Spontaneous cell loss during growth of postconfluent primary cultures from mammary adenocarcinomas

Growth properties of cells cultured from primary mammary tumors of C3H mice have been analyzed. Cells were seeded at 2 different densities (1 X 10(5) and 5 X 10(5)/sq cm) and were supported with a culture fluid containing 10% fetal calf serum and 5 mug insulin per ml. Mitosis continued after confluence was achieved, but cells did not accumulated in the monolayer; rather, certain cells were released into the culture fluid. Very few cells detached in this way from subconfluent cultures. Relased cells multiplied vigorously if replated. The release of these cells was strongly depressed by adrenal steroids, but other manipulations of culture conditions (hormones, culture substratum) influenced the release process much less. Analyses of release kinetics and observations of detachment with the scanning electron microscope suggested that tumor cells that became spheroid (including mitotic cells), and hence partly detached from the culture dish, were unable to reflatten into the monolayer because neighboring nonmitotic cells had spread onto the vacated culture surface. Eventually, such rounded cells apparently lost altogether their attachment to the culture dish. The release process may be related to the "critical phase" transition and to the sarcomatous transformation observed in long-term cultures from mouse epithelial tumors. The event could also reflect the tendency in vivo for cells of mammary tumors to slough into the lymphatics and blood vessels.