Different Clonal Drug Sensitivity of Murine Neuroblastoma Cells In Vivo By Shin-ichi Okuzono, Akira Nakagawara, Kohji Sue, Takahiko Fukushige, and Keiichi Ikeda Fukuoka, Japan 9 Four cloned murine neuroblastomas w e r e implanted intramuscularly into the left thigh of adult A / J a x mice. Cyclophosphamide, cisplatin, adriamycin, imidazole carboxamide, and vincristine w e r e administered intraperitoneally, in a dose of one third to one fourth of a median lethal dose, every w e e k after the implantation until all the mice died. The effects of continuous long-term chemotherapy, particularly on clonal differences, w e r e then assessed. Cyclophosphamide was most effective for four murine neuroblastomas, and cisplatin was the next most effective drug. Cisplatin was not effective in the NS-20 cell line, a cholinergic cloned neuroblastoma. The C 1300 cell line (wild type) was tolerant to adriamycin, imidazole carboxamide, and vincristine. The N-18 cell line (an inactive clone) exhibited tolerance of adriamycin and imidazole carboxamide. In the N I E - 1 1 5 cell line, an adrenergic clone, tumor, growth was inhibited by all the drugs given. W e conclude from this study that drug sensitivity differs with the clone, and that there are clones resistant to each drug. 9 1988 by Grune & Stratton, Inc INDEX WORDS: Nueroblastoma; cyclophosphamide; cisplatin; adriamycin; imidazole carboxamide; vincristine.
O I M P R O V E the prognosis for children with neuroblastoma, a great deal of experimental and clinical efforts have been made. However, a review of the impact of chemotherapy on the treatment of advanced neuroblastoma showed a lack of significant improvement in the survival of children with stage III and IV disease. 1-8 Moreover, neuroblastomas do not respond in the same manner as acute lymphocytic leukemia, Wilms' tumor, and rhabdomyosarcoma, to the new forms of drug therapy for children with these neoplastic diseases. To the present, one third to two thirds of patients with metastatic neuroblastoma temporarily achieve an apparent complete clinical response by multimodal therapy, but tumor growth resumes in many cases, and the tumor exhibits resistance to almost all chemotherapeutic agents. One possible cause is that neuroblastoma is a polyclonal tumor and the effects of antitumor agents differ according to the component clones of the tumor. The relevance of the C 1300 murine neuroblastoma
From the Department of Pediatric Surgery, Faculty of Medicine, Kyushu University, Fukuoka, Japan. Address reprint requests to Shin-ichi Okuzono, MD, Department of Pediatric Surgery, Faculty of Medicine, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812, Japan. 9 1988 by Grune & Stratton, Inc. 0022-3468/88/2310-0018503.00/0 962
system as a model for the study of disease in children has been under investigation in many laboratories. Finklestein et al L2 reported that nitrosourea (BCNU), cyclophosphamide, isophosphamide, and cytosine arabinoside, etc, were active against this C 1300 murine tumor. However, an in vivo study on clonal differences in the effects of antitumor agents has not been reported. We undertook a series of experimental works to evaluate the effects of chemotherapeutic agents on cloned murine neuroblastomas in vivo. Valuable information that can be used to develop new combination chemotherapy protocols and overcome the drug resistance was obtained. MATERIALS AND METHODS
T u m o r Cells The tumor cell lines used were transplantable C 1300 murine neuroblastoma9 provided by Dr E. Arima, 3 N-18 (an inactive clone), NS-20 (a cholinergic done), and N1E-115 (an adrenergic clone) established as a clone from the C 1300 neuroblastoma cell line. 1~ These clonal cell lines were provided by Dr T. Amano ~~(Mitsubishi Kasei Life Science Institute, Machida, Tokyo). The cells were cultured in a plastic tissue culture flask (Coming Glass Works, Coming, NY) with 25-cm 2 area; C 1300, N-18 and NS-20 cell lines were maintained in Eagle's minimal essential medium (Nissui Pharmaceutical Co Ltd, Tokyo) containing 10% newborn calf serum (NCS) (Grand Island Biological Co, Grand Island, NY) and 60 ttg/mL kanamycin, and the N1E-115 cell line was maintained in Dulbecco's modified Eagle's medium (Nissui Pharmaceutical Co Ltd, Tokyo) containing 10% NCS, 100 U / m L penicillin G potassium and 100 t~g/mL streptomycin sulfate. The cultures were incubated at 37~ in a humidified atmosphere of 5% C02. The exponentially growing cells were dispersed with 0.05% trypsin in phosphate buffered saline (PBS) and washed with the growth medium. The number of viable cells was calculated with the hemocytometer using 0.05% trypan blue dye exclusion. The final cell concentration was adjusted to the cell suspension of 5 • l0 s cells/0.3 mL in the culture medium.
Animals The A / J a x mice (Shizuoka Laboratory Animal Center, Shizuoka, Japan), 6- to 8-week-old females about 20g in body weight, were fed a specific pathogen free (SPF) diet during the entire time of the experiment. The mouse was given an inoculation of 0.3 mL of the cell suspension (5 x 105 cells) into the left thigh muscle. Control and treated groups were prepared; each group included seven to 17 mice.
Chemotherapeutic Agents Agents used were cyclophosphamide (CPA) and vincristine (VCR) (Shionogi Co Ltd, Osaka, Japan), adriamycin (ADM) and imidazole carboxamide (DTIC) (Kyowa Hakko Kogyo Co Ltd, Tokyo), and cisplatin (CDDP) (Bristol-Myers Co, Tokyo). Journal of Pediatric Surgery, Vol 23, No 10 (October), 1988: pp 962-966
DRUG SENSITIVITY OF MURINE NEUROBLASTOMA
Treatment for Tumor-Bearing Mice
Table 1. Doubling Time of Groups in Cell Lines
The therapeutic treatment, initiated on day 7 after inoculation of the tumor cells was given every week until all the mice died. All drugs were administered intraperitoneally and dosages of each drug were as follows: CPA, 100 mg/kg; CDDP, 4 m g / k g ; A D M , 4 mg/kg; DTIC, 200 mg/kg; VCR, 1 m g / k g . These dosages are equivalent to one third to one fourth of a median lethal dose (LDs0) in intraperitioneally injected mice. The control group was given normal saline, in an equal volume in the antitumor agents.
Evaluation of the Therapy Estimation of tumor volume (V) in cm 3 was calculated by a formula 12'j3 V = lr (a x b x c)/6, where a, b, and c were the values of three crossed diameters in centimeters of the mass. Survival curves were expressed by the Kaplan-Meier method. Statistical evaluation of differences in tumor volume and survival curve was carried out by the Student's t test and the generalized Wilcoxon's test, respectively.
Histologic Examination Some mice were killed, and the tumors and main organs were sampled and treated with formalin for hematoxylin-eosin staining. RESULTS
The tumor grew rapidly in the C 1300 murine neuroblastoma. As shown in Fig 1, the tumors in the control group were palpable about 3 weeks after the inoculation, and the tumors grew with 3.8 days of the doubling time. The mean survival time was 53.5 _+ 10.4 days (mean, _+ SD, n = 16). In this cell line, CPA and CDDP were the most effective drugs. In both groups treated with CPA and CDDP, tumor growth was markedly suppressed and survival time was prolonged, as shown in Fig 1. The doubling time of CPA and CDDP was 9.4 days and 8.6 days, respectively, and the ~20 I vo 2 10 E 50
3.8 (16) 
2.3 (16) 
1.7 (15) 
1.4 (17) 
9,4 (16) t
4.2 (16) *
2.8 (15) *
3.2 (9) t
8.6 (16) t
4.1 (13) *
1.9 (15) 
3.6 (9) t
2.7 (g) 
2.0 (10) 
2.5 (g) *
4.0 (8) t
2.4 (9) 
2.3 (10) 
4.6 (9) f
4.0 (9) t
2.4 (10) 
4.0 (10) *
3.2 (7) *
6.6 (10)  t
NOTE. Values in [ ] express percent of control group. P values express statistical difference in doubling time between the treatment group and the control group. * P < .01. t P < .001.
C 1300 Murine Neuroblastoma Cell Line (Wild Type)
Cell Lines days (n)
Days after Inoculation
mean survival time was 97.5 _+ 21.2 days and 73.5 _+ 15.5 days, respectively (Tables 1 and 2). CPA was more effective than CDDP, and in the CPA group, tumor growth was accelerated in the late stage while CDDP inhibited growth until the late stage. However, other antitumor agents (ADM, DTIC, and VCR) were not effective against the C 1300 murine neuroblastoma cell line, at least in the doses given.
N-18 Murine Neuroblastoma Cloned Cell Line (Inactive Clone) Tumor growth in this cell line was more rapid than in the C 1300 cell line, and tumors became palpable about 2 weeks after the inoculatin. The doubling time was 2.3 days. Among all the antitumor agents used, CPA, CDDP, and VCR inhibited tumor growth relatively well, but survival time in the VCR group was prolonged for a short time as compared with the control group (Fig 2; Tables 1 and 2). CPA and CDDP had marked inhibitory effects on tumor growth; A D M and DTIC were not as effective.
NS-20 Murine Neuroblastoma Cloned Cell Line (Cholinergic Clone)
Days after Inoculation Fig 1. (A) Tumor growth curves and (B) survival curves in C 1300 cell line. Symbols: --" --', control group; O - - - O , CPA group; H , CDDP group; I - i - - - r l , A D M group; H , DTIC group; A - - - A , VCR group.
All antitumor agents, except for CDDP, were effective against this cell line. CPA and DTIC, in particular, exhibited a remarkable antitumor effect with 2.8 days and 4.6 days compared with 1.7 days of doubling time in the control group. The mean survival time of the CPA and DTIC groups was 62.0 _+ 9.8 days and 59.8 +_ 14.3 days, respectively, and it was prolonged
OKUZONO ET AL Table 2. Mean Survival Time Cell Lines days (n) Group
53.5 4- 10.4 (16) [ 100]
35.1 4- 5.7 (16) [ 100]
32.1 • 7.5 (15) [ 100]
34.8 + 5.3 (17) [ 100]
97.5 4- 21.2 (16) [ 182]~:
54.4 4- 6.3 (16) [ 155]:1:
62.0 4- 9.8 (15) [ 1931:[:
50.3 4- 10.8 (9) t
73.5 4- 15.5 (16) [ 13711"
46.9 4- 10.1 (13) [ 134]'1"
24.8 4- 9.9 (15) 
25.9 + 2.9 (9) 
43.8 4- 3.5 (9) 
36.5 _+ 9.3 (10) 
42.6 4- 5.9 (9) *
36.9 -+ 6.6 (8) 
54.9 4- 10.4 (9) [ 103]
34.2 + 8.5 (10) 
59.8 _+ 14.3 (9) [ 186] 1"
28.6 4- 2.2 (9) 
51.4 4- 8.0(10) 
36.8 4- 5.9(10) 
47.4 4- 11.9(7) *
42.1 4- 5.5(10) *
NOTE. Data are expressed by mean days 4- SD. Values in [ ] express percent of control group. Pvalues express statistical difference in mean survival between the treatment group and the control group. *P < .01. 1'P < .001. ::I:P < .0001.
significantly in comparison with the mean survival time of 32.1 _+ 7.5 days in the control group. Tumor growth rate and survival time in the CDDP group were all but equal to findings in the control group (Fig 3; Tables 1 and 2).
NI E-115 Murine Neuroblastoma Cloned Cell Line (Adrenergic Clone) Growth rate in this cell line was markedly inhibited by all antitumor agents given, but CDDP, DTIC, and ~'~E20 o
A D M did not extend the lifespan. CPA was also most effective, with the next most effective drug being vincristine. Survival times were 50.3 _+ 10.8 days and 42.1 _+ 5.5 days, respectively, and were prolonged compared with 34.8 _+ 5.3 days of survival time in the control group (Fig 4; Tables 1 and 2).
Changes in Body Weight In the control group, the mean body weight, including tumor weight, increased with progression of the tumor. In the groups treated with CPA, ADM, DTIC, and VCR, the body weight was maintained near the ~" 20 E vo
-5 10 >
10 20 30 40 50 Days after inoculation
20 30 40 50 Days afterlnoculation
A 100 (B) -~ 50 I
20 30 40 50 Days after Inoculation
Fig 2. (A) Tumor growth curves and (B) survival in N-18 cell line. (Symbols are the same as in Fig 1 .)
30 40 50 60 Days afterlnoculatio~
Fig 3. (A) Tumor growth curves and (B) survival curves in NS-20 cell line. (Symbols are the same as in Fig 1 ,)
DRUG SENSITIVITY OF MURINE NEUROBLASTOMA
20 30 40 50 Days after Inocutatien
~100 r (B
20 30 40 50 Days after Inoculation
Fig 4. (A) Tumor growth curves and (B) survival curves in N I E - 1 1 5 cell line. (Symbols are the same as in Fig 1.}
level before chemotherapy in the early days, but it began to increase in the later stage. On the other hand, body weight loss was marked in the CDDP group. The maximum weight loss was 60% before chemotherapy, and the mean body weight was 80% of the body weight before chemotherapy. Thus, the toxicity of CDDP was the most severe of all antitumor agents given.
Histology and Metastasis There were no microscopic differences between the treated group and the control group. In groups treated with effective drugs, the greater part of tumors was necrosed, but viable cells were present in peripheral areas. There were no differences in the pattern of metastases among the groups. DISCUSSION
C 1300 murine neuroblastoma was established as a tumor that had originated spontaneously in the A / J a x mouse, 9 and many chemotherapy studies were done with a C 1300 murine neuroblastoma system, both in vitro ]4 and in vivo) 3'~5~ Finklestein et al ~'z studied the relevance of the C 1300 murine neuroblastoma system as a model for human disease, and they reported that CPA, BCNU, and isophosphamide were most effective, followed by cytosine arabinoside, ADM, and DTIC were effective. The VCR and actinomycin-D were not effective in vitro. In their study, the maximum percent increase in lifespan (%ILS) was 275% when 60 mg/kg of CPA was administered intraperitoneally three times every four days, on days 1, 5, and 9. However, studies on the effectiveness or resistance of several antitumor agents using several cloned cell lines have apparently not been reported. It is anticipated
that the antitumor activity of each drug may differ according to the clone. Moreover, according to studies done on the nude mouse xenograpft system by Tsuchida et al 4 and Makino et al, 5 chemotherapeutic effects depended on the cell line. They reported that CPA, CDDP, A C N U (a nitrosourea derivative), melphalan, and DTIC were effective when one third of the LD~o of the drug was administered three times every four days. These results suggested that the chemotherapeutic effects differed according to the component cells of the polyclonal xenografts. Indeed, in clinical experience a drug often shows different activities in different individuals. Thus, it is important to investigate differences of effects of antitumor agents on different clones that have a different catecholamine metabolic pathway. In almost all studies reported thus far, chemotherapeutic agents were administered in the early and short term. Costanza and Vaage ]9 indicated that continuous treatments begun early were more beneficial than short term chemotherapy. It is also thought that continuous chemotherapy resembles clinical treatment, so we undertook continuous long-term chemotherapy and observed the long-term effects. Our results confirmed that CPA is the most effective drug in the management of human and murine neuroblastomas. In our observation, CPA prominently prolonged the doubling time and the survival time of all four cell lines used. ADM was effective in the NS-20 and N1E-115 cell lines, but was inactive in C 1300 and N-18 cell lines. In evaluating these results, the following should be kept in mind. CPA, a masked compound, may be changed into an activated form, in a much larger amount in mice than in humans. 2~ Secondly, considerable amounts of ADM can be absorbed, particularly in the peritoneal surface and liver, when given intraperitoneally. 4,5 Despite these drawbacks, this murine neuroblastoma model is considered to be a useful system for testing sensitivity and resistance of drugs, because doses given to mice are approximately equal to those given clinically. There are differences between our results and those of the study by Finklestein et al) They reported that ADM and DTIC were effective, while we found no effects. This discrepancy may be due to differences in the dose of agents given and treatment schedule. Moreover, there were discrepancies between the doubling time and survival time. For example, in the N 1 E- 115 cell line, tumor growth was inhibited well by all antitumor agents, but this effect was not reflected in aspects of survival time, except in the CPA and VCR groups. An unknown tumor factor peculiar to this cell line might be involved. Our study showed that CDDP has a strong antitu-
OKUZONO ET AL
m o r activity almost equal to that of C P A on the C 1300, N - 1 8 , and N 1 E - 1 1 5 cell lines, and supports the proposal t h a t a protocol consisting m a i n l y of these two drugs ( C P A and C D D P ) is m o r e effective for patients with an a d v a n c e d n e u r o b l a s t o m a . On the other hand, C D D P was not effective on the N S - 2 0 cell line, w h i ch belongs to the cholinergic clone group. This cell line seems to be c o m p l e t e l y tolerant of C D D P , and to our knowledge, this is the first observation in c h e m o t h e r a peutic e x p e r i m e n t s for neuroblastoma. Therefore, the N S - 2 0 cell line can be used for research on the toleran ce of C D D P . Similarly, the C 1300 line was tolerant of A D M , D T I C , and V C R , and the N - 1 8 line was tolerant of A D M and D T I C . T h e presence of
h e t e r o g e n e i t y of m a l i g n a n t cells within neuroblastomas and o t h er solid cancers has been r e p e a t e d l y demonstrated in e x p e r i m e n t a l t u m o r models. 21,22 S o m e reporters 23,24 d e m o n s t r a t e d the varied potential for m e t a s t a s e s , biologic diversity, and h e t e r o g e n e i t y a m o n g the subpopulations of t h e parent tumor. Therefore, to o v e r c o m e the heterogeneity, these resistant clonal cell lines can be utilized for research on tolerance. ACKNOWLEDGMENT
We are indebted to Dr Y. Hachitanda, Second Department of Pathology, Faculty of Medicine, Kyushu University for his helpful guidance in the histologic examination, and to Ms M. Ohara for reading this paper.
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