Europ. J. CancerVol. 12, pp. 1025-I026. Pergamon Press 1976. Printed in Great Britain

Letter to the Editor

Survival of VX2 Carcinoma Cells In Vitro C. S. B. GALASKO* and D. W. HAYNES,

Nuffeld OrthopaedicCentre, Oxford, U.K.

Ta~. VX2 carcinoma is an animal tumour which is being used extensively to study different aspects of tumour behaviour. It is derived from a virus induced papilloma of rabbits [1], which subsequently progressed to a metastazing carcinoma [2]. In 1940 Kidd and Rous [3] showe.d that a squamous cell carcinoma, derived from the original papilloma, could be propagated easily in skeletal muscle. The loss of viral dependency of the tumour was reported in 1952 [4]. The VX2 carcinoma has since been free of viral characteristics and remains readily transmissible. It is maintained by serial passage in the skeletal muscle of New Zealand white rabbits. I f the tumour is lost, an inoculated rabbit has to be obtained from another laboratory. This may mean international transport of the animal with the problems of quarantine. We therefore decided to investigate whether there was any method of maintaining the VX2 carcinoma in vitro. In the first experiment, tumour cell suspensions of the VX2 carcinoma were obtained by finely dividing a cubic centimeter of turnout in 5 cm 3 of sterile saline. The fluid was filtered through gauze and 1 cm 3 of the turnout cell suspension dispensed into 3 glass ampoules. There were approximately 2-25 x 10 4 cells per cubic centimeter. Each ampoule was placed in ice and at intervals of 24, 48 and 72 hr the turnout cell suspension was injected into the thigh muscles of' New Zealand white rabbits. In the second experiment six ampoules, each containing 1 cc tumour cell suspension, which was obtained in the identical manner, were

frozen in liquid nitrogen. At intervals varying from immediately following freezing to f o u r weeks following freezing, the ampoules were removed from the liquid nitrogen, the tumour cell suspension allowed to thaw and then injected into the thigh muscles of New Zealand white rabbits. In the final experiment the piece of tumour was placed in a petri dish containing tissue culture medium (TC 199) with 10% D M S O added. A tumour cell suspension was again obtained by finely dividing the tumour in the medium, filtering it through gauze and dispensing it into six glass ampoules. The ampoules were sealed and placed in a - 20°C refrigerator which allowed the cell suspension to freeze at approximately 1°C/min. After 1 hr they were placed in a - 7 0 ° C refrigerator until required. When the tumour was reimplanted, at intervals varying from 4 hr to 13 months, the ampoules were allowed to thaw at room temperature. A fibrin clot had formed and it was impossible to resuspend the cells. The fibrin clot, with the VX2 cells enmeshed in it, was then implanted into the thigh muscle of the New Zealand white rabbit through a surgical incision. The supernatant fluid was injected into the thigh muscles of a different rabbit.

Cooling in ice In all instances, the tumour grew normally. There was no histological difference between the ice-cooled VX2 carcinoma and the normally passaged tumour (Fig. l a and l b). All the rabbits developed pulmonary metastases and died from these lesions at the same time as the rabbits in whom the tumour was being passaged normally.

Accepted 20 February 1976. *Current address: Orthopaedic Unit, Department of Surgery, Royal Postgraduate Medical School, London WI2 0HS, U.K.

Freezing in liquid nitrogen All specimens failed to grow.

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Letter to the Editor showed no evidence of growth several months after implantation. The results indicate that the VX2 carcinoma can be stored in vitro and does not necessarily have to be passaged from one animal to another. For periods of up to 72 hr the tumour cell suspension can be kept in ice. An attempt to store it for longer periods was made by freezing the tumour cell suspension in liquid nitrogen, but the tumour was destroyed. This may have been due to too rapid cooling or using saline as the suspension medium. Suspending the cells in T C 199 with 10% D M S O allowed the cells to remain viable when stored at - 7 0 ° C for up to 6 months. A fibrin clot was formed and this had to be implanted for successful take of the tumour. The supernatant fluid did not contain sufficient viable cells to propagate the lesions.

Tumour frozen with D M S O Tumours which were suspended in D M S O and stored at - 7 0 ° C for periods varying between 4 hr and 6 months grew when the fibrin clot was subsequently implanted into the muscle of New Zealand white rabbits. There was no histological difference between these tumours and the normally passaged tumours (Fig. l(c)) and these tumours were normally transmissible. The animals also developed pulmonary metastases but these took longer to develop and the animals survived longer than the passaged animals. In our laboratory, animals in whom the VX2 has been implanted usually die within six weeks. In this experiment some of the animals survived for eight to eleven weeks but all had pulmonary metastases at autopsy. T u m o u r which had been stored for 13 months

~ 1. 2. 3. 4.

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1~. E. SHOP~ and E. W. HURST, Infectious papillomatosis of rabbits with note on histopathology. J. exp. Med. ~ 607 (1933). P. Rous and J. W. B~ARD, Progression to carcinoma of virus-induced rabbit papillomas (Shope). J. exp. Med. 62, 523 (1935). J . G . KInD and P. Rous, Transplantable rabbit carcinoma originating in a virus-induced papilloma and containing the virus in masked or altered form. or. exp. Med. 71, 813 (1940). P. Rous, J. G. KIDD and W. E. SMITH, Experiments on cause of rabbit carcinomas derived from virus-induced papillomas. Loss by VX2 carcinoma of power to immunize hosts against papilloma virus. J. exp. Med, 96~ 159 (1952).