Human pancreatic carcinoma (mia paca-2) in continuous culture: Sensitivity to asparaginase

Int. J . Cancer: 19, 128-135 (1977)

HUMAN PANCREATIC CARCINOMA (MIA PaCa-2) IN CONTINUOUS CULTURE : SENSITIVITY TO ASPARAGINASE Adel A. YUNIS,Grace K. ARIMURA and David J. RUSSIN Departments of Medicine and Biochemistry, University of Miami School o j Medicine, and the Howard Hughes Medical Institute, Miami, Florida, USA

An undifferentiated human pancreatic carcinoma has been established in continuous culture and i s grown in Dulbecco's modified Eagle's medium fortified with 10% fetal calf serum and 2.5% horse serum. The established cell line ( M I A PaCa-2) has a doulbing time of 40 h. The cells are large with abundant cytoplasm, exhibit a high degree of aneuploidy and have a tendency to grow on top of other cells. M I A PaCa-2 grows in soft agar with a colonyforming efficiency of 19%. Both M I A PaCa-2 cells and a cell line from another pancreatic carcinoma obtained from National Cancer Institute ( N C I ) are sensitive to asparaginase, a property not shared by several other human tumor cell lines tested.

Pancreatic carcinoma is currently considered to be one of the major lethal cancers in the United States. It is usually a rapidly progressive and fatal disease, and is typically diagnosed late in its natural course. Various therapeutic modalities used to date including surgery, radiotherapy and chemotherapeutic agents have generally been unrewarding. Knowledge of the biological properties of this tumor has been limited due to lack of an experimental model. Thus, permanent cell lines established from human pancreatic cancer could provide important potential avenues of progress in this area. In this report we describe the establishment and partial characterization of a cell line from an undifferentiated pancreatic carcinoma. In addition, we include some recent comparative studies with another pancreatic carcinoma cell line (PANC-1) supplied to us by Lieber et al. (Lieber et al., 1975). MATERIAL AND METHODS

All media, fetal calf serum and horse serum were obtained from Grand Island Biological Co., Grand Island, N. Y . Tumor tissue was obtained at operation from a 65-year-old Caucasian man who presented to the hospital with recurrent abdominal pain of 6 months' duration, and a palpable upper abdominal mass. Upon abdominal exploration, a large tumorous mass was found involving the body and tail of the pancreas with induration and fixation to the periaortic area. Multiple biopsies, including two from the pancreatic mass, were taken for histology and tissue culture.

Method o j culture

A piece of tumor about 2.0cm in diameter was divided into two segments, each of which was placed in a 100mm Falcon tissue culture Petri dish and minced with a pair of sharp scissors into 1 to 2 mm pieces. The tissue fragments in each dish were dispersed in 5 ml of Dulbecco's modified Eagle's medium containing 10 % fetal calf serum and 2.5 % horse serum (DME-HSFC), and incubation was carried out at 37" C in 5 % COz in air. Serial transfer of primary and established cultures was carried out by trypsinization usiilg 0.1 % trypsin in phosphatebuffered saline. Cells were plated in 100 mm culture dishes containing 10 ml of medium. Culture in agar Established cells were cultured in agar according to the method of Bradley and M2tcalf (Bradley and Metcalf, 1966) as previously detailed (Yunis et al., 1975). Double-strength DME-HSFC was mixed with an equal volume of 0.6% agar in water. Cells were added to this medium-agar mixture to give concentrations ranging from 1,000 to 10,000 cells/ml and the mixture was plated in 1 ml aliquots. Incubation was carried out at 37" C in 10% COs in air. On day 6 the number of colonies was determined with a dissecting microscope at x 25. Histochemistry

For alkaline phosphatase stain freshly prepared smears were fixed in 95 %ethanol for 30 sec, followed by incubation for 30 min at room temperature in a substrate containing a-naphthyl phosphate, 0.1 mg/ ml, and fast red-violet LB salt, 1 mg/ml in 0.1 M Tris buffer PH 8.6. Histochemical staining for fat was carried out with Sudan Black B (Sheehan and Storey, 1947). Test for carcionoembryonic antigen The test was performed according to published procedures (Gold and Friedman, 1965, Coligan et al., 1972). Received: July 19, 1976 and in revised form September 29,1976.

129

PANCREATIC CARCINOMA IN CULTURE

Effect of Chemotherapeutic agents The sensitivity of established cultures to a number of chemotherapeutic agents was tested. Each individual drug was added to the cultures at the indicated final concentrations on the second or third day after plating. At intervals the cells in duplicate plates were collected by trypsinization and counted. RESULTS

Histological examination of the biopsy from the pancreatic mass revealed an undifferentiated carcinoma (Fig. 1 ~ ) .Dispersed small tissue pieces, cultured as described in “ Methods ”, adhered to the bottom of the plate and became surrounded within a week by fibroblasts with a few scattered round cells. After 2 weeks, the plates were filled with fibroblasts but with a few overlying compact colonies of epithelial-like cells. After about 1 month in primary culture with a change in medium every 3 days, the epithelial cells became predominant, replacing virtually all the fibroblasts. At this time the plates were trypsinized and the first transfer was carried out. The epithelial cells adhered immediately and began to form colonies, and fibroblasts were not seen

FIGURE 1

Histologic section of pancreatic tumor showing an undifferentiated carcinoma. x 440. Chromosome analysis Chromosome preparations were made by the method of Moorhead et al. (Moorhead et al., 1960). Actively growing cultures were exposed to colchicine for 3 h after which cells were removed by trypsinization, suspended in medium, centrifuged lightly, exposed to a hypotonic medium for 17min and fixed. One to two drops of the cell suspension were air-dried rapidly and the spreads were stained with Giemsa stain and mounted with Permount for examinat ion. Assay for fibrinolytic activity The cultured tumor cells were tested for their ability to secrete fibrinolytic activity as follows: when cell growth had covered more than 80 %of the surface area of the dishes, the culture medium was aspirated and the dishes were each rinsed three times with 5 ml Hanks’ BSS. Serum-free medium (3 ml/dish) was then added and incubation continued for an additional 48 h, after which the medium was harvested. The serum-free conditioned medium thus prepared was assayed for fibrinolytic activity by the heated and unheated fibrin-agar plate method (Nilsson et al., 1972, Lassen, 1952).

FIGURE 2

Six-day-old culture of MIA PaCa-2.

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YUNIS ET AL.

thereafter. The epithelial cell line (Fig. 2) thus established (MIA PaCa-2) has to date undergone over 100 transfers. It has a doubling time of 40 h (Fig. 3). Cells grow in large colonies, and some have a tendency to " round up " on top of others (Fig. 4) and then become free in suspension. The cells are large with abundant cytoplasm and nuclei containing from two to four nucleoli (Fig. 5 ) . Histochemical

0

.

1

2

1

4

8

6

10

12

I

14

DAYS

FIGURE 3

Growth curve of MIA PaCa-2. Cells were grown in DME-HSFC.

FIGURE 5

Wright-stained preparation of MIA PaCa-2. x 430.

stain for alkaline phosphatase activity was negative. Sudan Black B stain was strongly positive (Fig. 6). Growth in agar was used as an in vitro test of malignant potential of the cell line. MIA PaCa-2 formed discrete colonies (Fig. 7) with an average efficiency of 19 %. Chromosomal studies Chromosomal counts of MIA PaCa-2 revealed a highly aneuploid pattern with the number of chromosomes ranging between 58 and 71 (Table I).

FIGURE 4

Nine-day-oldculture of MIA PaCa-2 showing tendency of cells to grow on top of others.

Electron microscopy Electron microscopy was unrevealing except for the presence of fat droplets. No viral particles were observed.

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PANCREATIC CARCINOMA IN CULTURE

Phenotyping the cells for glucose 6-phosphate dehydrogenase revealed only type-B isoenzyme. Effect of chemotherapeutic agents In preliminary studies the sensitivity of MIA PaCa-2 to a selected number of chemotherapeutic agents was examined. The effect of 1, 3-bis-(2chloroethy1)-1-nitrosourea (BCNU), Ara C, vincristine and L-asparaginase is illustrated in Figure 8. Growth inhibition was greatest with vincristine &nd L-asparaginase. To determine whether this effect was specific for MIA PaCa-2 two other human tumor cell lines were txted: breast carcinoma and melanoma. MIA PaCa-2 did not differ in its sensitivity to vincristine from human breast carcinoma

FIGURE 6 Sudan B black stain of MIA PaCa-2 cells showing presence of fat granules. x 600.

Secretion of fibrinolytic activity

Secretion of fibrinolytic activity by cultured mammalian tumor cells has been described (Yunis et al., 1973). Conditioned serum-free medium prepared from MIA PaCa-2 and PANC-1 as described above contained fibrinolytic activity as assayed on unheated fibrin-agar plates. The presence of plasminogen activator in serum-free conditioned medium was established by direct activation of plasminogen (not illustrated). The purification and characterization of this plasminogen activator will be the subject of a future publication. The following assays were also carried out on conditioned medium and cell extracts of MIA PaCa-2 and were found to be negative: trypsin, chymotrypsin, and carcinoembryonic antigen.

FIGURE7 colony growth of MIA paCa-2 in soft agar.

TABLE I CHROMOSOME COUNTS FROM MIA PaCa-2 PREPARATIONS

Chromosome number

No. of cells analysed SS

128

4

59

1

60

1

61

2

2

63

62

4

8

64

2

2

6s

2

67

66

6

3

68

3

1

69

3

71

70

9

1

2

132

YUNIS ET AL.

10.0. 8.0 -

(Fig. 9~ and B), melanoma or rat breast carcinoma (not illustrated). However, the sensitivity of MIA PaCa-2 to L-asparaginase appeared to be relatively specific. The effect of various concentrations of L-asparaginase on the rate of growth of MIA PaCa-2 and human breast carcinoma is shown in Fig. 1 0 ~ and B. As little as 0.1 unit/ml of L-asparaginase caused significant inhibition of growth of MIA PaCa-2, with complete inhibition and extensive cell death occurring at concentrations of 0.5 and 1 unit/ ml. In contrast, these concentrations exerted little effect on the growth of human breast carcinoma cells. The comparative effect of 1 unit/ml of Lasparaginase on the growth of MIA PaCa-2, PANC-1, human melanoma, and human breast carcinoma is shown in Figure 11. It can be seen that the sensitivity of MIA PaCa-2 to L-asparaginase is

COHTROL

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1

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1

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8

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I

10

DAYS FIGURE 8 Effect of BCNU, Ara C, vincristine and L-asparaginase on the growth of MIA PaCa-2 in culture (see text for

methods).

S.Or

CONlROL

shared only by PANC-1, another pancreatic carcinoma cell line. Not only was the growth inhibited but there was also extensive cell killing in both cell lines. In contrast, L-asparaginase had relatively little effect on melanoma and breast carcinoma. This pattern of differential sensitivity of these cell lines to L-asparaginase has been consistent in the numerous experiments which have been carried out over the past year. DISCUSSION

Our primary objective in establishing h m a n pancreatic carcinoma in culture is to use it as an

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CONTROL

FIGURE 9 Effectof vincristine on thegrowth of MIA PaCa-2 (A) and human breast carcinoma cells (B) in culture.

0.01 1g/nl.

DAYS

DAYS

133

PANCREATIC CARCINOMA IN CULTURE

COHIROL 0 01 u,mI

20.0

0 1 ulml 10rnl

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FIGURE10

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Comparative effect of L-asparaginase on the growth of MIA PaCa-2 (A) and human breast carcinoma cells (B) in culture.

1.0 0.61

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experimental model t o learn more about the biological properties of the tumor and attempt to exploit such properties in approaches to early diagnosis and therapy. The pattern of initial growth and establishment of the carcinoma cells on top of human fibroblasts, eventually replacing them, is

typical of transformed cells. The growth o n fibroblasts, as well as formation of colonies in agar, are both considered as in vitro criteria for transformed cells. To our knowledge the only other human pancreatic carcinoma cell line currently available is that of Lieber et al. at the NCI, PANC-1 (Lieber et

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ASPARAGIWASE f I

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FIGURE 11

Comparative effect of L-asparaginase on the growth of MIA PaCa-2 (I), PANC-1 (11), human melanoma (111) and human breast carcinoma (IV) cells in culture.

134

YUNIS ET AL.

al., 1975). Through the courtesy of these workers we were able to examine their cell line. Although detailed comparative studies on the two lines are not yet completed, investigations thus far allow certain comparisons to be made. The tendency of MIA PaCa-2 cells to grow on top of others, round up and become free in suspension has some similarity to the “heaping u p ” of PANC-1 cells. The doubling time of MIA PaCa-2 is about 40 h, that of PANC-1 is approximately 52 h. Both cell lines grow in soft agar with comparable efficiency. The chromosomal pattern of MIA PaCa-2 exhibits more aneuploidy, with a chromosomal number ranging from 58 to 72 and a modal chromosome number of 67 compared to counts of 52-64 and a modal number of 63 for PANC-1. Neither cell line contained measurable amounts of carcinoembryonic antigen. We assayed both cell lines for trypsin and chymotrypsin activity but found none. The secretion of fibrinolytic activity by neoplastic or virus-transformed cells in culture has recently been described in a number of reports. Unkeless et al. (Unkeless et al., 1973 and 1974) described increased fibrinolytic activity in SV40-transformed mouse fibroblasts in culture. In a series of studies on the nature of this fibrinolytic activity these workers concluded that the cells produced a plasminogen activator. We have dzscribed the secretion of fibrinolytic activity by cultured rat ovarian tumor cells (Yunis et al., 1973). We have since observed the same phenomenon in all cultured mammalian tumor cell lines available in our laboratory. Detailed studies on “ fibrinolysin ” secreted by rat breast carcinoma R2426 (Wu et al., 1975; Schultz et al., 1975) in culture indicate that these cells secrete two types of activities: a plasminogen activator similar to that

described by Unkeless et al., and a direct fibrinolysin. Our studies indicate that both PANC-1 and MIA PaCa-2 secrete a plasminogen activator as shown by fibrin-agar plate assay. The nature and significance of this fibrinolysin is currently under intensive study. What may be an important observation is the apparent sensitivity of MIA PaCa-2 and PANC-1 to L-asparaginase. This has been a consistent feature of these two cell lines not shared by several other mammalian tumor cell lines including human breast carcinoma, three human melanomas and rat breast carcinoma. While the sensitivity of MIA PaCa-2 and PANC-1 to L-asparaginase was not expected, it is known that two of the major clinical toxicities due to L-asparaginase are pancreatitis and diabetes (Zubrod, 1970). The biochemical mechanism underlying the sensitivity of pancreatic tissue to L-asparaginase is not well understood and is currently being studied with our cultured cell lines. Perhaps the most important question regarding this observation is its possible application in the chemotherapy of human pancreatic carcinoma. To our knowledge L-asparaginase has not been used in the chemotherapy of this disease. Whether it will prove useful remains to be seen. ACKNOWLEDGEMENTS

The authors are indebted to Dr. Harold Haines of the Department of Dermatology for the electron microscopy, to Dr. Duane Schultz for the assay of carcinoembryonic antigen, and to Mrs. Mary A. Gross for expert technical assistance. Phenotyping for glucose-6-phosphates dehydrogenase was done in the laboratory of Dr. Philip Fialkow, V. A. Hospital, Seattle, Washington. A. A. Yunis was a Howard HughesInvestigator.

SENSlBlLlTC A L’ASPARAGINASE D’UN dPITHGLIOMA PANCReATlQUE H U M A l N ( M I A PaCa-2) E N CULTURE C O N T I N U E U n Cpithelioma pancreatique humain non diff6renci6 a Ct6 Btabli en culture continue e t maintenu dans un milieu de Eagle modifie selon Dulbecco e t additionne de 10% de sirum foetal de veau et 2.5% de sOrum de cheval. La lignee ainsi dtablie (MIA PaCa-2) a un temps de doublement de 40 h. Les cellules sont grandes, leur cytoplasme est abondant, elles sont fortement aneuploldes et ont tendance P se rassembler sur d’autres cellules. Elles ont 0th cultivees dans la g6lose molle e t le rendement en colonies a atteint 19%. Les cellules M I A PaCa-2 et une lignie provenant d’un autre cancer du pancreas obtenue au National Cancer Institute (NCI) sont sensibles P I’asparaglnase, propriCt6 que n’avaient pas plusieurs autres lignees de cellules tumorales humaines que I’on a testees.

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