Cancer Letters, 7 (1979) 197—202
`0 Else-vier/North-Holland Scientific Publishers Ltd
`
`197
`
`TRANSPLANTATION OF AZASERINE-INDUCLD CARCINOMAS OF
`PANCREAS IN‘lRATS*
`
`DANIEL S LONGNECKER. HERMAN S. LILJA, JANICE FRENCH, ELNA
`KUHLMAHN and WALTER NOLL
`Department of Pathology. Dartmouth Medical School, Hanover, NH 03755 (H 5.11 )
`(Received 26 March 1979}
`(Accepted 16 April 1979)
`
`SUMMARY
`
`Two pancreatic adenocarcinomas which had been induced in Wistar/
`Lewis rats by azuserine treatment were transplanted into rats of the same
`strain by subcutaneous and intraperitoneal injection of minced tumor.
`Subsequently, we have serially transplanted into non-rachated recipients.
`Transplanted tumors have maintained evidence of acinar cell differentiation
`including the presence of zymogen granules in tumors studied by electron
`microscopy, and of lipase, amylase and tiypsin activity in the supernatant
`of tumor homogenates. Histologically, the tumors vary from poorly dif-
`fei-en tiated solid carcinomas to well differentiated variants which form
`acini. Transplanted tumors are locally invasive and have metastasized to lung
`and liver in some recipients.
`
`INTRODUCTION
`
`Aziaiserine is a pancreatic carcinogen in rats which induces a spectrum of
`pancreatic lesions including nodules of atypical acinar cells, adenomas and
`adenocarcinomns [3]. Atypical acinar cell nodules (AACN) have been ob-
`served as early as 2 months after initial azaserine injection. Adenomas have
`developed by 6 months and adenocarcinomas by 9—12 months. We have
`attempted to establish transplantable neoplasms by implantation of minced
`AACN-bearing pancreas throughout the 2—18 month interval after initial
`azaserine treatment and by transplantation of induced adenocarcinomau.
`We have been successful only when we have transplanted from a grossly
`
`Addrosa all correspondence to Daniel S. Lonmiecker, NCI, NIH. Building 37. Room 5322,
`Bethesda MD 20205, U SA.
`*Supported by UBPHS (entrant No. 1 GP 33378 and grant (DA-20948 from the Division
`of Cancer Cause and Prevention. NCI/NIH
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`198
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`identifiable pancreatic carcinoma. The characteristics of 2 such tumors
`are described here.
`
`MATERIALS AND METHODS
`
`WisterlLewis rats (Charles River Breeding Laboratories, Wilmington, MA)
`were fed laboratory chow (except as noted) and water ad libitum. Azaserine
`(Calbiocliem. La Jolla, CA) dissolved in 0.9% NaCl was injected intraperi~
`mneally to induce AACN and carcinomas. A total of 12—15 injections,
`10 rug/kg, were given twice weekly, weekly, or monthly.
`Rats were autopsied 2—18 months after initial treatment. Pancreas or
`tumor from an area of necrosis was cxcisad and minced in sterile Hanks balanc-
`ed salt solution until fragments were small enough to pass through an 18
`gauge needle. The suspension of minced tissue was injected both subcuta-
`neously between the scapulae and intraperitoneally, depositing about 0.5 in]
`(estimated to contain 5—25 mg tissue) at each site. The recipients received
`425 rads of whole body radiation on the day prior to -ni'tia.l transplantation
`of pancreas from azaserine-treated rats. After transpianted tumors were
`established subsequent passages have been to non-radiated recipients. Re~
`cipiems were examined weekly for evidence of tumor growth for a period
`of 3 months when they were autopsied.
`During the early phase of transplantation attempts (i.e., 2—12 months),
`saline-mjecwd rats were also used as donors to control for the sumval and/or
`growth of transplanted ‘normal' pancreatic tissue. Tissue from one donor
`was injected into 5 recipients.
`Residual pancreas or portions of tumor of donor rats was fixed in Susa’s
`solution for histologic study. Portions of transplanted tumors have been
`fixed in 4% cacodylate-buffered glutaralclehyde (pH 7.4) for electron micro-
`scopy and prepared as described previously [2]. Evaluation of some recipi~
`ants has been limited to gross exam and histologic study of neoplasms.
`Portions of transplaan neoplasms from several rats were frozen at the
`time of autopsy for enzyme assays which were subsequently performed
`using the 2700 X g supernatant of 1 : 10 (w/v) homogenates prepared in
`N30! (0 .015 M)—NaCitrate (0.0015 M) buffer, pH 7.0. 'ii‘rypsm activity
`was assayed using the spectrophotometric method of Hummel [1] with
`p—tosyl-L-arginine methylester as substrate. Amylase activity was measured
`using a dyed starched method (Phadebas®, Phannacia Diagnostics, Piscata-
`way, NJ). Lipase activity was determmed by hydrolysis of an olive oil
`suspension and titrimetr'ic assay of liberated fatty acids using a modification
`[7] of the method of Tietz [8].
`RESU 'L'I‘S
`
`Data for the primary transplantation attempts'is summarized in Table 1.
`We failed in 10 attempts to establish transplants from AACN bearing pan-
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`TABLE 1
`SUMMARY OF ATTEMPTED TRANSPLANTATION FROM PANCREASES OF
`AZASERINETREATED WISTAE/LEWIS RATS
`'Takes’
`Donor rat
`Lesionh
`Interval”
`Number of
`Recxpients
`treatment ‘
`donors
`_.____'___________________,____._.________,.._
`A
`AACN
`2
`2
`12
`0
`A
`AACN
`4
`2
`10
`0
`A
`MON
`6
`2
`10
`0
`A
`AACN
`9
`2
`1 0
`0
`A
`MON
`1 2
`2
`1 0
`0
`A
`AACN, adenoma
`18
`2
`10
`0'1
`B
`Adenocarcmome
`1 5
`1
`5
`1.
`
` 12Adenccarcinoma Al 1 4‘
`
`C
`
`
`
`" Azssennc injections were given according to the followmg'Schedulcs: (A) 12 Imections,
`10 org/kg, twice weekly for 6 weeks, (B) 15 injections. 10 rug/kg. once weekly. The
`proper number (IF-33378, will be mud to denote transplants from this tumor, (0) 15
`injection:. 10 mglkg, once weekly for 6 weeks and then once monthly for 9 months.
`This group vva.I maintained on a modification of the defined diet recommended for rats
`by the American Institute for Nutrition which contained 18% corn oil. The prOJect
`number, CA-20948, will denote transplants from this tumor.
`1'A portion of the transplanted pancreas and/or neoplasm was processed for histology.
`The histologic diagnosis is indicated here.
`0The interval horn first azaserine injection until transplantation «nd autopsy
`“Four recipiean from one of these donors contained persustent ductal structures In the
`site of subcutaneous implantation. These were detected histologically. but they were not
`identified grossly. The resrdual donor pancreas contained both adenomas and AACN.
`aThese rel iplents did not receiva radiation.
`
`creasas. We were successful in 2 attempts to transplant grossly identified
`pancreatic neoplasms.
`The first successful transplant was from a 9-mm mass in the head of the
`panoreas and the second was from a 2.0-cm pancreatic mass. Histologically,
`the neoplasms were poorly differentiated adenocarcinomas which grew “1
`several patterns. The most common histologic appearance was as a solid
`mass ofrelatively small cells w1thout evidence of gland formation or zymogen
`production (Fig. 1). In limited areas, the neoplasm contained acinar or
`tubular structures composed of cells with apical, eosinophilic, coarsely
`granular cytoplasm cousu tent mth zy mogen granules (Fig. 2). In other
`areas the neoplasms were cystic and lived by cuboidal cells. Central necrosis
`was common in larger messes.
`Electron micrographs showed abundant rough endoplasmic retlculum and
`zymogen granules in some tumor cells (Fig. 3). Thus, the pnmary neoplasms
`and the transplants are Similar to prenously described azaserme-induced
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`Fig 1. Pancreatic carcinoma from a subcutaneous transplant of tumor CP 33378. This
`solid area shows httle evndence of acmar cell differenhaflon. H 8: E x 240
`Fig. 2 Pancreatic carcmomfl 1n liver from transplant recxplent of tumor CA 20948 The
`formation of tubules and mum Is evidence or acmar cell dlfferenhatmn H & E X 240
`
`
`
`
`Flt; 3. Ce
`ruticulm and zymogen granuleu Lend citrate and uranyl acetate )t 5000
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`201
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`neoplasms which have maintained evidence of aciiiar cell differentiation.
`Tumor homogenates demonstrated trypsin, amylase and lipase actiVity
`(Table 2). The level of activity was lower than that of normal pancreas, but
`evidence of these enzyme activ1ties persisted through 6 passages.
`Subcutaneously transplanted tumors achieved a maximum diameter of
`.5.4 cm in as short a period as 3.5 weeks. Larger neoplasms tended to ulcerate
`and become necrotic. Some of these neoplasms invaded the thorax.
`Intraperitoneal transplants grew in the small bowel mesentery, the omen-
`turn, the retroperitoneum, and in the region of the pancreas. Foci ii. kidney.
`liver, spleen and lung were apparently metastatic. The largest intraperitoneal
`mass weighed 18 g although most were nodules measuring 2—6 mm in
`diameter.
`The CP-33878 derived carcinoma has been transplanted into 49 recipients
`from 13 donors. Nine of the last 10 recipients have developed tumors. The
`CA-20948 derived tumor has been transplanted into 15 rats representing
`4 donors. Eleven of the last 12 recipients have had ‘takes.’
`Although none of the recipients which were implanted with AACN-
`bearing pancreas developed neoplasms, a few surviving well differentiated
`acmar cells and ductlike structures were found subcutaneously in the im-
`plantation site. It is not known if these cells were derived horn AACN or
`from normal pancreas, but we did not find such remnants in rats implanted
`with pancreas fragments from control rats.
`
`TABLE 2
`
`_ ENZYME ACTIVITY OF TRANSPLANTED TUMOR HOMOGENA'I‘ES“
`Tumor (generation)
`nb
`Amylase
`Lipase
`'Ih'ypsin
`CP 33378 (2)
`2
`2389
`120
`—
`(8)
`1
`1731
`100
`2.8
`(4)
`S
`7426
`290
`—
`(5)
`3
`4506
`1 20
`-
`(e)
`2
`4954
`25
`—-
`CA 209480)
`3
`1327
`810
`12.7
`(2)
`3
`1 583
`69'!
`-
`(4)
`1
`1333
`120
`-
`Rat pancreas
`1
`16667
`1200
`
`“Enuyme activity is expressed as units/ml of homogenate supernatant which represents
`100 mg of tissue (wei, wt). Amylase and trypsin activity is given in international units. and
`lipase activity is expressed as Sigma-Tana unim.
`bNumber of tumors assayed. Enzyme activities are the mean for n homogenates.
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`DISCUSSION
`
`The transplantable azaserine-induoed adenocarcinomas described in this
`report show ultrastructural and biochemical characteristics of acinar cell
`differentiation. They seem therefore to be similar to the transplantable
`acinar cell neoplasm reported by Reddy and Rao [4]. Recently, trans-
`plantable ductal adenocarcinomas of pancreas induced by N-nitrosobis(2-
`oxopropyliamine have been described in hamsters by 2 groups [5,6]. The
`availability of this spectrum of trmnsplantable pancreatic carcinomas in 2
`experimental species offers models for studies of tumor biology and ex-
`perimental chemotherapy.
`Our failure to establish neoplasms by implantation of minced pancreas
`containing AACN is consistent with the interpretation that these lesions,
`in general, do not have adequate growth potential and autonomy for ‘malig-
`nant’ growth. On the other hand, the failure to establish neoplasms in these
`experiments could reflect a technical barrier such as a low dose of AACN-
`deri'ved cells and poor cell survival under the transplant conditions. Pre-
`vious studies in rats have shown that the ratio of the number of AACN to
`the number of foci of carcinoma is high in carcinogen-treated animals. We
`estimate that this ratio is in the range of 100-1000. Thus, the failure to
`transplant could also reflect a specific failure to implant an AACN with
`malignant growth potential.
`REFERENCES
`
`1 Hurnmel, B.C.W. (1959) A modified spectrophotometnc determination of chymrx-
`tryuin, trypsin. and thrombin. Can. J. Biochem. Physiol., 37. 1893—1399.
`2 Longiecioer, D.S., Crawford, EC. and Nadier, DJ. (1975) Recovery of pancreas
`from mild puromycin‘inducsd injury. a histologic and ultrastructursi study in rats.
`Arch. Pathol., 99, 5—10.
`3 Lanznecker, BS. and Curphey, TJ. (1975) Adenocarcinoma of the pancreas in
`uzaserine treated rats. Cancer Res, 85, 2249-2257.
`4 Raddy, «Lit. and Rao, M.S. (1977) Transplantable pancreatic carcinoma of the nab.
`Science, 198, 78—80
`6 Scarpeiii, ILG. and Rao, M.S. (1979) Transplantable ductai alenocareinoma of
`Syrian hamster pancreas. Cancer Res, 39, 462—458.
`6 ’l‘akai-ashi, M., Rungc, R., Douneily, ’I‘. and Pour, P. (l979)’i‘he morpholognc and
`biologic chat .ictenstios of chemically-induced pancreatic ndenocarcinoma in Sy nan
`golden hammrs after homologous transplantation. Cancer Letters, in press
`Technical Bulletin No. 800, Sigma Chemical Company. St. Louis, MO
`'i‘ietz, N.W., Borden, T. and Stepieton, JD. (1959) An improved method for the
`determination of lipase in serum. Am. J. Clin. Pathol., 81. 14e—154.
`I
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`Gd
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