Clonality of pulmonary metastases from the bladder 6 subline of the B16 melanoma studied by Southern hybridization

The possible clonal origin of pulmonary metastases from the bladder 6 subline of the B16 melanoma (B16-BL6) was evaluated with the use of the technique of Southern hybridization. Genetic markers were introduced into B16-BL6 cells by the transfection of a plasmid containing a bacterial gene. Genetic markers were detected by hybridizing DNA extracted from tumors with a ³²P-labeled bacterial gene. Clones containing unique genetic markers were mixed and injected into the footpads of normal syngeneic C57BL/6 mice. Footpad tumors were amputated when they were 1 cm in diameter. Individual pulmonary metastases were harvested and expanded by implantation in nude mice. Although footpad tumors contained both genetic markers, virtually all (13/15) pulmonary metastases contained single unique genetic markers. Pulmonary metastases of both marker types were identified. Two examples of pulmonary metastases of mixed origin were detected. The Southern hybridization technique was sufficiently sensitive to detect a clone that was present at 5% of the total cell population.