Transplantation of azaserine-induced carcinomas of pancreas in rats

Cancer l,etters, 7 (1979) 197--202

197

Elsevier/North-HollandScientificPubhshersLtd

TRANSFLANTATION OF AZASERINE-INDUCI~D PANCREAS IN'RATS*

CARCINOMAS

OF

D A N I E L S L O N G N E C K E R ~ H E R M A N S. LILJA, JANICE FRENCH, E L N A KUHLMAI';N and W A L T E R N O L L Depar~men~ o f Pa~ologv, Dartmouth Medtcal School, Hanover, N H 03755 (U S.A )

(Received 26 March 1979) (Accepted 16 April 1979) SUMMARY Two pancreatic adenocarcinomas which had been induced m Wistar/ Lewis rats by azaserine treatment were transplanted into mrs of the same strah~ by subcutaneous and mtraperitone~l injection of minced tumor. Subsequently, we have serially transplanted into non-rachated recipients. Transplante,r] tumors have maintained evidence of acinar cell differentiation including the presence of zymogen granules in tumors stuched by electron microscopy, and of lipase, amylase and trypsin activity in the supernatant of tumor homogenates. Histologmally, the tumors vary from poorly differentiated solid carcinomas to well differentiated variants which form acini. Ttansplantad tumors are locally invasive and have metastasized to lung and liver in some recipients. INTRODUCTION Azaserine is a pancreatic carcinogen in rats which induc,es a spectrum of pancreatic lesions including nodules of atypical acinar cells, adenomas and adenocarcinomas [3]. Atypical acinar cell nodules (AACN) have been observed as early a~ 2 months after h~itial azaserine injection. Adenomas have develloped by 6 months and aclenoearcinomas 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 adenoc~rcinomas. We 13ave been successful only when we have transplanted from a grossly Address ¢dl correspondence to DarnelS. Longnecker, NCI, NIH, Bmldlng 37, R o o m 5B22,

B~thesda MD 20205, U I~.A. *Supporte,rlby USPHS contractNo. 1 CP 33378 and grant CA-20948 from the Divismn o£ Cm~cer Cause z~d Prevention,NCI/NIH

198

identifiable pancreatic carcinoma. The characteristics of 2 such tumors ~ e described here. MATERIALS AND METHODS Wister/Levris rats (Charles River Breeding Laboratories, Wilmington, MA) were fed laboratory cho~ (except as noted) and water ad libitura. Aza.,;erine (Calbiochem., La JoUa, CA) dissolved in 0.9% NaC1 was injected intraperitoneally to ~aduce AACN and carcinomas. A total of [2--15 .;njecticn% 10 mg/kg, were given ~wice weekly, weekly, or monthly. Rats were autopmed 2--18 months after irdtial treatment. Pancreas or tumor from an area oE necro,~ls was excised and minced in sterile Hanks balanced salt solution ~mtfl fragmenLs were small enough to pass thro~Jgh an 18 gauge needle. The suspension of minced tissue was injected both subcutaneously between the scapulae and intraperi~oneally, depositing about 0.5 ml (estimated to contaJ~ 5--25 mg tissue) at each .,'i/~e.Tbr~ recipients received 425 fads of whole body radiation on the day prior to .nitial transp][antation of pancreas from azaserine-treated rats. After transpmnted tumors were established subsequent passages have been to non-radiated recipienLs. Recipients were examined weekly for evidence of tumor growth for a pe~5od of 3 months when they were autopsied. During ~,he early phase of transpl~mtation attempts (i.e., 2--12 months), saline-mjecll~d rats were also used as donors to control for the survival and/or growth of ~ransplanted 'normal' p~ncreatic tissue° Tzssue from one donor was injected into 5 recipients. Residual pancre~ or portions of tumor of donor rats was fixed i,a Susa's solution for histologic study. Portions of trausplanted minors have been fixed in 4% cacodylate-buffered glutaraldehyde (pH 7.4) for electron microscopy ~md prepared as described previously [2]. Evaluatmn of some recipients has been limited to gross exam and histologic study of neoplasms. Portions of transplanted neopl.a,'~msfrom several rats were frozen at the time of ;autopsy for enzyme assays which were subsequently performed using ~he 27019 × g supernatant of 1 : 10 (w/v) homogenates prepared in NaC1 (0.015 M)-NaCitrate {0.0015 M) buffer, pH 7.0.. Trypsin activity was assayed using ~ne spectrophotomet~c method of Hummel [1] with p-tosyl-L-arginine methylester as substrate. ~mylase activity was measured using a dyed starched me~hod (Phadebas®, Pharmacia Diagnostics, Piscataway, NJ). LfLpaseactivity was determined by hydrolysis of an olive oil suspension and ~itrimetric assay of liberated fatty acids using a modification [7] of ~he method of Tiletz [8]. R E S D ,LTS

Data for the primary transpI~antation attempts is summanzea in Tahle 1. We fulled in 10 atte.npi, s to establish transplants from AACN b~aring pan-

199 TABLE 1 S U M M A R Y O F A T T E M P T E D T R A N S P L A N T A T I O N F R O M PANCREASES OF AZASERIN~E-TREATED WISTAR/LEWIS R A T S Donor rat treatment a

Les~on b

Interval c

Number of donors

Recipients

'Takes'

A A A A A A B C

AACN AACN AACN AACN AACN AACN, sdenoma Adenoearclnoma Adenocarcinoma

2 4 6 9 12 18 15 12

2 2 2 2 2 2 1 1

12 10 10 10 J0 10 5 4e

0 0 0 0 0 0d l 4

a Azaserme mject~ons were given according to the followmgschedules: (A) 12 mtections, 10 ,ng/kg, twice weekly for 6 weeks, (B) 15 mjectmns, 10 mg/kg, once weekly. The projec,* number CP-33378, will be used to denote transplants from th~s tumor, (C) 15 inlectionz. 10 mg/kg, once weekly for 6 week~ and then once monthly for 9 months. This group wa,-• maintained on a modffmation of the defined diet recommended for rats by the American Institute for Nutritmn which contained 18% cor~ od. The project number, CA-20948, will denote transplants from this turner. bA portion of the transphnted pancreas and/or neoplasm was proc,.~ssed for histology. The hmtologic diagnosis is indmated here. CThe interval from first azaserlne mjectmn until transpla1~tatic,,1 ~r,d autopsy dFour recipmnts from one of these donors contained persmtent dueta~ styuctures m the site of subcutaneous implantatmn. These were detected histologically, but they were not identified grossly. The residual donor pancreas contained both adenoma~ and AACN. eThese le~ ]pmnts did not receive radiatmn.

c r e a s e s . We w e r e s u c c e s s f u l in 2 a t t e m p t s t o t r a n s p l a n t g r o s s l y i d e n t i f i e d pancreatic neoplasms. T h e f i r s t s u c c e s s f u l t r a n s p l a n t w a s f r o m a 9 - r a m r, m s s in t h e h e a d o f t h e pancreas and the second was from a 2.0-cm pancreatic mass. Histologically, the neophsms were poorly differentiated adenocarcinomas which grew m several p a t t e r n s . T h e m o s t c o m m o n h~_stologic a p p e a r a n c e w~s as a s o l i d m a s s o f r e l a t i v e l y s m a l l cells w i t h o u t e w : d e n c e o f g l a n d f o r m a t , , o n o r z y m o g e n p r o d u c t i o n ( F i g . 1). In l i m i t e d a r e a s , t h e n e o p l a s m c o n t a i n e d a c i n a r o r t u b f f l a r s ~ r u c t u r e s c o m p o s e d o f cells w i t h a p i c a l , e o s i n o p h i l i c , c o a r s e l y g r a n u l a r c y t o p l a s m ~ol~sl: t e n t w i t h z:y m o g e n g r a n u l e s ( F i g . 2). I n o t h e r a r e a s t h e n e o p l a s m s w e r e c y s t i c a n d lived b y c u b o i d a l cells. C e n t r a l n e c r o s i s w a s c o m m o n in l a r g e r messes. Electron micrographs showed abundant rough endoplasmic retlculum and zy~mogen g r a n u l e s in s o m e t u m o r cells (Fig. 3). T h u s , t h e p r i m a r y n e o p l a s m s and the transplants are similar to pre~lously described azasertne-induced

200

Fig 1. Pancreatm carcir~oma from a subcutaneous transplant o f t ~ m o r CP 33378. Th~s solid area shows httle e ~ d e n c e o f acnnar cell dffferenhation. H & E × 240 F~g. 2 Pancreatnc carcinoma in liver from transplant rec~pmnt of t u m o r CA 20948 The formation o f tubules and acmn ns evidence ~ f acmar cell dffferen~natnon H & E × 240

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201 neoplasms which have maintained evideuce of acinar cell differentiation. T u m o r homogenates demonstrated trypsin, amylase and li_pase activity (Table 2). The level of activity was lower than that o f normal pancreas, but evidence o f these enzyme activxties persisted through 6 passages. Subcutaneausly transplanted t u m o r s achieved a m a x i m u m 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 neoplas,als invaded the ~horax. Intraperitoneal transplants grew in the small bowel mesentery, the omenturn, the retroperitoveum, and in the region ~f the pancreas. Foci fi. k/dney, liver, spleen and lung were apparently metastatic. The largest intraperitoneal mass weighed 18 g although most were nodules measuring 2--6 m m in diameter. The CP-33378 derived carcinoma has been transplanted into 49 recipients from 13 donors. Nine of the last 10 recipients have developed tumors. The CA-20948 derived t u m o r has been trans,.)lanted into 15 rats representing 4 ,donors. Eleven of 1;he last 12 recipients have had -'takes.' Although none of the recipients which were implanted with AACNbearing pancreas developed neoplasms, a few surviving well differentiated acmar cells and duc~like structures were found subcutaneously in the implantation site.It is not k n o w n if these cells were derived from A A C N or frum normal pancreas, but w e did not find such remnants in rats implanted with pancreas fragments from control rats.

TABLE2 E N Z Y M E ACTIVITY O F T R A N S P L A N T E D T U M O R H O M O G E N A T E S a

7~mor (generation)

nb

Amylase

Lipase

Trypsin

CP 33378 (2)

2 1 3

2389 1731 7426 4506 4'954 1327 1583 1333 16667

120 100 290 120 25 310 697 120 1200

--

(3) (4) (5)

(~) CA20948(1) (2) (4) Rat pancreas

3 2 3 3 1 1

2.8 --

--12.7 ----

aEm:yme activity is expressed as umts/ml of homogenate sup~xuatant which represents 100 mg of tissue (we1,wt). Amylase ~nd trypsin activity is given in international units, and lipase act~ivity is expressed as Slgma-Teitz units. bNumber of tumors assayed. Enzyme activities are the mean for n homogenates.

202

DISCUSSION

The transplantableazaserine-inducedadenocarcinomas described in this zeport show ultrastmctuml and biochemical characteristicsof acinar cell differentiai~Lon.They seem thereforeto be similarto the transplantable acinar cell iI)eoplasmreported by Reddy and Rao [4]. Recently, transphntable ductal adenocarcinomas of. pancreas induced by N-nitrosobi~.~(2oxoprop~d)amine have been describedin hamster~ by 2 groups [5,6].The availabili~ o f this spectrum o f transplantable pancreatic carcinomas in 2experimental species offers models for studies o f t u m o r biology and experimen~.7, chemotherapy. Our failure to establish neoplasms b y implantation o f minced pancreas containing AACN is consistent with t h e interpretation that these lesinns, in genera], do n o t have adequate growth potential and a u t o n o m y for 'malignant' gro~#th. On the other hand, the failure to establish neoplasms in these experiments could reflect a technical b a t ~ e r such as a low dose of AACNderived cells and p o o r cell survival under the: transplant conditions. Previous s~mdies in rats have s h o w n that the r a t m of the numl~er of AACN to the n u m b e r o f foci o f carch~oma is high in carcinogen-t£eated animals. We estimate t h a t this ratio is in the range o~ 100--1000. Thus: the failure to transplant could also reflect a specific failure to implant an .~ACN with malignant growth potential. REFERENCES

1 Hummel, B.C.W. (1959) A modified spectrophotometrm determination of chym~trypsin,trypsin, and thrombin. Can. J. B~ochem. Physiol., 37, 1393--1399. 2 I.~ugnecker, D.S., Crawford, B.C. and Nadler, D.J. (1975) Recovery of panc~ea~ from mild puromycin-indueed injury, a histologic and ultrastructural study in rats. l~ch. Pathol., 99, 5--10. 3 Y'~ngnecker,D.S. and Curphey, T.J. (1975) Adenocarcinoma of the pancreas in azsserine treated rats. Cancer Res., 35, 2249--2257. 4 Reddy, J.k. al~d P,~o, M.S. (19T7) Transplan.~ab~epancreatic carcinoma of the rat. Science, 198, 78--80 5 S~'arpelli~D.G. and Ran, M.S. (1979)Transplantable ductal at~enocascinoma nf Syrian hamsL~rpancreas. Cancer Res, 39, 452--458. 6 Takabashi, M.~ Runge, R., Do~melly, T. and Pour, P. (t979) The morphologm snd biologic cha~.mtenstics of ehemmally-induced pancrear~ic adenocarcinoma in Syrmn golden harn~ers a£~er homologous transplantation. Cancer Letters, in pres~. 7 Technical Blllletin No. 800, Sigma Chemical Company, St. Louis, Me 8 Tmtz, N.W., Borden, T. and Stepleton, J.D. (1959) An improved method for the determination of lipase in serum. Am. J. Clin. Pathol., 31,148--154.