An experimental study of human melanoma cells cutured in vitro
This thesis records the results of a series of experiments that were designed to examine the biology of human malignant melanoma cells cultured in vitro. The studies were so planned as to document phenotypic differences that exist between melanomas, to define respects in which melanoma cell differentiation could be modulated and to correlate biochemical variability with in vivo behaviour as measured in the nude mouse. Melanoma cell lines were established from biopsy material obtained from 7 patients at Groote Schuur Hospital. Two of these lines synthesized tyrosinase and melanin at a rate that was directly related to cell density. The five remaining lines did not pigment. ii All of the lines showed aneuploidy; 5 of the 7 showed anchorageindependent growth; and 6 of the 7 grew as lethal tumours in nude mice. As has been found with all other melanomas studied, these cells released a plasminogen activator that was chemically and immunologically identical to tissue activator. One of the lines proved to be an exception to this general rule in that it synthesized urokinase-type enzyme. Unlike most other human cells cultured in vitro, melanoma cells proved to be relatively refractory to hormonal stimuli. Addition of estrogen, progesterone, testosterone, dexamethasone or melanocyte-stimulating hormone to the culture medium had very little effect on cellular release of plasminogen activator, upon cell growth, or upon cellular morphology. Although remarkably resistant to hormonal influences, cellular release of plasminogen activator did appear to be inhibited to a striking degree by cocultivation with normal skin fibroblasts. This observation led to the discovery of a phenomenon in which fibroblasts of many types bound tissue-type plasminogen activator and so removed it from the medium. This was accompanied by an apparent change in molecular weight of the melanoma cell enzyme from 72K daltons to approximately 115K daltons, suggesting the presence of a 40-SOK binding molecule. iii In an attempt to influence in vitro differentiation, the tumour promoter tetradecanoylphorbol acetate, and the differentiation-inducing retinoid, retinoic acid, were added to the two pigmenting cell lines. The effects of these compounds on induction of tyrosinase activity, morphological change or plasminogen activator release differed. In the one cell line, tetradecanoylphorbol acetate caused morphological maturation with a decrease in the rate of plasminogen activator release and no obvious effect upon pigmentation. This line was relatively resistant to the action of retinoids. The other pigmenting line responded hardly at all to the tumour promoter. Retinoic acid, on the other hand, inhibited the induction of tyrosinase activity, yet caused an inhibition of growth and plasminogen activator release. A number of interesting observations could be made in experiments in which melanoma cells were inoculated into nude mice. Firstly, the growth rate of the tumours ~n vivo correlated poorly with the doubling times of the corresponding cells cultured in vitro. Secondly, despite a marked inhibitory effect of fibroblasts on plasminogen activator in vitro, coinjection of fibroblasts and melanoma cells in vivo greatly enhanced tumour growth when small tumour cell inocula were used and shortened the latent period for tumour appearance with larger inocula. Thirdly, melanomas growing in nude mice differed strikingly in their ability to elicit a desmoplastic response. Tumours in which large amounts of host connective tissue were deposited tended to be heavily contaminated with murine fibroblasts when re-established in vitro. This contamination was not seen with tumours that contained very little connective tissue. These results point to the existence of a melanoma-associated fibrogenic factor. Finally, by excision of the primary tumour, it was possible to avoid death of the animal from local complications and so allow time for metastases to develop. In three mice, metastatic melanoma deposits could be detected by this device, so establishing a protocol for the use of nude mice as valid models for the experimental study of metastatic spread of human tumours.
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