Transplantability of chemically induced skin tumors in syngeneic strains of mice, rats and guinea pigs

Exp. Path. 1i, 121-127 (1979) Dep1Htment of Pathology (Head: Prof. K. DAThDIERT, 1\1. D.), University of Quln, and Pathology Laboratory of the Finnish Oancer Foundation (Head: Assoc. Prof. Q. J{As;LxEN, NI. D.), QUill, Finland

Transplantability of chemically induced skin tumors in syngeneic strains of mice, rats and guinea pigs By. 0. RASA;\EN With 2 figures (Reeeived October 6, 1978) Address for correspondence: O. RAS;(N EN, Associate Professor, Department of Pathology, University of Oulu, SF - 90220 Oulu 22, Finland. Key w 0 r d s: skin tumors, chemically induced; transpla,ntability; basalioma; sq llamous eell careinoma: fibrosareoma: 9,10-dimethyl-1,2-benzanthracene; supporting agent in tumor transpl,tntation

Summary Syngeneic tumour transplants to inbred female animal strains were carried out with basaliomas, squamous cell carcinomas and fibrosarcomas of the skin, induced by DNIBA (9,10-dimethyl-l,2benz
J1 aterial and methods Tumor production The carcinogen used in !t\l the series was 9,10-dimethyl-1,2-benzanthracene (DNIBA), manufactured by Gnrr and diss:)lved in acetone. It was instilled on the dorsal skin of the experimental animals, over an interscapular area of 2 X 2 em which had been depilated. The numbers of animals, the amount of carcinogen, and the rates and periods of instillation were: n Exp. Path. 17, H. 3

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1. 10 mice, of the inbred NMRI strain, 20 pg DMBA instilled 3 times a week for 15 weeks. The result was one adnexal skin tumor that was tmnsplanted to 10 mice. 2. 10 mice, of the inbred brjHR strain, pilose and pigmented, were treated in the same way. Two squamous cell carcinomas suitable for transplantation were obtained and their suspensions were tmnsplanted to 15 mice altogether. 3. 10 rats, of the inbred Sprague-Dawley strain, were treated with a single dose of 200 pg DlVIBA 6 times a week for 15 weeks. Of the tumors that developed, two basaIiomas were suitable for transplantation, which were transplanted to altogether 25 rats, and three squamous cell carcinomas, which were transplanted to altogether 24 rats. 4. 5 rats, Sprague-Dawley strain, were given a single intracutaneolls injection of 100 f'g DiVIBA per animal. Of the resulting tumors, one fibrosarcoma was transplanted to 6 rats. 5. 10 guinea pigs, of ordinary laboratory strain, were treated with instillation 8f DlVIBA 20 pg on the dorsal skin, G times a week for 108 weeks. Only fibrosarcomas developed, and two of them were suitable for transplantation ,md they were gmfted on altogether 20 guinea pigs. All animals apart from the grey mice of the hrjHR strain, were obtained from lVIankkaa, Finland (Mankkaa Research Labomtory of Orion Oy, Medical Suppliers). The animals were 2-3 months old at the beginning of the experiments. They were fed on ordinary laboratory diet (The Hankkija Fodder llIixture, Hankkija Central Cooperative, Helsinki, Finland) and kept in plastic cages, 4-8 per cage. The grey mice of the hrjHR strain were obtalned from Prof. O. H. Iversen of the "Institut for Generell og Experimentell Patologi", Oslo, Norway. All animals used in the experiment were female. Tumors were transplanted into a total of 100 animals, and 14 animals were used as controls. Techniques and groups of transplantation The tumor was in each ease excised and grafted aseptically. For the grait, 0.5 g of oaeh tumour was taken from the most viable immediately after the animal had been killed. It was ground with 2 ml Ringer's solution by passing the suspension through a 0.34 mm mesh steel wire net. 0.5 ml of the tnmor suspension thus obtained was injected subcutaneously per animal as such, with a syngeneic myocardial suspension of a young (not more than seven days) animal (0.5 ml), or with a syngeneic embryonal skin suspension (0.5 ml). Prophylactically against the infection 10000 I. U. S-penicilline-G (lVI, S & D) per 0.2 ml transplantation suspension were given with the transplantate. To reduce the immunological defence mechanism, some groups of host animals were given X-ray irradiation one hour before the transplantation as follows: 4-G animals, depending on their size, were placed in a plastic cage which was irradiated with 370 rad from 11 distance of 55 cm. The filter was of copper and the current 230 kV ami 15 mA over an area of 26 X 26 cm. The treatment lasted for 9 minutes. The control animals were given only Ringer's solution, or together with that the myocardial suspension or the embryonal skin suspension. They were also exposed to X-ray irradiation in the corresponding groups. The general experimental armngements can be seen in table 1. Table 1. The geneml experimental arrangements Test groups: Tumor homogenate Tumor homogenate Tumor homogenate

+ + +

Ringer's solution Ringer's solution Ringer's solution

+ S-penicilline-G + S-penicilline-G + Myocardial suspension + S-penicilline-G + Embryonal skin suspension

Control groups: Ringer's solution + S-penicilline-G Ringer's solution + J\Iyocardial sllspension + S-penicilline-G Ringer's solution + Embryonal skin snspension + S-penicilline-G Both in test groups and in control groups to part of the animals X-ray irradiation was given one hOllr before the transplantation.

Results D.MBA was found to be a relatively effective inducer of tumors. But as it can be seen from the section on "Material and Methods", each group of animals yielded relatively few tumors suitable for transplantation, since many of the tumors contained necrotic areas and inflammation. After microscopic frozen section, a control viable tumor was chosen for transplantation. Tumors were grafted on a total of 100 animals, and in 8 of them a tumor

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Fig. 1. Fibrosarcoma in rat skin, co mposed of small and a few large cells of compact organization, growing with diffuse infiltration int o the surrounding tiss ues. HE st aining, x 125.

developed or the graft t ook in the co urse of the 18 months of follow-up. During the fullo w-lI 11 time it could be proved that one pleomorphic fibro sarcoma of the rat grew within one month in all the three rats grafted on into a distinctly palpabl e tumor at the site of grafting and it was histologically similar to the origiral tumor(fig. 1). This tumor plantation was accompanied by a myocardial suspension. In the parallel experimental gro up of three rats, in which onl y the tumor homogenate and Ringer's solution were grafted, no tumors developed during th e follow-up period. One of these rat s died after 35 days. No X -ray irradiation was given t o either of the experimental groups or the control group. The rest of the tumors proved in ho st animals to manifest themselves only after an interval of 10 mo nths or more and th erefore we cannot be sure if t hose were really tumors graft ed on or if they are to be regarded spontaneous despite the fact that two spino cellular carcinomas of the rat were histologically identi cal with the transplanted t umor (fig. 2, table 2, B) . Of the remaining three tumors, two were spinocellular carcinomas and one a.n adeno carcin oma, but they grew up in the co urse of 21 / 2 - 18 months in t he gronp of rats that had been grafted on with basalioma (tab le 2, C), which is why they mnst be interpreted as spontaneous tumors. The transplanted or grown tumor3 can be seen in table 2. No other tum ors grew neither in the other te3t gro ups or in the control groups cluring the follow-up time. 9*

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Fig. 2. Spinocellular carcino ma in rat skin, composed of tu mo r tissue presenting itself in th e form of large islets . Locally, a distinct tendenc y to keratinization can be seen. I-IE staining, X 125.

Discussion In the present study, transplantation of tumor was successful with the fibrosarcoma of rat skin (fig. 1) in all the grafts made with a tumor suspension to whi ch myocardial suspension of a yo ung, "immun ologically incompetent " rat had been added to serve as a kind of "supporting agent" . Thi s a.ppears to be a new interesting ph enomenon not reported previously. It has been reported earlier that t he transplantability of the tumors improved and their growth rate increased when the test animals were simultaneou sly inj eceted with homo-

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Table 2. Successfully transplan ted or dev eloped tu mo rs N umb er of experimental host anim als

A. Fi brosarcoma of rat s k i n Tumor homogenate + Ringer's sol ution myocardi,tl suspen+ sion B. S pinocellula r car cin oma o f ra t ski n T nmor h omogenate + Ringer's solution T Ullor homogenate + R inger's solution + myocardi a l sllspension of rat

X-ray

irrndiation

Tim!) after t he Tumor whi ch t ransplantatio n took or dev elo ped in host anillUtls

i3

1 month

10

12 months

1 spinocelJ nlar carcin oma

10 months

1 spinoeellnJar Cnl'CillOm l1

18 months

1 spinocelJ uiar carrino ma 1 spinoc ell uJ a r careinoma

()

+

:.1 fibrosarco illa s

C. Bas ali om<1

of rat s ldn Tumo r homogenate + Ringer's solution Tumor homogenate + Ringer's solution

T umor homogenate + Ringer's solution +- myocardial sllspension of r at

4

6

.L

ii

+

21/2 mou ths 10 month s

1 adeno car cinoma (in t he skin)

logous tissue homogenates of embry on al (SCH NEYER IDol) or gro wn (FISHE R (;t al. 1963) animals , but in t hese experiments the eardiac muscle was not exclusively used. Subcutaueous fibrosareomas of rats indueed by 3,4-b en zpyre ne (BP) also provided significantl y better resu It s in transplantations an d grew faster by this method th an t hose in the comparative groups (RJ. SAXE N and SKURNIK, to be published later) . The uns ure positive r esult. of the present experiment of grafting epider mal tumo rs (possibly two spinocellul ar carcinomas of the rat skin , table 2, TI) agrees well with the literature reports, a mong whieh I was not able to find any unambigu ously sueeessful transplantati on of a syngeneic epidermal tumor induce d with D MB A. A~ it can be seen from the literat ure of the introduction , successful t ransplantations ill several su ccessive generations of many inbred animal strains h ave bren accomp lish ed wit h several oth er car cinogenH a nd iu several other organs. The small numb er of the t um ors sUl:eessflllly transp lante d was hardly du e to the method of grafting, for benzpyrene-in duced ski n sareomas grafte d with t he same m ethod had a suecess rat e of ne arl y 100 % in a syngeneic stra.in of Long Evans rats ( RX. S _~N E N and SKURNIK, to be published). The test a.nimals we Ilsed ill t his work were also known inbred strains (brother - sister o"er 20 generations) . Theoretically sp eakjng, one reason might he the fact t hat th e transplantation of D:YIB:\indnred epidermal t u mors involves marked alterations of t he epider mal chalone mechani sms, possibly an en haneed chalonic effect of prevent ing cell division and proliferation, whi ch effect ep id ermal ehalc)]] es have been found to have in ani mal exp eriments (ELGJO and HE~N l "G S 1971; L AURE NCE and ELGJO 1£)71) and in human sq uamo us epithelial earcinoma. in vitro ( K ORSGAA RD et al. 1977). It m ust furth er be taken into aeeount that tum or-associate d t r ansplanta.tion antigenicity (T ATA) (DELU PORTA and PAKlflANI 1976) or " tumor

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specificity" (ALEKSANDER 197fi) may be more intensive and versatile in DlYIBA-induced epidermal tumors than in e. g. dermal fibrosarcomas, for mouse epidermis alone contains as many as four tissue-specific antigens, while rat epidermis contains eight sueh antigens (RASANEN 1968). It was recently noted in our laboratory that rat (,JARVINE:\T et al. 1\:)78) and human epidermis (RASA:\T'EN et al. 1978) as well a~ the other squamous epithelia of these speeies (RINNE et al. H178) contain a strongly antigenic low-molecular SH-protease inhibitor specific to these epithelia only; it does, however, appear in human squamous epithelial carcinoma of the lung (JAHVINEN et al. 1978), but not in other lung carcinomas. Such an antigen may also playa role in the poor transplantability of DlYIBA-induced epidermal tumors, because potent antigens probably activate the major histocompatibility complex, which at least in the monse is known as a H-2 complex associated with the transplantation rejection reaction (APELLA and LAW 1976). Our team is therefore pursuing studies on the occurrence of the aforesaid highly antigenic SH-protease inhibitor in e. g. DlVIBA-induced skin tumors of the rat and their possible transplants.

Acknowledgement My sincere thanks are dne to Mrs. Ranhu, Tolppanen who helped in the anirmd inoculations.

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