Effects of heat treatment in vitro and in vivo on human melanoma xenografts.

The heat response in vitro and in vivo of five human melanoma xenografts grown in athymic nude mice was studied. The melanomas differed significantly in terms of heat sensitivity both in vitro and in vivo. At least two different mechanisms governed the overall heat response of the melanomas in vivo: the primary cell death, induced during treatment, was due to direct cytotoxic effects of the heat; the secondary cell death, induced after completion of treatment, was due to heat-induced vascular damage. The activation energies for the melanomas were not significantly different in vitro and in vivo at temperatures above the inflection point of the Arrhenius curves. Below the inflection point, on the other hand, the activation energies were higher in vitro than in vivo, probably as a consequence of differences in the physiological conditions in vitro and in vivo. The heat responsiveness of the melanomas in vivo was not related to the radioresponsiveness, whether the heat treatment was given at a low or a high temperature. All melanomas developed thermotolerance after a priming heat treatment. The thermotolerance differed significantly in magnitude among the five melanomas. It was concluded from the thermotolerance data that clinical treatment protocols probably should not prescribe more than one hyperthermic treatment per week.

Radioresponsiveness of human melanoma xenografts given fractionated irradiation in vivo--relationship to the initial slope of the cell survival curves in vitro.

The radioresponsiveness of five human melanoma xenograft lines given fractionated irradiation in vivo was studied using specific growth delay and cell survival in vitro as endpoints. Superfractionation (3 fractions of 2.0 Gy with 4-h intervals each day) as well as conventional fractionation (1 fraction of 2.0 Gy each day) were used. The total dose was varied within the ranges 12 to 30 Gy and 10 to 30 Gy, respectively. The rankings of the melanomas in radioresponsiveness were almost identical, irrespective of the endpoint and the fractionation regime considered. The radioresponsiveness was found to be positively correlated to the initial slope of the in vitro cell survival curves, i.e. the alpha and the surviving fraction at 2.0 Gy (conventional dose rate; 3.0 Gy/min) and the D0 (low dose rate; 1.25 cGy/min). There was no relationship between the radioresponsiveness and any known growth or microenvironmental parameter. It is concluded that the differences in radioresponsiveness among the melanomas for the fractionation regimes studied here were governed mainly by the intrinsic repair capacity of the tumour cells and not by microenvironmental factors. The potential of in vitro cell survival curve parameters in predicting the clinical radioresponsiveness of tumours is discussed.

Radiation sensitivity in vitro of cells isolated from human tumor surgical specimens.

The radiation sensitivity of cells isolated directly from human tumor surgical specimens was studied using the Courtenay soft agar colony assay. Aerobic cell survival curves covering 2-3 decades were achieved for eight melanomas, seven ovarian, six cervix, five breast, four bladder, and four squamous cell carcinomas of the head and neck and two seminomas. Cell survival following exposure to 2.0 Gy was measured also for several other tumors of these histological types. Experiments repeated with cells stored in liquid nitrogen showed that the survival assay gave highly reproducible results. The D0 (0.61-1.65 Gy) as well as the surviving fraction at 2.0 Gy (0.12-0.66) differed considerably among individual tumors of the same histological type. Neither of these parameters was therefore significantly different for the seven tumor categories. However, about one-third of the melanomas showed a higher surviving fraction at 2.0 Gy than the highest value measured for the other tumors. Two of three seminomas showed surviving fractions at 2.0 Gy in the absolute lower range, i.e., below 0.20. Altogether the data were consistent with the suggestion recently put forward that the clinical radiocurability of tumors may be correlated to the cell surviving fraction in vitro at 2.0 Gy. However, it was not possible on the basis of individual tumors to investigate whether the surviving fraction at 2.0 Gy was correlated to the clinical radiocurability, since adequate clinical data were not available for the parent tumors. It is suggested that melanomas may be especially suitable for prospective studies aimed at establishing whether such a correlation really does exist. If a significant correlation can be verified, then a very important conclusion may be drawn from our data: the radiocurability of human tumors may differ almost just as much among individual tumors of the same histological type as among individual tumors of different histology.

Arrhenius analysis of the heat response in vivo and in vitro of human melanoma xenografts.

The response to heat treatment in vivo (40.5-44.0 degrees C) and in vitro (40.5-45.5 degrees C) of five human melanoma xenografts was studied. Specific growth delay was used as a measure of response after treatment in vivo. Colony-forming ability was assayed in soft agar after treatment in vitro. Dose-response curves were established and subjected to Arrhenius analysis. The Arrhenius curves were found to have an inflection point at 42.0-43.0 degrees C in vivo and 41.5-42.5 degrees C in vitro. The activation energies were in the ranges 426-771 kJ/mol in vivo and 676-739 kJ/mol in vitro above the inflection point and 774-1661 kJ/mol in vivo and 1118-2190 kJ/mol in vitro below the inflection point. Above the inflection point the activation energies in vivo and in vitro were not significantly different for any of the melanomas, and furthermore were similar to those reported for rodent tumours, normal tissues and cells in culture in the same temperature range. Below the inflection point on the other hand the activation energies were lower in vivo than in vitro. This difference was probably a consequence of differences in the physiological conditions in vivo and in vitro. The activation energies in vitro in this temperature range were comparable to those reported for normal tissues and cells in culture.

Radiation response of multicellular spheroids initiated from five human melanoma xenograft lines. Relationship to the radioresponsiveness in vivo.

Multicellular spheroids, initiated from five human melanoma xenograft lines (E.E., E.F., G.E., M.F., V.N.) and grown in liquid-overlay culture, were characterised with regard to radiation response. The principal aim of the work was to search for possible correlations with the radioresponsiveness in vivo of the parent xenografts. The spheroids were 100 +/- 5 microns in diameter at irradiation and did not contain radiobiologically hypoxic cells. Single-cell survival measured in soft agar, specific growth delay and spheroid cure were used as end-points. The cellular radiosensitivity was the same whether a melanoma was grown as spheroids or as xenografts. An intercellular contact effect was found for spheroids of the G.E. melanoma but not for spheroids of the E.E., E.F., M.F. and V.N. melanomas, in agreement with previous observations from studies of the corresponding xenografts in vivo. A positive correlation was found between the radiation response of the spheroids, measured as cell survival after 6 Gy or as specific growth delay after 6 Gy, and the radiation response of the parent tumours, measured as specific growth delay after 15 Gy. There was no correlation between the SCD50 (the dose required to cure 50% of the spheroids) and the radioresponsiveness in vivo. It is concluded that differences in radioresponsiveness in vivo among tumours may be identified from studies of the corresponding multicellular spheroids grown in liquid-overlay culture.

Heterogeneity in radiosensitization by heat of cloned cell lines derived from a single human melanoma xenograft.

Heterogeneity in radiosensitization by heat was studied using one uncloned and five cloned cell lines isolated from a single tumour of a human melanoma xenograft. Cells from passages 7-12 in vitro were given heat treatments of 42.5 degrees C (45 min), 43.5 degrees C (45 min) or 44.5 degrees C (45 min) immediately after exposure to graded doses of radiation. The survival curves after irradiation alone had similar D0 values but differed in the size of the shoulder. The heterogeneity in heat radiosensitization was reflected in differences in decrease of the D0 values. The thermal enhancement ratios, calculated from the D0 values, were in the ranges 1.2 +/- 0.2-1.7 +/- 0.2 (42.5 degrees C), 1.4 +/- 0.3-2.4 +/- 0.4 (43.5 degrees C) and 2.3 +/- 0.4-3.4 +/- 0.4 (44.5 degrees C). Moreover, at 43.5 degrees C the heterogeneity was also reflected in different modifications of the shape of the survival curves. Two lines showed survival curves with a significant shoulder and a relatively low D0 value whereas two other lines had lost the shoulder almost completely but showed relatively high D0 values. All lines showed survival curves with a broad shoulder after heating at 42.5 degrees C, whereas none of the lines showed survival curves with a significant shoulder after heating at 44.5 degrees C.

Radiation response of human melanoma multicellular spheroids measured as single cell survival, growth delay, and spheroid cure: comparisons with the parent tumor xenograft.

The radiation response of multicellular spheroids, initiated from a human melanoma xenograft (E.E.) propagated in athymic mice, was studied using cell survival, growth delay, and spheroid cure as endpoints. The relationship between these endpoints was analyzed, and the radiation response of the spheroids was compared with the parent xenograft. At irradiation, the spheroids were 100 +/- 5 micron in diameter and did not contain radiobiologically hypoxic cells. Growth delay of the spheroids mainly depended on the fraction of surviving cells as measured in soft agar, that is, there was a good correlation between these two endpoints. Moreover, Do-values calculated from spheroid cure curves were similar to those of the cell survival curves measured in soft agar. However, the number of stem cells per spheroid, calculated from SCD50-values (the doses required to cure 50% of the spheroids), was at least a factor of seven lower than the clonogenicity of cells from disaggregated spheroids would indicate. The cellular radiosensitivity of the spheroids was similar to the parent xenograft. An intercellular contact effect was not found for the spheroids, in agreement with observations from studies of xenografted tumors. Moreover, specific growth delays, as well as Do-values calculated from cure curves were similar for spheroids and tumors when the data for the latter were corrected for the presence of hypoxic cells. The high degree of conformity in the results indicates that multicellular spheroids and xenografted tumors may complement one another in studies of human tumor radiobiology.

Differences in thermosensitization among cloned cell lines isolated from a single human melanoma xenograft.

Differences in thermosensitization (effect of step-down heating) among one uncloned and five cloned cell lines isolated from a single tumor of a human melanoma xenograft were studied. Cells from passages 7-12 in vitro were exposed to graded heat treatments at 41.5 degrees C immediately, 1 h, and 2 h after a conditioning treatment of 43.5 degrees C (90 min). The thermosensitization was largest immediately after the conditioning treatment and then decayed exponentially. The differences among the cell lines were reflected in the maximum magnitude as well as in the rate of decay of the thermosensitization. The maximum thermosensitization ratios (TSR), calculated as the ratio of the D0 values at 41.5 degrees C for single-heated and preheated cells, ranged from 5.3 +/- 1.5 to 14.9 +/- 5.2 and were not correlated to the surviving fractions after the conditioning treatment. The half-times for the decay of the thermosensitization ranged from 1.5 +/- 0.3 h to 3.1 +/- 0.5 h and were not correlated to the maximum TSR. Moreover, there was no correlation between the magnitude of the maximum thermosensitization at 41.5 degrees C and the magnitude of the maximum thermotolerance at 43.5 degrees C, as induced by the same treatment (43.5 degrees C for 90 min).

Growth characteristics of human melanoma multicellular spheroids in liquid-overlay culture: comparisons with the parent tumour xenografts.

The growth characteristics of multicellular spheroids, derived from human melanoma xenografts and cultivated in liquid-overlay culture, were studied and compared with those of the parent tumours. Six of the seven melanomas investigated formed spheroids, which grew exponentially up to a volume of 1-2 X 10(7) microns 3 (a diameter of 270-340 microns) before the growth rate tapered off. The morphology of the spheroids varied considerably among the melanomas; some spheroids grew as densely packed, spherical structures of cells whereas others were loosely packed and showed an irregular shape. Central necrosis developed when the spheroids attained a diameter of 150-200 microns. The histological and cytological appearance of the spheroids was remarkably similar to that of the parent xenograft in five of the six cases. The sixth melanoma contained two subpopulations with distinctly different DNA content, one of which was predominant in the spheroids, the other in the tumours. This gave rise to clear histological and cytological differences. The volume-doubling time of the spheroids during the exponential growth phase ranged from 1.7 +/- 0.2 to 2.7 +/- 0.4 days and the fraction of cells in S from 13 +/- 1 to 28 +/- 2%. The volume-doubling time decreased with increasing fraction of cells in S, indicating that the differences in growth rate were due mainly to differences in the growth fraction or to differences in the duration of G1. The spheroid volume-doubling times did not correlate with those of the parent xenografts (Td = 4.2-22.5 days at V = 200 mm3), possibly because the cell loss factors of the xenografts were large and varied among the melanomas. The fractions of cells in G1/G0, S and G2 + M in the spheroids and the xenografts did not correlate either, but were found to be within the same narrow ranges in the spheroids and the xenografts--i.e. 50-80% (G1/G0), 10-30% (S) and 10-20% (G2 + M).

Primary and secondary cell death in human melanoma xenografts following hyperthermic treatment.

The response to hyperthermic treatment (42.5 degrees C for 60 min) of 5 human malignant melanomas grown in athymic mice (BALB/c/nu/nu/BOM) was studied. Local hyperthermia was given by immersing the tumor-bearing leg of the mice into a thermostatically regulated water bath. Growth delay studies indicated that the melanomas were different in heat responsiveness. The differences were confirmed by measuring the fraction of clonogenic cells in the melanomas as a function of time after treatment. The latter experiment showed that some tumor cells were inactivated during the treatment, while others lost clonogenicity first after completion of the treatment. Examinations of histological sections from tumors fixed 1 h after treatment revealed considerable vascular occlusion in all 5 melanomas. This indicates that the observed delayed cell death might be due to a number of factors, e.g., insufficient supply of oxygen and nutrients, increased tumor acidity, and accumulation of toxic metabolic products. It is concluded that at least two different mechanisms govern the overall heat response of the melanoma xenografts: the primary cell death, induced during treatment, is due to direct cytotoxic effects of the heat; the secondary cell death, induced after completion of treatment, is due to heat-induced vascular damage. The differences among the melanomas in overall heat responsiveness appeared mainly to be a consequence of differences in secondary cell death. The secondary cell death was shown to be least pronounced for those melanomas in which most of the larger vessels were embedded in broad bands of connective tissue.

Survival curves after X-ray and heat treatments for melanoma cells derived directly from surgical specimens of tumours in man.

X-ray and heat survival curves were established for melanoma cells derived directly from surgical specimens of tumours in man by using the Courtenay soft agar colony assay. The plating efficiency for 11 of the 14 melanomas studied was sufficiently high (PE = 0.3-58%) to measure cell survival over at least two decades. Experiments repeated with cells stored in liquid nitrogen showed that the survival assay gave highly reproducible results. The melanomas exhibited individual and characteristic survival curves whether exposed to radiation or heat (43.5 degrees C). The Do-values were in the ranges 0.63-1.66 Gy (X-rays) and 33-58 min (heat). The survival curves were similar to those reported previously for human melanoma xenografts. The radiation sensitivity of the cells was not correlated to the heat sensitivity. Since the melanomas appeared to be very heterogeneous in radiation response in vitro as melanomas are known to be clinically, it is suggested that melanomas may be suitable for prospective studies aimed at establishing whether clinical radioresponsiveness somehow is related to in vitro survival curve parameters.

Heat response of human melanoma multicellular spheroids: comparisons with single cells and xenografted tumors.

Multicellular spheroids were grown from cells derived directly from a human melanoma xenograft propagated in athymic mice. The histological appearance of the spheroids was similar to that of the parent xenograft. The spheroids were heated in culture medium (42.5-44.5 degrees C); growth delay and single cell survival measured in soft agar were used as end points. There was a good correlation between the results obtained with these two end points, indicating that growth delay depended mainly on cell survival. Large spheroids (200 +/- 12 microns in diameter) were found to be more heat sensitive than small ones (100 +/- 5 microns in diameter), probably because the physiological conditions in large spheroids were more favorable for cell inactivation. The cells were more resistant when heated as spheroids than as single cells. This effect was not a secondary effect of differences in cell-cycle distribution. Spheroids were also found to be more heat resistant than xenografted tumors. In the tumors, heat treatment caused vascular damage which resulted in delayed cell death due to hypoxia and/or nutrient deficiency. It is concluded that spheroids seem well suited for studies of primary heat-induced cytotoxic effects. However, they appear not to mirror the complex heat response of tumors since that response also includes secondary effects, related to heat-induced reduced perfusion.

Vascular structure of the C3H mammary carcinoma, the B16 melanoma and the Lewis lung carcinoma in syngeneic, conventional mice and congenitally athymic mice.

The vascular system of three commonly used murine experimental tumours, the C3H mammary carcinoma, the B16 melanoma and the Lewis lung carcinoma, in syngeneic (C3D2F1/Bom or C57BL/6J/Bom) and athymic (BALB/c/nu/nu/Bom) mice was studied. The main vascular characteristics of each tumour, i.e. the mean vessel diameter, the total vessel volume and the distribution of the total vessel volume among vessels with different diameters, did not change upon transplantation from conventional to athymic mice. However, the length of vessels with diameters in the range 5-15 micron was for all tumours shorter in athymic than in conventional mice. The vascular volume of the B16 melanoma per unit histologically intact tumour volume in athymic mice (0.040 +/- 0.004) was considerably larger than that of five human melanoma xenografts previously studied (0.009 +/- 0.001 to 0.022 +/- 0.002). This difference was mainly due to occurrence of vessels with diameters in the range 55-145 micron in the B16 melanoma; vessels which were generally not observed in the human melanoma xenografts.

Tumour growth delay following single dose irradiation of human melanoma xenografts. Correlations with tumour growth parameters, vascular structure and cellular radiosensitivity.

The radiation response of 5 different lines of human melanoma xenografts was studied. Tumours grown s.c. in the flanks of athymic mice were exposed to single doses of 5-25 Gy and subsequently analysed with respect to specific growth delay. The variation in radiation response among these melanoma lines was almost as large as that reported for human tumour xenografts differing in histological type. The most radioresistant melanomas showed longer volume-doubling times, lower growth fractions, higher cell loss factors and lower vascular density than the most radiosensitive ones. The radiation response was not correlated to the fraction of cells in S-phase or the DNA content of the tumour cells. Cell suspensions prepared from the different melanomas, irradiated under aerobic conditions and assayed in soft agar, also showed large variability in radiation response. Specific growth delay after 15 Gy was found to be correlated to the surviving fraction measured in vitro after 6 Gy, but not clearly to the Do value. It is suggested that tumour growth characteristics in vivo as well as radiation response in vitro may be of prognostic value for prediction of radioresponsiveness of melanomas.

Heterogeneity in heat sensitivity and development of thermotolerance of cloned cell lines derived from a single human melanoma xenograft.

One uncloned and five cloned cell lines were isolated from a single human melanoma xenograft in passage 39 in athymic mice. Cells from passages 7-12 in vitro were heated at 42.5, 43.5 or 44.5 degrees C and the colony forming ability of the cells was assayed in soft agar. The six cell lines showed individual and characteristic responses to heat treatment. The D0 values of the survival curves were in the ranges 76 +/- 5 to 131 +/- 13 min (42.5 degrees C), 12.5 +/- 1.1 to 22.2 +/- 1.9 min (43.5 degrees C) and 9.4 +/- 1.0 to 15.6 +/- 1.5 min (44.5 degrees C). Cells from all lines developed thermotolerance during protracted treatments at 42.5 degrees C. Thermotolerance was also studied by giving the cells a priming treatment of 43.5 degrees C for 90 min and then, after different fractionation intervals at 37 degrees C, second graded treatments at 43.5 degrees C. Thermotolerance ratio (TTR), i.e. the ratio of the slopes of the survival curves for preheated and single-heated cells, was used as a quantitative measure of the thermotolerance. Thermotolerance developed rapidly for all lines, reached a maximum at 12 or 16 h, and then decayed slowly. Maximum TTR varied among the lines from 4.2 +/- 0.5 to 6.0 +/- 0.9, i.e. within a factor of about 1.4. The survival curves and the TTR-curve for the uncloned line were positioned in the midst of those of the cloned lines. A linear correlation between maximum TTR and heat sensitivity was found for the six lines; maximum TTR decreased with increasing D0 value at 43.5 degrees C. Nevertheless, the lines which were most resistant before thermotolerance developed were also most resistant at maximum thermotolerance.

Response to heat treatment (42.5 degrees C) in vivo and in vitro of five human melanoma xenografts.

The response to heat (42.5 degrees C) of five human melanoma xenografts was studied. Tumours grown subcutaneously in the hind leg of athymic mice were heated in a water-bath and specific growth delay was used as a measure of response. In other experiments, cells from the same xenografts were heated in vitro and the colony-forming ability was assayed in soft agar. The slopes of the in-vivo dose-response curves (specific growth delay versus heating time) varied within a factor of about three among the five melanomas. The Do values of the in-vitro heat survival curves ranged from 44 +/- 3 to 123 +/- 15 min. The response to heat in vivo was not positively correlated with the tumour volume-doubling time, the growth fraction, the cell loss factor or the intrinsic heat sensitivity of the tumour cells, i.e., the Do values of the in vitro heat survival curves. If the results obtained in the present work are representative for clinical practice, they indicate that the response to heat may vary considerably among tumours in different patients. This variability can probably not be predicted from measurements of cytokinetic parameters of the tumours. The lack of correlation between the response to heat in vivo and in vitro demonstrates that extrapolations of results from studies in vitro to tumours are highly speculative and, when attempted, should be executed only with extreme caution.

Tumour growth delay, cell inactivation and vascular damage following hyperthermic treatment of a human melanoma xenograft.

The effect of hyperthermia at 42.5 degrees C on a human melanoma xenograft in athymic mice was studied. The tumours were heated in vivo in a water-bath. Tumour growth delay and single-cell survival in vitro were used as endpoints. Qualitative information regarding heat-induced vascular damage was obtained from microangiographic analysis. Tumour growth delay after a given treatment was considerably longer than that expected from the cell survival measured in vitro immediately after treatment. Experiments in which removal of the tumours was delayed revealed that tumour cells were continuously dying for at least 24 hr after heat treatment. The volume of the tumour vasculature was considerably reduced after treatment, suggesting that the delayed cell death was attributed to vascular occlusion which resulted in an insufficient supply of oxygen and nutrients and an increased tumour acidity. The present work indicates that at least two mechanisms may be involved in heat-induced cell inactivation in our xenograft: firstly, direct cytotoxic effect of heat; secondly, indirect effect following heat-induced vascular damage.

Heat sensitivity and thermotolerance in cells from five human melanoma xenografts.

The heat sensitivity and the development of thermotolerance in cells from five human melanoma xenografts were studied. The cells were heated in vitro in a water bath, and the colony-forming ability of the cells was assayed in soft agar. The DO values of the heat survival curves were in the ranges of 44 to 123 min (42.5 degrees), 22.3 to 63.3 min (43.5 degrees), and 6.0 to 22.1 min (44.5 degrees). The order of the heat sensitivity of the five melanomas was not the same at the three temperatures studied. Cells from all melanomas developed thermotolerance during protracted treatments at 42.5 degrees. Thermotolerance was also studied by exposing cells to a priming heat treatment of 43.5 degrees for 60 min and, after different fractionation intervals at 37 degrees, to graded heat treatments at 43.5 degrees. The ratio of the slopes of the survival curves for preheated and single-heated cells, i.e., the thermotolerance ratio (TTR), was used as a quantitative measure of the thermotolerance. For all melanomas, the TTR reached a maximum at 24 hr and then decayed slowly. The maximum TTR values ranged from about 2.9 to about 10.2. There was no correlation between the maximum TTR and the heat sensitivity for the five melanomas. Thus, the magnitude and the kinetics of thermotolerance in tumors can probably not be predicted from the surviving fraction after the priming treatment. The heat sensitivity of the tumor cells and their ability to develop thermotolerance are probably among the factors which are decisive for the response of tumors to fractionated heat treatments. The large variability in these parameters observed in the present work indicates that the response to heat may vary considerably among tumors of the same histological type in different patients.

Hyperthermic radiosensitization of cells from a human melanoma xenograft.

Cells derived directly from a human melanoma xenograft were exposed to radiation and/or hyperthermia under aerobic conditions in vitro. Single cell survival was assayed in soft agar. The activation energies for heat treatment alone were 420 +/- 40 kcal/mole (41.5-42.5 degrees C) and 170 +/- 10 kcal/mole (43.0-45.5 degrees C). Heat doses of 41.5 degrees C (30 min) or 43.5 degrees C (30 min) did not cause a reduction in the shoulder of the X-ray survival curve of the cells, but the D0 value was reduced. The thermal enhancement ratios (the ratio of the D0 values) were in the ranges 1.0-1.3 (41.5 degrees C, 30 min) and 1.1-1.7 (43.5 degrees C, 30 min), depending on the sequence and the time between the treatments. Repair of sublethal radiation damage was not significantly inhibited when treatments with 41.5 degrees C (30 min) or 43.5 degrees C (30 min) preceded irradiation, but was inhibited when 44.5 degrees C (30 min) was given immediately before radiation and when 41.5 degrees C (120 min) was given immediately after radiation. Implications of the results for clinical treatment of malignant melanomas are discussed.