Isolation and visualization of Met-72-positive, metastatic variants present in B16 melanoma tumor masses

Metastatic variants of the B16 melanoma displaying high experimental metastatic potential have been shown to express high levels of a 72,000-dalton glycoprotein (Met-72) on their cell surface (Kimura AK, Xiang J: J Nat Can Inst 76:1247-1253, 1986). Monoclonal antibodies (MoAb) directed against the Met-72 determinant have been used in this study as immunohistochemical reagents on preparations of fresh B16 melanoma tumors and their metastases. These immunohistochemical analyses have utilized frozen sections, impression smears, and cytospin preparations of fresh tumors harvested at various time points during tumor growth, to view the presence and location of Met-72-positive metastatic variants within tumor masses. Biotinylated anti-Met-72 MoAbs were reacted with freshly dissociated tumor cells from a B16 melanoma ovarian metastasis. These cells were then reacted with fluorescein isothiocyanate (FITC)-streptavidin and analyzed by flow cytometry. A discrete population of positively staining cells was detected and isolated by cell sorting techniques. Met-72-positive cells were then cloned and reanalyzed after several weeks of in vitro expansion and found to have high experimental metastatic potential to ovaries. Frozen sections of subcutaneous tumors and their metastases were analyzed by immunoperoxidase techniques. A consistent finding in these studies has been that the few tumor cells which showed high intensity of Met-72 staining were positioned perivascularly and at the invading front of B16 melanoma tumors.