Transplantable Acinar Cell Carcinoma of the Rat Pancreas iorphologic and Biochemical Characterization M. Sambasiva Rao, MD, and Janardan K. Reddy, MD
An acinar cell carcinoma of the pancreas, which developed in a F-344 rat after longterm nafenopin administration, was serially transplanted into inbred weanling rats by subcutaneous and intraperitoneal routes. The transplantability rate was 95. or more by both routes. The tumor implants became palpable in 20 to 30 days after subcutaneous transplantation, increasing in size rapidly thereafter during the next 25 to 30 days. In intraperitoneal recipients the abdomen was markedly distended within 1 month. No metastases were observed in this series of transplantations. Amvlase and lipase levels in serum and tumor homogenates increased with tumor size. Morphologically, only a few cells contained zymogen granules immediately after the appearance of a palpable tumor; at later intervals, however, these granules were observed in manv tumor cells. Seventy-two hours after the surgical removal of tumors, the serum amylase and lipase levels returned to control values. This transplantable pancreatic acinar cell carcinoma can be dissociated into functionally viable single cells by a simplified enzyme digestion and divalent cation chelation procedure. By light microscopic autoradiographv, approximatelv 209 of these isolated cells were found to incorporate 3H-thymidine in vitro into nuclear DNA. The data presented in this paper should serve as a baseline for future studies on this transplanted tumor. (Am J Pathol 94:333-348, 1979)
Hi \IAN PXNCRE_TIC CARCINONMA iS classified histologically as ductal adenocarcinoma or acinar cell carcinoma based on the cell of origin. Cubilla and Fitzgerald 1 reported that a majority of pancreatic carcinomas are ductal in origin whereas acinar cell carcinomas constitute approximately 1 % of all pancreatic tumors. However, there are other reports which indicate that the incidence of acinar cell carcinoma may be as high as 13% .2 One probable reason for the reported low incidence of acinar cell carcinoma 1 is the failure of recognizing, by light microscopy, the acinar cell origin of poorly differentiated tumors, which are often classified under a separate category of anaplastic and/or unclassified variety.' To study the histogenesis of pancreatic carcinoma, -arious experimental models have been developed using a variety of chemical carcinogens in different species of animals. In hamsters, a high percentage of tumors From the Department of Pathology. Northmvestern University \Medical School. Chicago. Illinois Supported by Grant CA 2:305.5 from the National Cancer Institute. U. S Department of Health. Education and 'Welfare Accepted for publication October 16. 1978 Address reprint requests to Dr MI S Rao. Department of Patholoey. Northwestern University Mledical School. Chicago. IL 60611 0002-9440/79/0208-0333$01.00 333 ( 1979 American Association of Pathologists
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appeared to arise from ductal or ductular epithelium,3'4 whereas studies in guinea pigs 6 and rats 6,7 suggested that pancreatic tumors are most likely derived from the acinar cell. Irrespective of the histologic type, prognosis of pancreatic carcinoma is very poor: the 1-year and 5-year relative survival rates, in general, are 10% and 2%, respectively.8 Of several factors that may be responsible for the poor prognosis of pancreatic carcinoma, the delayed diagnosis as well as inadequate knowledge of its biologic behavior appear to be of primary importance. This paper deals with the functional and morphologic features of a transplantable acinar cell carcinoma of the rat pancreas developed in this laboratory.9 Materials and Methods Transplantation Procedure
The morphologic features of the primary acinar cell carcinoma of the pancreas, which served as a source for this transplantable tumor, were described previously.9'10 For transplantation of tumor, weanling Fischer-344 rats weighing 40 to 60 g were used. The initial transplantation from the original tumor into 5 male rats was carried out intraperitoneally after laparotomy under sterile conditions. Subsequent transplantations were carried out intraperitoneally in groups of 2 to 3 rats and subcutaneously in groups of 12 to 20 rats at 40- to 60-day intervals. The technique for subcutaneous transplantation was previously described."' To determine the growth pattern of the subcutaneous transplants, tumor size was measured with vernier calipers on alternate days for 30 days after the tumors became palpable. Morphologic Studies
Morphologic features were studied by light and electron microscopy on intact tissue and by transmission as well as scanning electron microscopy on dissociated cells. Sequential morphologic changes were studied at 30, 45, and 70 days after transplantation of tumor during the seventh transplant generation. All other transplant generations were studied prior to succeeding transplantation. For light microscopy, the tumor tissue was fixed in neutral buffered formalin and embedded in paraffin. Five-micron-thick sections were stained routinely with hematoxylin and eosin. Selected sections were stained with permanganate-high-iron-diamine technique 12 for zymogen granules. For transmission electron microscopy, both the tissue pieces and dissociated cells were fixed for 30 minutes in 2.5% glutaraldehyde buffered with 0.1 M sodium cacodylate at pH 7.4 and were postfixed for 1 hour in 1% osmium tetroxide buffered with sym-collidine at pH 7.4. For scanning electron microscopy, the dissociated cells were fixed in 2.5% glutaraldehyde for 4 hours, dehydrated through graded series of alcohol, and dried by the CO2 critical point method. Chromosome Preparation
Chromosome analysis of the 10th transplant was done 50 days after transplantation in 3 rats. Colchicine (0.1 mg/100 g body wt) was injected intramuscularly 4 hours prior to sacrifice. One gram of tumor tissue was minced into small fragments in phosphatebuffered saline (PBS), pH 7.4, and washed three times to remove erythrocytes. The tissue fragments were then incubated in 15 ml of PBS containing 0.25% trypsin and 0.05% EDTA for 20 minutes at 37 C with frequent swirling. The resulting cell suspension was filtered through four layers of gauze, and the filtrate was centrifuged at 1000 rpm for 5
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minutes. The pellet was resuspended in .3 ml of 1% sodium citrate and incubated for 3 minutes at 37 C. The incubation was terminated by the addition of 1 ml of fixative (1 1) methanol: acetic acid, and the tubes were kept at 4 C for 30 minutes. The cell suspension was centrifuged at 1000 rpm for 3 minutes. and the supernatant was discarded. The pellet was washed two more times with the methanol :acetic acid mixture. and final suspension was made in 2 ml of fixative. Two to three drops of cell suspension were placed on a microscopic slide, dried on a flame, and stained with Giemsa stain. Cell Dissociation Technique
Normal pancreatic cells were dissociated by using the procedure of Amsterdam and Jamieson."3 For dissociation of tumor cells from tumor transplants. a single enzymatic digestion and divalent cation chelation was used; two enzyme digestions mvere used in studies of normal pancreas. In Vitro
H-Thymlidine Labeling of Dissociated Tumor Cells
The tumor cells (10" :ml) were incubated in Krebs-Ringer bicarbonate (KRB) containing 1 %E bomine plasma albumin (BPA). a complete L-amino acid supplement. glucose. Ca. M.g. and soya bean trvpsin inhibitor (STI)4 with 100 MCi ml of 3H-thvmidine for 1 hour (specific activit,. 43 pCi millimole) (Research Products International Corporation. Elk Grove Village. Ill). At the end of incubation. the cells were washed three times with KRB solution and processed for electron microscopy. One-micron-thick sections were prepared for autoradiography using Ilford L-4 emulsion as described earlier."'
Amylase and Upase Determinations The amrnlase and lipase levels in the serum of tumor-bearing and control rats and in tumor homogenates were measured by the methods of Caraway "I and Roe and Bvler,17 respectively. at different intervals after transplantation. Enzyme contents of the tumor tissue were measured from homogenates as described previously.'0 On 4 animals, the serum amrvlase and lipase values were measured 72 hours after the surgical removal of subcutaneous tumor.
Results The tumor transplants were carried subcutaneously in 200 male rats (11 passages) and 10 female rats (1 passage) and intraperitoneally in 24 males (8 passages). The take-up rate wvas 95% and 100% after the subcutaneous and intraperitoneal transplantation, respectively, in both sexes. All the animals wvere observed for 3 months after transplantation before they were considered negative for tumor growth. The subcutaneous transplants grew to a palpable size in 20 to 30 days. From the time the tumors became palpable, they grew rapidly and reached a maximum size in approximatelv 30 days (Text-figure 1). The subcutaneous transplants grew only locally and did not metastasize in the 200 rats that received transplants. The intraperitoneal transplants caused marked distention of abdomen within a month after transplantation, with accumulation of varying amounts of hemorrhagic fluid rich in amvlase and lipase activity. The tumor nodules grew on the mesentery and involved different abdominal
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TEXT-FIGURE 1-Growth pattern of acinar cell carcinoma transplants in syngeneic rats after subcutaneous implantation. Each value represents mean tumor growth in 6 to 8 rats. /The tumor size is plotted as mean of longest and smallest diameter.
3 5 7 9 11 1315 25 30 DAYS AFTER APPEARANCE OF TUMOR
organs by local extension. Even in this group, no distant metastases were encountered. Generally, all the animals with subcutaneous transplants died within 4 to 6 months after the appearance of tumor, and intraperitoneal recipients died within 2 to 3 months after transplantation, with cachexia and pulmonary hemorrhage. Gross and Microscopic Morphology
Immediately after the tumors became palpable, they were firm and brownish. As the size increased more than 1 cm, they became soft and hemorrhagic and contained cystic spaces filled with dark brown fluid rich in amylase and lipase activity. In large tumors, areas of necrosis were common, and viable tumor tissue was present mostly at the periphery. By light microscopy, the tumors, 3 days after their appearance, showed large oval to polyhedral cells arranged in sheets. The nuclei were prominent and pleomorphic, occupied most of the cell, and contained one or more nucleoli. The cytoplasm was scanty and only a few cells contained zymogen granules. Mitotic figures were frequent. Thirty days after appearance, the tumor was highly vascular. The nuclear cytoplasmic ratio was markedly decreased, and many cells contained abundant granular eosinophilic cytoplasm rich in zymogen granules (Figure 1). Areas of necrosis were frequently seen. The morphologic appearance of tumors was the same at later intervals except for extensive cavitation and necrosis. The cytoplasmic granules stained positively for zymogen granules with permanganate-high-iron-diamine technique. No morphologic changes were observed in the pancreata of host animals. The ultrastructural features differed markedly depending on the age of the tumor. The tumor cells from a 3-day palpable tumor contained large nuclei with an irregular nuclear outline and indentations. The nucleoplasm appeared homogeneous. Some cells contained a prominent nucleolus with granular and fibrillar areas. The cytoplasm of all the cells
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contained well-formed rough endoplasmic reticulum arranged in parallel arrays. The Golgi complex appeared well developed. The mitochondria are round to oval X ith X ell-formed cristae. Some cells had few or no zvrmogen granules (Figure 2), while other cells contained several prozymogen granules or a few well-formed zymogen granules (Figure 3). The plasma membranes of adjacent cells are closely apposed with distinct intercellular junctions. The cells are normally arranged in sheets with an occasional lumen formation. Fifteen to 20 days after the appearance of tumor, and at later intervals, many cells showed increased amounts of cytoplasm with various well-developed organelles. Wtell-formed zymogen granules were readily visible (Figure 4). Most of the zymogen granules appeared round and contained homogenous material, while some were of irregular shape and contained fibrillar material. The microfilaments were prominent and arranged as irregular bundles. ChOomosome Analysis
The results of the chromosome counts are summarized in Text-figure 2. The chromosome distribution was wide and ranged from hvpodiploid to hypertetraploid without any definitive mode. Serum and Tumor Enzyme Studies
The amvlase and lipase values of serum and tumor homogenates at different intervals after the seventh transplant are summarized in Textfigure 3. Within 3 days (30 dayrs after transplantation) after the appearance of tumor, the serum levels of both amvlase and lipase showed marked increase over the control levels. There was progressive increase in 35 30
TEXT-FIGtRE 2-Pattern
of chromosome distribution in acinar cell carcinoma of the rat pancreas after 10 serial transplantations in inbred rats (a total of 100 cells were counted)
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TEXT-FIGURE 3-Amylase and lipase levels of serum and tumor homogenate at different intervals after transplantation. Each value represents the mean ± SEM of 3 to 5 animals. Broken lines represent amylase and lipase levels 72 hours after surgical removal of tumor. Normal amylase and lipase values in control animals were 863 ± 61 and 100 + 10 units/100 ml of serum, respectively. Open circles, units/100 ml serrum; closed circles, units/gram of tissuie.
the serum content of these enzymes with increase in tumor size. Likewise, amylase and lipase content of the tumor tissue also increased with tumor size and age. The values of amylase and lipase in serum remained high even in the 11th transplant and measured 34,400 ± 7220 and 1513 ± 227 units for 100 ml, respectively. In 4 animals in which tumor was surgically resected, the serum amylase and lipase levels measured 1110 ± 153 and 283 + 100 for 100 ml, respectively, after 72 hours. These levels are comparable to the levels found in control animals. Morphologic Appearance of Dissociated Tumor Cells
The dissociation of tumor cells is relatively easy and required only one enzymatic digestion for 15 minutes, followed by EDTA treatment for 10 minutes, compared with dissociation of normal exocrine pancreas cells described by Amsterdam and Jamieson."3 At the end of dissociation, the cell yield was good and consisted of a uniform population of tumor cells. With this procedure, 92 to 96% of the dissociated tumor cells were found viable, as determined by trypan blue exclusion test. Transmission Electron Microscopy
The isolated cells were rounded up, and the morphologic features were well preserved (Figure 5). The plasma membrane was intact, with irregularly distributed short microvilli and a few cytoplasmic blebs. The nucleus occupied a basal or parabasal position. Zymogen granules, Golgi complex, microfilaments, and mitochondria were placed at one pole of the cell. The endoplasmic reticulum appeared abundant at the periphery.
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Scwing Eectr Mc
Surface morphology of the tumor cells and normal cells is shown in Figures 6 and 7, respectively. The tumor cells contained irregularly distributed multiple microvilli of varying lengths and diameter; the normal cells had rows of microplicae and a few short microvilli. 'H-Thymdn lIcopratio
After 1 hour of incubation with 3H-thymidine at a concentration of 100 uCi/ml, 15 to 20% of the cells showed nuclear labeling as determined by autoradiography (Figure 8). Many of the labeled cells contained wellformed zvrnogen granules. Discssion
The transplantable acinar cell carcinoma described here is a moderatelv differentiated tumor, both morphologicallv and functionally. The morphologic features of the tumor did not markedly differ from the original tumor 9,10 during the 11 passages in inbred rats. It remains to be seen if this transplantable acinar cell carcinoma continues to produce amylase and lipase during the several succeeding generations. The transplantable acinar cell carcinoma did not metastasize in animals thus far transplanted, although the original tumor metastasized to liver. Ultrastructurally, the tumor cells differed from normal pancreatic cells by having irregularnuclei with nuclear inclusions. As in the normal acinar cells, the rough endoplasmic reticulum and the Golgi complex are well developed and the intercellular plasma membrane between tumor cells appeared straight with well-developed junctions. In atvpical acinar nodules developed in rats after 4-hydroxyaminoquinoline oxide treatment, the intercellular plasma membrane showed papillary projections forming interdigitations.18 Another striking feature that differed from normal acinar cell was increased occurrence of microfilaments as irregular bundles. A similar increase in the number of microfilaments was described in acinar cells of mouse pancreas treated with vinblastine 19 and in pancreatic tumors induced in rats by azaserine ' and 7,12-dimethylbenz(a)anthracene.7 Using the modified procedure of Amsterdam and Jamieson, ' a high yield of single viable cells with well-preserved morphologic and functional features was obtained, as demonstrated by trypan blue dye exclusion test, electron microscopy, and thymidine incorporation studies. On transmission electron microscopy, the surface plasma membrane showed irregularly distributed short microvilli, in contrast to normal pancreatic acinar cells which had microvilli at one place, representing former apical plasma membrane.13 The distribution of various cytoplasmic organelles was sim-
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ilar to that observed by Amsterdam and Jamieson 19 in normal pancreatic acinar cells, except for increased number of microfilaments. Under the scanning electron microscope, the cell surface contained irregularly distributed, somewhat pleomorphic microvilli. This feature markedly contrasted to observations in normal acinar cells, in which the surface showed rows of microplicae and a few short microvilli.21'22 Similar surface alterations were described in preneoplastic and neoplastic bladder mucosa in rats treated with chemical carcinogens.2'24Autoradiography of plasticembedded 1-,u-thick sections showed a nuclear labeling index of 15 to 20% of the cells, a percentage similar to that observed in intact tumor tissue labeled in vivo.26 Several tumor cells that showed nuclear labeling had zymogen granules in their cytoplasm. Detailed analyses of the nuclear labeling and zymogen granule synthesis are expected to yield valuable information about differentiation in tumor cells.26 Functionally, the transplantable acinar cell carcinoma produced the enzymes amylase and lipase, as demonstrated by increased serum levels and their presence in tumor homogenates. The capacity to produce these enzymes is retained through 11 transplant generations. The morphologic features of the tumor correlate well with the functional aspects at different periods during the growth of the tumor. The transplantable acinar cell carcinoma will be useful in the delineation of differences between normal pancreatic acinar cells and malignant acinar cells both morphologically and functionally. Because of the capacity of this tumor to synthesize exocrine pancreatic enzymes, it will provide an opportunity to study the transport of secretory proteins in tumor cells. Because of the easy dissociability, these tumor cells will be useful for a number of biologic and biochemical studies.27 References 1. Cubilla AL, Fitzgerald PJ: Morphological patterns of primary nonendocrine human pancreas carcinoma. Cancer Res 35:2234-2247, 1975 2. Webb JN: Acinar cell neoplasms of the exocrine pancreas. J Clin Pathol 30:103112, 1977 3. Pour P, Kruger FW, Althoff J, Cardesa A, Mohr U: Cancer of the pancreas induced in the Syrian golden hamster. Am J Pathol 76:349-358, 1974 4. Pour P, Althoff J, Kruger FW, Mohr U: A potent pancreatic carcinogen in Syrian hamsters: N-nitrosobis (2-oxopropyl) amine. J Natl Cancer Inst 58:1449-1453, 1977 5. Reddy JK, Rao MS: Pancreatic adenocarcinoma in inbred guinea pigs induced by N-methyl-N-nitrosourea. Cancer Res 35:2269-2276, 1975 6. Dissin J, Mills LR, Main DL, Black 0, Webster PD: Experimental induction of pancreatic adenocarcinoma in rats. J Natl Cancer Inst 55:857-864, 1975 7. Bockman DE, Black 0, Mills LR, Mainz DL, Webster PD: Fine structure of pancreatic adenocarcinoma induced in rats by 7,12-dimethylbenz(a)anthracene. J Natl Cancer Inst 57:931-936, 1976
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8. Axtell LM. Cuttler SJ Myers NIH (editors): End Results in Cancer. Report 4. Biometrv Branch. National Cancer Institute. US Department of Health. Education and W'eifare Publication No. (NIH) 73-272, 1972 9. Reddy JK. Rao NMS: Malignant tumors in rats fed nafenopin, a hepatic peroxisome proliferator. J Nat] Cancer Inst 59:1645-1650. 19,77 10. Reddy JK. Rao NMS: Transplantable pancreatic carcinoma of the rat. Science 198:78-80. 1977 11. Rao MS, Reddy JK: Malignant neoplasms in rats fed lasiocarpine. Br J Cancer 37:289-293. 1978 12. Klessen C: Histochemical staining of zymogen granules of pancreatic acinar cells using a permanganate-HID technique. Histochemie 30:365-366. 1972 13. Amsterdam A, Jamieson JD: Studies on dispersed pancreatic exocrine cells. J Cell Biol 63:1037-1056, 1974 14. Amsterdam A, Jamieson JD: Studies on dispersed pancreatic exocrine cells. J Cell Biol 63:1057-1073, 1974 15. Reddy JK, Svoboda D: The relationship of nucleolar segregation to ribonucleic acid and svnthesis following the administration of selected hepatocarcinogens. Lab Invest 19:132-143, 1968 16. Carawav WNT: A stable starch substrate for the determination of am-lase in serum and other bod- fluids. Am J Clin Pathol 32:97-99, 1957 17. Roe JH, Byler RE: Serum lipase determination using a one-hour period of hydrolysis. Anal Biochem 6:451-460, 1973 18. Shinozuka H, Popp JA, Konishi Y: Ultrastructures of atypical acinar cell nodules in rat pancreas induced by 4-hv-droxvaminoquinoline-1-oxide. Lab Invest 34:501-509. 1976 19. Nevalainen TJ: Cytotoxicity of \inblastine and \incristine to pancreatic acinar cells. V'irchows Archiv (Cell Pathol) 18:119-127, 1975 20. Longnecker DS, Curphey TJ: Adenocarcinoma of the pancreas in azaserine-treated rats. Cancer Res 35:2249-2258, 1975 21. Motta P. Andrews PM, Caramia F, Correr S: Scanning electron microscopy of dissociated pancreatic acinar cell surfaces. Cell Tissue Res 176:493-504. 1977 22. Reddy JK. Rao NMS, Warren JR. Minick OT: Concanavalin A agglutinability and surface microvilli of dissociated normal and neoplastic pancreatic acinar cells of the rat. Exp Cell Res (In press) 23. Hicks RNM, W'akefield JSJ: Nlembrane changes during urothelial hyperplasia and neoplasia. Cancer Res 36:2502-2507, 1976 24. Arai NI, Kani T. Sugihara S, Nlatsumura K, NMiyata Y, Shinohara Y, Ito N: Scanning and transmission electron microscopy of changes in the urinary bladder in rats treated wvith N-buty l-N-(4-h. droxv-buty l ) nitrosoamine. Gann 65:529-340, 1974 25. Rao NIS, Reddy JK: Unpublished data 26. Pierce GB, Nakane PK, Martinez-Hernandez A, Ward JN: Ultrastructural comparison of differentiation of stem cells of murine adenocarcinomas of colon and breast with their normal counterparts. J Natl Cancer Inst 58:1329-1345, 1977 27. Emmelot P: Biochemical properties of normal and neoplastic cell surface: A review. Eur J Cancer 9:319-33, 1973
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[Illustrations follow]
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An acinar cell carcinoma of the pancreas, which developed in a F-344 rat after longterm nafenopin administration, was serially transplanted into inbred weanling rats by subcutaneous and intraperitoneal routes. The transplantability rate was 95. or more by both routes. The tumor implants became palpable in 20 to 30 days after subcutaneous transplantation, increasing in size rapidly thereafter during the next 25 to 30 days. In intraperitoneal recipients the abdomen was markedly distended within 1 month. No metastases were observed in this series of transplantations. Amvlase and lipase levels in serum and tumor homogenates increased with tumor size. Morphologically, only a few cells contained zymogen granules immediately after the appearance of a palpable tumor; at later intervals, however, these granules were observed in manv tumor cells. Seventy-two hours after the surgical removal of tumors, the serum amylase and lipase levels returned to control values. This transplantable pancreatic acinar cell carcinoma can be dissociated into functionally viable single cells by a simplified enzyme digestion and divalent cation chelation procedure. By light microscopic autoradiographv, approximatelv 209 of these isolated cells were found to incorporate 3H-thymidine in vitro into nuclear DNA. The data presented in this paper should serve as a baseline for future studies on this transplanted tumor. (Am J Pathol 94:333-348, 1979)
Hi \IAN PXNCRE_TIC CARCINONMA iS classified histologically as ductal adenocarcinoma or acinar cell carcinoma based on the cell of origin. Cubilla and Fitzgerald 1 reported that a majority of pancreatic carcinomas are ductal in origin whereas acinar cell carcinomas constitute approximately 1 % of all pancreatic tumors. However, there are other reports which indicate that the incidence of acinar cell carcinoma may be as high as 13% .2 One probable reason for the reported low incidence of acinar cell carcinoma 1 is the failure of recognizing, by light microscopy, the acinar cell origin of poorly differentiated tumors, which are often classified under a separate category of anaplastic and/or unclassified variety.' To study the histogenesis of pancreatic carcinoma, -arious experimental models have been developed using a variety of chemical carcinogens in different species of animals. In hamsters, a high percentage of tumors From the Department of Pathology. Northmvestern University \Medical School. Chicago. Illinois Supported by Grant CA 2:305.5 from the National Cancer Institute. U. S Department of Health. Education and 'Welfare Accepted for publication October 16. 1978 Address reprint requests to Dr MI S Rao. Department of Patholoey. Northwestern University Mledical School. Chicago. IL 60611 0002-9440/79/0208-0333$01.00 333 ( 1979 American Association of Pathologists
334
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American Journal of Pathology
appeared to arise from ductal or ductular epithelium,3'4 whereas studies in guinea pigs 6 and rats 6,7 suggested that pancreatic tumors are most likely derived from the acinar cell. Irrespective of the histologic type, prognosis of pancreatic carcinoma is very poor: the 1-year and 5-year relative survival rates, in general, are 10% and 2%, respectively.8 Of several factors that may be responsible for the poor prognosis of pancreatic carcinoma, the delayed diagnosis as well as inadequate knowledge of its biologic behavior appear to be of primary importance. This paper deals with the functional and morphologic features of a transplantable acinar cell carcinoma of the rat pancreas developed in this laboratory.9 Materials and Methods Transplantation Procedure
The morphologic features of the primary acinar cell carcinoma of the pancreas, which served as a source for this transplantable tumor, were described previously.9'10 For transplantation of tumor, weanling Fischer-344 rats weighing 40 to 60 g were used. The initial transplantation from the original tumor into 5 male rats was carried out intraperitoneally after laparotomy under sterile conditions. Subsequent transplantations were carried out intraperitoneally in groups of 2 to 3 rats and subcutaneously in groups of 12 to 20 rats at 40- to 60-day intervals. The technique for subcutaneous transplantation was previously described."' To determine the growth pattern of the subcutaneous transplants, tumor size was measured with vernier calipers on alternate days for 30 days after the tumors became palpable. Morphologic Studies
Morphologic features were studied by light and electron microscopy on intact tissue and by transmission as well as scanning electron microscopy on dissociated cells. Sequential morphologic changes were studied at 30, 45, and 70 days after transplantation of tumor during the seventh transplant generation. All other transplant generations were studied prior to succeeding transplantation. For light microscopy, the tumor tissue was fixed in neutral buffered formalin and embedded in paraffin. Five-micron-thick sections were stained routinely with hematoxylin and eosin. Selected sections were stained with permanganate-high-iron-diamine technique 12 for zymogen granules. For transmission electron microscopy, both the tissue pieces and dissociated cells were fixed for 30 minutes in 2.5% glutaraldehyde buffered with 0.1 M sodium cacodylate at pH 7.4 and were postfixed for 1 hour in 1% osmium tetroxide buffered with sym-collidine at pH 7.4. For scanning electron microscopy, the dissociated cells were fixed in 2.5% glutaraldehyde for 4 hours, dehydrated through graded series of alcohol, and dried by the CO2 critical point method. Chromosome Preparation
Chromosome analysis of the 10th transplant was done 50 days after transplantation in 3 rats. Colchicine (0.1 mg/100 g body wt) was injected intramuscularly 4 hours prior to sacrifice. One gram of tumor tissue was minced into small fragments in phosphatebuffered saline (PBS), pH 7.4, and washed three times to remove erythrocytes. The tissue fragments were then incubated in 15 ml of PBS containing 0.25% trypsin and 0.05% EDTA for 20 minutes at 37 C with frequent swirling. The resulting cell suspension was filtered through four layers of gauze, and the filtrate was centrifuged at 1000 rpm for 5
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minutes. The pellet was resuspended in .3 ml of 1% sodium citrate and incubated for 3 minutes at 37 C. The incubation was terminated by the addition of 1 ml of fixative (1 1) methanol: acetic acid, and the tubes were kept at 4 C for 30 minutes. The cell suspension was centrifuged at 1000 rpm for 3 minutes. and the supernatant was discarded. The pellet was washed two more times with the methanol :acetic acid mixture. and final suspension was made in 2 ml of fixative. Two to three drops of cell suspension were placed on a microscopic slide, dried on a flame, and stained with Giemsa stain. Cell Dissociation Technique
Normal pancreatic cells were dissociated by using the procedure of Amsterdam and Jamieson."3 For dissociation of tumor cells from tumor transplants. a single enzymatic digestion and divalent cation chelation was used; two enzyme digestions mvere used in studies of normal pancreas. In Vitro
H-Thymlidine Labeling of Dissociated Tumor Cells
The tumor cells (10" :ml) were incubated in Krebs-Ringer bicarbonate (KRB) containing 1 %E bomine plasma albumin (BPA). a complete L-amino acid supplement. glucose. Ca. M.g. and soya bean trvpsin inhibitor (STI)4 with 100 MCi ml of 3H-thvmidine for 1 hour (specific activit,. 43 pCi millimole) (Research Products International Corporation. Elk Grove Village. Ill). At the end of incubation. the cells were washed three times with KRB solution and processed for electron microscopy. One-micron-thick sections were prepared for autoradiography using Ilford L-4 emulsion as described earlier."'
Amylase and Upase Determinations The amrnlase and lipase levels in the serum of tumor-bearing and control rats and in tumor homogenates were measured by the methods of Caraway "I and Roe and Bvler,17 respectively. at different intervals after transplantation. Enzyme contents of the tumor tissue were measured from homogenates as described previously.'0 On 4 animals, the serum amrvlase and lipase values were measured 72 hours after the surgical removal of subcutaneous tumor.
Results The tumor transplants were carried subcutaneously in 200 male rats (11 passages) and 10 female rats (1 passage) and intraperitoneally in 24 males (8 passages). The take-up rate wvas 95% and 100% after the subcutaneous and intraperitoneal transplantation, respectively, in both sexes. All the animals wvere observed for 3 months after transplantation before they were considered negative for tumor growth. The subcutaneous transplants grew to a palpable size in 20 to 30 days. From the time the tumors became palpable, they grew rapidly and reached a maximum size in approximatelv 30 days (Text-figure 1). The subcutaneous transplants grew only locally and did not metastasize in the 200 rats that received transplants. The intraperitoneal transplants caused marked distention of abdomen within a month after transplantation, with accumulation of varying amounts of hemorrhagic fluid rich in amvlase and lipase activity. The tumor nodules grew on the mesentery and involved different abdominal
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TEXT-FIGURE 1-Growth pattern of acinar cell carcinoma transplants in syngeneic rats after subcutaneous implantation. Each value represents mean tumor growth in 6 to 8 rats. /The tumor size is plotted as mean of longest and smallest diameter.
3 5 7 9 11 1315 25 30 DAYS AFTER APPEARANCE OF TUMOR
organs by local extension. Even in this group, no distant metastases were encountered. Generally, all the animals with subcutaneous transplants died within 4 to 6 months after the appearance of tumor, and intraperitoneal recipients died within 2 to 3 months after transplantation, with cachexia and pulmonary hemorrhage. Gross and Microscopic Morphology
Immediately after the tumors became palpable, they were firm and brownish. As the size increased more than 1 cm, they became soft and hemorrhagic and contained cystic spaces filled with dark brown fluid rich in amylase and lipase activity. In large tumors, areas of necrosis were common, and viable tumor tissue was present mostly at the periphery. By light microscopy, the tumors, 3 days after their appearance, showed large oval to polyhedral cells arranged in sheets. The nuclei were prominent and pleomorphic, occupied most of the cell, and contained one or more nucleoli. The cytoplasm was scanty and only a few cells contained zymogen granules. Mitotic figures were frequent. Thirty days after appearance, the tumor was highly vascular. The nuclear cytoplasmic ratio was markedly decreased, and many cells contained abundant granular eosinophilic cytoplasm rich in zymogen granules (Figure 1). Areas of necrosis were frequently seen. The morphologic appearance of tumors was the same at later intervals except for extensive cavitation and necrosis. The cytoplasmic granules stained positively for zymogen granules with permanganate-high-iron-diamine technique. No morphologic changes were observed in the pancreata of host animals. The ultrastructural features differed markedly depending on the age of the tumor. The tumor cells from a 3-day palpable tumor contained large nuclei with an irregular nuclear outline and indentations. The nucleoplasm appeared homogeneous. Some cells contained a prominent nucleolus with granular and fibrillar areas. The cytoplasm of all the cells
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contained well-formed rough endoplasmic reticulum arranged in parallel arrays. The Golgi complex appeared well developed. The mitochondria are round to oval X ith X ell-formed cristae. Some cells had few or no zvrmogen granules (Figure 2), while other cells contained several prozymogen granules or a few well-formed zymogen granules (Figure 3). The plasma membranes of adjacent cells are closely apposed with distinct intercellular junctions. The cells are normally arranged in sheets with an occasional lumen formation. Fifteen to 20 days after the appearance of tumor, and at later intervals, many cells showed increased amounts of cytoplasm with various well-developed organelles. Wtell-formed zymogen granules were readily visible (Figure 4). Most of the zymogen granules appeared round and contained homogenous material, while some were of irregular shape and contained fibrillar material. The microfilaments were prominent and arranged as irregular bundles. ChOomosome Analysis
The results of the chromosome counts are summarized in Text-figure 2. The chromosome distribution was wide and ranged from hvpodiploid to hypertetraploid without any definitive mode. Serum and Tumor Enzyme Studies
The amvlase and lipase values of serum and tumor homogenates at different intervals after the seventh transplant are summarized in Textfigure 3. Within 3 days (30 dayrs after transplantation) after the appearance of tumor, the serum levels of both amvlase and lipase showed marked increase over the control levels. There was progressive increase in 35 30
TEXT-FIGtRE 2-Pattern
of chromosome distribution in acinar cell carcinoma of the rat pancreas after 10 serial transplantations in inbred rats (a total of 100 cells were counted)
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American Journal of Pathology
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TEXT-FIGURE 3-Amylase and lipase levels of serum and tumor homogenate at different intervals after transplantation. Each value represents the mean ± SEM of 3 to 5 animals. Broken lines represent amylase and lipase levels 72 hours after surgical removal of tumor. Normal amylase and lipase values in control animals were 863 ± 61 and 100 + 10 units/100 ml of serum, respectively. Open circles, units/100 ml serrum; closed circles, units/gram of tissuie.
the serum content of these enzymes with increase in tumor size. Likewise, amylase and lipase content of the tumor tissue also increased with tumor size and age. The values of amylase and lipase in serum remained high even in the 11th transplant and measured 34,400 ± 7220 and 1513 ± 227 units for 100 ml, respectively. In 4 animals in which tumor was surgically resected, the serum amylase and lipase levels measured 1110 ± 153 and 283 + 100 for 100 ml, respectively, after 72 hours. These levels are comparable to the levels found in control animals. Morphologic Appearance of Dissociated Tumor Cells
The dissociation of tumor cells is relatively easy and required only one enzymatic digestion for 15 minutes, followed by EDTA treatment for 10 minutes, compared with dissociation of normal exocrine pancreas cells described by Amsterdam and Jamieson."3 At the end of dissociation, the cell yield was good and consisted of a uniform population of tumor cells. With this procedure, 92 to 96% of the dissociated tumor cells were found viable, as determined by trypan blue exclusion test. Transmission Electron Microscopy
The isolated cells were rounded up, and the morphologic features were well preserved (Figure 5). The plasma membrane was intact, with irregularly distributed short microvilli and a few cytoplasmic blebs. The nucleus occupied a basal or parabasal position. Zymogen granules, Golgi complex, microfilaments, and mitochondria were placed at one pole of the cell. The endoplasmic reticulum appeared abundant at the periphery.
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Scwing Eectr Mc
Surface morphology of the tumor cells and normal cells is shown in Figures 6 and 7, respectively. The tumor cells contained irregularly distributed multiple microvilli of varying lengths and diameter; the normal cells had rows of microplicae and a few short microvilli. 'H-Thymdn lIcopratio
After 1 hour of incubation with 3H-thymidine at a concentration of 100 uCi/ml, 15 to 20% of the cells showed nuclear labeling as determined by autoradiography (Figure 8). Many of the labeled cells contained wellformed zvrnogen granules. Discssion
The transplantable acinar cell carcinoma described here is a moderatelv differentiated tumor, both morphologicallv and functionally. The morphologic features of the tumor did not markedly differ from the original tumor 9,10 during the 11 passages in inbred rats. It remains to be seen if this transplantable acinar cell carcinoma continues to produce amylase and lipase during the several succeeding generations. The transplantable acinar cell carcinoma did not metastasize in animals thus far transplanted, although the original tumor metastasized to liver. Ultrastructurally, the tumor cells differed from normal pancreatic cells by having irregularnuclei with nuclear inclusions. As in the normal acinar cells, the rough endoplasmic reticulum and the Golgi complex are well developed and the intercellular plasma membrane between tumor cells appeared straight with well-developed junctions. In atvpical acinar nodules developed in rats after 4-hydroxyaminoquinoline oxide treatment, the intercellular plasma membrane showed papillary projections forming interdigitations.18 Another striking feature that differed from normal acinar cell was increased occurrence of microfilaments as irregular bundles. A similar increase in the number of microfilaments was described in acinar cells of mouse pancreas treated with vinblastine 19 and in pancreatic tumors induced in rats by azaserine ' and 7,12-dimethylbenz(a)anthracene.7 Using the modified procedure of Amsterdam and Jamieson, ' a high yield of single viable cells with well-preserved morphologic and functional features was obtained, as demonstrated by trypan blue dye exclusion test, electron microscopy, and thymidine incorporation studies. On transmission electron microscopy, the surface plasma membrane showed irregularly distributed short microvilli, in contrast to normal pancreatic acinar cells which had microvilli at one place, representing former apical plasma membrane.13 The distribution of various cytoplasmic organelles was sim-
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ilar to that observed by Amsterdam and Jamieson 19 in normal pancreatic acinar cells, except for increased number of microfilaments. Under the scanning electron microscope, the cell surface contained irregularly distributed, somewhat pleomorphic microvilli. This feature markedly contrasted to observations in normal acinar cells, in which the surface showed rows of microplicae and a few short microvilli.21'22 Similar surface alterations were described in preneoplastic and neoplastic bladder mucosa in rats treated with chemical carcinogens.2'24Autoradiography of plasticembedded 1-,u-thick sections showed a nuclear labeling index of 15 to 20% of the cells, a percentage similar to that observed in intact tumor tissue labeled in vivo.26 Several tumor cells that showed nuclear labeling had zymogen granules in their cytoplasm. Detailed analyses of the nuclear labeling and zymogen granule synthesis are expected to yield valuable information about differentiation in tumor cells.26 Functionally, the transplantable acinar cell carcinoma produced the enzymes amylase and lipase, as demonstrated by increased serum levels and their presence in tumor homogenates. The capacity to produce these enzymes is retained through 11 transplant generations. The morphologic features of the tumor correlate well with the functional aspects at different periods during the growth of the tumor. The transplantable acinar cell carcinoma will be useful in the delineation of differences between normal pancreatic acinar cells and malignant acinar cells both morphologically and functionally. Because of the capacity of this tumor to synthesize exocrine pancreatic enzymes, it will provide an opportunity to study the transport of secretory proteins in tumor cells. Because of the easy dissociability, these tumor cells will be useful for a number of biologic and biochemical studies.27 References 1. Cubilla AL, Fitzgerald PJ: Morphological patterns of primary nonendocrine human pancreas carcinoma. Cancer Res 35:2234-2247, 1975 2. Webb JN: Acinar cell neoplasms of the exocrine pancreas. J Clin Pathol 30:103112, 1977 3. Pour P, Kruger FW, Althoff J, Cardesa A, Mohr U: Cancer of the pancreas induced in the Syrian golden hamster. Am J Pathol 76:349-358, 1974 4. Pour P, Althoff J, Kruger FW, Mohr U: A potent pancreatic carcinogen in Syrian hamsters: N-nitrosobis (2-oxopropyl) amine. J Natl Cancer Inst 58:1449-1453, 1977 5. Reddy JK, Rao MS: Pancreatic adenocarcinoma in inbred guinea pigs induced by N-methyl-N-nitrosourea. Cancer Res 35:2269-2276, 1975 6. Dissin J, Mills LR, Main DL, Black 0, Webster PD: Experimental induction of pancreatic adenocarcinoma in rats. J Natl Cancer Inst 55:857-864, 1975 7. Bockman DE, Black 0, Mills LR, Mainz DL, Webster PD: Fine structure of pancreatic adenocarcinoma induced in rats by 7,12-dimethylbenz(a)anthracene. J Natl Cancer Inst 57:931-936, 1976
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8. Axtell LM. Cuttler SJ Myers NIH (editors): End Results in Cancer. Report 4. Biometrv Branch. National Cancer Institute. US Department of Health. Education and W'eifare Publication No. (NIH) 73-272, 1972 9. Reddy JK. Rao NMS: Malignant tumors in rats fed nafenopin, a hepatic peroxisome proliferator. J Nat] Cancer Inst 59:1645-1650. 19,77 10. Reddy JK. Rao NMS: Transplantable pancreatic carcinoma of the rat. Science 198:78-80. 1977 11. Rao MS, Reddy JK: Malignant neoplasms in rats fed lasiocarpine. Br J Cancer 37:289-293. 1978 12. Klessen C: Histochemical staining of zymogen granules of pancreatic acinar cells using a permanganate-HID technique. Histochemie 30:365-366. 1972 13. Amsterdam A, Jamieson JD: Studies on dispersed pancreatic exocrine cells. J Cell Biol 63:1037-1056, 1974 14. Amsterdam A, Jamieson JD: Studies on dispersed pancreatic exocrine cells. J Cell Biol 63:1057-1073, 1974 15. Reddy JK, Svoboda D: The relationship of nucleolar segregation to ribonucleic acid and svnthesis following the administration of selected hepatocarcinogens. Lab Invest 19:132-143, 1968 16. Carawav WNT: A stable starch substrate for the determination of am-lase in serum and other bod- fluids. Am J Clin Pathol 32:97-99, 1957 17. Roe JH, Byler RE: Serum lipase determination using a one-hour period of hydrolysis. Anal Biochem 6:451-460, 1973 18. Shinozuka H, Popp JA, Konishi Y: Ultrastructures of atypical acinar cell nodules in rat pancreas induced by 4-hv-droxvaminoquinoline-1-oxide. Lab Invest 34:501-509. 1976 19. Nevalainen TJ: Cytotoxicity of \inblastine and \incristine to pancreatic acinar cells. V'irchows Archiv (Cell Pathol) 18:119-127, 1975 20. Longnecker DS, Curphey TJ: Adenocarcinoma of the pancreas in azaserine-treated rats. Cancer Res 35:2249-2258, 1975 21. Motta P. Andrews PM, Caramia F, Correr S: Scanning electron microscopy of dissociated pancreatic acinar cell surfaces. Cell Tissue Res 176:493-504. 1977 22. Reddy JK. Rao NMS, Warren JR. Minick OT: Concanavalin A agglutinability and surface microvilli of dissociated normal and neoplastic pancreatic acinar cells of the rat. Exp Cell Res (In press) 23. Hicks RNM, W'akefield JSJ: Nlembrane changes during urothelial hyperplasia and neoplasia. Cancer Res 36:2502-2507, 1976 24. Arai NI, Kani T. Sugihara S, Nlatsumura K, NMiyata Y, Shinohara Y, Ito N: Scanning and transmission electron microscopy of changes in the urinary bladder in rats treated wvith N-buty l-N-(4-h. droxv-buty l ) nitrosoamine. Gann 65:529-340, 1974 25. Rao NIS, Reddy JK: Unpublished data 26. Pierce GB, Nakane PK, Martinez-Hernandez A, Ward JN: Ultrastructural comparison of differentiation of stem cells of murine adenocarcinomas of colon and breast with their normal counterparts. J Natl Cancer Inst 58:1329-1345, 1977 27. Emmelot P: Biochemical properties of normal and neoplastic cell surface: A review. Eur J Cancer 9:319-33, 1973
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[Illustrations follow]
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