INT. J. HYPERTHERMIA,
1991,
VOL.
7,
NO.
3, 511-518
Augmented antitumour effects of combined treatment with hyperthermia and tumour necrosis factor on human gastric cancer xenotransplanted into nude mice
Int J Hyperthermia Downloaded from informahealthcare.com by Flinders University of South Australia on 01/08/15 For personal use only.
S. FUJIMOTO, C. KONNO, K. KOBAYASHI, M. KOKUBUN, R. D. SHRESTHA, S . KIUCHI, M. TAKAHASHI, M. OHTA and K. OKUI First Department of Surgery, School of Medicine, Chiba University, 1-8-1, Inohana, Chiba 280, Japan
(Received 9 July 1990; revision received 6 September 1990; accepted 12 September 1990) Hyperthermia combined with recombinant human tumour necrosis factor (rH-TNF) was evaluated for antitumour efficacy in vivo. Use was made of human gastric cancer tissues xenografted into nude mice. When 100, 300, 600, and 1200 units of rH-TNF (2.4 x lo6 units/mg protein) were given twice intraperitoneally, tumour regression did not occur in any animal. In contrast, a remarkable suppression of tumour growth was observed when 600 and 1200 units of rH-TNF was given in combination with hyperthermia at 43.5 & O . 1"C. No effects were evident with the regimen of 100 and 300 units of rH-TNF plus hyperthermia at the same temperature, as compared with evidence obtained with hyperthermia alone. The tumoral blood flow, determined by the hydrogen diffusion method, decreased immediately after hyperthermia alone or hyperthermia plus 1200 units of rH-TNF, whereas a slight decrease was seen after rH-TNF alone. When hyperthermia plus 1200 units of rH-TNF were given, there was a remarkable delay in reversion to pretreatment values of tumoral blood flow, as compared to findings with rH-TNF only or heat only. These results are discussed in relation to the antitumour and side-effects of rH-TNF.
1. Introduction The in vivo cytocidal effect of hyperthermia is related to poor or little blood flow in cancer tissues, as caused by high temperature (Bicher et al. 1980, Dudar and Jain 1984, Song 1984). We examined events related to the combined use of hyperthermia and misonidazole, as a thermosensitizing drug, to treat advanced gastric cancer (Fujimoto et al. 1988 a). We also reported a marked decrease in tissue oxygen tension due to the combination of hyperthermia and misonidazole, compared with findings when hyperthermia was given only (Kokubun et al. 1989). Tumour necrosis factor (TNF) was originally reported by Carswell et al. (1975) to be a substance present in serum of laboratory animals treated with Bacillus Calmette-Guerin or Corynebacterium parvum and with endotoxin. TNF is defined as a protein producing haemorrhagic necrosis of experimental tumours, and has cytocidal effects on tumour cells in vitro (Helson et al. 1975, Matthews and Watkins 1978, Sugarman et al. 1985, Creasey et al. 1987, Shine et al. 1989). This compound has a cytotoxic effect on cultured bovine aortic endothelial cells, as well as a promoting effect on clot formation, in vitro (Kull and Cuatrecasas 1981, Nawroth and Stern 1986). Watanabe et al. (1988 b) reported that intravenous administration of TNF to tumour-bearing mice resulted in blockage of the blood circulation only in newly formed blood vessels in the tumours. However, the susceptibility of TNF to tumours is limited, particularly in gastric cancer (Watanabe et al. 1985). The current study was done to determine whether the antitumour efficacy of hyperthermia on xenoplanted human gastric cancer would be enhanced with recombinant human tumour necrosis factor (rH-TNF) and if the effect of rH-TNF on tumour vessels would be intensified by hyperthermia. 0256-6736/91 $3.00 01991 Taylor & Francis Ltd
512
S. Fujimoto et al.
Int J Hyperthermia Downloaded from informahealthcare.com by Flinders University of South Australia on 01/08/15 For personal use only.
2. Materials and methods 2.1 . Animals BALB/c nu/nu mice (Japan Clea Laboratories, Tokyo, Japan) aged 5-6 weeks were kept under specific-pathogen-free conditions. The animals had free access to aseptic food and water, and were assigned randomly to groups of 10-12, except for 41 mice for the controls and 17 given rH-TNF of 600 units. 2.2 Turnours A human gastric cancer, H-23, which had been maintained in our laboratory by serial transplantation, was a moderately differentiated adenocarcinoma. Fragments of the tumour were inoculated into the subcutaneous space of the external root in the right hindleg, to avoid liyperthermic damage to intra-abdominal organs. The rate of successful transplantation wa\ 9 3-95 %) and spontaneous rejection never occurred. 2.3. Recombinant human turnour necrosis factor (rH-TNF) rH-TNF was kindly provided by the Asahi Chemical Industry Co. (Tokyo, Japan). Thi\ preparation has a specific activity of 2 . 4 x lo6 unitslmg protein, as determined by cytotoxic activity against mouse L-M cells (Yamazaki et al. 1986). 2.4. 7ieutment When the transplanted tumour grew to about 100 m3the treatment was initiated. An injection of 50 mg/kg of Nembutal (pentobarbital-Na; Abbott Laboratories, North Chicago, Ill.. USA) was made intraperitoneally (i.p.) and subsequently rH-TNF, 100, 300, 600, and 1200 units, was given i.p. Successively, the right hindleg was placed in a watcr bath at 4 3 . 5 + 0 . I “C, for 23 min. The temperature in the centre of the tumour equilibrated with that of the water bath within 2-3 min of heating (Fujimoto et al. 1988 b). The body temperature of nude mice in the experimental room at 24-25°C was about 28°C. This procedure was carried out twice at a 7-day interval in due consideration of findings that the thermotolerance of the H-23 tumour reached a maximum at a 3-day interval and disappeared completely after a 7-day interval (Fujimoto et ul. 1989).
2.5. Ewluation qf treatments Two perpendicular diameters (length and width) of the transplanted tumours were measured every other day using vernier sliding calipers, and tumour volume was calculated as 112 xab’, where N and b are the longest and shortest diameters, respectively. ‘To assess the effects of rH-TNF on microcirculatory factors in the H-23 tumours, blood flow in the H-23 tumours was measured consecutively in each mouse, by a hydrogen diffusion method (Aukland et al. 1964). The method is based on the polarographic determination of the amount of hydrogen gas reaching a platinum electrode. Hydrogen saturation of the tissues was achieved by allowing the animal to breathe hydrogen gas together with air, which was regulated and checked by a hydrogen gas controller (Model SHI- 102. Unique Medical Co., Tokyo, Japan). A hydrogen clearance curve in cancer tissue was computed by a digital UH-meter (Model MHG-D1, Unique Medical Co.) based on ; IZicrlcr’s theory (1965), and the value of tissue blood flow was given by a digital computer o f the UH-meter every 1 s. Statistical analysis was made using Student’s t-test.
3. Results The H-23 tumour grew at a doubling time of 5.3 days when the volume was between 100 and 300 mm’. The time course of tumour growth in the heat-only administered
Effects of hyperthermia and TNF on xenografts
513
Int J Hyperthermia Downloaded from informahealthcare.com by Flinders University of South Australia on 01/08/15 For personal use only.
groups is shown in Figure 1 . The mean tumour volume 6 days after the second hyperthermia was smaller than that of the controls (0.0139 < p < 0 . 0 4 5 3 ) . 3 . 1 . Eflect of rH-TNF only on tumour growth As shown in Figure 1, no difference in the tumour volumes was observed among four groups given 100,300,600, and 1200 units of rH-TNF only. Findings in these four groups did not differ from those in the controls, and in the four groups given 100, 300, 600, and 1200 units of rH-TNF, the tumour was no smaller than in the heat only administered group.
3 . 2 . Effect of rH- TNF plus hyperthermia on tumour growth Tumour growth in mice given hyperthermia plus 100 and 300 units of rH-TNF was unchanged compared to that in mice given heat only (Figure 2 ) . On the other hand, in mice given 1200 units of rH-TNF plus hyperthermia, a marked suppression occurred all through the study at 0 . 0 0 2 1 7 < p < 0 . 0 2 1 8 , compared with findings in the case of hyperthermia only. In case of administration of 600 units plus heat, a positive delay in tumour growth was observed at 0.0153
1991,
VOL.
7,
NO.
3, 511-518
Augmented antitumour effects of combined treatment with hyperthermia and tumour necrosis factor on human gastric cancer xenotransplanted into nude mice
Int J Hyperthermia Downloaded from informahealthcare.com by Flinders University of South Australia on 01/08/15 For personal use only.
S. FUJIMOTO, C. KONNO, K. KOBAYASHI, M. KOKUBUN, R. D. SHRESTHA, S . KIUCHI, M. TAKAHASHI, M. OHTA and K. OKUI First Department of Surgery, School of Medicine, Chiba University, 1-8-1, Inohana, Chiba 280, Japan
(Received 9 July 1990; revision received 6 September 1990; accepted 12 September 1990) Hyperthermia combined with recombinant human tumour necrosis factor (rH-TNF) was evaluated for antitumour efficacy in vivo. Use was made of human gastric cancer tissues xenografted into nude mice. When 100, 300, 600, and 1200 units of rH-TNF (2.4 x lo6 units/mg protein) were given twice intraperitoneally, tumour regression did not occur in any animal. In contrast, a remarkable suppression of tumour growth was observed when 600 and 1200 units of rH-TNF was given in combination with hyperthermia at 43.5 & O . 1"C. No effects were evident with the regimen of 100 and 300 units of rH-TNF plus hyperthermia at the same temperature, as compared with evidence obtained with hyperthermia alone. The tumoral blood flow, determined by the hydrogen diffusion method, decreased immediately after hyperthermia alone or hyperthermia plus 1200 units of rH-TNF, whereas a slight decrease was seen after rH-TNF alone. When hyperthermia plus 1200 units of rH-TNF were given, there was a remarkable delay in reversion to pretreatment values of tumoral blood flow, as compared to findings with rH-TNF only or heat only. These results are discussed in relation to the antitumour and side-effects of rH-TNF.
1. Introduction The in vivo cytocidal effect of hyperthermia is related to poor or little blood flow in cancer tissues, as caused by high temperature (Bicher et al. 1980, Dudar and Jain 1984, Song 1984). We examined events related to the combined use of hyperthermia and misonidazole, as a thermosensitizing drug, to treat advanced gastric cancer (Fujimoto et al. 1988 a). We also reported a marked decrease in tissue oxygen tension due to the combination of hyperthermia and misonidazole, compared with findings when hyperthermia was given only (Kokubun et al. 1989). Tumour necrosis factor (TNF) was originally reported by Carswell et al. (1975) to be a substance present in serum of laboratory animals treated with Bacillus Calmette-Guerin or Corynebacterium parvum and with endotoxin. TNF is defined as a protein producing haemorrhagic necrosis of experimental tumours, and has cytocidal effects on tumour cells in vitro (Helson et al. 1975, Matthews and Watkins 1978, Sugarman et al. 1985, Creasey et al. 1987, Shine et al. 1989). This compound has a cytotoxic effect on cultured bovine aortic endothelial cells, as well as a promoting effect on clot formation, in vitro (Kull and Cuatrecasas 1981, Nawroth and Stern 1986). Watanabe et al. (1988 b) reported that intravenous administration of TNF to tumour-bearing mice resulted in blockage of the blood circulation only in newly formed blood vessels in the tumours. However, the susceptibility of TNF to tumours is limited, particularly in gastric cancer (Watanabe et al. 1985). The current study was done to determine whether the antitumour efficacy of hyperthermia on xenoplanted human gastric cancer would be enhanced with recombinant human tumour necrosis factor (rH-TNF) and if the effect of rH-TNF on tumour vessels would be intensified by hyperthermia. 0256-6736/91 $3.00 01991 Taylor & Francis Ltd
512
S. Fujimoto et al.
Int J Hyperthermia Downloaded from informahealthcare.com by Flinders University of South Australia on 01/08/15 For personal use only.
2. Materials and methods 2.1 . Animals BALB/c nu/nu mice (Japan Clea Laboratories, Tokyo, Japan) aged 5-6 weeks were kept under specific-pathogen-free conditions. The animals had free access to aseptic food and water, and were assigned randomly to groups of 10-12, except for 41 mice for the controls and 17 given rH-TNF of 600 units. 2.2 Turnours A human gastric cancer, H-23, which had been maintained in our laboratory by serial transplantation, was a moderately differentiated adenocarcinoma. Fragments of the tumour were inoculated into the subcutaneous space of the external root in the right hindleg, to avoid liyperthermic damage to intra-abdominal organs. The rate of successful transplantation wa\ 9 3-95 %) and spontaneous rejection never occurred. 2.3. Recombinant human turnour necrosis factor (rH-TNF) rH-TNF was kindly provided by the Asahi Chemical Industry Co. (Tokyo, Japan). Thi\ preparation has a specific activity of 2 . 4 x lo6 unitslmg protein, as determined by cytotoxic activity against mouse L-M cells (Yamazaki et al. 1986). 2.4. 7ieutment When the transplanted tumour grew to about 100 m3the treatment was initiated. An injection of 50 mg/kg of Nembutal (pentobarbital-Na; Abbott Laboratories, North Chicago, Ill.. USA) was made intraperitoneally (i.p.) and subsequently rH-TNF, 100, 300, 600, and 1200 units, was given i.p. Successively, the right hindleg was placed in a watcr bath at 4 3 . 5 + 0 . I “C, for 23 min. The temperature in the centre of the tumour equilibrated with that of the water bath within 2-3 min of heating (Fujimoto et al. 1988 b). The body temperature of nude mice in the experimental room at 24-25°C was about 28°C. This procedure was carried out twice at a 7-day interval in due consideration of findings that the thermotolerance of the H-23 tumour reached a maximum at a 3-day interval and disappeared completely after a 7-day interval (Fujimoto et ul. 1989).
2.5. Ewluation qf treatments Two perpendicular diameters (length and width) of the transplanted tumours were measured every other day using vernier sliding calipers, and tumour volume was calculated as 112 xab’, where N and b are the longest and shortest diameters, respectively. ‘To assess the effects of rH-TNF on microcirculatory factors in the H-23 tumours, blood flow in the H-23 tumours was measured consecutively in each mouse, by a hydrogen diffusion method (Aukland et al. 1964). The method is based on the polarographic determination of the amount of hydrogen gas reaching a platinum electrode. Hydrogen saturation of the tissues was achieved by allowing the animal to breathe hydrogen gas together with air, which was regulated and checked by a hydrogen gas controller (Model SHI- 102. Unique Medical Co., Tokyo, Japan). A hydrogen clearance curve in cancer tissue was computed by a digital UH-meter (Model MHG-D1, Unique Medical Co.) based on ; IZicrlcr’s theory (1965), and the value of tissue blood flow was given by a digital computer o f the UH-meter every 1 s. Statistical analysis was made using Student’s t-test.
3. Results The H-23 tumour grew at a doubling time of 5.3 days when the volume was between 100 and 300 mm’. The time course of tumour growth in the heat-only administered
Effects of hyperthermia and TNF on xenografts
513
Int J Hyperthermia Downloaded from informahealthcare.com by Flinders University of South Australia on 01/08/15 For personal use only.
groups is shown in Figure 1 . The mean tumour volume 6 days after the second hyperthermia was smaller than that of the controls (0.0139 < p < 0 . 0 4 5 3 ) . 3 . 1 . Eflect of rH-TNF only on tumour growth As shown in Figure 1, no difference in the tumour volumes was observed among four groups given 100,300,600, and 1200 units of rH-TNF only. Findings in these four groups did not differ from those in the controls, and in the four groups given 100, 300, 600, and 1200 units of rH-TNF, the tumour was no smaller than in the heat only administered group.
3 . 2 . Effect of rH- TNF plus hyperthermia on tumour growth Tumour growth in mice given hyperthermia plus 100 and 300 units of rH-TNF was unchanged compared to that in mice given heat only (Figure 2 ) . On the other hand, in mice given 1200 units of rH-TNF plus hyperthermia, a marked suppression occurred all through the study at 0 . 0 0 2 1 7 < p < 0 . 0 2 1 8 , compared with findings in the case of hyperthermia only. In case of administration of 600 units plus heat, a positive delay in tumour growth was observed at 0.0153
