C sequence) and step-down (44 leads to 42 degrees C sequence) heating were studied on the skin of C3H/He mouse feet. Skin damage in mouse feet exposed to 44 degrees C alone became proportionally more severe with increasing exposure time, while it was slight or not observed at 42 degrees C for up to 2 hr. Preheating to 42 degrees C for 60 or 120 min (step-up) had little effect on the damage from 30-60 min exposure at 44 degrees C, but increased the damage seen with 90 min at 44 degrees C. However, skin damage was markedly enhanced by exposure to step-down heating. On the other hand, thermal resistance was induced in the mouse skin by 44 degrees C fractionated treatments. The induced thermal resistance reached a maximum with a 1 day interval, and then disappeared after a 3 day interval. In another fractionation schedule of step-down heating, the enhanced skin damage which was observed when here was no interval, recovered rapidly and attained an additive response within a 1 day interval. No thermal resistance was observed.     

Effect of chronic thermotolerance on thermosensitization in Chinese hamster ovary cells studied at various temperatures

The effect of chronic thermotolerance on the thermal responses of Chinese hamster ovary (CHO) cells to single and step-down heating was studied. Thermotolerance was induced by pre-heating exponentially growing cells at 39°C for 9 h, followed by test treatments for variable times at temperatures ranging from 39 to 43 °C. In the temperature range studied, the heat sensitivity of thermotolerant CHO cells was characterized by an Arrhenius activation energy of E_a=1175 ± 40 kJ/mol. This value agreed well with E_a = 1180 ± 45 kJ/mol measured after single heating, indicating that the induction of chronic thermotolerance did not affect the activation energy for cell killing by heat. Thermosensitization was studied after a priming treatment at 43°C for 50 min followed by step-down heating at temperatures ranging from 39 to 43°C. The temperature dependence of the thermal response after step-down heating was characterized by an activation energy of E_a =490 ± 17 kJ/mol. When the cells were pre-treated for 1–16 h at 39°C prior to step-down heating (43°C, 50 min, followed by graded exposure to 39–43°C), the activation energy was gradually enhanced and approached E_a = 825 ± 42 kJ/mol for 39 z°C, 16 h. This change in E_a reflects the effect of thermotolerance on the priming treatment at 43°C for 50 min, whereas the effect on the final test treatment resulted in a parallel shift of the Arrhenius curve without changing the slope, indicating that the effect of thermotolerance on the priming and the test treatment is expressed in the Arrhenius diagram in different ways.     

Effect of chronically induced thermotolerance on thermosensitization in CHO cells

Chronic thermotolerance was induced in Chinese hamster ovary (CHO) cells by pretreatment at 40 degrees C for various times ranging from 15 min to 16 h. The thermotolerant cells were either exposed to single heat treatments at 43 degrees C or subjected to step-down heating consisting of a priming treatment at 43 degrees C for 90 min immediately followed by a graded test treatment at 40 degrees C. The results showed that chronic thermotolerance affected the thermal sensitivity of step-down-heated CHO cells in two ways: by lowering the effectiveness of the priming treatment at 43 degrees C and by reducing the response to the test treatment at 40 degrees C. The effect on the priming treatment corresponds to a reduction in the effective heating time, i.e. the thermotolerant cells respond as if they were exposed to 43 degrees C for times shorter than 90 min. It was further shown that, for a given conditioning treatment, the effectiveness of both the priming and the test treatment was reduced by the same factor; the thermotolerance ratios determined for 43 degrees C and 40 degrees C showed an identical dependence on the duration of the thermotolerance-inducing conditioning treatment. Since thermotolerance development did not reverse heat sensitization by step-down heating, it is concluded that thermotolerance and thermosensitization are distinct phenomena which act independently.     

Significance of additive heat effect in the therapeutic gain factor in combined hyperthermia and radiotherapy: murine tumor response and foot reaction

The thermal enhancement ratio (TER) and therapeutic gain factor (TGF) were evaluated for combined hyperthermia and radiation treatments of a murine fibrosarcoma, FSa-II. The TER is the ratio of the radiation dose that induces a given reaction without hyperthermia to that with hyperthermia. The TGF is defined as the ratio of TER for tumor response to TER for normal tissue response. Tumors in the subcutaneous tissue of the right foot were irradiated with graded radiation doses when they reached an average diameter of 6 mm (110 mm3). Hyperthermia was given by immersing animal feet in a constant temperature water bath 10 min before or after irradiation. The tumor growth time to reach 500 mm3 was obtained for each tumor and the median tumor growth time was calculated for each treatment group. For the normal tissue study, the non-tumor bearing murine foot was treated, as was the tumor, and the foot reaction was scored after treatment, according to our numerical score system for radiation damage, until the 35th post-treatment day and averaged. Using the fraction of animals showing a given average foot reaction score in a treatment group, the RD50, or the radiation dose to induce the given foot reaction or greater, was calculated. A single heating at 45.5 degrees C for 10 min and a step-down heating (first heat at 45.5 degrees C for 10 min immediately followed by the second heat at 41.5 degrees C for 60 min) prolonged the tumor growth time, indicating that hyperthermia per se resulted in some cell killing. The prolongation was greater following step-down heating than following single heating. These heat treatments alone induced no noticeable heat damage on the foot, but decreased the threshold dose observed on the radiation dose response curves for the foot reaction. Accordingly, TER and TGF were evaluated with or without normalizing this thermal effect. TER's for both tumor and foot responses without normalization were greater than the TER's after normalization and decreased with increasing radiation dose (between 1.9 and 7.1 or greater for tumor and between 1.3 and 4.3 or greater for foot reaction), whereas the normalized TER's were relatively constant (between 1.6 and 1.7 for tumor and between 0.7 and 1.5 for foot reaction). TGF's without normalization were greater than those obtained after normalization. The former was large at small doses and decreased with increasing radiation dose (between 1.5 and 4.0 or greater), whereas the latter was within 0.8 and 1.3 and relatively independent of radiation dose.(ABSTRACT TRUNCATED AT 400 WORDS)     

Step-down heating in a C3H mammary carcinoma in vivo: effects of varying the time and temperature of the sensitizing treatment

The effect of step-down heating (SDH), consisting of an initial sensitizing treatment (ST) performed at either 44.5 degrees C or 43.5 degrees C followed by a lower temperature test treatment (TT), was investigated in a C3H mammary carcinoma in vivo. A linear relationship between heating time and tumour growth delay was observed for all temperature combinations applied. At a given TT temperature, SDH increased the slope of the dose-response curve compared to the curve for tumours, single-heated without an initial ST. The slope of the SDH curves increased asymptotically towards a plateau value as the ST time at 44.5 degrees C was increased. The time-temperature relationship for single heating was described by a biphasic Arrhenius curve with activation energies of 1361 +/- 34 and 666 +/- 54 kJ/mol below and above an inflection point at 42.5 degrees C, respectively. For SDH, the Arrhenius curve gradually became straight with increasing ST time, and the activation energy saturated at a value of 425 +/- 25 kJ/mol. The reduction of the activation energy at an ST temperature of 43.5 degrees C was due rather to the extent of ST heat damage than to the ST time or temperature used. These results may be relevant for calculations of thermal doses, since even a short temperature peak (e.g. 44.5 degrees C/5 min) significantly changed the time-temperature relationship.     

Step-down heating of CHO cells at 37.5-39 degrees C

The thermosensitizing effect of step-down heating was studied using Chinese hamster ovary (CHO) cells. Exponentially growing cells were given priming heat treatments at 43 degrees C for 45 or 90 min, immediately followed by a second exposure to a temperature ranging from 37.5 to 39 degrees C. The measured rates of cell killing, 1/D0, increased exponentially with temperature; the slopes correspond to Arrhenius activation energies of Ea = 1200 +/- 150 kJ mol-1 and Ea = 1275 +/- 125 kJ mol-1 for cells preheated at 43 degrees C for 45 or 90 min, respectively. For the temperature range 39-43 degrees C an activation energy of Ea = 561 +/- 24 kJ mol-1 was obtained for step-down heated cells (43 degrees C, 45 min followed by T = 39-43 degrees C). These results indicate that there is a 'second inflection point' at 39 degrees C on the Arrhenius curve for step-down heating of CHO cells. Data evaluation using a mathematical model published previously (H. Jung, Radiation Research, 106, 56-72, 1986) showed that the rate constant c for the conversion of nonlethal lesions into lethal events increased with an activation energy of Ea = 1520 +/- 140 kJ mol-1 in the temperature range from 37.5-39 degrees C. For 39-45 degrees C the activation energy for c was Ea = 360 +/- 26 kJ mol-1, indicating that the temperature dependence of c shows a break at 39 degrees C similar to that observed on the 1/D0 Arrhenius plot.     

Effects of tumor necrosis factor and hyperthermia on Meth-A tumors

The combined effects of purified human natural tumor necrosis factor (TNF) and hyperthermia were investigated in a transplanted TNF-sensitive Meth-A tumor model. We assessed the sequence and interval for the two treatments, the temperature that caused maximal heat sensitization, and the effects of pH modification on this combination therapy. Tumor response was evaluated by means of a tumor growth delay assay. TNF at a dose of 50 JRU/g caused significant tumor growth delay. A synergistic effect of TNF and hyperthermia was observed when TNF was administered 10 min before heating. This thermal enhancement of the action of TNF became more prominent with an increase in the heating temperature. Tumor growth delay was maximal when TNF was given immediately before or after hyperthermia. However, after an increase in the time interval to more than 2 h, there was no enhancement of growth delay. Injection of glucose (5 g/kg) caused a significant fall in pH at 10 and 30 min after administration. Further enhancement in tumor growth delay was seen with the trimodality of glucose, TNF, and heat compared to combined treatment with heat and either TNF or glucose at a hyperthermia of 42 degrees C. This effect was not obtained with heating to 40 degrees C. TNF appears to be a potent heat sensitizer when an appropriate temperature and time interval between hyperthermia and TNF administration are used. Trimodality treatment with hyperthermia, TNF, and glucose may be a new method of anticancer therapy.     

The effect of step-down heating on mouse small intestinal mucosa

Step-down heating (SDH) was investigated in mouse small intestine by giving a primary (conditioning) treatment at or above 43 degrees C followed by a test treatment below 43 degrees C. Crypt dose-response curves following SDH were compared with those obtained using the test treatment alone; the SDH effect was characterized by a reduction in shoulder (an additive effect) and an increase in slope (thermosensitization). The thermosensitization ratio, defined as slope SDH-heated/slope single-heated, was independent of the conditioning temperature but increased to a maximum of approximately three as the duration of conditioning increased. Thermosensitization was eliminated when the conditioning treatment was itself sufficient to cause significant crypt loss and, also, when the interval between the two treatments was 0.5 h or longer. This period was less than that required for either recovery of the 'shoulder' on the crypt dose-response curve or the development of thermotolerance following the primary treatment. Thermotolerance which develops in intestine during prolonged hyperthermia (after approximately 100 min) was not affected by SDH and Arrhenius analysis indicated that the activation energy for temperatures below 43 degrees C was not significantly altered by SDH. In summary, the SDH effect on small intestine, assessed using the crypt loss endpoint, was similar to thermosensitization observed in vitro. However, the lower magnitude of the effect and its complex dependence on the primary heat treatment suggest either that crypt cells respond to SDH in a unique and characteristic manner or that the crypt assay in vivo and reproductive survival in vitro do not reflect the same endpoint.     

Effects of step-up and step-down heating on a transplantable murine tumor

The effects of step-up (42----44 degrees sequence) and step-down (44----42 degrees sequence) heating were studied on a transplantable mammary adenocarcinoma of C3H/He mouse. Tumor-bearing legs were immersed in a water bath and the response to hyperthermia was evaluated in terms of the delay in tumor growth. Tumor growth was delayed greatly with increase in the duration of treatment with 44 degrees hyperthermia, whereas with 42 degrees hyperthermia of up to 180 min, tumor growth was delayed only slightly. The effects of step-up heating were similar to those of 44 degrees hyperthermia alone and the response was enhanced by a factor of 0.9-1.1 with the 60-min treatment at 42 degrees followed by treatment at 44 degrees. Thermal resistance developed when the preheating time at 42 degrees was longer than 30 min. On the other hand, the tumor response was markedly enhanced by step-down heating by a factor of 1.8-2.4 with the treatment at 44 degrees followed by 60-min treatment at 42 degrees. Since the enhancement factor for skin damage found previously was similar to that for the tumor, therapeutic gain cannot be expected by the use of these combined heat treatments.     

Effects of step-up and step-down heating combined with radiation on murine tumor and normal tissues

Radiosensitizing effects of step-up heating (SUH) and step-down heating (SDH) on the tumor and skin were studied by using mammary adenocarcinoma transplanted to the foot of the C3H/He mouse. The tumor and skin responses were assessed by the tumor growth delay method and the skin reaction scoring method, respectively. Neither SDH (44 degrees, 10 min----42 degrees, 30 min) nor SUH (42 degrees, 30 min----44 degrees, 10 min) alone caused a substantial tumor or skin response. When the heat treatment was given immediately after irradiation, the thermal enhancement ratio (TER) was higher in SDH than in SUH for tumors as well as the skin. A therapeutic gain factor (TGF) of 1.2 was obtained in SUH, while no therapeutic benefit was found in SDH. SDH was applied at various times (0 to 3 hr) before or after irradiation. When SDH was given before irradiation, the TER was consistently high to almost the same degree for tumors and the skin regardless of the time interval, resulting in minimal or no therapeutic gain. With SDH after irradiation, the TER for the skin decreased with increase in the time interval, while the TER for the tumor was moderately enhanced. Therefore, the TGF increased with increase in the time interval and reached 2.2 when SDH was given 3 hr after radiation. SUH is slightly advantageous over SDH in terms of the TGF, and SDH should be given 3 hr after irradiation when selective tumor heating is not possible.     

Step-down heating of human melanoma xenografts: effects of the tumour microenvironment.

Thermosensitisation by step-down heating (SDH) has previously been demonstrated in experimental rodent tumours. The purpose of the study reported here was to investigate whether the SDH effect in tumours in part may be attributed to heat-induced alterations in the capillary network and/or the microenvironment. Two human melanoma xenograft lines differing substantially in vascular parameters were selected for the study. A thermostatically regulated water bath was used for heat treatment. The conditioning treatment (44.5 degrees C or 45.5 degrees C for 15 min) was given in vivo, whereas the test treatment (42.0 degrees C for 45, 90, 135 or 180 min) was given either in vitro or in vivo. Treatment response was measured in vitro using a cell clonogenicity assay. Fraction of occluded vessels following heat treatment was assessed by examination of histological sections from tumours whose vascular network was filled with a contrast agent. Tumour bioenergetic status and tumour pH were measured by 31P magnetic resonance spectroscopy. The conditioning heat treatments caused significant vessel occlusion, decreased tumour bioenergetic status and decreased tumour pH in both tumour lines. The SDH effect measured when the test treatment was given in vivo was significantly increased relative to that measured when the test treatment was given in vitro. The magnitude of the increase showed a close relationship to fraction of occluded vessels, tumour bioenergetic status and tumour pH measured 90 min after treatment with 44.5 degrees C or 45.5 degrees C for 15 min. The increased SDH effect in vivo was probably attributable to tumour cells that were heat sensitive owing to the induction of low nutritional status and pH during the conditioning treatment. Consequently, the SDH effect in some tumours may in part be due to heat-induced alterations in the microenvironment. This suggests that SDH may be exploited clinically to achieve increased cell inactivation in tumours relative to the surrounding normal tissues.     

Effect of chronically induced thermotolerance on thermosensitization in CHO cells

Chronic thermotolerance was induced in Chinese hamster ovary (CHO) cells by pretreatment at 40°C for various times ranging from 15 min to 16 h. The thermotolerant cells were either exposed to single heat treatments at 43 °C or subjected to step-down heating consisting of a priming treatment at 43 °C for 90 min immediately followed by a graded test treatment at 40°C. The results showed that chronic thermotolerance affected the thermal sensitivity of step-down-heated CHO cells in two ways: by lowering the effectiveness of the priming treatment at 43 °C and by reducing the response to the test treatment at 40°C. The effect on the priming treatment corresponds to a reduction in the effective heating time, i.e. the thermotolerant cells respond as if they were exposed to 43°C for times shorter than 90 min. It was further shown that, for a given conditioning treatment, the effectiveness of both the priming and the test treatment was reduced by the same factor; the thermotolerance ratios determined for 43°C and 40°C showed an identical dependence on the duration of the thermotolerance-inducing conditioning treatment. Since thermotolerance development did not reverse heat sensitization by step-down heating, it is concluded that thermotolerance and thermosensitization are distinct phenomena which act independently.