Loss of malignancy during serial passage of human carcinoma in culture and discordance between malignancy and transformation parameters

Human epidermoid carcinoma (HEp-3), a tumor which grows on chorioallantoic membrane and metastasizes with high efficiency into the organs of chick embryo, was adapted to growth in culture. Upon serial passage in vitro, a progressive loss of metastatic potential and of tumorigenicity was noted. The metastatic potential was completely lost within the first 2 to 10 weeks in culture (4 to 14 passages), while tumorigenicity declined rapidly during a period of 50 passages, as reflected in the progressively larger inoculum required for development of detectable tumors. Beyond this time, macroscopically visible tumors were not observed during the standard 7-day growth period even when the inoculum was increased 100-fold to 10⁷ cells. Identical results were obtained with six individual tumors adapted to grow in vitro. Metastatic potential could be recovered from cell populations that retained some degree of tumorigenicity. This required at least two sequential passages on the chorioallantoic membrane. Loss of metastatic ability and tumorigenicity was accompanied by change in hormone responsiveness and by a distinct improvement in growth efficiency in culture as manifested by shortened doubling time and increased saturation density. Further, anchorage independence and serum independence two properties that are generally correlated with the transformed phenotype, were inversely correlated with malignancy. The malignant and the nontumorigenic cells were compared with respect to production of plasminogen activator, an enzyme associated with malignancy and transformation. Malignant cells produced larger amounts of plasminogen activator, and enzyme production was resistant to modulation by several hormonal and nonhormonal effectors; in contrast, plasminogen activator synthesis in nontumorigenic cells was stimulated by cholera toxin and inhibited by glucocorticoids.