This paper describes the growth of HEp-3, a human epidermoid carcinoma, in the chick embryo. When inoculated onto the chorioallantoic membrane, the tumor grows locally with a doubling time of 21 to 24 hr and disseminates widely in the embryo proper, metastasis to the lung and heart being especially prominent. The tumor secretes large amounts of plasminogen activator (PA) both in vivo and in vitro. This enzyme of human origin can be easily identified in a mixture containing both human and

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... PA's, since each PA has a marked preference for the homologous plasminogen. Thus the presence of human PA in tissues and body fluids can be used as a quantitative marker for HEp-3 metastasis both in embryos and in newly hatched chicks. Two assays for metastasis are described; their respective sensitivities are less than 4 x 10(4) and less than 500 HEp-3 cells per 17-day chick embryo lung (or approximately 1 HEp-3 cell per 400 and per 3 x 10(4) lung cells), respectively. These assays revealed that tumor growth and metastasis are sensitive to embryo age, inoculation onto the chorioallantoic membrane at 10 days being optimal for both. Tumor size, the length of the latent period that precedes the appearance of lung metastasis, and the number of metastatic cells in the lungs are all influenced by inoculum size. Generally, but not uniformly, an increased level of lung metastasis is correlated with tumor size on the chorioallantoic membrane. The attractive features of this system for quantitative study of metastasis are reproducibility rapidity, sensitivity, convenience, and cost.