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(American Journal of Pathology. 2000;156:439-443.)
© 2000 American Society for Investigative Pathology

Short Communications

In Vitro Induction of Giant Cell Tumors from Cultured Hamster Islets Treated with N-Nitrosobis(2-Oxopropyl)amine

Hosei Matsuzaki*{dagger}, Bruno M. Schmied*{ddagger}, Alexis Ulrich*§, Surinder K. Batra and Parviz M. Pour*||

From the UNMC Eppley Cancer Center,*
University of Nebraska Medical Center, Omaha, Nebraska; the Department of Surgery II,{dagger}
Kumamoto University School of Medicine, Kumamoto, Japan; the Department of Visceral and Transplantation Surgery,{ddagger}
Insel Hospital, Bern, Switzerland; the Department of Surgery,§
Rheinische Friedrich-Wilhelms-University, Bonn, Germany; and the Departments of Biochemistry and Molecular Biology
and Microbiology,||
University of Nebraska Medical Center, Omaha, Nebraska


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Giant cell carcinoma of the pancreas is a rare tumor. Its histogenesis is still controversial. In a Syrian hamster pancreatic cancer model, tumors similar to human giant cell carcinomas have been induced at an extremely low rate of incidence and after the use of high doses of pancreatic carcinogens. Thus far no tumors of giant cell type have been induced by the in vitro treatment of hamster pancreatic ductal cells with the potent pancreatic carcinogen N-nitrosobis(2-oxopropyl)amine (BOP). In the present study we report the induction of giant cell carcinoma from hamster islets treated with BOP in vitro. The results suggest that in hamsters some component of islet cells, probably stem cells, are the origin of giant cell carcinoma.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
It is generally believed that over 90% of pancreatic cancers originate from ductal/ductular cells. Studies of the hamster pancreatic cancer model, which in biological and morphological aspects mimics human tumors,1-3 however, have shown that pancreatic ductal adenocarcinoma originates not only from ductal/ductular cells but primarily from within islets, most probably from precursor (stem) cells.1-3 In this model, tumors resembling human giant cell carcinoma have been induced only after prolonged treatment with high doses of pancreatic carcinogen N-nitrosobis(2-oxopropyl)amine (BOP).4 In humans, giant cell carcinoma is also a rare tumor and appears as two different phenotypes, osteoclastic and pleomorphic. The histogenesis of these tumors from epithelial or mesenchymal cells has not yet been settled. Treatment of isolated hamster pancreatic ductal cells with BOP or methylnitrosourea (MNU) in vitro resulted in the induction of anaplastic cancer, but no giant cell tumors have been observed.5,6 A similar treatment scheme of isolated purified hamster pancreatic islets with BOP, however, resulted in formation of anaplastic invasive cancers, which, in vitro and in vivo, presented patterns consistent with human pancreatic giant cell tumor. In this report we describe some biological patterns of this tumor compared to the human tumor.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

Female and male Syrian golden hamsters (all 8–10 weeks old) from the Eppley colony and 8-week-old female BEIGE DX 1 nude mice from Harlan Comp. (Indianapolis, IN) were used in this experiment. The animals were housed in cages with Sani-cell bedding and kept under standard laboratory conditions (temperature 20 ± 2°C, 10 air changes/minute, 12 hour/12 hour light/dark cycle). They received a pelleted diet (Wayne, Indianapolis, IN) and water ad libitum.

Chemicals

BOP was synthesized at our institute. The culture medium (M3:5) was a gift from InCell Corporation (San Antonio, TX). Penicillin, streptomycin, trypsin, EDTA, and RPMI-1640 were purchased from Life Technologies (Gaithersburg, MD). Fetal bovine serum was purchased from Summit Biotechnology (Ft. Collins, CO). Collagenase P was obtained from Boehringer Mannheim (Mannheim, Germany).

Islet Cell Culture and BOP Treatment

Pancreatic islets of 10 male Syrian golden hamsters were isolated, purified, and cultured as described earlier.7 Briefly, surgically removed pancreata were digested with 5 mg/ml collagenase P for 10 minutes, and islets were hand-picked under a dissecting light microscope. For the first 14 days the islets were maintained on a rocker (20 rpm) in 60-mm Petri dishes containing 8 ml M3:5 medium in a humid atmosphere and 5% CO2. While the islets were floating, the fibroblasts attached to the bottom of the dishes. Islets were hand-picked every other day and placed in a new Petri dish with fresh M3:5 medium. At day 15, after the initial isolation, pure islets were plated in T-25 flasks (about 100 islets per flask) and allowed to attach to the bottom of the flask. From attaching islets, epithelial cells radiated into the surrounding area and formed a monolayer. The cells were trypsinized and subcultured when subconfluent. Half of the islets were treated with BOP from the first day of culture at a concentration of 0.25 mmol/L every other day, three times a week, and were designated as MS7B. The other half served as an untreated control group (MS7N). After 27 weeks in culture, the cells were gradually shifted from M3:5 medium to 1640-RPMI containing 10% fetal bovine serum. Cell growth speed and anchorage-independent cell growth were determined as reported.5

In Vivo Cell Growth

A total of 6 x 106 MS7B and MS7N cells from the same passages were injected subcutaneously into two sites of the abdomen of four Syrian golden hamsters and four nude mice. The tumor growth was measured weekly and bidimensionally with a caliper. Tumor-bearing animals were euthanized after 4 weeks. In animals with MS7N, transplants were observed for 10 weeks. The tumors were freed from soft tissues, weighed, measured, fixed in 10% buffered formalin for 16 hours, and processed for histology by conventional methods.

Histology/Immunocytochemistry

At weeks 27 and 40, 5.0 x 106 MS7B and MS7N cells were washed in 1x phosphate-buffered saline, fixed in 10% buffered formalin for 1 hour, embedded in paraffin, and cut into 4-µm serial sections. Cells of both groups were also cultured in four-well chamber slides (Nalge Nunc International, Naperville, IL) and in 12-well plates on Thermanox plastic coverslips (Nalge Nunc International) and fixed with 10% buffered formalin for 1 hour. The cells were stained with hematoxylin and eosin (H&E). For immunocytochemistry, the avidin-biotin peroxidase complex (ABC) method was used, and the antibodies were visualized with diaminobenzidine (DAB) peroxide (KPL, Gaithersburg, MA). The primary antibodies used in this study were as follows: insulin, glucagon, somatostatin, and p53 (PAb240) purchased from Zymed (San Francisco, CA), neuron-specific enolase (NSE), vimentin and {alpha}-1-antitrypsin from Biogenex (San Ramon, CA), pancytokeratin and laminin from Sigma (St. Louis, MO), and transforming growth factor-{alpha} (TGF-{alpha}) and epidermal growth factor receptor (EGFR) from Oncogene Science (Cambridge, MA). Tomato lectin and Phaseolus vulgaris leucoagglutinin (L-PHA) were purchased from Vector (Burlingame, CA). The primary antibodies were diluted as shown in Table 1 . A normal hamster pancreas served as a control. Negative control sections were processed similarly, except that nonimmunized serum of the host animal or type-specific immunoglobulin was used instead of the primary antibody. The staining intensity was scored as negative (-), weak (+), moderate (++), and strong (+++).


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  		Table 1. Immunohistochemical Reactivity of Antibodies to Hamster-Islet-Derived Cell Line MS7N and MS7B at 40 Weeks in Vitro and in Vivo

 
Electron Microscopy

At weeks 27 and 40, 1.0 x 106 MS7B and MS7N cells were each prepared for transmission electron microscopic examination as reported.8

Examination of Ki-ras Mutation

Mutation of the Ki-ras gene in MS7B and MS7N cells at weeks 27 and 40 by reverse transcriptase–polymerase chain reaction, and the sequence analysis was performed as reported.5,9 The primer sequences used are listed elsewhere.9


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The patterns of BOP-treated and untreated islets in culture and their subsequent cytological changes were similar to those published earlier.7,10 The sizes of the treated and untreated islets varied between 0.12 and 0.32 mm without significant difference between the two groups. After the islets were transferred into the flasks, all islets attached to the bottom of the flasks within a week and a large number of epithelial cells radiated from the islet cores into the surrounding area as reported.7,10 These epithelial cells were gradually replaced by monomorphic undifferentiated cells. At 11 weeks and later, contrary to the monomorphic appearance of the MS7N cells, the MS7B cells became pleomorphic, with abundant eosinophilic cytoplasm and a few hyperchromatic nuclei surrounded by fine granules.

After 36 weeks in culture, mononucleated or polynucleated giant cells appeared for the first time. The polynucleated cells had three to eight overlapping nuclei in a large polygonally shaped cytoplasm that was 10–20 times larger in size than the surrounding undifferentiated cells (Figure 1) . MS7N cells retained their monomorphic phenotype throughout the whole culture.



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  		Figure 1. MS7B cells at 40 weeks in culture, grown on a chamber slide. a: Scattered between small cells in haphazard order, there are scattered mononucleated or polynucleated giant cells with large cytoplasm. H&E; original magnification, x75. b: Higher power view of polynucleated giant cells, showing multiple nuclei that were either separated from each other (top) or presented more than 10 overlapping nuclei (bottom). H&E; original magnification, x140.

 
After 13 weeks, the growth of MS7B cells accelerated significantly compared to the MS7N cells, showing a doubling time of 30 hours compared to 36 hours for the MS7N cells. The doubling time of untreated and treated cells further decreased with time in culture. After 40 weeks in culture the cell growth of both groups remained fairly constant, with a doubling time of 11 hours for the MS7B cells and 21 hours for the MS7N cells.

At 27 weeks in culture BOP-treated cells but not MS7N cells formed for the first time 12 colonies per dish in soft agar. In in vivo experiments, these MS7B cells also formed subcutaneous tumors for the first time. At this stage, tumors grew to an average size of 0.32 cm3 within 3 weeks in both species. Cells of later stages grew more aggressively and formed tumors of 1.72 cm3 within 3 weeks on average. MS7N cells at the same stages as the MS7B cells did not form tumors within a period of 10 weeks. There were no differences between the tumors grown in hamsters and nude mice. Histologically, both hamster and mouse tumors were composed primarily of pleomorphic spindle-shaped cells, forming an anaplastic tumor but no giant cell tumor. Tumors from MS7B cells treated with BOP for 40 weeks were primarily composed of anaplastic cells with scattered pleomorphic multinucleated giant cells (Figure 2) . In nude mouse and hamster, eight tumors were found, and all eight were giant cell tumors.



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  		Figure 2. MS7B cells at 40 weeks in culture, grown in a nude mouse. The tumor is composed of pleomorphic small cells with interspersed mononucleated or polynucleated cells, some with large and almost homogeneous cytoplasm. H&E; original magnification, x210.

 
Immunohistchemical results of MS7N and MS7B cells at 40 weeks in culture in vitro are shown in Table 1 . MS7N and MS7B cells, the MS7B cells growing as tumors in vivo, as well as the giant cells in vitro and in vivo, stained positively with anti-pancytokeratin, -NSE, -TGF-{alpha}, -vimentin, -laminin, and bound to tomato lectin and L-PHA. Reactivity to EGFR staining was found only in MS7B cells after 40 weeks in culture. p53 overexpression was found only in MS7B at week 40. None of the cells reacted to anti-insulin, -glucagon, or -somatostatin.

Electron microscopically, the cells treated with BOP for 24 weeks had a pleomorphic shape and were 5–200 µm in diameter. They were loosely arranged and occasionally attached to each other. They also presented oval or round mitochondria, chromatin-rich irregular nuclei, and large nucleoli. Some cells had intracytoplasmic cystic spaces filled with debris. After 40 weeks in culture, scattered polynucleated giant cells 600–800 µm in diameter were observed. They contained three to five large nuclei, many lysosomes, and sparse rough endoplasmic reticulum (Figure 3) .



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  		Figure 3. Electron microscopically, the giant cells had several elongated nuclei with irregular contours, small hyperchromatic nucleoli, and sparse organelles and lysosomes. At week 40 in culture. Original magnification, x4500.

 
Mutation of the K-ras oncogene was found in the MS7B giant cells in culture but not in the MS7N cells. The mutation was in codon 12 (GGT -> GAT).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Giant cell tumor of the pancreas is a rare tumor with an incidence of 2.1–12.8%11,12 and has a grave prognosis.13 Histologically, two types of giant cell tumors are distinguished: pleomorphic and osteoclastic. Pleomorphic giant cell tumor, also termed carcinosarcoma, is considered to be a sarcomatous metaplasia of ductal adenocarcinoma14-16 ; the origin of osteoclastic giant cell tumor from epithelial or mesenchymal cells is still debated.14 Their differential diagnoses include malignant fibrous histiocytoma, amelanotic melanoma, soft tissue sarcoma, and hepatocellular carcinoma. Because of their morphological and biological patterns these tumors were initially regarded as a separate type of pancreatic cancer. Although the new classification system includes these tumors in the anaplastic category, their etiology and histogenesis have remained obscure.

In the hamster pancreatic cancer model, which in many aspects resembles the human disease,1,2,9,17 both pleomorphic giant cell tumor and osteoclastic giant cell tumor have been induced.4 However, the cell origin of these neoplasms could not be ascertained. Although in humans most cancer types, including pleomorphic giant cell tumors, are thought to derive from ductal cells, in the hamster model many cancers originate from within islets,1-3 most probably from stem cells.10 In contrast to tumors arising from ducts (intraductal carcinomas), which remain within the ductal boundary for a long time before invasion, tumors developing within islets (intrainsular cancers) are invasive from their inception.10 We and others have shown that in vitro treatment of hamster ductal cells with pancreatic carcinogens leads to malignant cell transformation and adenocarcinoma in vivo.5,6 No giant cell carcinomas have ever been produced. On the contrary, treatment of isolated and purified hamster islets with BOP produced tumors of giant cell morphology. Similarities between the human and hamster tumors exist also on immunohistochemical and biological levels, including the coexpression of the epithelial marker cytokeratin, mesenchymal marker, vimentin,14,18,19 neuroepithelial marker, NSE,13 the mutation of K-ras18,19 and overexpression of p53.20 P16 and DPC4 mutations reported in human pancreatic cancer21,22 could not be examined because of the unavailability of the respective primers for hamsters (see comments in Ref. 22 ).

Opinions on the progenitor cells of giant cell tumor are divided. Although most investigators believe that the giant cells derive from ductal cells,23 others assume that pancreatic precursor cells are the origin of the tumor.23 The latter possibility is supported by the occurrence of mixed ductal adenocarcinoma-pleomorphic giant cell tumor on the one hand and osteoclastic and pleomorphic giant cells on the other.15,24 Our finding supports the precursor (stem) cell derivation of giant cell carcinoma. Because the tumor was induced only when islet and not ductal cells were treated with the carcinogen, we concluded that some cells within islets present the tumor progenitor cells. We have identified these cells in culture and have shown their response to BOP.7,10 The reactivity of giant cells with NSE (a marker for neuroendocrine differentiation) and {alpha}1-antitrypsin, which has been shown to be expressed in human cancer cells assumed to derive from pancreatic stem cells, such as pancreatoblastoma and solid cystic tumors,25-27 also points to their stem cell derivation.

Nevertheless, we have shown for the first time the induction of pleomorphic giant cell cancer from cultured hamster islets treated with a potent pancreatic carcinogen that they derive from stem cells embedded within islets.


   Footnotes
 
Address reprint requests to Dr. Parviz M. Pour, The Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 986805 Nebraska Medical Center, Omaha, NE 68198-6805. E-mail: ppour{at}unmc.edu

Supported by National Cancer Institute grants CA60479 and CA367127, SPORE grant P5O CA72712, and a Special Institution grant of the American Cancer Society. A. U. is a recipient of a scholarship of the Deutsche Forschungsgemeinschaft, Germany.

Accepted for publication October 26, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Pour P: Islet cells as a component of pancreatic ductal neoplasms. I. Experimental study: ductular cells, including islet cell precursors, as primary progenitor cells of tumors. Am J Pathol 1978, 90:295-316[Abstract]
  2. Pour PM: Histogenesis of exocrine pancreatic cancer in the hamster model. Environ Health Perspect 1984, 56:229-243[Medline]
  3. Pour PM: Modification of tumor development in the pancreas. Prog Exp Tumor Res 1991, 33:108-131[Medline]
  4. Pour PM: Induction of unusual pancreatic neoplasms, with morphologic similarity to human tumors, and evidence for their ductal/ductular cell origin. Cancer 1985, 55:2411-2416[Medline]
  5. Ikematsu Y, Liu G, Fienhold MA, Cano M, Adrian TE, Hollingsworth MA, Williamson JE, Sanger W, Tomioka T, Pour PM: In vitro pancreatic ductal cell carcinogenesis. Int J Cancer 1997, 72:1095-1103[Medline]
  6. Mangold KA, Hubchak S, Mangino MM, Laconi S, Scarpelli DG: In vitro carcinogenesis of hamster pancreatic duct cells: cellular and molecular alterations. Carcinogenesis 1994, 15:1979-1984[Abstract/Free Full Text]
  7. Schmied B, Liu G, Matsuzaki H, Hernberg S, Moyer MP, Weide L, Murphy L, Batra S, Pour P: Transformation of hamster pancreatic endocrine cells into exocrine cells. Pancreas 1999 (in press)
  8. Takahashi T, Moyer MP, Cano M, Wang QJ, Mountjoy CP, Sanger W, Adrian TE, Sugiura H, Katoh H, Pour PM: Differences in molecular biological, biological and growth characteristics between the immortal and malignant hamster pancreatic cells (published erratum appears in Carcinogenesis 1995, 16: 1257). Carcinogenesis 1995, 16:931-939[Abstract/Free Full Text]
  9. Fujii H, Egami H, Chaney W, Pour P, Pelling J: Pancreatic ductal adenocarcinomas induced in Syrian hamsters by N-nitrosobis(2-oxopropyl)amine contain a c-Ki-ras oncogene with a point-mutated codon 12. Mol Carcinog 1990, 3:296-301[Medline]
  10. Schmied B, Liu G, Moyer MP, Hernberg IS, Sanger W, Batra S, Pour PM: Induction of adenocarcinoma from hamster pancreatic islet cells treated with N-nitrosobis(2-oxopropyl)amine in vitro. Carcinogenesis 1999, 20:317-324[Abstract/Free Full Text]
  11. Sommers S: Unusual carcinoma of the pancreas. Arch Pathol 1954, 58:101-111
  12. Cubilla AL, Fitzgerald PJ: Morphological patterns of primary nonendocrine human pancreas carcinoma. Cancer Res 1975, 35:2234-2248[Abstract/Free Full Text]
  13. Tsutsumi M, Morohoshi T, Konishi Y, Lewandrowski KB, Yong WH: Anaplastic (undifferentiated) Carcinoma: Atlas of Exocrine Pancreatic Tumors. Edited by PM Pour, Y Konishi, G Klöppel, DS Longnecker. Tokyo, Springer-Verlag, 1994, pp 175–184
  14. Watanabe M, Miura H, Inoue H, Uzuki M, Noda Y, Fujita N, Yamazaki T, Sawai T: Mixed osteoclastic/pleomorphic-type giant cell tumor of the pancreas with ductal adenocarcinoma: histochemical and immunohistochemical study with review of the literature. Pancreas 1997, 15:201-208[Medline]
  15. Cubilla A, Fitzgerald P: Tumors of the exocrine pancreas. Cubilla A Fitzgerald P eds. Atlas of Tumor Pathology, 2nd Series, Fascicle 19. 1984, :pp 155-167 Armed Forces Institute of Pathology, Washington DC
  16. Silverman JF, Finley JL, MacDonald KG, Jr: Fine-needle aspiration cytology of osteoclastic giant-cell tumor of the pancreas. Diagn Cytopathol 1990, 6:336-340[Medline]
  17. Pour PM, Egami H, Takiyama Y: Patterns of growth and metastases of induced pancreatic cancer in relation to the prognosis and its clinical implications. Gastroenterology 1991, 100:529-536[Medline]
  18. Westra WH, Sturm P, Drillenburg P, Choti MA, Klimstra DS, Albores-Saavedra J, Montag A, Offerhaus GJ, Hruban RH: K-ras oncogene mutations in osteoclast-like giant cell tumors of the pancreas and liver: genetic evidence to support origin from the duct epithelium. Am J Surg Pathol 1998, 22:1247-1254[Medline]
  19. Imai Y, Morishita S, Ikeda Y, Toyoda M, Ashizawa T, Yamamoto K, Inoue T, Ishikawa T: Immunohistochemical and molecular analysis of giant cell carcinoma of the pancreas: a report of three cases. Pancreas 1999, 18:308-315[Medline]
  20. Mogaki M, Hirota M, Chaney WG, Pour PM: Comparison of p53 protein expression and cellular localization in human and hamster pancreatic cancer cell lines. Carcinogenesis 1993, 14:2589-2594[Abstract/Free Full Text]
  21. Ceha HM, Clement MJ, Polak MM, Offerhaus GJ, Slebos RJ: Mutational analysis of the P16-binding domain of cyclin-dependent kinase 4 in tumors in the head region of the pancreas. Pancreas 1998, 17:85-88[Medline]
  22. Hahn SA, Schutte M, Hoque AT, Moskaluk CA, da Costa LT, Rozenblum E, Weinstein CL, Fischer A, Yeo CJ, Hruban RH, Kern SE: DPC4, a candidate tumor suppressor gene at human chromosome 18q21.1. Science 1996, 271:350-353[Abstract]
  23. Hoorens A, Prenzel K, Lemoine NR, Kloppel G: Undifferentiated carcinoma of the pancreas: analysis of intermediate filament profile and Ki-ras mutations provides evidence of a ductal origin. J Pathol 1998, 185:53-60[Medline]
  24. Lewandrowski KB, Weston L, Dickersin GR, Rattner DW, Compton CC: Giant cell tumor of the pancreas of mixed osteoclastic and pleomorphic cell type: evidence for a histogenetic relationship and mesenchymal differentiation. Hum Pathol 1990, 21:1184-1187[Medline]
  25. Mao C, Guvendi M, Domenico DR, Kim K, Thomford NR, Howard JM: Papillary cystic and solid tumors of the pancreas: a pancreatic embryonic tumor? Studies of three cases and cumulative review of the world’s literature. Surgery 1995, 118:821-828[Medline]
  26. Hoorens A, Gebhard F, Kraft K, Lemoine NR, Kloppel G: Pancreatoblastoma in an adult: its separation from acinar cell carcinoma. Virchows Arch 1994, 424:485-490[Medline]
  27. Morohoshi T, Klöppel G: Pancreatoblastoma: Atlas of Exocrine Pancreatic Tumors. Edited by PM Pour, Y Konishi, G Klöppel, DS Longnecker. Tokyo, Springer-Verlag, 1994, pp 199–206




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