The plasminogen activator (PA) content of metastatic malignant melanoma was determined in Triton X-100 extracts of 11 surgical specimens and adjacent normal tissue, using azocaseinolysis with added plasminogen. The mean PA content of the tumors was 8.4 ± 10 (SD) CTA u/g tissue (6 times that of the surrounding tissue), lower than found earlier in lung, colon and breast tumors. Inhibition by goat IgG against purified human urokinase showed that the predominant activator was of the UK type, as was the case with the tumors examined earlier, except those of the prostate. This is in contrast with recent reports which showed that human melanoma-derived cells secrete into the culture fluid almost exclusively the “vascular type” PA (Wilson et al., Cancer Res. 40, 933, 1980; Roblin and Young, Ibid. 40, 2706, 1980). While this type of PA was present in all extracts here examined, in 3 of them only trace amounts (<1%) could be found. The vascular type PA, when present, could be inhibited by rabbit IgG against human melanoma cell culture-derived vascular PA (kind gift of Dr. E. Dowdle). SDS-gel electrophoresis in conjunction with fibrin-agar overlay zymography showed multiple activator bands ranging in mol. wt. from 100,000 to 30,000. All but the 70,000 mol. wt. vascular type PA band were inhibited by inclusion of anti-UK IgG into the fibrin-agar mixture.The discrepancy between the tumor extracts and the culture media may be due to a selective advantage of vascular type PA-secreting cells in culture, or to the turning off of the UK gene in the culture environment. It is also possible that production of vascular type PA is suppressed iii vivo.

Quantitation of plasminogen activators present in tissue may depend to a large extent on the extraction procedure used to solubilize the enzymes. Potassium thiocyanate solution is known to be an efficient solubilizer, but it can inhibit assay systems other than fibrin plates. An equally effective acetate-detergent extractant is reported here which can be used with the highly sensitive azocaseinolytic assay procedure. The results indicate that a three-fold increase in activator activity can be extracted from selected tissues relative to that previously reported for a phosphate-detergent extractant. The extraction medium contains 75 mM K acetate, 0.3 M NaCl, 0.1 M L-arginine, 10 mM EDTA, 0.25% Triton X-100, final pH 4.2.

Conditioned media from explants of human colorectal and gastric tumors in short-term organ culture were analysed for plasminogen activator activity, activity toward the synthetic urokinase substrate, Spectrozyme-UK, and for the presence of urokinase antigen using monospecific goat antibody, by enzyme-linked immunosorbent assay. Comparisons were made between primary tumors, adjacent normal mucosa and metastatic lesions. These analyses were carried out on unfractionated culture fluids and on fractions obtained by fast protein liquid chromatography separation using Superose 6 gels. Plasminogen activator activity, tested by azocaseinolysis in the presence of added plasminogen, was restricted to peaks of 55 kD and 155 kD. These were of the urokinase type as shown by specific immunoinhibition and by absorption by an antiurokinase antibody-Affigel 10 column. Spectrozyme-UK, in addition to these peaks, detected a series of higher molecular weight activities, the largest of which appeared in the void volume, and were therefore of greater than 10(6) molecular weight. These activities were greatly increased by inclusion of trace plasmin indicating that these components were mostly in their proenzyme forms. The characteristics of these very large enzymes were similar to those isolated earlier from a human lung cancer cell line. Comparison of the primary and metastatic tumors confirmed earlier observations showing that urokinase secretion by the metastatic tumors was greatly reduced in comparison with the primary tumors: in the colon carcinomas it was 10 per cent of the value for the primary, in the gastric tumors 3 per cent, whether means or medians were compared (P less than 0.0001). This large difference was characteristic only of plasminogen activator secretion assayable by azocaseinolysis; activities toward Spectrozyme-UK, and antigen reacting with anti-urokinase antibody, were considerably less different in the two groups. In individual tissues, no correlation was found between the amount of extractable plasminogen activator and amounts secreted, or between the latter and the amount of lactic acid released. It is postulated that the greatly reduced plasminogen activator secretion by explants of metastatic tumors may be a phenotypic characteristic of distinct advantage for cancer cells destined to initiate metastatic foci, and may contribute to the ability of circulating cancer cells to lodge in the blood vessels of the target organ.

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