American Journal of Pathology, Vol. 138, No. 3, March 1991 Copright © American Association of Pathologists

Development of Intrapancreatic Transplantable Model of Pancreatic Duct Adenocarcinoma in Syrian Golden Hamsters

Hiroshi Egami,* Tsutomu Tomioka,* Margaret Tempero,t David Kay,t and Parviz M. Pour*t From The Eppley Institute for Research in Cancer and Allied

Dseases,* and the Departments of Internal Medicine,t Pathology, and Microbiology,t University of Nebraska Medical Center, Omaha, Nebraska

Intrapancreatic and subcutaneous (SC) inoculation of cultured pancreatic cancer cells, derived from an induced primary pancreatic cancer in a Syrian hamster, resulted in tumor take in all recipient hamsters. The intrapancreatic allografts grew rapidly, were invasive, and metastasized into the lymph nodes and liver in 2 of 9 cases. In comparison, SC tumors grew relatively slower and formed a large encapsulated mass without invasion and metastases. Histologically, tumors of both sites showed fairly welldifferentiated adenocarcinomas of ductal/ductular type resembling the induced primary cancer. Similar to the primary induced pancreatic cancers, tumor cells of both allografts expressed blood-grouprelated antigens, including A, B, H, Le", Ley, Le, and tumor-associated antigen TAG-72. The tumor cells did not express Lea, CA 19-9, 1 7-1A orDU-PAN-2. The expression of these antigens was retained in the metastases and presented the same patterns of reactivity as the allografts. Thus intrapancreatic transplantation provides a rapid model for production of pancreatic cancer with morphologic similarities to human pancreatic cancer. (Am JPathol 1991, 138:55 7-

561) Pancreatic ductal adenocarcinoma is a lethal disease. Presently, effective therapeutic modalities are unavailable. Although experimental pancreatic cancer models have been useful in understanding carcinogenesis,1 the long latency for tumor induction and uncertainty about tumor development made it difficult to use chemically in-

duced pancreatic cancers in preclinical studies. The disadvantages of existing preclinical models using tumors inoculated subcutaneously (SC) or in cheek pouch tissue2' is that these tumors grow in a different and perhaps privileged environment and, hence, may undergo a selection process. An intrapancreatic location seems to be desirable for these studies. We have shown that the newly established pancreatic cancer cell line (PC-1) from a primary induced pancreatic cancer in Syrian hamster6 can be grown successfully in the subcutaneous and pancreatic tissue of recipient hamsters. We have observed differences, however, in the growth and metastatic potential of the neoplasms between the two transplanted sites. In the present study, we are reporting on the growth, morphology, metastatic patterns, and antigenicity of intrapancreatic allografts of pancreatic cancer cells in comparison with subcutaneous allografts.

Materials and Methods

Animals Syrian golden hamsters (Eppley Colony), 4 to 12 weeks of age, were housed in plastic cages (Macrolon) on granular cellulose bedding (Bed-o-Cobs, Anderson Cob Co., Maumee, OH). They were kept under standard laboratory conditions (temperature, 22 ± 3°C; relative humidity 40% ± 5%; light/dark cycle 12 hours/1 2 hours) and given Wayne pelleted diet (Allied Mills, Chicago, IL) and water ad libitum. Supported by grant 90-25 from the Nebraska Health Department, Laboratory Cancer Research Support grant CA36727 from NCI/NIH, and ACS Special Institutional grant SIG-16. Accepted for publication October 16, 1990. Address reprint requests to Parviz M. Pour, The Eppley Institute for Research in Cancer, University of Nebraska Medical Center, 600 South 42nd St., Omaha, NE 68198-6805.

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Cell Line Pancreatic cancer cell line, PC-1,6 derived from a pancreatic cancer induced by N-nitrosobis (2-oxopropyl)amine (BOP) in a Syrian golden hamster, was established as reported.6 Serial subcultures were performed at a 1:2 or 1:3 split by treatment with 0.05% trypsin and 0.02% ethylenediaminetetra-acetic acid (EDTA) solution (GIBCO, Grand Island, NY). They were maintained in culture medium, RPMI1 640, supplemented with 5% fetal calf serum (FCS), penicillin G (100 lU/ml), amphotericin B (250 ,ug/ml), and streptomycin (100 mg/ml) (antibiotic antimycotic solution, GIBCO) at 300C in a 5% C02 incubator in the plastic culture flasks (Corning 25 cm2/tissue culture flask, Corning, NY). At the time of this report, the cells of PC-1 cells had been maintained in culture for 2 years.

Transplantation of Cultured Cells into the Pancreas and Subcutaneous Tissue of the Hamster Cultured cells were treated with 0.05% trypsin and 0.02% EDTA solution at 37C and detached from the bottom of the culture flasks. The cells were washed with normal saline three times and cell number was counted using a hemocytometer. For intrapancreatic transplantation, 1 06 cultured cells were resuspended in normal saline and injected under Nembutal (Abbott Laboratories, North Chicago, IL) anesthesia (75 mg/kg body weight) into the gastric lobe of nine recipient hamsters by a 26-gauge needle. The same procedure was used for inoculation of tumor cells into the interscapular area of nine other recipient hamsters using a 26-gauge needle. The whole pancreas and the intrapancreatic and subcutaneous tumors were fixed in Bouin's solution, embedded in paraffin, serially sectioned (20 consecutive sections), and processed for histology and immunohistochemistry (see below). In addition, the liver, lung, and axillary and abdominal lymph nodes of tumor-bearing hamsters were examined histologically.

Growth Studies We established the growth curve of intrapancreatic transplanted tumors by measuring the length, width, and

height of the tumors by successive laparotomy, 7, 14, and 21 days after tumor cell inoculation. Incisions were made under Nembutal anesthesia and the wounds were closed by absorbable sutures. Wound healing was rapid and without complication. The use of antibiotics was not necessary. The tumor volume in cubic centimeters was calculated as reported.4

Immunohistochemistry Monoclonal Antibodies

Commercially available monoclonal antibodies (MAbs) against the synthetic trisaccharide of blood groups A, B, and H determinants were used (Dako Co., Santa Barbara, CA). Monoclonal antibodies against Lewis-related antigens were generated and characterized as reported.78 CO19-9 CO17-1 vided by Dr. Z. Steplewski (Wistar Institute, Philadelphia, PA). Productions and characterizations of these antibodies have been reported.913 DU-PAN-2 was a gift of Dr. R. Metzgar (Duke University, Durham, NC) and B72.3 was provided by Dr. J. Schlom (NIH, Bethesda, MD). Productions and characterizations of DU-PAN-2 and B72.3 have been reported.1117

Immunoperoxidase Procedure An avidin-biotin complex (ABC) immunohistochemical method with Vectastain ABC kit (Vector Lab., Burlingame, CA) was used as reported.18 Control slides were examined as follows: 1) tissues were incubated with PBS instead of primary antibodies; and 2) tissues were incubated with nonspecific immunoglobulin, MOPC-21 (Litton Bionetics, Charleston, SC) as IgGl, UPC-10 (Litton Bionetics, Charleston, SC) as IgG2a, and P3 x 68Ag8 myeloma cell culture supernatant as IgM instead of primary MAbs. The staining was scored as described previously6 on the basis of the number of cells showing staining, and samples were placed into one of the following categories: 0% (-); less than 5% (1 +); 5% to 30% (2 +); 30% to 70% (3 +); and more than 70% (4+).

Table 1. Patterns of Tranplanted Pancreatic Cells of PC-1 Cells in Syrian Hamsters Incidence of Incidence of Incidence of Injection tumor growth liver metastasis lymph node metastases site (%)t (%)t Pancreas 9/9 (100) 2/9 (22) 1/9 (1 1) Subcutaneous 9/9 (100) 0/9 (0) 0/9 (0) * No, of hamsters with tumors/No. of hamsters inoculated. t No. of hamsters with metastases/No. of hamsters with tumors.

Incidence of carcinomatosis

(%)t 1/9 (1 1) 0/9 (0)

Intrapancreatic Transplantation of Cancer Cells

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2.0

_

: Intrapancreatic

Transplants (n=9)

-

E

- -o :Subcutaneous

0 a

Transplants (n=9)

E0

h-

1 .0

0 N

c',

0

Figure 1. Growth rate of intrapancreatic and subcutaneous allografts of PC-1 cells.

Time after Transplantation

Results

ate the epidermis and all animals were killed. Also at this stage no evidence for metastases was found.

(days)

Tumorigenicity and Growth Characteristics Table 1 summarizes the frequency of tumor take and metastases of intrapancreatic and SC transplants. Metastases into the liver and regional lymph nodes and peritoneal carcinomatosis were found only in the host hamsters with intrapancreatic transplants. Other tissues examined were free of metastases. The growth curve of transplanted tumors is shown in Figure 1. The intrapancreatic allografts grew rapidly and were readily visible 7 days after tumor inoculation. After 3 weeks, the average size of tumors reached 1.8 cm3. Because of the advanced tumor growth and metastases, all hamsters with intrapancreatic tumors were euthanized after 3 weeks. The growth of SC allografts was slower than that of intrapancreatic transplants. Tumors were palpable after 7 days and reached a size of about 1 cm3 after 21 days. Because tumors were thought to be well demarcated and showed no evidence of metastasis, they were allowed to grow for 8 weeks, at which time they reached a size of about 13 cm3. At this size, tumors began to ulcer-

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Morphology A typical macroscopic appearance of an intrapancreatic transplant is depicted in Figure 2. Tumors were firm, round, or lobulated; yellowish white when small, and dark red when more than 0.5 cm3. Areas of small cystic pattems and hemorrhage were encountered in the cut surface of large tumors. The SC transplants were well circumscribed, elastic, and yellowish-white; large tumors showed extensive cystic and necrotic areas on sectioning.

Histologically, the principal structure of all tumors was of ductal/ductular adenocarcinoma, indistinguishable from a primary induced cancer in this species (Figure 3). Areas of mucus production and fibrosis were seen primarily in SC transplants, which also exhibited inflammation and necrosis, particularly extensive in large tumors more than 1 cm3 in size. Metastases to the regional lymph node and into the

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Figure 2. Intrapancreatic allograft of PC-1 cells 3 weeks after tumor cell inoculation. PT, intrapancreatic tumor (arrows depict the extent of cancer); LM, liver metastasis; PM, peritoneal metastases.

Figure 3. Histologic appearance of intrapancreatic allograft exbibiting a well-differentiated cancer with entrapped acinar cells between the malignant glands (H&E stain, x200).

560 Egami et al AJP March 1991, Vol 138, No. 3

Table 2. Expression of Antigens in the Allografts of PC-1 Cells and in TheirMetastases Site of tumor

Antigen

Pancreas

SC

A B H Leb Lex Ley

++++ ++ ++ +

++++ ++ ++ +

+ +

+ +

+

+

TAG-72

Liver metastasis

LN metastasis

++++

++++

++

+

++ + -

+ + +

+

+

+

-

The reactivities with anti-Le*, -CA19-9, -DU-PAN-2, and -17-lA were negative.

liver, and particularly, perineural invasion and peritoneal carcinomatosis were found in two hamsters with intrapancreatic tumors (Figure 2). The SC transplants did not show any invasion or metastases in any tissues examined, even after 8 weeks of growth and ulceration of the skin.

Expression of Antigens Immunohistochemical findings are listed in Table 2. The cells of both allografts expressed A (100%, Figure 4), B, and H (each 20%), Leb, Ley, Lex, and TAG-72 antigens (each

Development of intrapancreatic transplantable model of pancreatic duct adenocarcinoma in Syrian golden hamsters.

Intrapancreatic and subcutaneous (SC) inoculation of cultured pancreatic cancer cells, derived from an induced primary pancreatic cancer in a Syrian h...
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