Biotherapy 3: 337-344, 1991, © 1991 Kluwer Academic Publishers, Printed in the Netherlands.
In vivo effects of human recombinant tumor necrosis factor alone and in combination with other biological response modifiers on human digestive organ cancer xenografts transplanted in nude mice Yoshinori Nio', Takahiro Shiraishi l, Michihiko Tsubono 1, Hideki Morimoto ~, Chen-Chiu Tseng ~, Kazuya Kawabata ~, Yoshikazu Masai 1, Thein Tun ~, Manabu Fukumoto 2 & Takayoshi Tobe ~
JFirst Department of Surgery; 2First Department of Pathology, Kyoto University Faculty of Medicine, Kyoto 606, Japan Received 8 November 1990; accepted 12 December 1990
Key words: human tumor xenograft, interferon (IFN), interleukin-2 (IL-2), nude mouse, Picibanil (OK-432), tumor necrosis factor (TNF) Abstract
The present study was designed to evaluate the effect of rTNF alone or in combination with other BRMs on human digestive organ cancers. Six kinds of human digestive organ cancer xenografts (esophageal, stomach, colonic, pancreatic, bile duct, and liver cancers: EC-YO, GC-YN, CC-KK, PC-HN, BDC-SN and Li-7, respectively) were transplanted in nude mice, and rTNF was administered at 103, 5 x 103, or 104U/head directly into the tumor 3 times a week for 2 weeks. EC-YO was the most sensitive to rTNF, and intratumoral administration of rTNF at 103 U/head caused tumor regression. PC-HN, CC-KK and GC-YN were relatively sensitive to rTNF, and their growth was significantly inhibited by rTNF at 5 x 103 U/head, however, the tumors regrew after treatment. Li-7 and BDC-SN were resistant to rTNF. The effects of rTNF in combination with recombinant interferon-7 (rIFN-7), recombinant interleukin-2 (rIL-2), or streptococcal preparation OK-432 were assessed in mice transplanted with GC-YN. All combinations of rTNF at 5 x 103 U/head and other BRMs were more effective than rTNF alone, and GC-YN tumors were completely regressed after treatment with a combination of rTNF and rIFN-~ or rTNF and OK-432. However in all cases, the combination of rTNF at 103 U/head and any other BRM did not improve the effect. Furthermore, the adverse effects of the combinations were more serious than those of rTNF alone. TNF may still be a useful cytokine, because it can induce the regression of tumors. However, for its clinical application, a method should be developed to reduce its side effects.
Abbreviations: BRM: biological response modifier; IFN: interferon; IL-2: interleukin-2; PBS: phosphate buffer saline; rIFN-7: recombinant interferon-gamma; rIL-2: recombinant interleukin-2; rTNF: recombinant tumor necrosis factor; TNF: tumor necrosis factor.
338 Introduction
Tumor necrosis factor (TNF) has a variety of biologic activities, and its cytotoxic activity against tumor cells especially has attracted the interest of oncologists and immunologists. TNF lyses a variety of tumor cells, however, non-malignant cells are insensitive to the cytotoxicity of TNF [1-3]. The mechanism of TNF-mediated cytotoxicity has not been fully clarified, although it has been suggested that TNF molecules may affect proteolytic enzymes in lysosomes and fragment of DNA after they are internalized into cells [4-6]. TNF was originally thought to be a revolutionary agent for cancer therapy, and recombinant TNF technology has made it possible to produce sufficient amount of recombinant TNF (rTNF) for clinical application [7-9]. However in its clinical application, intravenous administration of rTNF causes serious side effects, such as shock, hypotension, chills and fever, although the response rate to rTNF has been comparatively low [10-12]. Furthermore, some authors have reported that TNF is the same molecule as cachectin, which causes cachexia, and that TNF may be a major cause of endotoxin shock [ 13, 14]. In order to reduce the adverse effects of rTNF in clinical application, intratumoral administration has recently been chosen, experimentally as well as clinically [15-17]. In addition, in order to augment the cytotoxic effect of TNF, combination with various therapies, including interferon (IFN), interleukin-2 (IL-2), and hyperthermia, have been studied [1,2,19,20]. However there have been only a few reports on the susceptibility of various human tumors, especially of digestive organ cancers, to rTNF and these combination therapies. In the present study various human digestive organ cancer xenografts were transplanted in nude mice and the effects of rTNF alone and in combination with other biological response modifiers (BRM), recombinant interferon-7 (rlFN-7),
recombinant IL-2 (rIL-2), and streptococcal preparation OK-432 (Picibanil) were assessed.
Materials and methods
Nude mice Eight-week-old male BALB/c nude mice were employed in this study. The nude mice were purchased from CLEA Japan Inc., Tokyo, Japan, and were bred and housed in SPF conditions at the Kyoto University Experimental Animal Center.
Biological response modifiers (BRM) Human recombinant tumor necrosis factor (rTNF : PAC-4D, Lot # L637111-1) was obtained from Asahi Chemical Industry Co., Ltd., Tokyo, Japan. One unit of PAC-4D is approximately equivalent to 50 units of rTNF (Genentech Corp). Human recombinant interferon-gamma (rlFN-~, Lot # GTA-001) was obtained from Tore Co., Ltd., Tokyo, Japan. Human recombinant interleukin-2 (rlL-2: TGP-3, Lot # 1-6-258) was obtained from Takeda Pharmaceutical Co. Ltd., Osaka, Japan. Streptococcal preparation OK-432 (Picibanil R, Lot # Z7G05) was supplied by Chugai Pharmaceutical Co., Ltd., Tokyo, Japan. One KE of OK-432 contains 0.1 mg dry streptococci. All the agents were dissolved in 0.02 ml phosphate buffer saline (PBS) for purposes of injection. In combination therapy the agents were mixed and dissolved in 0.02 ml PBS for injection.
Human tumor xenografts Six lines of human tumors were employed in this study, and the profiles of the lines are summarized in Table 1. EC-YO (esophageal cancer), GC-YN (gastric cancer), CC-KK (colonic cancer), PC-HN (pancreas cancer) and BDC-SN (bile duct cancer) were established from specimens, which had been
339 Table 1. Human tumor xenografts. Line
Origin
Histology
Doubling time (day)
EC-YO GC-YN CC-KK PC-HN BDC-SN Li-7
Esophageal ca. Gastric ca. Colonic ca. Pancreatic ca. Bile duct ca. Hepatoma
Squamous cell carcinoma Tubular adenocarcinoma Tubular adenocarcinoma Adenocarcinoma Undifferentiated carcinoma Hepatocellular carcinoma
6.6 8.3 7.5 I2.4 6.9 4.0
resected from patients at Kyoto University Hospital. Li-7 is a liver cancer line and was kindly supplied by Dr. T. Kubota (Department of Surgery, Keio University). All the lines were maintained by subcutaneous transplant into nude mice in SPF conditions at the Kyoto University Experimental Animal Center.
Protocol of drug administration and evaluation of antitumor activity Each tumor was cut into 2-3 mm cubes with scissors and the fragments were subcutaneously transplanted into the backs of nude mice. After transplantation, tumor size was determined by a series of measurements using calipers. When the tumor grew to reach 5001000 mm 3, the agents were administered directly into the tumor 3 times a week for 2 weeks (on day 0, 2, 4, 7, 9 and 1t). At the end of the administration period (on day 14), the tumor growth rate recorded for each group was compared with the corresponding values for the control group receiving only PBS. The estimated volume of the tumor (V) and tumor growth rate (TGR) were calculated according to the following formulae. V=LxW
~xl/2,
L is the length and W is the width of the tumor TGR = Vn/Vo.
Vn is the tumor volume recorded n days after initial administration, and Vo is the tumor volume recorded at the start of administration.
Statistics All results were expressed as the mean + standard deviation, and Student's t-test was applied to determine p values. All data were analysed by computer using Medical Plan II computer program (Sankyo Co. Ltd., Tokyo, Japan).JJ °
Results
rTNF was administered directly into the tumors at 103, 5 x 103, o r 10 4 U/head, 3 times a week for 2 weeks. The effects of rTNF on the growth of human tumors are summarized in Table 2. EC-YO was most sensitive to rTNF and intratumorat injection of rTNF at 103 U/ head caused tumor necrosis. The tumors completely regressed and did not regrow after cessation of rTNF treatment. PC-HN was also sensitive to rTNF and the growth of the tumors was significantly inhibited by intratumoral injection of rTNF at 103 and 5 x 103 U/head, compared with the growth of the tumors which were injected with PBS alone, rTNF at these doses also caused central necrosis of the tumor, although the marginal parts of the tumors gradually regrew after the cessation of rTNF treatment.
340 Table 2. Effect of rTNF on the growth of human tumor xenografts. Tumor
EC-YO GC-YN CC-KK
Tumor size a on day 14 after intratumoral injection of rTNF (U/head) b Control (PBS, 0.02 ml)
103
5 x 103
104
2.45 __+0.55 (n:4) 1,82 +__0.58
0.85 + 0.17 *~ (n:4) 1.80 _ 0.64
0.80 + 0.17 *¢ (n:4) 1.11 __+0.40 *d
(n : 8)
(n : 8)
(n : 9)
1.99 + 0.56
(n : 4) PC-HN
2.66 + 0.70
(n : 5) BDC-SN Li-7
8.14 + 4.16
1.21 + 0.49 **d
all died (n:4) 0.93 + 0,82 *d (n : 9) 0.40 ___0.25 *d (n:4) 0.80 __+0.24 **~
(n : 5)
(n : 5)
(n: 5)
11.15 + 5.97
n,t.
2.92 __.0.55
5.68 + 2.38 (n : 7)
rlA.
2.02 __+0.73
0.65 + 0.30 *d
(n : 4)
(n : 3)
1.21 + 0.73 *~
(n : 6)
(n : 4)
10.04 + 2.96 (n : 6)
7.95 _+ 4.77 (n : 6)
(n : 3)
n.t., not tested. * P < 0.05; **P < 0.01.
a Tumor size was expressed in tumor growth rate
tumor volume on day ~'~ - tumor volume on day 0 / "
b rTNF was administered 6 times on days 0, 2, 4, 7, 9 and 11, c Tumors did not regrow after treatment on day 28. d Tumors regrew after treatment on day 28.
P C - H N tumors regressed after injection of rTNF at 1 0 4 U/head without rebound growth after cessation o f rTNF administration. GCY N and CC-KK were also comparatively sensitive to rTNF, and their growth was not inhibited by intratumoral injection o f rTNF at 103 U/head, however their growth was significantly inhibited by rTNF at 5 x 10 3 and 10 4 U/head. rTNF caused only slight tumor necrosis, and the tumors regrew after rTNF treatment. Li-7 and B D C - S N were resistant to rTNF. The intratumoral administration of rTNF resulted in the regression of some tumors, however, rTNF caused serious side effects, resulting in a high rate o f mouse mortality: 13% at 103 U/head, 14% at 5 × 103 U/head and 24% at 104 U/head (Table 3). The effects o f combining rTNF with rIFNy, rIL-2 or OK-432 were assessed in mice transplanted with the relatively resistant line G C - Y N (Table 4). Combination o f rTNF at 103 U/head with other BRMs did not augment the cytotoxic effect of rTNF, however,
combinations o f rTNF at 5 x 103 U/head and other BRMs were more effective than rTNF alone, and tumors completely regressed after treatment with a combination o f rTNF and rlFN-~ (at 103 U/head), or rTNF and OK432 (at 0.1 or 1.0 KE/head). These combinations caused central tumor necrosis and most
Table 3. Mortality after intratumoral injection of rTNF (U/head). Tumor
Mortality rate of tested mice (% mortality) on day 28
103
5 × 103
104
EC-YO GC-YN CC-KK PC-HN BDC-SN Li-7
1/4 1/8 0/4 0/5 I/4 1/6
1/4 2/9 1/3 2/5 n.t. 1/7
4/4 0/9 I/4 I/5 0/3 n.t.
Overall
4/31 (12.9%)
4/28 (t4.3%)
6/25 (24.0%)
n.t., not tested.
341 Table 4. Effect of rTNF in combination with other BRMs on the growth of GC-YN.
Protocol
Tumor size a on day 14
Regrowth on day 28
Mortality (%) on day 28
Control
(PBS 0.02 ml/head)
3.76 + 0.86 (n:7)
rTNF ( 103 U/head) rTNF (103 U/head) rTNF (103 U/head) rTNF ( 103 U/head) rTNF (103 U/head)
alone
1.27 + 0.15 (n : 5) 1.77 + 0,52 (n : 5) 1.97 ± 0.83 (n : 5) 1.65 + 0.64 (n : 5) 1.68 _+0.57 (n: 5)
+ +
1/5 (20%)
++
I/5 (20%)
++
0/5
++
2/5 (40%)
++
0/5
rTNF (5 x 103 U/head) rTNF (5 x l03 U/head) rTNF (5 x 103 U/head) rTNF (5 x I03 U/head) rTNF (5 x 103 U/head) rTNF (5 × 103 U/head)
alone
1.06 + 0.59 (n : 7) 0.60 + 0.50 (n: 7) 0.47 + 0.43 (n : 7) 0.61 _+0.53 (n : 7) 0.92 + 1.05 (n : 7) 0.29 + 0.21 (n : 7)
+ +
2/7 (29%)
-
0/5
-
3/7 (43%)
+
2/7 (29%)
+
2/5 (40%)
-
4/7 (57%)
+rlFN- 7 ( t02 U/head) + rlFN-y ( 103 U/head) rlL-2 ( 10 U/head) OK-432 (0.1 KE/head)
rIFN 7 (102 U/head) rlFN-y (103 U/head) rlL-2
(10 U/head) OK-432 (0.i KE/head) OK-432 (1.0 KE/head)
tumor volume on day 14~
"Tumor size was expressed in tumor growth rate \ = t--~-omor~ e
of the remaining tumors regressed, although tumors which were injected with rTNF plus rlL-2 regrew. Unfortunately these combinations resulted in more adverse effects as well as an augmented tumoricidal effect, and the combination of rTNF + OK-432 caused the most serious side effects (Table 4).
Discussion The present study shows that some human tumors are sensitive to the cytotoxic effect of rTNF. In clinical studies, intravenous administration of rTNF has resulted in about a 5% rate of response. Renal cancer, colorectal cancer, liver cancer, breast cancer, gastric cancer, non-Hodgkin's lymphoma,
~n daay 0-,]'
leukemia, multiple myeloma, bladder cancer, and osteosarcoma have responded to the intravenous rTNF therapy [10-12, 15-17]. However the adverse effects are also serious, and the main side effects of intravenous rTNF are fever, chills, anorexia, nausea and hypotension [10-12, 15-17]. By contrast, intratumoral administration of rTNF resulted in better responses than intravenous rTNF, and a 30-50% response rate has been reported. Various primary and metastatic skin tumors, breast cancer, oropharynx cancer, osteosarcoma and lung cancer have responded to intratumoral rTNF, and it has also been reported that the side effects of intratumoral administration are transient [15-17]. These reports suggest that rTNF may be useful for cancer therapy. However, the response rates
342 for rTNF therapy do not seem to be so prominent, and the size of the tumor decreased < 2 5 % in many patients. One reason for this inefficacy of rTNF therapy may be that human tumors are more resistant to TNF than murine tumors, however, the antitumor spectrum of T N F against human tumors, especially against digestive organ cancers, has never been studied in detail. This study suggests that esophageal and pancreatic cancers may be more sensitive to rTNF than gastric, colonic, bile duct and liver cancer although we can not say conclusively on the basis of results with some examples of tumors. Pancreatic cancer is resistant to current cancer therapies, and has been one of the most difficult tumors to treat. Accordingly, rTNF may be considered one new treatment for pancreatic cancer. Another reason for the inefficacy of rTNF therapy may be that the adverse effects of rTNF limit rTNF doses to levels which may be less than the optimum. In order to apply rTNF in clinical cancer therapy it may be necessary to determine which human cancers are sensitive to rTNF, and to develop methods to reduce rTNF's side effects and to improve its cytotoxic effect. It has been reported that rlFN-v augments the cytotoxicity of rTNF, mediated by an increase in number of TNF-receptors [1, 21, 22]. Combining rTNF with rlFN-~ or rlL-2 also produces a synergistic increase in the cytotoxicity of rTNF in murine models [19, 28]. A protein synthesis inhibitor, cycloheximide, augments the cytotoxicity of rTNF against TNF-resistant cells in vitro [23, 24]. Hyperthermia also augments the cytotoxicity of rTNF both in vitro and in vivo [25-27]. Conventional use of the chemotherapeutic agent Mitomycin C also augments the in vivo inhibitory effect on human tumors transplanted in nude mice [29]. Likewise, it has been reported that combining of rTNF with other BRMs or with cytotoxic agents augments the cytotoxic effect of rTNF, however further analysis of these reports reveals that the augmentation may not be so prominent,
because regrowth of the tumors after the cessation of combination therapy was discernible. In the present study the effect of rTNF, alone and in combination with BRMs on human digestive organ cancers was assessed. Combining rTNF with OK-432, as well as with rlFN- 7 and rlL-2, augmented the cytotoxic effect of rTNF on the relatively resistant line, GC-YN. The mechanism of the augmentation by OK-432 is unclear. However it has been reported that OK-432 induces various lymphokines and cytokines, including IFN, IL-2, TNF, NKCF, etc. [3033]. These substances may be released from tumor infiltrating lymphocytes after stimulation by OK-432, and may be responsible for this augmentation. On the other hand it has been reported that OK-432 also has a direct cytotoxic and cytostatic effect on murine and human tumors [34-36]. Accordingly, the direct effect of OK-432 on tumor cells may also be responsible for TNF cytotoxicity augmentation. Three BRMs augmented the cytotoxic effect of rTNF, however, this augmentation was seen only when rTNF was administered at effective but lethal doses, which were equivalent to LDlo-LD20. Combinations of rTNF at lower doses and BRMs were not beneficial. Furthermore, the side effects of these combinations were also more serious than for rTNF alone, as shown in Table 4. These results suggest that combinations of rTNF and rlFN-~ or OK-432 can induce complete regression of tumors, which are relatively resistant to rTNF alone. Accordingly, rTNF may still be a useful cytokine for induction of tumor regression, if a method can be developed to reduce its side effects.
Acknowledgement We gratefully acknowledge Mrs. Hiroko Higashikawa for her excellent technical assistance.
343
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Address for correspondence: Yoshinori Nio, M.D., Division of Surgical Oncology, First Department of Surgery, Kyoto University Faculty of Medicine, 54 Shogoin-Kawara-cho, Sakyoku, Kyoto 606, Japan.
In vivo effects of human recombinant tumor necrosis factor alone and in combination with other biological response modifiers on human digestive organ cancer xenografts transplanted in nude mice Yoshinori Nio', Takahiro Shiraishi l, Michihiko Tsubono 1, Hideki Morimoto ~, Chen-Chiu Tseng ~, Kazuya Kawabata ~, Yoshikazu Masai 1, Thein Tun ~, Manabu Fukumoto 2 & Takayoshi Tobe ~
JFirst Department of Surgery; 2First Department of Pathology, Kyoto University Faculty of Medicine, Kyoto 606, Japan Received 8 November 1990; accepted 12 December 1990
Key words: human tumor xenograft, interferon (IFN), interleukin-2 (IL-2), nude mouse, Picibanil (OK-432), tumor necrosis factor (TNF) Abstract
The present study was designed to evaluate the effect of rTNF alone or in combination with other BRMs on human digestive organ cancers. Six kinds of human digestive organ cancer xenografts (esophageal, stomach, colonic, pancreatic, bile duct, and liver cancers: EC-YO, GC-YN, CC-KK, PC-HN, BDC-SN and Li-7, respectively) were transplanted in nude mice, and rTNF was administered at 103, 5 x 103, or 104U/head directly into the tumor 3 times a week for 2 weeks. EC-YO was the most sensitive to rTNF, and intratumoral administration of rTNF at 103 U/head caused tumor regression. PC-HN, CC-KK and GC-YN were relatively sensitive to rTNF, and their growth was significantly inhibited by rTNF at 5 x 103 U/head, however, the tumors regrew after treatment. Li-7 and BDC-SN were resistant to rTNF. The effects of rTNF in combination with recombinant interferon-7 (rIFN-7), recombinant interleukin-2 (rIL-2), or streptococcal preparation OK-432 were assessed in mice transplanted with GC-YN. All combinations of rTNF at 5 x 103 U/head and other BRMs were more effective than rTNF alone, and GC-YN tumors were completely regressed after treatment with a combination of rTNF and rIFN-~ or rTNF and OK-432. However in all cases, the combination of rTNF at 103 U/head and any other BRM did not improve the effect. Furthermore, the adverse effects of the combinations were more serious than those of rTNF alone. TNF may still be a useful cytokine, because it can induce the regression of tumors. However, for its clinical application, a method should be developed to reduce its side effects.
Abbreviations: BRM: biological response modifier; IFN: interferon; IL-2: interleukin-2; PBS: phosphate buffer saline; rIFN-7: recombinant interferon-gamma; rIL-2: recombinant interleukin-2; rTNF: recombinant tumor necrosis factor; TNF: tumor necrosis factor.
338 Introduction
Tumor necrosis factor (TNF) has a variety of biologic activities, and its cytotoxic activity against tumor cells especially has attracted the interest of oncologists and immunologists. TNF lyses a variety of tumor cells, however, non-malignant cells are insensitive to the cytotoxicity of TNF [1-3]. The mechanism of TNF-mediated cytotoxicity has not been fully clarified, although it has been suggested that TNF molecules may affect proteolytic enzymes in lysosomes and fragment of DNA after they are internalized into cells [4-6]. TNF was originally thought to be a revolutionary agent for cancer therapy, and recombinant TNF technology has made it possible to produce sufficient amount of recombinant TNF (rTNF) for clinical application [7-9]. However in its clinical application, intravenous administration of rTNF causes serious side effects, such as shock, hypotension, chills and fever, although the response rate to rTNF has been comparatively low [10-12]. Furthermore, some authors have reported that TNF is the same molecule as cachectin, which causes cachexia, and that TNF may be a major cause of endotoxin shock [ 13, 14]. In order to reduce the adverse effects of rTNF in clinical application, intratumoral administration has recently been chosen, experimentally as well as clinically [15-17]. In addition, in order to augment the cytotoxic effect of TNF, combination with various therapies, including interferon (IFN), interleukin-2 (IL-2), and hyperthermia, have been studied [1,2,19,20]. However there have been only a few reports on the susceptibility of various human tumors, especially of digestive organ cancers, to rTNF and these combination therapies. In the present study various human digestive organ cancer xenografts were transplanted in nude mice and the effects of rTNF alone and in combination with other biological response modifiers (BRM), recombinant interferon-7 (rlFN-7),
recombinant IL-2 (rIL-2), and streptococcal preparation OK-432 (Picibanil) were assessed.
Materials and methods
Nude mice Eight-week-old male BALB/c nude mice were employed in this study. The nude mice were purchased from CLEA Japan Inc., Tokyo, Japan, and were bred and housed in SPF conditions at the Kyoto University Experimental Animal Center.
Biological response modifiers (BRM) Human recombinant tumor necrosis factor (rTNF : PAC-4D, Lot # L637111-1) was obtained from Asahi Chemical Industry Co., Ltd., Tokyo, Japan. One unit of PAC-4D is approximately equivalent to 50 units of rTNF (Genentech Corp). Human recombinant interferon-gamma (rlFN-~, Lot # GTA-001) was obtained from Tore Co., Ltd., Tokyo, Japan. Human recombinant interleukin-2 (rlL-2: TGP-3, Lot # 1-6-258) was obtained from Takeda Pharmaceutical Co. Ltd., Osaka, Japan. Streptococcal preparation OK-432 (Picibanil R, Lot # Z7G05) was supplied by Chugai Pharmaceutical Co., Ltd., Tokyo, Japan. One KE of OK-432 contains 0.1 mg dry streptococci. All the agents were dissolved in 0.02 ml phosphate buffer saline (PBS) for purposes of injection. In combination therapy the agents were mixed and dissolved in 0.02 ml PBS for injection.
Human tumor xenografts Six lines of human tumors were employed in this study, and the profiles of the lines are summarized in Table 1. EC-YO (esophageal cancer), GC-YN (gastric cancer), CC-KK (colonic cancer), PC-HN (pancreas cancer) and BDC-SN (bile duct cancer) were established from specimens, which had been
339 Table 1. Human tumor xenografts. Line
Origin
Histology
Doubling time (day)
EC-YO GC-YN CC-KK PC-HN BDC-SN Li-7
Esophageal ca. Gastric ca. Colonic ca. Pancreatic ca. Bile duct ca. Hepatoma
Squamous cell carcinoma Tubular adenocarcinoma Tubular adenocarcinoma Adenocarcinoma Undifferentiated carcinoma Hepatocellular carcinoma
6.6 8.3 7.5 I2.4 6.9 4.0
resected from patients at Kyoto University Hospital. Li-7 is a liver cancer line and was kindly supplied by Dr. T. Kubota (Department of Surgery, Keio University). All the lines were maintained by subcutaneous transplant into nude mice in SPF conditions at the Kyoto University Experimental Animal Center.
Protocol of drug administration and evaluation of antitumor activity Each tumor was cut into 2-3 mm cubes with scissors and the fragments were subcutaneously transplanted into the backs of nude mice. After transplantation, tumor size was determined by a series of measurements using calipers. When the tumor grew to reach 5001000 mm 3, the agents were administered directly into the tumor 3 times a week for 2 weeks (on day 0, 2, 4, 7, 9 and 1t). At the end of the administration period (on day 14), the tumor growth rate recorded for each group was compared with the corresponding values for the control group receiving only PBS. The estimated volume of the tumor (V) and tumor growth rate (TGR) were calculated according to the following formulae. V=LxW
~xl/2,
L is the length and W is the width of the tumor TGR = Vn/Vo.
Vn is the tumor volume recorded n days after initial administration, and Vo is the tumor volume recorded at the start of administration.
Statistics All results were expressed as the mean + standard deviation, and Student's t-test was applied to determine p values. All data were analysed by computer using Medical Plan II computer program (Sankyo Co. Ltd., Tokyo, Japan).JJ °
Results
rTNF was administered directly into the tumors at 103, 5 x 103, o r 10 4 U/head, 3 times a week for 2 weeks. The effects of rTNF on the growth of human tumors are summarized in Table 2. EC-YO was most sensitive to rTNF and intratumorat injection of rTNF at 103 U/ head caused tumor necrosis. The tumors completely regressed and did not regrow after cessation of rTNF treatment. PC-HN was also sensitive to rTNF and the growth of the tumors was significantly inhibited by intratumoral injection of rTNF at 103 and 5 x 103 U/head, compared with the growth of the tumors which were injected with PBS alone, rTNF at these doses also caused central necrosis of the tumor, although the marginal parts of the tumors gradually regrew after the cessation of rTNF treatment.
340 Table 2. Effect of rTNF on the growth of human tumor xenografts. Tumor
EC-YO GC-YN CC-KK
Tumor size a on day 14 after intratumoral injection of rTNF (U/head) b Control (PBS, 0.02 ml)
103
5 x 103
104
2.45 __+0.55 (n:4) 1,82 +__0.58
0.85 + 0.17 *~ (n:4) 1.80 _ 0.64
0.80 + 0.17 *¢ (n:4) 1.11 __+0.40 *d
(n : 8)
(n : 8)
(n : 9)
1.99 + 0.56
(n : 4) PC-HN
2.66 + 0.70
(n : 5) BDC-SN Li-7
8.14 + 4.16
1.21 + 0.49 **d
all died (n:4) 0.93 + 0,82 *d (n : 9) 0.40 ___0.25 *d (n:4) 0.80 __+0.24 **~
(n : 5)
(n : 5)
(n: 5)
11.15 + 5.97
n,t.
2.92 __.0.55
5.68 + 2.38 (n : 7)
rlA.
2.02 __+0.73
0.65 + 0.30 *d
(n : 4)
(n : 3)
1.21 + 0.73 *~
(n : 6)
(n : 4)
10.04 + 2.96 (n : 6)
7.95 _+ 4.77 (n : 6)
(n : 3)
n.t., not tested. * P < 0.05; **P < 0.01.
a Tumor size was expressed in tumor growth rate
tumor volume on day ~'~ - tumor volume on day 0 / "
b rTNF was administered 6 times on days 0, 2, 4, 7, 9 and 11, c Tumors did not regrow after treatment on day 28. d Tumors regrew after treatment on day 28.
P C - H N tumors regressed after injection of rTNF at 1 0 4 U/head without rebound growth after cessation o f rTNF administration. GCY N and CC-KK were also comparatively sensitive to rTNF, and their growth was not inhibited by intratumoral injection o f rTNF at 103 U/head, however their growth was significantly inhibited by rTNF at 5 x 10 3 and 10 4 U/head. rTNF caused only slight tumor necrosis, and the tumors regrew after rTNF treatment. Li-7 and B D C - S N were resistant to rTNF. The intratumoral administration of rTNF resulted in the regression of some tumors, however, rTNF caused serious side effects, resulting in a high rate o f mouse mortality: 13% at 103 U/head, 14% at 5 × 103 U/head and 24% at 104 U/head (Table 3). The effects o f combining rTNF with rIFNy, rIL-2 or OK-432 were assessed in mice transplanted with the relatively resistant line G C - Y N (Table 4). Combination o f rTNF at 103 U/head with other BRMs did not augment the cytotoxic effect of rTNF, however,
combinations o f rTNF at 5 x 103 U/head and other BRMs were more effective than rTNF alone, and tumors completely regressed after treatment with a combination o f rTNF and rlFN-~ (at 103 U/head), or rTNF and OK432 (at 0.1 or 1.0 KE/head). These combinations caused central tumor necrosis and most
Table 3. Mortality after intratumoral injection of rTNF (U/head). Tumor
Mortality rate of tested mice (% mortality) on day 28
103
5 × 103
104
EC-YO GC-YN CC-KK PC-HN BDC-SN Li-7
1/4 1/8 0/4 0/5 I/4 1/6
1/4 2/9 1/3 2/5 n.t. 1/7
4/4 0/9 I/4 I/5 0/3 n.t.
Overall
4/31 (12.9%)
4/28 (t4.3%)
6/25 (24.0%)
n.t., not tested.
341 Table 4. Effect of rTNF in combination with other BRMs on the growth of GC-YN.
Protocol
Tumor size a on day 14
Regrowth on day 28
Mortality (%) on day 28
Control
(PBS 0.02 ml/head)
3.76 + 0.86 (n:7)
rTNF ( 103 U/head) rTNF (103 U/head) rTNF (103 U/head) rTNF ( 103 U/head) rTNF (103 U/head)
alone
1.27 + 0.15 (n : 5) 1.77 + 0,52 (n : 5) 1.97 ± 0.83 (n : 5) 1.65 + 0.64 (n : 5) 1.68 _+0.57 (n: 5)
+ +
1/5 (20%)
++
I/5 (20%)
++
0/5
++
2/5 (40%)
++
0/5
rTNF (5 x 103 U/head) rTNF (5 x l03 U/head) rTNF (5 x 103 U/head) rTNF (5 x I03 U/head) rTNF (5 x 103 U/head) rTNF (5 × 103 U/head)
alone
1.06 + 0.59 (n : 7) 0.60 + 0.50 (n: 7) 0.47 + 0.43 (n : 7) 0.61 _+0.53 (n : 7) 0.92 + 1.05 (n : 7) 0.29 + 0.21 (n : 7)
+ +
2/7 (29%)
-
0/5
-
3/7 (43%)
+
2/7 (29%)
+
2/5 (40%)
-
4/7 (57%)
+rlFN- 7 ( t02 U/head) + rlFN-y ( 103 U/head) rlL-2 ( 10 U/head) OK-432 (0.1 KE/head)
rIFN 7 (102 U/head) rlFN-y (103 U/head) rlL-2
(10 U/head) OK-432 (0.i KE/head) OK-432 (1.0 KE/head)
tumor volume on day 14~
"Tumor size was expressed in tumor growth rate \ = t--~-omor~ e
of the remaining tumors regressed, although tumors which were injected with rTNF plus rlL-2 regrew. Unfortunately these combinations resulted in more adverse effects as well as an augmented tumoricidal effect, and the combination of rTNF + OK-432 caused the most serious side effects (Table 4).
Discussion The present study shows that some human tumors are sensitive to the cytotoxic effect of rTNF. In clinical studies, intravenous administration of rTNF has resulted in about a 5% rate of response. Renal cancer, colorectal cancer, liver cancer, breast cancer, gastric cancer, non-Hodgkin's lymphoma,
~n daay 0-,]'
leukemia, multiple myeloma, bladder cancer, and osteosarcoma have responded to the intravenous rTNF therapy [10-12, 15-17]. However the adverse effects are also serious, and the main side effects of intravenous rTNF are fever, chills, anorexia, nausea and hypotension [10-12, 15-17]. By contrast, intratumoral administration of rTNF resulted in better responses than intravenous rTNF, and a 30-50% response rate has been reported. Various primary and metastatic skin tumors, breast cancer, oropharynx cancer, osteosarcoma and lung cancer have responded to intratumoral rTNF, and it has also been reported that the side effects of intratumoral administration are transient [15-17]. These reports suggest that rTNF may be useful for cancer therapy. However, the response rates
342 for rTNF therapy do not seem to be so prominent, and the size of the tumor decreased < 2 5 % in many patients. One reason for this inefficacy of rTNF therapy may be that human tumors are more resistant to TNF than murine tumors, however, the antitumor spectrum of T N F against human tumors, especially against digestive organ cancers, has never been studied in detail. This study suggests that esophageal and pancreatic cancers may be more sensitive to rTNF than gastric, colonic, bile duct and liver cancer although we can not say conclusively on the basis of results with some examples of tumors. Pancreatic cancer is resistant to current cancer therapies, and has been one of the most difficult tumors to treat. Accordingly, rTNF may be considered one new treatment for pancreatic cancer. Another reason for the inefficacy of rTNF therapy may be that the adverse effects of rTNF limit rTNF doses to levels which may be less than the optimum. In order to apply rTNF in clinical cancer therapy it may be necessary to determine which human cancers are sensitive to rTNF, and to develop methods to reduce rTNF's side effects and to improve its cytotoxic effect. It has been reported that rlFN-v augments the cytotoxicity of rTNF, mediated by an increase in number of TNF-receptors [1, 21, 22]. Combining rTNF with rlFN-~ or rlL-2 also produces a synergistic increase in the cytotoxicity of rTNF in murine models [19, 28]. A protein synthesis inhibitor, cycloheximide, augments the cytotoxicity of rTNF against TNF-resistant cells in vitro [23, 24]. Hyperthermia also augments the cytotoxicity of rTNF both in vitro and in vivo [25-27]. Conventional use of the chemotherapeutic agent Mitomycin C also augments the in vivo inhibitory effect on human tumors transplanted in nude mice [29]. Likewise, it has been reported that combining of rTNF with other BRMs or with cytotoxic agents augments the cytotoxic effect of rTNF, however further analysis of these reports reveals that the augmentation may not be so prominent,
because regrowth of the tumors after the cessation of combination therapy was discernible. In the present study the effect of rTNF, alone and in combination with BRMs on human digestive organ cancers was assessed. Combining rTNF with OK-432, as well as with rlFN- 7 and rlL-2, augmented the cytotoxic effect of rTNF on the relatively resistant line, GC-YN. The mechanism of the augmentation by OK-432 is unclear. However it has been reported that OK-432 induces various lymphokines and cytokines, including IFN, IL-2, TNF, NKCF, etc. [3033]. These substances may be released from tumor infiltrating lymphocytes after stimulation by OK-432, and may be responsible for this augmentation. On the other hand it has been reported that OK-432 also has a direct cytotoxic and cytostatic effect on murine and human tumors [34-36]. Accordingly, the direct effect of OK-432 on tumor cells may also be responsible for TNF cytotoxicity augmentation. Three BRMs augmented the cytotoxic effect of rTNF, however, this augmentation was seen only when rTNF was administered at effective but lethal doses, which were equivalent to LDlo-LD20. Combinations of rTNF at lower doses and BRMs were not beneficial. Furthermore, the side effects of these combinations were also more serious than for rTNF alone, as shown in Table 4. These results suggest that combinations of rTNF and rlFN-~ or OK-432 can induce complete regression of tumors, which are relatively resistant to rTNF alone. Accordingly, rTNF may still be a useful cytokine for induction of tumor regression, if a method can be developed to reduce its side effects.
Acknowledgement We gratefully acknowledge Mrs. Hiroko Higashikawa for her excellent technical assistance.
343
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Address for correspondence: Yoshinori Nio, M.D., Division of Surgical Oncology, First Department of Surgery, Kyoto University Faculty of Medicine, 54 Shogoin-Kawara-cho, Sakyoku, Kyoto 606, Japan.
