Experimental intra-oral carcinoma

165 J. max.-fac. Surg. 7 (1979) 165-171

Experimental Intra-Oral Carcinoma Hugh CANNELL

Department of Oral and Maxillo-Facial Surgery (Head: Prof. G. R. Seward, M.D.S., F.D.S., M.B., B.S.), London Hospital Medical College, London, U.K.

Summary

3. Antigenicity

Fundamental studies of tumour host relationships in oral cancer cases would be assisted if a suitable animal model could he found. Current concepts in the use of animal models for the study of intra-oral carcinoma are reviewed and the development of a unique transplantable tumour system which had been induced and maintained only in syngeneic animals, is recorded.

The tumour should be antigenically unique as is usual with tumours that originally have been induced by chemical carcinogens.

Key-Words: Intra-oral carcinoma; Animal models; New transplantable tumour. Introduction

Mortality figures from intra-oral carcinoma have not improved in the U.K. during the last few years (Binnie et al. 1972, Langdon et al. 1977), despite developments in the fields of surgery and of radiotherapy. Yet the lack of suitable animal models or oral cancer has prevented basic studies of tumour-host relationships which ultimately might help in the management of the disease. The purpose of this article is to record experience in the field of experimentally induced oral cancers and to report the successful evolution of a transplantable oral carcinoma. Criteria U s e d for the Studies

The criteria used for the studies demanded that: 1. Tumour type and derivation The tumour system should be a differentiated squamous cell carcinoma and therefore analogous to intra-oral cancer in humans (Lucas 1972). The primary tumour should have arisen in oral mucosa and should retain its differentiated characteristics through subsequent passages in vivo. 2. Syngeneic animals The turnout should have arisen and then be maintained in vivo, only within syngeneic animals as histo-incompatibility factors were then excluded. 0£01--0503/79

1300--0165

$ 03.00

4. Growth in oral sites The malignancy should be slow-growing in order to allow suitable periods of time for experiments. It should be capable of transfer to various oral sites and non-infected when growing sub-mucosally. 5. Regional lymph node involvement The tumour should spread late in the course of the disease to draining regional lymph nodes but distant metastases should be uncommon. The spread of the tumour would thus have features in common with the disease in humans (Lucas 1972). Material and Methods

Experiments were carried out to examine: 1. Tumour induction in various strains of the hamster (Mesocricetus auratus)

Induction was applications of 0.5 % 7,12-dimethylbenz-(a)-anthracene (DMBA) in pure paraffin oil (Salley 1957). The D M B A was applied thrice weekly to the left cheek pouches of three in-bred strains of the animals. The concurrent application of normal saline to right cheek pouches was used as a control. Continued growth into non-immunologically privileged oral sites of histologically verified carcinomata was used as an experimentl end-point (Cannell 1973). 2. Transfer of an allogeneic skin tumour to oral sites or subcutaneous sites

The purpose of the experiment was to test the feasibility of transplants to an oral site. A squamous carcinoma of allogeneic hamster skin (Chernozemski 1969) was used. Transplantation was by

@ Georg Thieme Publishers

166

H. Cannell

Table 1 Frequency of papillomata per treated pouch, 13 weeks after commencement of DMBA treatment in three inbred hamster strains. CLAC strain animals had signficantly more lesons than CA strain animals. (p < 0.01, Fisher's exact test.) Inbred strain

Colour

Total no.

No. assessed

Deaths (all causes)

CA CBC CLAC

Albino Cream Cream

24 25 20

9 7 7

1 0 4

Table 2 Mean times of appearances of first tumours in pouches of one inbred and two random-bred strains of hamsters following treatment by DMBA (Reishin and Berry 1968). Hamster strain

Colour

Tumour latent p.eriods Weeks + S.E.

Inbred DEA Random bred Random bred

Partial albino Golden Cream

7.3 -+ 2.8 10.0 _+ 0.49 10.7 _+ 0.44

subcutaneous inoculation of tumour fragments by trocar to available in-bred strains of hamster. Repeated passages in vivo allowed one strain to be selected for further studies. I n this strain (CBC), investigations were then carried out which compared the growth of transplanted tumour material at skin and oral sites. 3. Transfer of a syngeneic oral tumour to oral or subcutaneous sites

The experiment used data obtained from experiment (1). The strain of animal employed was the tumour susceptible but tumour burden resistant cream C L A C - L H . This strain of hamsters are maintained at our laboratories and had been continuously inbred for at least 27 generations so as to remain a genetically homozygous line. One D M B A induced differentiated squamous cell carcinoma which had invaded the oral cavity was selected for transplantation experiments. A t first the tumour was passaged only to subcutaneous sites on the flank, but later, experience gained in experiment (II) allowed comparison of tumour sizes to be made after growth at both flank and oral sites.

No. of lesions per treated pouch < lO > 10 8 3 1

1 4 6

Results 1. Strain differences

Asa result of treatment by DMBA, the left cheek pouches developed papillomata in each of tile three inbred strains. T h e cheek pouches treated with saline did not develop lesions. The number of papillomata was greatest in C L A C strain animals (Table 1) at 13 weeks after treatment commenced. The time of appearance of the first papillomata (latency) was not separately recorded but the susceptibility of cream animals to papilloma formation confirmed work by Reiskin and Beny (1968) (Table 2). W h e n papillomata were allowed to develop further, the CLAC strain animals developed invasive tumours into the oral cavity more slowly than the other two strains. 2. Allogeneie skin tumour growth

The experiments demonstrated the relative homogenicity of hamster strains as the allogeneic skin tumour (Chernozemshi 1969) from r a n d o m - b r e d golden strain animals was successfully transplanted to three different in-bred strains. Table 3 shows the results of inter-strain passages. Attempted transplants to oral sites resulted in fewer successful takes than similar transplants to subcutaneous sites. Nevertheless, tumour sizes were measured at 8, 19, 28 and 36 days after passage (Table 4), thus demonstrating that the techniques used with this allogeneic system were reproducible and of interest when similar experiments using the syngeneic tumours were to be attempted. 3. Syngeneic tumour growth

The syngeneic tumour material was successfully transplanted in 52 cases to subcutaneous sites in the flank. It was observed that successful tumour takes occurred more often when tumours were left to grow for some months before attempted passage

(Table 5).

Experimental Intra-Oral Carcinoma

167

'Fable 3 Results of 286 transplants of Chernozemski's allogeneic squamous carcinoma during passages, within and between available strains of hamster. CA strain Albino

Golden random-bred

CLAC strain cream

CBC strain cream

Skin 4/14 (28%)

Skin 8/14 (57%)

Skin 15/25 (60%)

Skiv 12/23 (52%)

Orat 5/28 (19%)

Oral 1/3 (33%)

Oral 22/58 (38%)

Oral 74/121 (61%)

After the seventh passage in vivo, transfer of material was accomplished regularly to submucosal sites in the tongue (Fig. 1). A f t e r several weeks growth the intraoral tumours were found to spread by direct extension along lymph channels to cervical nodes (Figs. 2+3). The growth of the tumours at either oral or flank sites was progressive. A n i mals were killed and examined post-mortem in all cases before tumour burdens became large. Temporal changes in the size of tumours within each passage were plotted as individual measurements and a graph of tumour volume against time was constructed for passages 1 to 6 (Fig. 4). The slope of the curve was then fitted to plotted data by the least squares regression method (Rees and Westwood 1974) and allowed an estimate of the tumour doubling time (fifteen days) to be made.

Table 4 CBC strain animals: allogeneic tumour volumes at skin (flank) and oral (tongue) sites. Note some regression of size after 28 days due to central necrosis of growing tumours and onset of rejection in others.

Age of donor tumour from CLAC - LH hamster

Succesful passages to CLAC - LH hamsters

Discus,sion Carcinogenesis studies

Over 100 days growth Under 100 days growth

23/35 (71°/o) 29/62 (47°/o)

Totals

52/97 (56%)

Historically, most of the experimental work on oral cancers has studied changes in the mouths of rodents in response to chemical carcinogens. These studies of carcinogenesis have used two approaches: A) Production of exophytic growths in the protected and immunologically privileged site at the cheek-pouch of the hamster. B) Production of p r e - m a l i g n a n t and occasional malignant changes in the oral cavity itself, in mice, rats or hamsters. A)

The hamster cheek pouch

The mucosa of the cheek pouch of the hamster has been found to be an immunologically privileged site in that isologous, homologous or heterologous transplants are accepted r e a d i l y in an untreated animal (Handler and Shepro 1968). The immunologically privileged status results from the paucity of lymphatic drainage and the presence of an areolar connective tissue barrier beneath the pouch mucosa. Eventually the regional

Days after rtansfer

Size after subcutaneous inoculation : log. vol. (mm ~) flank -+ S.E. tongue -+ S.E.

8 19 28 36

0.13 1.62 2.98 2.85

_+ 0.39 -+ 0.18 +_ 0.17 -t- 1.0

0.14 2.35 2.15 1.94

+ 0.27 -I- 0.18 +_ 0.17 _+ 0.16

Table 5 The syngeneic tumour. Successful tumour passages by age of donor tumour (subcutaneous sites).

lymph node nearest to the pouch, the superior cervical lymph node, produces an immune response but only after sufficient diffusion of antigen. The delay in that response ensures that a transplant survives long enough to become established (Shepro et al. 1960). These unique features of the cheek pouch site have also assisted studies in the induction of p r i m a r y tumours of cheek-pouch oral mucosa by chemical carcinogens. Two carcinogens have mainly been used, 3-methyl cholanthrene (MCA) and 7,12dimethylbenz-(a)-anthracene (DMBA). Most authors have used D M B A and histological studies (Salley 1957), modifying factors studies (Morris 1961, Siegel and Shhlar 1969) and latent period studies (Woods 1969) have each been reported.

168

H. Ca~mell

Fig. 1 Carcinoma of the tongue. Transplants to many syngeneic animals enable precision experiments to be performed. HE, x28.

Fig. 2 Direct spread of tumour along lymph vessel of tongue. HE, x84.

Strain DiHerences

(1972). They reported that following an initial acute inflammatory response which resolved, thickening of the epidermis occurred. Eventually discrete papillomatous lesions arose which proliferated on the surface of the mucosa. Despite some uncertainty as to the validity of the term "malignancy" to describe similar lesions, many workers then used the growths as the end point of carcinogenesis studies (Salley 1957, Levij and Polliak 1970, Siegel and Shklar 1969) or for the assessment of surgical techniques for the removal of carcinomata (Poswillo 1971). Terms such as careinoma-in-situ or intra-epithelial carcinoma have been used to describe the papillomatous lesions but Homburger (1972) warned that these early lesions may be benign. Confirmation of this was found during the present series of investigations when it became clear that many papillo-

More recently, work by Homburger et al. (1972) has shown that the appearance of malignant tumours following exposure of animals to polynuclear hydrocarbons, such as DMBA, was a genetically determined phenomenon. Reishin and Berry (1968) found that genetic factors implicit in the strain of the animals produced differences in the latent period of tumours arising in the cheek-pouch site (Table 1). The present investigation showed that even minor differences in strain resulted in differences in both papilloma and in formed tumour formation. Interpretation of histopathological changes in the cheek pouch The changes that occur in the hamster cheek pouch in response to a chemical carcinogen were reviewed by Homburger (1972) and by Shklar

Experimental Intra-Oral Carcinoma

169

mata, which had appeared in response to treatment with DMBA, ultimately regressed. Lappe and Prehn (1969) had previously noted that MCA-induced papillomata on the skin of mice regressed, but that a few progressed to frank carcinomata. The same authors also found that the antigenicity of the papillomata and their malignant potential were independent characteristics. Thus the evidence from several laboratories pointed out that papillomatous lesions, produced in response to topical applications of a carcinogen, may sometimes behave biologically as benign lesions and that occasional progression to malignancy appeared to be a random event. Biological behaviour of cheek-pouch tumours

The biological behaviour of formed turnouts of the cheek pouch has been noted by Homburger (1972) and Cannell (1973) to be of great importance in the assessment of a potential malignancy. It appeared therefore that in the hamster, turnouts confined to the cheek pouch had several serious objections to their use as models of intra-oral carcinoma. Nevertheless the model has been useful for kinetic studies of developing pre-malignancies (Brown and Berry 1968):but biological criteria of malignancy (Cannell 1973) should be included if experiments attempt to provide evidence of use of a true cancer rather than a possible malignancy. B) Intra-Oral Carcinogenesis Bonne (1927) was the first to claim that carcinoma could be induced in the palates of mice following repeated applications of coal-tar. The difficulties he found were that the presence of saliva tended to wash away the carcinogen and that malignant changes of oral epithelium were induced in very few of the animals. Similar problems were experienced by Al-Ani and Shklar (1966). A large study by Yamamura et al. (1975) utilized a specially created oral mueosal lined labial pouch in random bred rats of different strains in order to compare the carcinogenetic activity of various polycyclic hydrocarbons. They concluded that the turnout types produced by their system were not predictable although a few carcinomata were found. Injection of standard doses of carcinogen beneath skin or mucosa has also been performed in attempts to create malignancies. Unfortunately such attempts whilst avoiding the difficulty of maintenance of long-term contact of the carcino-

Fig. 3 Cervical lymph node invaded by tumour. H E, x28.

gen with the epithelium, also resulted in the production of fibro-sarcomata rather than carcinomata (Homburger and Hsueh 1970). An overview of these attempts to induce malignant changes within the oral cavity of rodents suggested that occasionally it was possible to produce single primary tumours. Unfortunately, due to experimental difficulties, both the histological appearances of formed tumours and their latent periods were variable. Reproduceability of the experiments was poor and the need for an animal model system of intra-oral carcinoma of use for surgical research had not been fulfilled. Transplantable tumours

These are tumours which are capable of being transferred to many recipient animals of the same strain (or in the case of genetically homozygous hamsters to a closely similar strain). The virtue of a transplantable system being that each recipient animal would bear a similarly sized tumour which

H. Cannell

170 TUMOUR VOLUME Log. pl 20,000

10,000

x

x

x x X x ~ x/~ /

x

1,00C

x

×

x

S

x

x'X

/

X

~ / X

X

x 100

x

××x

7

x×/ Xx ~X

,.0 ,0' 2'o

X

x

×

x

Sx." 10

X

x x

The syngeneic tumour system

×x x )0<

4'o s'0 6o' 7'0

subcutaneous passages in vivo (Table 3) before transfer to oral sites was attempted. The success of the latter experiments showed that standard sized tumours within the oral cavity could be produced in many animals simultaneously. 3?he usefulness of this allogeneic model was therefore to test the feasibility of future experiments with a syngeneic tumour but was still subject to the important criticism that the slight transplantation differences between the original donor sto& and the recipient strain must have influenced results. Furthermore, the original tumour had been induced by Chernozernski (1969) on hair-bearing skin rather than oral mucosa. The experience gained in the use of the model was however most valuable.

;0 i;0 '

' .40' ' DAYS

Fig. 4 Growth of transplantable oral tissue derived carcinoma at subcutaneous sites in syngeneic hosts. Fitted linear regression line (least squares method) of log. volume in FI on growth time in days, from pooled data. Points plotted after 30 days less accurate due to central necrosis of tumours (see text).

had been transferred from the original source at the same time. It would then become possible therefore to compare different surgical treatments in matched groups of animals. Until recently, few transplantable squamous cell carcinomata have been available for use in small laboratory animals (Roberts and Drobycz 1975). The three tumour systems that had been developed in rodents were each of skin rather than oral mucosal origin (Flan: nery et al. 1973, Peters 1975, Chernozemshi 1969). There appeared to be no reports of attempted transfer of these skin malignancies to oral sites.

Chernozemshi (1969) reported that a new squamous carcinoma of hamster skin had been successfully passaged in vivo and he was kind enough to send some laboratory bred Golden strain tumourbearing animals to this department. The tumour was adapted to available strains of hamster by

Growth of transplanted fragments from donor oral primary syngeneic tumours was at first prevented by infection. Several hundred attempts were made before treatment of fragments, prior to transfer, with antibiotics and an anti-fungal agent contained in serum free tissue culture medium, resulted in successful takes. When one non-infected tumour became established and grew at a subcutaneous site, further transplants from this one syngeneic source were continued only to other syngeneic animals. Nevertheless, a high yield of successful tumour transplants from subcutaneous tumours was not always achieved until progressively growing "old" tumours were used. It was concluded that a degree of immunoselection had occurred in "older" turnouts, whereby slow growing cells of high antigenicity had been eliminated by natural host defences. This also suggested that slightly more anaplastic cells now comprised the tumour cell population. Despite this degree of immunoselection, passaged tumours retained their differentiated characteristics. A most important feature of a malignancy is the ability to spread by direct invasion and by metastasis. In human-intra-oral carcinoma, the spread of the tumour is mainly by the slow but inevitably progressive direct involvement of the tightly pa&ed lymph node field of the ne& (McKelvie 1976). In the experimental system described above, tumours within the oral cavity were also found to have spread by invasion of the regional cervical lymph nodes.

Experimental Intra-Oral Carcinoma Distant metastases to the lungs or the liver of the experimental animals were not detected, although if they had occurred as microscopic foci they could have been missed at histological examination of the organs. The absence (or the non-detection) of distant metastases was thought to be an example of the complex inter-relationships of tumour and host factors which influence the spread of malignancies (Hellman and Carter 1974, Currie and Alexander 1974).

Conclusion The system appears to be the first available example of a reproducible model which can be used in the detailed study of intra-oral cancer.

References Al-Ani, S., G. Shklar: Effects of a chemical carcinogen applied to hamster gingiva. J. Periodont. 37 (1966) 36 Binnie, W. H., R. A. Cawson, G. B. Hill, A. Soaper: Oral Cancer in England and Wales. HMSO, London 1972 Bonne, C.: Ober Geschwi_ilste bei Seetieren. S. Krebsforsch. 25 (1927) 1 Brown, ]. M., R. J. Berry: Effects of X-irradiation on cell proliferation in normal epithelium and in tumours of the hamster cheek pouch. In: Effects of radiation on cellular proliferation and differentiation. Int. Atomic Energy A~ency, Vienna 1968 Cannell, H.: Oral Cancer and Immunity. Brit. J. Oral Surg. 11 (1973) 171 Chernozemski, I. N.: New transplantable hamster tumour: a squamous carcinoma of the skin. J. Natl. Ca. Inst. 36 (1969) 1189 Currie, G. A., P. Alexcmder: Spontaneous shedding of TSTA by viable sarcoma cells. Brit. J. Cancer 29 (1974) 72 Flannery, G. R., P. J. Chalmers, T. M. Rolland, R. C. Nairn: Immune response to a syngeneic rat tumour: development of regional lymph node anergy. Brit. J. Cancer 28 (1973) 118 Handler, A. H., D. Shepro: Cheek pouch technology: uses and application. In: The Golden Hamster. Iowa State Univ. Press 1968 Hellman, K., R. Carter: Experimental metastases. Brit. J. Cancer 30 (1974) 168 Homburger, F.: Chemical carcinogenesis in Syrian hamsters. In: Pathology of the Syrian hamster. Progr. exp. tumour Res. Karger, Basel 1972 Homburger, F., S. S. Hseuh: Rapid induction of subcutaneous fibrosarcoma by DMBA in an inbred line of Syrian hamsters. Cancer Res. 20 (1970) 1449 Homburrger, F., S. S. Hseuh, C. S. Kerr, A. B. Russfield: Inherited susceptibility of inbred strains of Syrian hamsters to induction of gastro-intestinal carcinomas by subcutaneous and gastric adminis-

171 tration of polynuclear hydrocarbons. Cancer Res. 32 (1972) 360 Langdon, ]. D., P. W. Harvey, A. D. Rapidis, M. ]. Patel, N. W. Johnson, R. Hopps: Oral Cancer the behaviour and response to treatment of 200 cases. J. max.-fac. Surg. (1977) 221 Lappe, M. A., R. T. Prehn: Immunologic surveillance at the macroscopic level: non-selective elimination of premalignant skin papillomas. Cancer Res. 29 (1969) 2374. Levij, L S., A. Polliack: Inhibition of chemical carcinogenesis in the hamster cheek pouch by topical chlorpromazine. Nature 228 (1970) 1096 Lucas, R. B. In: Pathology of tumours of the oral tissue 2nd ed. Livingstone, Edinburgh 1972 McKelvie, P.: Penetration of the cervical lymph node system by cancer. Proc. roy. Soc. Med. 69 (1976) 409 Morris, A. L.: Factors influencing experimental carcinogenesis in the hamster cheek pouch. J. dent. Res. 40 (1961) 3 Peters, L. ].: A study of the influence of various diagnostic and therapeutic procedures applied to a murine squamous carcinoma on its metastatic hehaviour. Brit. J. Cancer 32 (1975) 355 Poswillo, D. E.: Cryosurgery and electrosurgery compared in the treatment of experimentally induced oraI carcinoma. Brit. dent. J .131 (1971) 347 Rees, ]. A., M. Westwood: Method of comparing differences in tumour growth rates applied to a study of the increasing growth capacity of mouse carcinomata. Brit. J. Cancer 29 (1974) 151 Reiskin, A. B., R. ]. Berry: Cell proliferation and carcinogenesis in the hamster cheek pouch. Cancer Res. 28 (1968) 898 Roberts, D. C., B. Drobycz: Research using transplanted tumours in laboratory animaIs. XII I.C.R.F. Mill Hill, London (1975) SaUcy, ]. ].: Histologic changes in the hamster cheek pouch during early hydrocarbon carcinogenesis. J. dent. Res. 36 (1957) 48 Shepro, D., L. P. Eidelhoch, D. 1. Patt: Lymph node responses to malignant homo- and heterografts in the hamster. Anat. Rec. 136 (1960) 193 Shklar, G.: Experimental oral pathology in the Syrian hamster, In: Pathology of the Syrian hamster. Progr. exp. turnout Res. Karger, Basel 1972 Siegel, W. V., G. Shklar: The effect of dimethyl suitoxide and topical triamcinolone on chemical carcinogenesis of hamster buccal pouch. Oral. Surg. 27 (1969) 772 Woods, D. A.: Influence of ALS on DMBA induction of oral carcinoma. Nature 224 (1969) 226 Yamamura, T., Y. Nishida, S. Eda, M. Shimono, H. Yamane, T. Tachikawa, H. Koike, T. Ichikawa, M. Yoshida, 0. Watanabe, H. Matsuyama: An experimental study of intraoral carcinogenesis in rats. Oral Surg. 39 (1975) 87 Hugh CannellFDSRCS.,MRCS., LRCP., MSc., MD. Reader in Oral Surgery The London Hospital Medical College Turner Street, London, E1