Cancer Lelters. 5 (1978) 333--~37 © Else~¢ier/No~'th-Hollaad Scientific Publishers Ltd.
HETEIROTRANSPLANTATION CARCINOMA INDUCED IN HAMSTERS
33~3
INTO :NUDE MICE OF PANCREATIC BY N.BIS(2-HYDROXYPROPYL)NITROSAMIN~',
YOICHI KONISHI, SEIICHI TAKAHASHI, MASAOKI SUNAGAWA, NOBUYUKI MIYAGI aad HIROMU KONDO Depar~!men~ of Oneological Pathology, Cancer Center. Nara Med&al Uniuersi¢], 840 Shi]o-cho. K~hihara, Nara 634 (Japan)
(Received 29 May 1978} (Revised version received 4 August 1978} (P.ccept,ed 15 August 1978}
SUMIvI.ARY Heterotransplantation into nude mice by the s.ubcut~meous (s.c.) and intraperitoneal (i.p.) routes of hamster pancreatic ductal cell carcinomas induced by N-bis(:~-hydroxypropyl)nitrosamine (DHPN) was studied. By s.c. transplantation, tumors occurred in 1 o£ 4 mice at t:he first trial, and the t u m o r incidence was ! 0 0 % J:'or shorter periods by serial 1;ransfer. By i.p. transplantation, the mali~l~ant potential of hamster pancreatic dttctal cell carcinoma was expressed nz intra-abdominal invasive tumors with bloody ascites (carcinomatous peritonitis). Tumors in nude mice basically retair, ed tIhe histology of the o r ~ n a t hamster pancreatic ductal cell carcinoma.
][NTR()DUCTION Pancreatic carcinomas are induced by DI=iPN in hamsters [ 3 f i ] , and by ~zaserine [4] and 4-hydroxyaminoqtfinoline 1-oxide (4-HAQO) [2] in rats. However, DHPN-induced p,~ncreatic carcinoma in the hamster is ~he only available experillnent.~2 model for ductal eel! carcinoma, which has the highes~ incidence among the pancreatic carcinomas occurring in humans [ 1]. Nude mice tlave been widely used for the heterotransplan~;ation animal model since riCe initial report by Kygaard and Povlsen [8] ; however, no reports ,on the establishment of 1;ransDlantable pancreatic carcinoma in nude mice and t~he resulting expresAbbrevhTtions: DHPN, N-bis(2-hydroxypropyl)aitrosamine; quinoHn.a 1-oxlde; i.p., intraperi~oneal ; s.c., subcutarmous.
4-HAQO, 4-hydrox.yarni.no-
334
sion of potential have been shown. This paper describes briefly the results of heterotransplantation into made mice of pancreatic carcinoma induced by DHPN. MATERIALS AND METHODS Tumors used for the s.c. transplantation were obtained from ~:he hamster pancreatic carcinoma induced by .,;.c. injections of DHPN 'Nakarai Chemicals Ltd., Kyoto, Japan) as previously described [6]. Tumor cells used for the i.p. transplantation were obtained from subcutaneous tumors at passage 3 in nude mice that had receive,c! serial transplants of the hamster pancreatic carcinoma. T u m o r cells were counted by hemocytometer. Athymic nude BALB/c mice, 4--8 weeks old, were purchased through Nihon CLEA Co., Tokyo~ Japan. Mice were maintained under conventional conditions with CLEA No. 2 Chow for Ge~nf]cee Mice (Nihon CLEA Co., Tokyo, Japan) and sterilized water. For the s.c. transplantation, the tumors were divided into 2 pieces, 1 fi)r the transplantation and 1 for histological confirmation of ductal cell carcinoma of the pancreas. The piece for the transpl:antation was minced into ~.pprox. 2 × 2 mm ~ fragments in re.fxigerated Eagle's minimum ea~;ential mediltm solution containing 10% calf serum, 100 mg streptomycin/liter, and 1 × 10 s U penic'illin/liter. Several fragments were t:ransplanted with a trocar into the s.c. space of the bilateral back o f nude mice. For the i.p. transplar~.tation, ~he tumors were minced hy scissors and squashed on a cytosieve (100 pores/cm z) by a rubber plug in Eagle's minimum essential medium solution containing the same substances as for s.c. transplantation. The suspension contaiifing 1 × I06 cells/0.5 ml was i.p. tl~aasplanted. Nude mice were: killed by cervical dislocation, completeIy autopsied, and macroscopically examined. Appropriate organs were fixed in 10% buffered formalin and the tissues processed by routine histological examinatic, n. T m n o r rake was confirmed by histologicaI examination. RESULTS The results of the s.c. transplantation of hamster pancreatic carcinoma into nude mice are summarized in Tal)le 1. Tumor take was 1 of 4 mice at the first trial, and it was 100% (t/4) for shorter periods by serial tr~,nsfer. '['he largest tumor grown in the s.c. space of nude mice reached 722 ram" at 5 week'. Macroscopically, nQ predi]lected area was seen, for induced durra] c¢~[]carcinoma in the pancreas of hamsters, and! the hi,~tolo~w (Fig. 1) was similar to that p r e v i o ~ l y described [~;~7]. Tumors grown in the s.c. space of nude mice showed ~,Lodules {F~g. 2 ), which did not i~vade the surrounding skin or muscular tissues. Etristology of the transple0ated tumors was similar to the original pancreatic ductal cell carcinoma in hamsters (Fig. 3). Carcinomatous peritonitis (Fig. 4) developed in 2 of 2 mice receiving i.p. transplantation of the tumor cell suspension for 15 days. [nvasiorLs were seen in the peritoneum, diaphragm,
335 TABLE 1 HETEROTRANSPLANTATION INTO N U D ~ MICE O F P A N C R E A T I C C A R C I N O M A I N D U C E D IN H A M S T E R S B Y D H P N Passages
No. of tumors taken/ No. of tumors transptm~ted
Incubation period (weeks)
Tumor size (ram:)
1 2 3 4 5
1/4 4/4 414 4/4 4/4
10 11 7 5 R
64--110 49--6 '58 153~722 120~162
Fig. 1. Original histolo6,W of ductal cell carcinoma of pancreas il:~duc~d by Syrian golden hamster~ by DHPN. H & E x 160. Fi~:. 2 Macros,~ople view of transplanted pancreatic carcinoma at the 3rd passage in nude mice. Fi~$. 3. t'lis~ology of transplanted pancreatic carcinoma at the 2nd p~msage in nude mice. H& E X 160. Fi~. 4. Macroscopic view o1' carcinomatous peritonitis induced by tumor cell suspensio;n pr~pa~ed from seri.~dly transplanted pan,:~ea~ie carcinoma 0! ;!,e 3rd passage in nude mice
336 o m e n t u m , meseatedes, perirenal fatty ~;issues, and pancreas and metastasis occurred in the liver. DISCUSSICIN
Pour e t a l . [7] reported a L~igh inci4ence o f pancreatic neoplasms indt~ced by weekly s.c, application of D H P N in hamsters. The morp:holo~cal patterns and biological behavior of the i n d u c e d neoplasms resembled those in man. In the ;~resent e x p e r i m e n t we transplanted hamster pancreatic ductal cell carcinoma .:.'or the pu;rpose of establi~hir~g a m o d e l for the s t u d y of the characteristic ~.~owth behavior of the pancreatic ductal carcinoma. The malignant potential ,).f the restS!ring neoplasms wa:; expressed as carcinomatous peritonitis in nude :nice. Previously, we succeeded in converting a p o o r l y differentiated h u m a n ~.~denocarcinoma to ascites fo~Ln with invasion and metastasis in n u d e mice [9]. the present ,experiment, carcinomatous: perit,~nitis developing in nude mice was usually accompanied w i t h b l o o d y asci~es. A conversion experiment for the ~ransplantable ascites f o r m o f the pancreatic carcinoma in n u d e mice is also required. Mechanisms of t h e d:ifference in growth behavior of the tumors, with or w i t h o u t invasion, b e t w e e n t h e abdo~nirtal cavity and the s.c. space o f nude mice is unexplained. Experimer~t in this c o n t e x t :is required also. A transplantable pancreatic ductal cell c ~ c i n o m a line ~,;hould be established to obtain basic i~ffor~ation on cancer c h e m o t h e r a p y , since h u m a n par~creatic cancer shows o.oe of the lowes~ 5-year s~trviw~t rates afte~ diagnosis [ 5]. ACKNOWLEDGEMENT The authors would like t o acknowledge Professor K. Kojima for his contia~uous e n c o u r a g e m e n t and hell~ful discussion. The'se studies were supported in part by Grant.in-Aid for Cancer Research from the Ministry of Education, Science and Culture in Japan. REFERENCES 1 Ct.Lbilta,A.L. and Fit;zl;eralcl~P.J. (]975) Morpi~ological patterns of primary nonendoerine human pancreas .~arcirJoma. Canner Res., 35, 22,34--224.B~ 2 Kc,nishi, Y., Denda, A., Inui, S., Takahashi, S. and Kondo, H. (I976) Pancreatic carcinoma induced by 4-hydroxyaminoqu~no[ine 1-oxide after p~rtial panereatec~omy and splcneetor~y in rat's. Gann, 67, 9 t 9--920. 3 Kr~l]ger.F.W, PoLir, P. and Altho.rf, ,J. (t974) Induction of pancrea~ ~urnours by di~ isopropylniLrosamJne. Naturwisse~schafl;en, 61,328. 4 Longnecker, D.S. and Curphey, T.J. (1975) Aclenoca:ccinoma .of the pancreas in azaserme-treated rats. Cancer Res., 35, 2249--2255. 5 Pledger, R,A., Bates~ R.R, and Saffiot~i, U. (1975) ~:;urrent progress in pancreatic carcinogenesis research. Irtrod~ction: National C~ncer Institute Pancreatic Carcino.~enesis Program. Cancer Res., 35, ~!226--2227. (~ Pour, P., Kriiger, F.W., Althoff, J . Carde:~a, ,~. and Mohr, U. (19'74) Cancer of" the pancreas induced in the Syrian g~lden hamster. Arn. J. PathoI., 76,349--358.
337 7 Pour, P., Mohr, U., Cardesa, A., Althoff, J. and Kruger, F.W, {1975) Pancreatic neoplasms in an animal model: Morphological, biological+ and comparative ~)ldies. Cancer, 36, 379--389. 8 Rygaard, J. and Povlsen, C.O. (1969) Heterotransplantatioi~ ~f a hum~r~ malignant turnout to "nude" mice. Acta Pathol. Microhiol. Scand., 77,758,--7f~0. 9 Takahashi, S., Konishi, Y., Nakatar~i, K., Inui, S., Kojima, K, and Stliratori, T. (197,3) Conversion of poorly differentiated human adenoeare~noma to aseites form ~ t h invasion and metastasis in nude mice. J. Natl. Cancer Inst., 60,925--929.
HETEIROTRANSPLANTATION CARCINOMA INDUCED IN HAMSTERS
33~3
INTO :NUDE MICE OF PANCREATIC BY N.BIS(2-HYDROXYPROPYL)NITROSAMIN~',
YOICHI KONISHI, SEIICHI TAKAHASHI, MASAOKI SUNAGAWA, NOBUYUKI MIYAGI aad HIROMU KONDO Depar~!men~ of Oneological Pathology, Cancer Center. Nara Med&al Uniuersi¢], 840 Shi]o-cho. K~hihara, Nara 634 (Japan)
(Received 29 May 1978} (Revised version received 4 August 1978} (P.ccept,ed 15 August 1978}
SUMIvI.ARY Heterotransplantation into nude mice by the s.ubcut~meous (s.c.) and intraperitoneal (i.p.) routes of hamster pancreatic ductal cell carcinomas induced by N-bis(:~-hydroxypropyl)nitrosamine (DHPN) was studied. By s.c. transplantation, tumors occurred in 1 o£ 4 mice at t:he first trial, and the t u m o r incidence was ! 0 0 % J:'or shorter periods by serial 1;ransfer. By i.p. transplantation, the mali~l~ant potential of hamster pancreatic dttctal cell carcinoma was expressed nz intra-abdominal invasive tumors with bloody ascites (carcinomatous peritonitis). Tumors in nude mice basically retair, ed tIhe histology of the o r ~ n a t hamster pancreatic ductal cell carcinoma.
][NTR()DUCTION Pancreatic carcinomas are induced by DI=iPN in hamsters [ 3 f i ] , and by ~zaserine [4] and 4-hydroxyaminoqtfinoline 1-oxide (4-HAQO) [2] in rats. However, DHPN-induced p,~ncreatic carcinoma in the hamster is ~he only available experillnent.~2 model for ductal eel! carcinoma, which has the highes~ incidence among the pancreatic carcinomas occurring in humans [ 1]. Nude mice tlave been widely used for the heterotransplan~;ation animal model since riCe initial report by Kygaard and Povlsen [8] ; however, no reports ,on the establishment of 1;ransDlantable pancreatic carcinoma in nude mice and t~he resulting expresAbbrevhTtions: DHPN, N-bis(2-hydroxypropyl)aitrosamine; quinoHn.a 1-oxlde; i.p., intraperi~oneal ; s.c., subcutarmous.
4-HAQO, 4-hydrox.yarni.no-
334
sion of potential have been shown. This paper describes briefly the results of heterotransplantation into made mice of pancreatic carcinoma induced by DHPN. MATERIALS AND METHODS Tumors used for the s.c. transplantation were obtained from ~:he hamster pancreatic carcinoma induced by .,;.c. injections of DHPN 'Nakarai Chemicals Ltd., Kyoto, Japan) as previously described [6]. Tumor cells used for the i.p. transplantation were obtained from subcutaneous tumors at passage 3 in nude mice that had receive,c! serial transplants of the hamster pancreatic carcinoma. T u m o r cells were counted by hemocytometer. Athymic nude BALB/c mice, 4--8 weeks old, were purchased through Nihon CLEA Co., Tokyo~ Japan. Mice were maintained under conventional conditions with CLEA No. 2 Chow for Ge~nf]cee Mice (Nihon CLEA Co., Tokyo, Japan) and sterilized water. For the s.c. transplantation, the tumors were divided into 2 pieces, 1 fi)r the transplantation and 1 for histological confirmation of ductal cell carcinoma of the pancreas. The piece for the transpl:antation was minced into ~.pprox. 2 × 2 mm ~ fragments in re.fxigerated Eagle's minimum ea~;ential mediltm solution containing 10% calf serum, 100 mg streptomycin/liter, and 1 × 10 s U penic'illin/liter. Several fragments were t:ransplanted with a trocar into the s.c. space of the bilateral back o f nude mice. For the i.p. transplar~.tation, ~he tumors were minced hy scissors and squashed on a cytosieve (100 pores/cm z) by a rubber plug in Eagle's minimum essential medium solution containing the same substances as for s.c. transplantation. The suspension contaiifing 1 × I06 cells/0.5 ml was i.p. tl~aasplanted. Nude mice were: killed by cervical dislocation, completeIy autopsied, and macroscopically examined. Appropriate organs were fixed in 10% buffered formalin and the tissues processed by routine histological examinatic, n. T m n o r rake was confirmed by histologicaI examination. RESULTS The results of the s.c. transplantation of hamster pancreatic carcinoma into nude mice are summarized in Tal)le 1. Tumor take was 1 of 4 mice at the first trial, and it was 100% (t/4) for shorter periods by serial tr~,nsfer. '['he largest tumor grown in the s.c. space of nude mice reached 722 ram" at 5 week'. Macroscopically, nQ predi]lected area was seen, for induced durra] c¢~[]carcinoma in the pancreas of hamsters, and! the hi,~tolo~w (Fig. 1) was similar to that p r e v i o ~ l y described [~;~7]. Tumors grown in the s.c. space of nude mice showed ~,Lodules {F~g. 2 ), which did not i~vade the surrounding skin or muscular tissues. Etristology of the transple0ated tumors was similar to the original pancreatic ductal cell carcinoma in hamsters (Fig. 3). Carcinomatous peritonitis (Fig. 4) developed in 2 of 2 mice receiving i.p. transplantation of the tumor cell suspension for 15 days. [nvasiorLs were seen in the peritoneum, diaphragm,
335 TABLE 1 HETEROTRANSPLANTATION INTO N U D ~ MICE O F P A N C R E A T I C C A R C I N O M A I N D U C E D IN H A M S T E R S B Y D H P N Passages
No. of tumors taken/ No. of tumors transptm~ted
Incubation period (weeks)
Tumor size (ram:)
1 2 3 4 5
1/4 4/4 414 4/4 4/4
10 11 7 5 R
64--110 49--6 '58 153~722 120~162
Fig. 1. Original histolo6,W of ductal cell carcinoma of pancreas il:~duc~d by Syrian golden hamster~ by DHPN. H & E x 160. Fi~:. 2 Macros,~ople view of transplanted pancreatic carcinoma at the 3rd passage in nude mice. Fi~$. 3. t'lis~ology of transplanted pancreatic carcinoma at the 2nd p~msage in nude mice. H& E X 160. Fi~. 4. Macroscopic view o1' carcinomatous peritonitis induced by tumor cell suspensio;n pr~pa~ed from seri.~dly transplanted pan,:~ea~ie carcinoma 0! ;!,e 3rd passage in nude mice
336 o m e n t u m , meseatedes, perirenal fatty ~;issues, and pancreas and metastasis occurred in the liver. DISCUSSICIN
Pour e t a l . [7] reported a L~igh inci4ence o f pancreatic neoplasms indt~ced by weekly s.c, application of D H P N in hamsters. The morp:holo~cal patterns and biological behavior of the i n d u c e d neoplasms resembled those in man. In the ;~resent e x p e r i m e n t we transplanted hamster pancreatic ductal cell carcinoma .:.'or the pu;rpose of establi~hir~g a m o d e l for the s t u d y of the characteristic ~.~owth behavior of the pancreatic ductal carcinoma. The malignant potential ,).f the restS!ring neoplasms wa:; expressed as carcinomatous peritonitis in nude :nice. Previously, we succeeded in converting a p o o r l y differentiated h u m a n ~.~denocarcinoma to ascites fo~Ln with invasion and metastasis in n u d e mice [9]. the present ,experiment, carcinomatous: perit,~nitis developing in nude mice was usually accompanied w i t h b l o o d y asci~es. A conversion experiment for the ~ransplantable ascites f o r m o f the pancreatic carcinoma in n u d e mice is also required. Mechanisms of t h e d:ifference in growth behavior of the tumors, with or w i t h o u t invasion, b e t w e e n t h e abdo~nirtal cavity and the s.c. space o f nude mice is unexplained. Experimer~t in this c o n t e x t :is required also. A transplantable pancreatic ductal cell c ~ c i n o m a line ~,;hould be established to obtain basic i~ffor~ation on cancer c h e m o t h e r a p y , since h u m a n par~creatic cancer shows o.oe of the lowes~ 5-year s~trviw~t rates afte~ diagnosis [ 5]. ACKNOWLEDGEMENT The authors would like t o acknowledge Professor K. Kojima for his contia~uous e n c o u r a g e m e n t and hell~ful discussion. The'se studies were supported in part by Grant.in-Aid for Cancer Research from the Ministry of Education, Science and Culture in Japan. REFERENCES 1 Ct.Lbilta,A.L. and Fit;zl;eralcl~P.J. (]975) Morpi~ological patterns of primary nonendoerine human pancreas .~arcirJoma. Canner Res., 35, 22,34--224.B~ 2 Kc,nishi, Y., Denda, A., Inui, S., Takahashi, S. and Kondo, H. (I976) Pancreatic carcinoma induced by 4-hydroxyaminoqu~no[ine 1-oxide after p~rtial panereatec~omy and splcneetor~y in rat's. Gann, 67, 9 t 9--920. 3 Kr~l]ger.F.W, PoLir, P. and Altho.rf, ,J. (t974) Induction of pancrea~ ~urnours by di~ isopropylniLrosamJne. Naturwisse~schafl;en, 61,328. 4 Longnecker, D.S. and Curphey, T.J. (1975) Aclenoca:ccinoma .of the pancreas in azaserme-treated rats. Cancer Res., 35, 2249--2255. 5 Pledger, R,A., Bates~ R.R, and Saffiot~i, U. (1975) ~:;urrent progress in pancreatic carcinogenesis research. Irtrod~ction: National C~ncer Institute Pancreatic Carcino.~enesis Program. Cancer Res., 35, ~!226--2227. (~ Pour, P., Kriiger, F.W., Althoff, J . Carde:~a, ,~. and Mohr, U. (19'74) Cancer of" the pancreas induced in the Syrian g~lden hamster. Arn. J. PathoI., 76,349--358.
337 7 Pour, P., Mohr, U., Cardesa, A., Althoff, J. and Kruger, F.W, {1975) Pancreatic neoplasms in an animal model: Morphological, biological+ and comparative ~)ldies. Cancer, 36, 379--389. 8 Rygaard, J. and Povlsen, C.O. (1969) Heterotransplantatioi~ ~f a hum~r~ malignant turnout to "nude" mice. Acta Pathol. Microhiol. Scand., 77,758,--7f~0. 9 Takahashi, S., Konishi, Y., Nakatar~i, K., Inui, S., Kojima, K, and Stliratori, T. (197,3) Conversion of poorly differentiated human adenoeare~noma to aseites form ~ t h invasion and metastasis in nude mice. J. Natl. Cancer Inst., 60,925--929.
