[CANCER RESEARCH 46. 5810-5815, November 1986]

Morphological, Biological, Biochemical, and Karyotypic Characteristicsof Human Pancreatic Ductal Adenocarcinoma Capan-2 in Tissue

Culture and the Nude MouseAikaterini A. Kyriazis, Andreas P. Kyriazis,1 Cora N. Sternberg, Nathan H. Sloane, and James D. Loveless

Department of Pathology, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey 07103 [A. P. K„A, A. K.J; MemorialSloan-Ketlering Cancer Center, New York, New York 10021 fC. N. SJ; Department of Biochemistry, University of Tennessee, Memphis, Tennessee 38163 fN. H. SJ;and Sloan-Kettering Institute for Cancer Research, Rye, New York 10580 [J. D. LJ

ABSTRACT

Human pancreatic ductal adenocarcinoma Capan-2, derived from a 56-yr-old male Caucasian, has been studied in both tissue culture and thenude mouse. In tissue culture, tumor cells showed epithelial-like features,whereas in the nude mouse, the tumor grew as a well-differentiatedadenocarcinoma, resembling histopathologically the original neoplasm.Ultrastructurally, the neoplastic cells showed characteristics of ductalepithelium. The allozyme phenotypic profile of Tumor Capan-2 wasdetermined in eight genetically determined loci, and chromosome studiesshowed a hypotetraploid pattern with a number of morphological andnumerical changes. Carcinoembryonic antigen was produced in traceamounts, and lactate dehydrogenase was represented only by Isoenzyme5, regardless of environmental conditions. The characteristics of Capan-2 tumor make it a valuable addition to the small number of availablepancreatic tumor lines in studies aiming at clarifying certain aspects ofthe biology of this type of malignancy.

INTRODUCTION

The usefulness of the nude mouse as an experimental modelin cancer research is becoming increasingly evident. This isprimarily due to its ability to sustain a large number of humanneoplasms following transplantation of established tumor celllines and primary xenografts removed at surgery. The fact thatnude mouse transplanted human tumors preserve their morphological and biological integrity through successive transplant generations would facilitate an in-depth study of variousfactors which may determine tumor biological behavior andtumor response to various therapeutic modalities.

With these objectives in mind, efforts have been made byvarious investigators to establish in vitro and in vivo lines ofadenocarcinomas of the human exocrine pancreas. The importance of these efforts becomes evident considering that pancreatic cancer is an extremely aggressive tumor ranking fourthas a cause of cancer deaths, exceeded only by cancer of thelung, large bowel, and breast (1). More than 21,000 cases arediagnosed each year (2) with a 5-yr survival rate less than 1%

(3).As of today, only a small number of established pancreatic

tumors have been reported (4-11), and fewer have been adequately characterized. Furthermore, of these only occasionalcases have been studied in the nude mouse.

As a continuation of our efforts to establish and characterizea series of ductal adenocarcinomas of the human pancreasencompassing a wide spectrum of histopathological and biological features, we report our studies on Tumor Capan-2 whichwas derived from a pancreatic adenocarcinoma of ductal origin.Tumor Capan-2 has been studied in tissue culture and the nudemouse with regard to growth characteristics, histopathological

Received 3/10/86; revised 8/4/86; accepted 8/5/86.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1To whom requests for reprints should be addressed.

appearance, ultrastructural morphology, and production of biological markers such as LDH2 and CEA. The karyotype of the

line and its allozyme phenotypic profile were also evaluated.

MATERIALS AND METHODS

Cell line Capan-2 was established at Sloan-Kettering Cancer Center,Rye, NY, by Dr. J. Fogh and J. D. Loveless from a pancreatic adenocarcinoma of a 56-yr-old male Caucasian. The patient underwent totalpancreatectomy and cholecystectomy, partial gastrectomy and largeand small bowel omentectomy, and splenectomy. The tumor involvedthe head of the pancreas and infiltrated the muscularis of the duodenalwall distal to the ampula and the peripancreatic fibroadipose tissueposteroinferiorly. Histopathologically the tumor was characterized as"carcinoma of the head of the pancreas, ductal type." Preoperatively,serum CEA was 7.3 ng/ml; a-1 fetoprotein was negative. The patient's

ABO blood group was B positive. Postoperatively the patient receivedchemotherapy; he died 6 yr and 9 mo later.

At the present time cell line Capan-2 is maintained in RPMI-1640medium containing 15% fetal calf serum and supplemented with penicillin (100 lU/ml) and streptomycin (100 Mg/ml) (GIBCO). Tumorcells grow in monolayer and are passed every 3 to 4 wk. The culture isfree of Mycoplasma contamination (Mycoplasma medium; GIBCO).

Plating Efficiency. Plating efficiency was determined by plating 2.5x IO5 viable cells in 25-cm2 tissue culture flasks using RPMI-1640

medium. Fifteen hr later, the medium was discarded, and the attachedcells were collected following trypsinization and counted. The experiment was run in triplicate.

Cell Doubling Time. Cell doubling time was determined by countingthe number of viable cells from freshly trypsinized monolayers. Sixteen25-cm2 tissue culture flasks, each receiving 5 x IO5 cells, were used.

Cell countings, in duplicate, were performed at 24 li intervals for 8days. Throughout the entire procedure cell viability was determined bymeans of the trypan blue exclusion method.

Determination of Human LDH and CEA. The Corning agarose universal electrophoresis system (Corning, Palo Alto, CA) was used todifferentiate and quantitate human LDH isozymes in tissue culturemedia, cell extracts, and plasma of tumor-bearing mice. Human andmouse isozymes were separated on the basis of different electrophoreticmobilities (9, 12, 13), and quantitation was done on a colorimetriedensitometer (Helena Quick Scan, Jr.; Helena Laboratories, Beaumont,TX). CEA assays were performed on the plasma of tumor-bearing miceby the Abbott CEA-EIA diagnostic kit (Abbott Laboratories, NorthChicago, IL).

Enzyme Phenotypic Profile. The genetically determined enzyme phenotypic profile of cultured cells was determined according to the methodof Halton et al. (14).

Cytogenetics. Cytogenetic analysis, including G banding, on culturedtumor cells was done by the methods described by Peterson et al. (15)and Seabright (16).

Nude Mice. Female athymic mice of Swiss background from ournude mouse colony were maintained in a laminar air flow clean roomunder controlled temperature and humidity.

Collection of Mouse Plasma Samples. For the determination of

2The abbreviations used are: LDH, lactate dehydrogenase; LDH-1, LDH-2,LDH-3, LDH-4, LDH-5, láclatedehydrogenase isoenzymes 1 to 5; CEA, carci-noembryonic antigen; GIBCO, Grand Island Biological Co.

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PANCREATIC CANCER CAPAN 2 IN TISSUE CULTURE AND NUDE MICE

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Fig. 1. In /(, cell line Capan-2 grown in tissue culture is characterized by large epithelial-like cells with large, oval, round, or indented nuclei: chromatin clumping;and basophilic granulated cytoplasm. Phase contrast, x 200 (passage 16). B, tumor cell line Capan-2 grown in the nude mouse, fourth transplant generation.Microscopically, the neoplasm is made up of duct-like structures lined with tall columnar epithelial cells showing mild nuclear pleomorphism, hyplerchromasia, andoccasional mitoses. Stratification of neoplastic cells is also observed. H & E, x 250.

Table I Enzyme phenotypic profile of Tumor Capan-2

CelllineCapan-2

HelaPOMI*1 1PGM32 1ESD1 1Me-22 1-2AK-11 1GLO12 2G6PDB ALDHHuman*Unni.Ili'Phenotype

frequency0.0006

0.000236°PGM, phosphoglucomutase, EC 2.7.5.1; ESD, esterase D, EC 3.1.1.1; Me-2, malate dehydrogenase, EC 1.1.1.40; AK-1, adenylate kinase, EC 2.7.4.3; GLO1,

glyoxylase 1, EC 4.4.1.5; G6PD, glucose-6-phosphate dehydrogenase, EC 1.1.1.49; LDH, EC 1.1.1.27.* Only Isoenzyme 5 was present.c All isoenzymes present.

o>

CAPAN-2

10i r12 13

WEEKS

\14 15 17

I16

Fig. 2. Growth curve of Capan-2 tumor grown s.c. in the nude mouse (fourthtransplant generation). Points, mean of 6 animals, bars, S.D. Arrow, time oftransplantation.

human LDH and CEA present in the plasma of tumor-bearing mice,blood was drawn by orbital venipuncture with heparinized microcapil-lary tubes. Plasma was separated by centrifugation and stored at —80°C

until used.Determination of LDH in tissue culture growing cells was done as

follows. Cultured cells were harvested, washed 3 times with serum-freemedium, and packed by centrifugation. To the packed cells Tris-HCI:EDTA buffer was added in the ratio of 1:1 (v/v, 0.05 M Tris-

HC1:0.001 M disodium EDTA). Cells suspended in buffer were disrupted by freezing-thawing in dry ice:acetone and centrifuged to removeinsoluble matter, and the supernatant containing the cytosol was usedfor LDH determination. To determine the presence of LDH releasedinto the culture medium, triplicate cultures growing in 25-cm2 flasks

were used. At approximately 75% culture confluency the culture medium was removed, monolayers were washed repeatedly with serum-free medium, and then incubated for 24 h with the same serum-freemedium. Twenty-four-h culture media were collected, centrifuged toremove particulate matter, and concentrated to 100 x using a minicon-centrator unit (Amicon Corp., Lexington, MA). Concentrated mediawere assayed for LDH immediately or were kept frozen at -80°Cuntil

used.Solid Tumors in Nude Mice. For injection into nude mice cultured

tumor cells were dispersed with 0.5% trypsin:0.2% EDTA in Hanks'balanced salt solution (GIBCO) and adjusted to 1 x IO6viable cells/

0.2 ml. Cell viability was determined by the trypan blue dye exclusiontest. Initially, s.c. tumors were established by giving the animals injections of 1 x IO6viable tumor cells. In subsequent experiments, tumor

propagation was accomplished by transplanting s.c. through a skinincision in the anterior lateral thoracic wall a small piece of tumormeasuring approximately 6 mm' (17). The size of nude mouse-grown

tumors was measured in 2 dimensions with calipers, and tumor volumewas calculated weekly by using the following formula (18):

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Fig. 3. Electron micrograph of Tumor Capan-2 grown in tissue culture (A). The neoplastic cells are characterized by surface microvilli, interdigitation of cellmembranes, and tight junctions connecting adjacent cells. The cytoplasm contains a Golgi complex, short profiles of smooth and rough endoplasmic reticulum,mitochondria, few free ribosomes, and occasional mucigen granules. Uranyl acetate:lead citrate, x 7100. Electron micrograph of Tumor Capan-2 grown in the nudemouse (/'}. The apical plasma membrane shows well-developed surface microvilli. The cytoplasm contains short profiles of smooth and rough endoplasmic reticulum,mitochrondria, few free ribosomes, and occasional mucigen granules. Adjacent cells show infoldings of the basal cytoplasm with the presence of terminal bars, tightjunctions, and well-developed desmosomes. Uranyl acetate-lead citrate, x 9150.

Length x (width)2 x 0.4

Nude mouse-grown tumors were removed, fixed in buffered formalin,embedded in paraffin, and cut and stained with hematoxylin:eosin,periodic acid-Schiff and mucicarmine.

For transmission electron microscopy, tumor tissue was cut intofragments 0.5- to 1-mm thick and fixed in cold 2.5% glutaraldehyde in0.1 M sodium phosphate buffer, pH 7.4, immediately after removal.They were embedded in LX-112 resin, and thin sections were preparedwith a Leitz ultramicrotome, stained with uranyl acetate:lead citrate,and examined in a Philips EM-301 electron microscope. Studies ofTumor Capan-2 in tissue cultures were performed on passages 14through 25, cytogenetic studies were on passages 16 and 20, and nudemouse growth characteristics were followed for a period of 10 successivetransplant generations (1 through 10).

RESULTS

Tissue Culture Characteristics. Tumor Capan-2 grown intissue culture was composed of cells exhibiting epithelial-likemorphological characteristics. They were ovoid or round characterized by round, oval, or indented nuclei with prominentnucleoli, chromatin clumping, and moderately basophilic cytoplasm (Fig. 1). Histochemical studies for cytoplasmic mucinwere positive. The plating efficiency of the cultured cells was55%, and doubling time was calculated at 96 h.

The allozyme phenotypic profile of Capan-2 tumor was studied at eight genetically determined loci, and comparison wasmade with HeLa cells. The results of these studies are presentedin Table 1.

Tumor Growth in Nude Mice. Transplantation of 1 x IO6

viable tumor cells s.c. in nude mice resulted in tumor growthhaving a latency period, the time between transplantation andfirst positive evidence of tumor growth, of 4 to 6 wk. To furtherestablish the growth characteristics of nude mouse-grown tumors, solid pieces of growing tumors measuring approximately0.3 x 0.2 cm were transplanted s.c. as described in "Materialsand Methods." The follow-up of many consecutive transplant

generations showed a consistently reproducible growth pattern(Fig. 2).

Microscopically, s.c. growing tumors exhibited characteristics of a well-differentiated adenocarcinoma, similar to that ofthe original neoplasm. They were made up of glandular structures supported by fibrovascular stroma. The neoplastic cellswere tall columnar, showing mild nuclear polymorphism, hy-perchromasia, and occasional mitoses. Stratification of neoplastic cells was also observed (Fig. 1). The cytoplasm wasamphophilic or slightly basophilic. Histochemical studies revealed cytoplasmic mucin in the form of fine granules occupyingthe apical portion of the cytoplasm. The tumor invaded itspseudocapsule with evidence of micro- and macroinvasion.Criteria for invasion were the same as described previously (19).

Ultrastructurally, the characteristics of tumor cells growingin both tissue culture and the nude mouse were those of ductalepithelium (20, 21) and, with the exception in the amount ofintracytoplasmic mucin, showed close similarity to those reported for Capan-1 and SW-1990 tumor lines (8, 9). The apicalplasma membrane consisted of well-developed microvilli. Thecytoplasm contained short profiles of smooth and rough endoplasmic reticulum, well-developed mitochondria, and few free

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PANCREATIC CANCER CAPAN-2 IN TISSUE CULTURE AND NUDE MICE

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Fig. 4. LDH ¡soenzymeelectrophoretogram. Pattern 1 represents the humanisoenzyme profile. The slower migrating band (extreme left) is human LDH-S.Other visible bands are, from left to right, human LDH-4, -3, -2, and -I. Pattern2 shows the mouse LDH isoenzyme profile. Note that LDH-5, which is thedominant LDH isoenzyme in the mouse, occupies a position between humanLDH-4 and LDH-3. Pattern 3 represents the LDH profile of plasma of nudemouse-bearing Tumor Capan-2. In this pattern only human LDH-S is recognized.Pattern 4 represents the LDH isoenzyme profile of cell extracts of Tumor Capan-2, and Pattern 5, that of culture medium of cells Capan-2. In both patterns onlyisoenzyme LDH-5 is represented. LDH-4, -3, -2, and -1 are not identifiable.Arrows, point of origin.

Si«H

2O->

006 at 10TUMOR WEIGHT . c

Fig. 5. Relationship between tumor weight and circulating human LDH inthe nude mouse-bearing Tumor Capan-2. Points, single animal. U/L, units/liter.

Table 2 Chromosome frequency distribution of cell line Capan-2

Of the 100 metaphases scanned for ploidy, 13 metaphases had 46 to 48chromosomes, 81 metaphases had 60 to 68 chromosomes, 2 metaphases had 90+chromosomes, and 4 metaphases had 120+ chromosomes.

No. ofmetaphasesNo.

ofchromosomespreciselycounted1461602661671668

ribosomes. Typical mucigen granules were also seen in a number of cells. Adjacent cells showed extensive infoldings of thebasal cytoplasm with the presence of terminal bars, tight junctions, and well-developed desmosomes (Fig. 3).

Production of LDH and CEA. Our studies indicated thattumor cell line Capan-2 preserved the ability to elaborate LDH

5813

under different environmental conditions. LDH expressed entirely at LDH-5 was present in measurable amounts in bothtissue culture media and cell extracts. The ability of tumor cellsto produce LDH continued in the nude mouse where it wasdetected over a wide range of tumor burden. Fig. 4 shows theLDH isozyme profile under varying experimental conditions,and the existing relationship between tumor weight and circulating LDH in the nude mouse is given in Fig. 5.

Studies of CEA production showed only trace amounts circulating in the serum of tumor-bearing mice.

Chromosome Studies. In Tumor Capan-2, chromosomecounts ranged from 45 to 68 with most chromosome countsbeing in the hypotetraploid range (Table 2). Most of the chromosomes were trisomie with the exception of chromosomes 14,15, and 21. Chromosome 14 was absent in 4 of 6 karyotypesand monosomic in 2 of 6. Chromosome 15 was monosomic in4 of 6 karyotypes, absent in 1, and 2 copies of it were presentin only 1 karyotype. Monosomic, also, was chromosome 21 inmost of the karyotypes. There were 5 identifiable marker chromosomes. The marker ml was a lp~, m2 was an ¡(14),m3 was

a translocation between 12q and 6q arms, ni4 was a chromosome 9 with part of another chromosome translocated to the qarm, and m5 appeared to be an llp~q~. In addition, 4 to 7

unidentifiable marker chromosomes were present in all karyotypes studied (Fig. 6).

DISCUSSION

There are two main concerns regarding tumors propagatedin tissue culture or the nude mouse:tumor integrity and tumorbiological stability. Studies of glucose-6-phosphate dehydrogen-ase identified Tumor Capan-2 as type B, thus confirming itsorigin from a Caucasian patient. Furthermore, the enzymephenotypic profile made it distinct from any other tumor line,providing at the same time a reliable internal control againstany accidental inter- or intraspecies contamination. CulturedCapan-2 cells preserved their epithelial character regardless ofthe number of passages, and in the nude mouse the tumorexhibited a well-differentiated pattern similar to that of theoriginal tumor. We have followed Capan-2 through a numberof successive transplant generations without any evidence ofchanges in growth pattern and histopathological appearance,an observation we have reported for a number of nude mouse-grown human tumors of different histogenetic backgrounds (8,9, 13) and which has been confirmed in more recent reports(22, 23).

With regard to cytogenetic studies, of interest was the observation that all chromosomes were of human origin showing aconsistent pattern which also reflected both qualitative andquantitative changes in a number of karyotypic profiles. Inaddition, marker chromosomes 1 to 5 were present in allkaryotypes examined. It is of interest that marker lp~ has been

reported previously for another pancreatic adenocarcinoma line(20). Whether, however, the observed morphological and numerical changes relate to those of other pancreatic carcinomasis not clear at the present time. The number of pancreaticcarcinomas in which detailed chromosome analysis has beenperformed is very small (9, 10, 24), thus precluding any generalization of the reported findings. It is hoped that, as morecases are studied, analysis of accumulated data may permit thedistinction between primary and secondary karyotypic changes.

As in previous cases, LDH was selected as a tumor markerbecause: (a) it is present in all human cells; (h) qualitative andquantitative determinations are easy to perform; and (c) the

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PANCREATIC CANCER CAPAN-2 IN TISSUE CULTURE AND NUDE MICE

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Fig. 6. Karyotype of cell line Capan-2 in the near-Iriploid range. Note absence of chromosome 14 and monosomic IS and 21 chromosomes. Y chromosome ismissing. X chromosomes are present.

difference in electrophoretic mobility between human andmouse LDH makes possible their separation and quantitation.This latter property makes LDH a useful marker in identifyingthe human origin of any tumor grown in the nude mouse, andunder certain conditions, it may be used as an approximateindicator of tumor burden. It is worth noting that TumorCapan-2 was represented exclusively by LDH-5 regardless ofthe environmental conditions (Fig. 4). This, most probably,accounts for the high correlation coefficient between theamount of LDH in the plasma of tumor-bearing mice andtumor burden (Fig. 5). It should be recalled here that mouseLDH-5, which is the dominant mouse LDH isozyme, moveselecrophoretically between human LDH-4 and LDH-3 (Fig. 4),and in a mixture of mouse and human LDHs, broad overlappingof isozymes of different origin does occur in this region. Thisis probably one of the obstacles in successfully correlating theamount of circulating human LDH and tumor burden in tumorselaborating the entire LDH isozyme spectrum (9, 13). Capan-

2 tumor is the second pancreatic adenocarcinoma of ductalorigin we are reporting as elaborating only LDH-5 (8). Whetherthis represents a characteristic of this type of malignancy iscurrently under investigation in our laboratory.

CEA associated with pancreatic adenocarcinomas has beenreported in pancreatic cancer patients (24) and in pancreaticcancer lines in vivo and in vitro (9, 25, 26). Capan-2 tumorproduces CEA, although in very small quantities. This, undoubtedly reflects the degree of its differentiation and correlateswell with the clinical history of the patient from whom thetumor originated.

In conclusion, Capan-2 tumor derived from a human pancreatic adenocarcinoma of ductal origin shows histopathologi-

cal, biological, and biochemical characteristics similar to thatof the original tumor. It grows well in tissue culture and thenude mouse and exhibits a distinct enzyme phenotypic profile.The low amount of CEA produced, most probably, reflects itswell-differentiated character. In addition Capan-2 presents theunique feature of elaborating only LDH-5, an observation madepreviously on another pancreatic carcinoma line (8). In view ofthese characteristics Tumor Capan-2 may contribute in improving our understanding of certain aspects of the biology of humanpancreatic cancer, and it may be used, along with other well-characterized pancreatic carcinoma lines, in furthering ourknowledge of response to treatment of this type of malignancy(27, 28).

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1986;46:5810-5815. Cancer Res   Aikaterini A. Kyriazis, Andreas P. Kyriazis, Cora N. Sternberg, et al.   Capan-2 in Tissue Culture and the Nude MouseCharacteristics of Human Pancreatic Ductal Adenocarcinoma Morphological, Biological, Biochemical, and Karyotypic

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