Conference to Reevaluate the Toxicity of Vinyl Chloride Monomer, Poly(vinyl Chloride) and Structural Analogs. Bethesda, Md., March 20-21, 1980.

Canine melanoma has been transplanted to allogeneic neonatal recipients receiving continuous immunosuppression with anti-lymphocyte serum. One spontaneous melanoma was directly transplanted into 8 recipients, 6 of which developed tumours. 5/5 melanoma cell cultures were transplantable, with 19 tumour takes in 31 allogeneic recipients. Serial passage was performed in the case of two melanomas. Tumour development required continuous immunosuppression and the site was dependent upon the route of inoculation and other factors. Transplanted cell cultures were all amelanotic in vitro and in vivo, except in the case of one melano-ma which reverted to a melanotic morphology after in vivo growth. CANINE spontaneous neoplasms have been infrequently transplanted, and success rates were low when irradiation or drug immunosuppression were employed Intrafoetal inoculation or immunosuppression with anti-lymphocyte serum (ALS) have yielded better results with canine osteosarcoma (Owen, 1969) and lymphosarcoma (Owen and Nielsen, 1968). Canine oral papil-loma, mast cell leukaemia (Lombard, Moloney and Rickard, 1963) and the transmissible venereal tumour (Epstein and Bennett, 1974) are transplantable without immunosuppression. In many respects canine melanoma provides a valuable comparative model but has not previously been transplanted. Successful allogeneic transplantation into newborn ALS-treated dogs is now reported. MATERIALS AND METHODS Melanoma cells.-Spontaneous cases of canine melanoma, involving the oropharynx or metastasizing to the regional lymph nodes, were excised aseptically under general anaesthesia. Cell suspensions for inoculation were prepared by finely cutting the melanoma tissue and pressing the cells through a 150-,tm aperture sieve. Cells were either injected immediately after preparation or after cryopreservation at-196TC in medium containing 10% DMSO. Melanoma cells for in vitro cultivation were dissociated by sieving or by trypsini-zation. Cell cultures were grown as adherent monolayers in medium 199 or RPMI 1640 plus penicillin/streptomycin and 10% calf or foetal bovine serum and serially subcultured. Prior to inoculation, melanoma cells were detached with trypsin plus EDTA solution, washed and resuspended in Hanks' BSS.

CANINE spontaneous neoplasms have been infrequently transplanted, and success rates were low when irradiation or drug immunosuppression were employed (Allam et al., 1954;Nielsen and Cole, 1961;Moldovanu et al., 1966). Intrafoetal inoculation or immunosuppression with antilymphocyte serum (ALS) have yielded better results with canine osteosarcoma (Owen, 1969) and lymphosarcoma (Owen and Nielsen, 1968). Canine oral papilloma, mast cell leukaemia (Lombard, Moloney and Rickard, 1963) and the transmissible venereal tumour (Epstein and Bennett, 1974) are transplantable without immunosuppression. In many respects canine melanoma provides a valuable comparative model but has not previously been transplanted. Successful allogeneic transplantation into new-born ALS-treated dogs is now reported.

MATERIALS AND METHODS
Melanoma cells.-Spontaneous cases of canine melanoma, involving the oropharynx or metastasizing to the regional lymph nodes, were excised aseptically under general anaesthesia. Cell suspensions for inoculation were prepared by finely cutting the melanoma tissue and pressing the cells through a 150-,tm aperture sieve. Cells were either injected immediately after preparation or after cryopreservation at -196TC in medium containing 10% DMSO.
Melanoma cells for in vitro cultivation were dissociated by sieving or by trypsinization. Cell cultures were grown as adherent monolayers in medium 199 or RPMI 1640 plus penicillin/streptomycin and 10% calf or foetal bovine serum and serially subcultured. Prior to inoculation, melanoma cells were detached with trypsin plus EDTA solution, washed and resuspended in Hanks' BSS.
Recipients.-Pregnant mongrel bitches were barrier-maintained and, following parturition, the newborn recipient dogs were placed on an immunosuppressive course of ALS treatment as previously reported (Owen, 1969)  inoculation. s.c. = subcutaneous inoculation.

RESULTS
Transplantation directly from spontaneous melanomas In a series of 16 dogs inoculated with viable cells from 5 canine melanomas, tumour growth was observed in 6 animals. All successful transplants were derived from one donor (VI) either as fresh or cryopreserved cell suspensions (Fig. 1). J.v. injection produced miliary lung tumours and myocardial infiltration along the coronary vessels. Pulmonary tumours exhibited various degrees of pigmentation. Some dogs also developed melanomas in the pancreas, mesentery, pleura and lymph nodes. S.c. inoculation produced large melanotic tumours which were locally invasive and, in one recipient, present in distant sites. Development time for detectable growth ranged from 3-13 weeks, except for one dog which remained clinically normal until euthanasia at 7 months of age, when pulmonary melanoma deposits were found. All tumours were confirmed histologically as melanomas. Subsequently, three serial in vivo passages were obtained with inoculation by the i.v. and s.c. routes (Fig. 1). All the remaining melanomas failed to grow after i.v. or s.c. inoculation of 108 tumour cells into ALStreated recipients.   (Betton, 1975). The RVC 347 melanoma cell line (Kasza, 1964) was also generously provided for study.
The results of inoculation of 5 canine melanoma cultures into immunosuppressed recipients are summarized in the Table. All the melanoma cultures tested showed in vivo growth in a proportion of recipients, although V1 tumours showed regression after the initial 3 week growth phase.
Iv. inoculation produced tumour growth in the lungs and frequently the myocardium and in some cases in the pleura, peritoneum, bone, pancreas, thymus (Fig.3), pelvic and perirenal lymph nodes and the central nervous system. Miliary s.c. deposits of melanoma have also been observed. Histopathological examination revealed the presence of anaplastic melanoma cells with frequent mitotic figures and devoid of melanin except in the case of H73-858 cells. Although the latter cells had become amelanotic during growth in vitro, the transplanted tumours were all heavily pigmented in vivo in s.c. and disseminated visceral sites.
S.c. tumour inoculation produced rapidly-growing localized tumours (Fig. 4), often locally invasive and sometimes ulcerating. Distant tumours were not observed, except in the case of H73-858 cells, when some lung tumours also developed. In the case of H71-1843, melanoma cells from transplanted tumour tissue were maintained in tissue culture for two passages and were subsequently capable of further growth in vivo when reinoculated.  Immunosuppression with ALS was found to be essential for tumour growth, as rapid regression of pulmonary and s.c. transplanted melanomas was observed when ALS treatment was withdrawn. In the case of RVC 347, 0/4 recipients not receiving ALS developed melanomas, whereas 4/7 immunosuppressed recipients became tumour bearers (Table). An unusual feature of transplanted RVC 347 cells was the long latent period in 3 recipients, in which development of lung tumours was minimal, permitting survival until the development of tumours in the bone, central nervous system and other sites was observed. Osteolytic tumours were observed in the ribs and in the maxilla eroding the dental alveoli, closely resembling spontaneous primary oral melanomas.

DISCUSSION
Using immunosuppression of newborn dogs with ALS in the present series, it was possible to transplant 1/5 spontaneous melanomas directly (6/16 dogs inoculated) and 5/5 melanoma cell cultures produced growth in vivo in 19/31 recipients. The apparent higher transplantability of melanoma cells after subculture in vitro may have resulted from selection of more rapidly dividing cells in tissue culture, but the direct and cell culture transplantation series were not directly comparable. Some considerable variation in development time was observed between tumours. As no effort was made to match histocompatibility antigens between doniors and recipients, random variations in antigenic disparity were to be expected. Possible failure of immunosuppression during long periods of ALS administration could therefore have produced litter-associated failures in transplantation following inactivation of the ALS by antiglobulin produced by the recipients. Similarly, cytotoxic anti-DL-A antibodies of the donor specificities could be produced in the xenogeneic ALS, according to the DL-A type of the immunizing lymphoid cells. Such antibodies could thus selectively inhibit tumour cell growth without producing toxicity in the recipients dependent on DL-A specificities. This aspect will be investigated in future studies.
The distribution pattern of tumours after i.v. inoculation indicated the cardiopulmonary capillary bed as the principal site of tumour cell lodgement. This finding was supported by the distribution of i.v. injected 51Cr-labelled melanoma cells (Betton, 1975) after 1 h. Similar findings were reported by Fisher and Fisher (1967) for other species. The disseminated spread of cells to extrapulmonary sites in some dogs could have resulted from the degree of trypsinization and the presence of cell aggregates, as reported for the murine B16 melanoma by Hagmar and Norrby (1973). In the case of the RVC 347 melanoma, some recipients failed to develop rapidly fatal pulmonary tumours, but later produced tumours in sites such as bone and neural tissue.
The reversion of H73-858 cells to melanin synthesis in vrivo indicated that loss of pigment formation in vitro may be produced by deficiencies in culture conditions and is not an irreversible change. The progressive growth in vivo, following inoculation of canine melanoma cells cultivated in vitro for various numbers of passages, in a substantial proportion of recipients was considered confirmatory of the malignant nature of such cells. In vitro features of malignancy, such as morphology and agglutination by the lectin Concanavalin A (Betton, in preparation) support this conclusion. No definite effect of passage number on the transplantability of melanoma cells was observed. Primary cultures or cell lines of normal canine cells have never produced tumours after transplantation into identically treated allogeneic recipients in this laboratory.
The availability of spontaneous canine melanoma in vitro and in vivo will facilitate comparative studies on melanoma in progress.
The enthusiastic co-operation of Mr D. E. Bostock in providing clinical material and histological diagnosis is gratefully acknowledged. Dr L. Kasza and Professor W. Plowright generously made the RVC 347 line available for study. The work was supported by grants from the Cancer Research Campaign and the Medical Research Council of Great Britain. G. R. B. was in receipt of a Horserace Betting Levy Board Scholarship and is currently supported by the Wellcome Trust.