The soft agar clonogenicity and characterization of cells obtained from human solid tumors by mechanical and enzymatic means

A two-step procedure for releasing cells from solid tumors has been applied to specimens of human melanoma, sarcoma, lung, colon, and breast carcinoma. The first population released mechanically has been compared with the population subsequently released enzymatically in tests of dye exclusion, ribonucleoside triposphate pool sizes, intactness of DNA, and clonogenicity in soft agar. While greater numbers of dye-excluding cells are released in the enzymatic step, and these cells have higher ribonucleoside triphosphate pools and more intact DNA, both populations contain clonogenic cells in approximately equal numbers. Several semisolid media were employed in tests of clonogenicity, and all methods employing an agar underlayer appeared satisfactory and approximately equivalent in cloning efficiency. The methyl cellulose upper layer system facilitated implanting of pooled colonies into nude mice, which resulted in growth in the nude host and marked increase in cloning efficiency when the cells were replanted into soft agar-methyl cellulose plates. A comparison of four different areas of individual tumor specimens was made with cells released enzymatically and measuring cell yield, dye exclusion, ATP pool size, and uptake and metabolism of 5-fluoropyrimidines. Only relatively small variations were seen from one area to the next, with trypan blue exclusion exhibiting the least variation, and metabolism of fluorinated pyrimidines showing the most.