Human squamous cell carcinoma in culture: a defect in terminal differentiation and its relation to malignancy

Thirteen primary squamous cell carcinomas of the epidermis and of the oral and pharyngeal epithelium were cultured with a 3T3 fibroblast feeder layer, a system originally developed for clonal growth and long-term serial cultivation of normal human keratinocytes. Six of these tumors could be propagated indefinitely as established cell lines. They formed rapidly growing well-differentiated squamous cell carcinomas when injected sc into athymic (nude) mice. The squamous cell carcinoma lines possessed different aneuploid karyotypes. They displayed subtle differences in colony morphology such that the were visually distinguishable from one another as well as from normal keratinocytes. The lines also varied greatly in their dependence on the fibroblast feeder layer for clonal growth in surface culture. Only 1 line could form large colonies with high efficiency in semisolid medium; the others grew only abortively under this condition and eventually differentiated terminally to form cornified envelopes. Progressive growth in semisolid medium, therefore, was not a useful in vitro marker of malignant transformation for these cancer cells of keratinocyte origin. However, a property shared by all the squamous cell carcinoma lines was a subnormal rate of commitment to terminal differentiation during incubation in suspension culture. Deprivation of anchorage triggers commitment so rapidly in normal keratinocyte populations that no cell remain viable after 2 days in semisolid medium. In contrast, after this same-period, all 6 lines retained more than 20% of their original colony-forming ability when replated in surface culture. This phenotype of increased survival capacity in semisolid medium promises to be a useful selective marker for the detection of rare malignant keratinocytes within large normal keratinocyte populations.