THE THERMOSENSITIVITY OF HUMAN GINGIVAL SQUAMOUS CARCINOMA Ca9－22 CELLS WITH ONCOGENE erbB－1／EGFR

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Thermosensitization by step-down heating in mouse testis

Step-down heating (SDH) was investigated in mouse testis by giving an initial treatment of 3 min at 43.0°C followed immediately by a treatment in the temperature range 38.0–42.0°C. The dose-response curves for testis weight loss as a function of duration of hyperthermia were compared with those obtained using single-temperature treatments. In all cases the curves were linear, allowing the use of Arrhenius analysis. For single- temperature treatments the Arrhenius relationship showed an inflection at approximately 41°C with a small, but significant, increase in activation energy for hyperthermal temperatures below the transition. SDH increased the thermal sensitivity in this lower range, by approximately 1 °C, but the activation energy was not significantly altered. The results support the view that in vivo thermosensitization by SDH is not due solely to inhibition of development of thermotolerance.     

Time-temperature relationships for step-down heating in normal and thermotolerant cells

Normal and thermotolerant H35 cells were submitted to step-down heating (SDH). SDH can significantly reduce the induction and expression of thermotolerance. For SDH a sensitizing treatment (ST) at 44 · 6°C was followed by a test treatment (TT) at a lower hyperthermic temperature. The comparison between the thermotolerant and non-thermotolerant condition was based on isosurvival ST doses. For both conditions dose-effect relationships were obtained by plotting the ST-surviving fraction against the D₀ of a TT. The TT was at either 41 or 42–5°C, representing respectively, a permissive or a non-permisive condition for chronic induction of thermotolerance (CIT). The complex dose-effect relationships are partly exponential. In non-thermotolerant cells tested at 42–5°C the dose-effect relationship between ST and TT is relatively weak. At 41°C, however, the expression of CIT is strongly inhibited after a ST that kills < 20% of the cells. At higher ST doses the response is comparable with that at 42–5°C. In thermotolerant cells a high degree of thermosensitization is also observed for relatively low ST doses, but in contrast with non-thermotolerant cells a stronger dose-effect relationship remains at the higher ST doses. Ultimately this results in a comparatively higher degree of thermosensitization that can be achieved in non-thermotolerant cells. For example, at an isosurviving fraction of 0–15 the reduction of D₀ in non-thermotolerant cells at 42 · 5°C is less than five times, whereas in thermotolerant cells, the D₀ reduction is between 40 and 50 times. A similar reduction is found in non-thermotolerant cells tested at 41 °C. Subsequently, an isosurvival ST dose of about 40% was used in combination with a TT that was varied between 39 and 44°C. D0's were plotted in an Arrhenius diagram to obtain a time-temperature relationship for the effect of SDH on thermotolerant and non-thermotolerant cells. The four plots are all biphasic with a downward inflection. Thermotolerance causes an upward shift of the inflection point of 2°C relative to single-heated cells, whereas SDH causes a downward shift of 1 °C in single-heated cells and of 2°C in thermotolerant cells. For most of the temperature range, i.e. 39–43 · 5°C, SDH decreases the activation energies.     

Evidence for an upper temperature limit for thermotolerance development in L1A2 tumour cells in vitro

The response of LI A2 cells in vitro to water bath hyperthermia was investigated. In previous studies in the temperature range 40–5–45 0°C., an Arrhenius plot for heat killing of L1A2 cells was linear, and thermotolerance did not develop during continuous heating. The present Arrhenius analysis was extended to the range 38–45°C., and the Arrhenius plot was biphasic with an inflection point at 40–5°C., below which the activation energy was significantly increased from 724 to 923 kJ/mol (P< 0 001). In order to test for the development of thermotolerance during continuous heating, the cells were heated in the range 38–41°C for 10 h, followed immediately by a graded test treatment at 42°C. Thermotolerance developed below 40–5°C as shown by an increased D₀ of the 42°C survival curve, but not at 40 5 and 4FC. Preheating for 90min at 42°C followed by a 10 h incubation at 37°C resulted in maximal thermotolerance with a thermotolerance ratio of approximately 4–3, a ratio also obtained if the cells were incubated for 10 h at temperatures of 38–40°C. No thermotolerance was observed at incubation temperatures of 40 5°C and above. Thus, in the L1A2 cells 40°C is the upper temperature permissive for thermotolerance development, and the data support the assumption that the inflection point on the Arrhenius plot reflects the upper limit for thermotolerance development.     

Winner of the Lund Science Award 1992 Thermosensitization induced by step-down heating: A review on heat-induced sensitization to hyperthermia alone or hyperthermia combined with radiation

A few minute's exposure to a high temperature (sensitizing treatment, ST) may substantially increase the cytotoxic and the radiosensitizing effect of a subsequent heating at a lower temperature (test treatment, TT). This phenomenon, which is known as step-down heating (SDH) or thermosensitization, has been observed both in cultured cells in vitro and in tumours and normal tissues in vivo. The effect of SDH increases with a lowering of TT temperature, but it is rapidly lost at temperatures very close to 37°C. SDH-induced thermosensitization decays within a few hours, when an interval is inserted between ST and TT. In vitro results suggest an exponential decay of the SDH effect with half times ranging from 1·5- to 3·1 h. The effect of SDH increases with increasing ST time or temperature. For single heating, the Arrhenius plot is biphasic with activation energies of 500–800 and 1200–1700 kJ/mol above and below a break point temperature in the region 42·5–43·0°C, respectively. For SDH, the Arrhenius plot gradually becomes monophasic with increasing severity of ST and it approaches asymptotically to an activation energy of about 400 kJ/mol. The reduction of the activation energy depends on cell survival after the priming ST and not on the specific ST heating time or temperature. SDH strongly enhances hyperthermic radiosensitization with a 5–6-fold reduction of the radiation dose required to achieve tumour control. The thermosensitizing and the radiosensitizing effects of SDH have several features in common. Both effects become more prominent when the TT temperature is decreased and when the ST heating time or temperature increases. In addition, the decay kinetics for both effects are comparable. For heat alone, the effect of SDH in tumour and normal tissue seems to be quantitatively similar. However, the therapeutic ratio may be increased by combining SDH with radiation. Biologically, the critical subcellular targets involved in the SDH effect have not been revealed. However, the ability of SDH to inhibit the clearance of heat-induced aggregation of proteins in the nucleus is interesting. Blockage of the nuclear function by proteins is a central theory in the present molecular biological models for both cell kill by heat and heat radiosensitization. Clinically, SDH may be an advantage since even a short exposure to high temperature increases the effect of an otherwise inadequate heat treatment. The disadvantages are that SDH complicates thermal dose calculations, and may cause unacceptable damage to normal tissue.     

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.     

Thermosensitization by step-down heating in mouse testis

Step-down heating (SDH) was investigated in mouse testis by giving an initial treatment of 3 min at 43.0 degrees C followed immediately by a treatment in the temperature range 38.0-42.0 degrees C. The dose-response curves for testis weight loss as a function of duration of hyperthermia were compared with those obtained using single-temperature treatments. In all cases the curves were linear, allowing the use of Arrhenius analysis. For single-temperature treatments the Arrhenius relationship showed an inflection at approximately 41 degrees C with a small, but significant, increase in activation energy for hyperthermal temperatures below the transition. SDH increased the thermal sensitivity in this lower range, by approximately 1 degree C, but the activation energy was not significantly altered. The results support the view that in vivo thermosensitization by SDH is not due solely to inhibition of development of thermotolerance.     

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.     

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°C or 43·5°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°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°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°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°C/5 min) significantly changed the time-temperature relationship.     

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.     

Thermotolerance in human glioma cells

Human glioma cells held in plateau phase were tested for the development of chronic and acute thermotolerance. Long duration, mild hyperthermia at 39–42°C for up to 48 h showed no development of chronic thermotolerance. Heating at 45°C immediately after mild hyperthermia showed that acute thermotolerance did develop for 40–42°C heating. This thermotolerance developed at about the same rate for the three inducing temperatures (40–42°C) but the decay characteristics were temperature dependent. In fact, for 42°C heating thermosensitization to subsequent 45°C heating was achieved after 48 h of heating. These data show that chronic and acute thermotolerance may be different in human glioma cells.     

Cell cycle progression during chronic hyperthermia in S phase CHO cells

S phase CHO cells chronically exposed to 41.5°C exhibited an apparent resistance to further cell killing after 4–6 h. During the treatment interval, cells were observed to progress out of S phase and into G₂, mitosis, and the next cell cycle. Progression through S was delayed by about 2 h and an equivalent mitotic delay was observed. After entry into mitosis, heated cultures showed an altered nuclear morphology, presumably as the result of abnormal division occurring during the treatment. Tritium suicide experiments at this temperature showed that clonogenic, as well as non-clonogenic, cells progressed during this period. When S phase cultures were chronically heated at 42°C, however, the delay in transit through S phase was 8–10 h, and an accumulation in G₂ phase was observed. Therefore, our results show that, in S phase cultures heated at 42°C, cell killing continues while cells progress through S, suggesting that chronic thermotolerance cannot be expressed in S phase. Furthermore, at 41.5°C, cells which progress out of S during the treatment express resistance to heat killing in subsequent cell cycle phases. In summary, our results indicate that, although chronic tolerance is not expressed during heating in S phase, it is expressed after the cells progress out of S phase.     

Protection against heat-induced cell killing by alanine

When L-alanine was added either to full growth medium or to Hanks″ balanced salt solution (HBSS) prior to hyperthermia, survival of heated cells was significantly increased in a concentration-dependent manner. Maximal heat protection was not immediate, but required at least 1 h at 37°C incubation prior to heating. Heat protection was principally reflected in an increased D_q on the 45°C survival curve; for example, with 100 mM L-alanine, the D_q increased from ≈20 (control) to 30 min at 45°C. Hyperthermia of 1 h at temperatures between 42°C and 45°C indicated that 100 mM alanine had shifted the isotoxic temperature by 0.5°C. Comparable heat protection was also observed with D-alanine and amino acid dimers, such as alanyl-alanine or alanyl-leucine. Leucine at similar concentrations by itself, without alanine, did not protect cells against heat killing, but increased cellular heat sensitivity. The data suggest that heat protection by alanine does not require incorporation of alanine into cellular protein, but is mediated by the free amino acid.     

Sensitization to cisplatin action by step-down heating in cddp-sensitive and -resistant cells

Hyperthermia treatment (≥ 43°C) has been shown to be able to (partially) reverse acquired cDDP resistance. However, such heat treatment is difficult to achieve in the clinic. Short pre-treatment at a high temperature (>42°C), immediately before a treatment at a lower temperature (<42°C) can enhance the heat toxicity of the lower temperatures. This “step-down heating schedule” was explored for its possible drug-sensitizing potential in in vitro-cultured cDDP-sensitive and -resistant murine and human tumour cells. A 10-min pre-treatment at 44°C enhanced the cytotoxicity of 41°C hyperthermia alone. It also enhanced sensitivity to cDDP when given at 37°C. However, it did not increase the 41°C-induced cDDP sensitization. Thus, no correlation was found between heat kill and cDDP sensitization for step-down heating schedules. The observed effects of step-down heating were comparable in sensitive and in resistant cells, so the step-down heating schedule, unlike the 43°C treatment, did not lead to a decrease of the cDDP-resistance factor. Yet the total cytotoxicity caused by this treatment protocol was 10-fold more than for cDDP with 41°C alone, due to the extra hyperthermic cell killing and the cDDP-sensitizing effect of the pre-treatment. This treatment could have a substantial impact on cDDP efficacy in the clinic even when cDDP resistance has developed. © 1995 Wiley-Liss, Inc.     

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.     

Acute extracellular acidification reduces intracellular pH, 42°C-induction of heat shock proteins and clonal survival of human melanoma cells grown at pH 6.7

Two human melanoma cell lines, SK-Mel-28 and DB-1, were used for in vitro studies of the mechanisms underlying heat resistance of human tumour cells adapted to growth in acidic environments. Adaptation to growth at low pH was characterized by resistance to 42°C cytotoxicity and accompanied by an increase in endogenous levels of Hsp70 and/or Hsp27. Acute extracellular acidification to levels below pH 6.5 was required to sensitize the melanoma cells to 42°C. Furthermore, cells grown at low pH were more resistant to sensitization by acute acidification than cells grown at pH 7.3. The intracellular pH (pH_i) of cells grown at pH 6.7 was less than the pH_i of cells grown at pH 7.3 both before and after acute acidification. A pH_i threshold existed for melanoma cells growing at pH 7.3 below which they became sensitized to 42°C. This pH_i threshold differed between the SK-Mel-28 and DB-1 cells. In contrast, a pH_i threshold for heat sensitization did not exist for cells growing at pH 6.7: any reduction in pH_i before heating resulted in increased cell killing. Since cells grown at low pH lack a pH_i threshold for heat sensitization, they are sensitized more to 42°C per unit decrease in pH_i than cells grown at pH 7.3. Acute acidification abrogated the 42°C-induction of Hsp70 and Hsp27 in the melanoma cells. The pH_i thresholds for abrogation of these HSPs are slightly higher than or comparable with the thresholds for cytoxicity for each cell line grown at pH 7.3, but abrogation occurred over a narrower range of pH_i compared with cytotoxicity. Abrogation of heat-induced expression of these HSPs correlates with cytotoxicity in both cell lines with the exception of Hsp27 expression in SK-Mel-28 cells. In conclusion, strategies that reduce pH_i in melanoma cells growing at low pH, such as in acidotic regions of tumours, could selectively sensitize them to hyperthermia because they lack a pH_i threshold for heat sensitization.     

The relationship between treatment duration and temperature for hyperthermia induced lethality of cultured murine cells: Influence of medium conditions

The heat sensitivity and the time-temperature relationship of non-tolerant and thermotolerant M8013 cells treated at different pHs in either culture medium (including serum) or Hanks' salts solution (HBSS) were compared. The cells were growing asynchronously. Arrhenius plots for non-tolerant cells heated in culture medium pH 7.35 showed two linear parts below and above the transition temperature (T_trans). The inactivation energies below and above T_trans were respectively 2980 and 490 kJ/mole. With thermotolerant cells under the same conditions the inactivation energy was approximately constant over the range 42–46°C at 890 kJ/mole. The cells were more sensitive to heat treatment at low pH or in HBSS. Moreover, it appeared that the expression of thermotolerance was strongly dependent on medium conditions: the thermotolerance ratio (TTR, ratio between slopes of survival curves of thermotolerant and normal cells) was much lower at low pH or in cells heated in HBSS. Generally a high TTR observed in experiment with fractioned hyperthermia at temperatures above T_trans correlated fairly well with a high inactivation energy below T_trans from the Arrhenius plot derived from data from experiments with the same cells that were not made thermotolerant before treatment.     

Thermal sensitivity and kinetics of thermotolerance in bovine aortic endothelial cells in culture

The effect of heat on the clonogenicity of bovine aortic endothelial (B AE) cells in vitro was measured. Continuous heating of cells at 43°C or 43–5°C produced survival curves exhibiting thermoresistant tails. When heated at 44°C the survival curve of BAE cells was exponential except for a small shoulder. The BAE cells heated at 44°C and 45°C had D₀ values of 33 min and 19 min, respectively. The development of thermotolerance in BAE cells was studied by measuring the sensitivity of cells to a 44°C heating at various times following a priming heat treatment at 43°C or 44°C for 30 min. The thermotolerance ratio in BAE cells preheated at 43°C for 30 min reached a peak of 3.8 at 3 h and declined to 1.9 at 24 h after the prime heating. After prime heating at 44°C for 30 min the thermotolerance ratio increased rapidly to 5.4 in 5 h, remained elevated at 12 h and then declined to a value of 2.1 at 24 h. Thermotolerance in endotheliaJanuary-February 1991 cells may be partially responsible for the thermotolerance in blood vessels of normal tissues and tumours.     

Enhancement of nitrocaphane cytotoxicity by hyperthermia in vitro

HEp-2 cells were treated with hyperthermia (39–44°C) and nitrocaphane (NC) at various time intervals. A 1 h exposure to 39°C and 41 °C was non-lethal to cells, but it did potentiate the cell killing of NC (1.0 μg/ml). It was further shown that the sequence between heat and the drug can affect cell survival. Cell killing effect was decreased when heat was given before or after administration of drug. In contrast the simultaneous administration of these two modalities was synergistic. Maximal thermotolerance of HEp-2 cells was developed using an 8-h interval at 37 °C between the cell exposures to two equal thermal doses (44°C, 30 min). HEp-2 cells became thermotolerant when preheated for 30 min at 44°C followed by a 10-h interval at 37°C. The thermotolerant cells showed resistance to subsequent heat at 44°C (D₀=2.26 h, control D₀=0.38 h), to subsequent NC treatments, and to heat combined with NC. However, in the thermotolerant cells, cytotoxicity of NC was still enhanced by hyperthermia.     

Thermal sensitisation by lonidamine of human melanoma cells grown at low extracellular pH

Purpose: This study tested the ability of lonidamine (LND), a clinically applicable inhibitor of monocarboxylate transporters (MCT), to thermally sensitise human melanoma cells cultured at a tumour-like extracellular pH (pH_e) 6.7.

Materials and methods: Human melanoma DB-1 cells cultured at pH_e 6.7 and pH_e 7.3 were exposed to 150?µM LND for 3?h, beginning 1?h prior to heating at 42?°C (2?h). Intracellular pH (pH_i) was determined using 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) and whole spectrum analysis. Levels of heat shock proteins (HSPs) were determined by immunoblot analysis. Cell survival was determined by colony formation.

Results: Treatment with LND at pH_e 6.7 reduced pH_i to 6.30?±?0.21, reduced thermal induction of HSPs, and sensitised cells growing at pH_e 6.7 to 42?°C. When LND was combined with an acute acidification from pH_e 6.7 to pH_e 6.5, pH_i was reduced to 6.09?±?0.26, and additional sensitisation was observed. LND had negligible effects on cells cultured at pH 7.3.

Conclusions: The results show that LND can reduce pH_i in human melanoma cells cultured at a tumour-like low pH_e so that the 42?°C induction of HSPs are abrogated and the cells are sensitised to thermal therapy. Cells cultured at a normal tissue-like pH_e 7.3 were not sensitised to 42?°C by LND. These findings support the strategy that human melanoma cells growing in an acidic environment can be sensitised to thermal therapy in vivo by exposure to an MCT inhibitor such as LND.