A collagen gel assay will be used to select from hyperplastic alveolar nodule (HAN)-derived CL-Sl mouse mammary epithelial cells those variants that grow in the substratum. The progressively increased growth of these cells in collagen implies adaptive changes that may duplicate in culture the changes associated with tumor progression in the host animal. The basic hypothesis is that collagen favors retention of hyaluronic acid, the major glycosaminoglycan (GAG) synthesized by the cells, and that HA accumulating in the extracellular matrix stimulates growth. The perpetuation of the two mutually reinforcing processes, mitosis and HA synthesis, is seen as the underlying mechanism for the rapid proliferation of cell within the collagen stroma and a valid model of tumor growth in vivo. Furthermore, the accumulation of HA along the basement membrane-collagen boundary may lessen adhesion of the cell aggregates to the stroma and explain how cords of malignant mouse mammary cells invade the lungs while completely invested in an unbreached basement membrane. Because collagen gels model the stromal environment far more closely than does soft agar, the gels are the system of choice for the discrimination of fast-growing HAN-derived cells and, by implication, those with the highest rates of HA synthesis. The selected cells will be passaged in unenriched collagen and then labeled with (3H) glucosamine, after which the GAGs will be collected and analyzed to determine whether HA has accumulated in the basement membrane. The cells will also be plated on plastic to determine whether, after passaging, they have acquired characteristics of the +SA tumorigenic cells, namely, equivalent rates of growth on plastic and in collagen, and continuous synthesis of HA irrespective of cell density. Cells will also be tested for the acquisition of anchorage-independent growth in soft agar. The importance of HA synthesis to CL-Sl cell growth will be assessed by inhibiting hyaluronate synthase. The collagen assay system is ideal for selecting cell variants and for revealing the underlying mechanisms that support growth and enhance motility of cell aggregates in the stroma, factors associated with tumor progression. The project should test our original hypotheses and provide subpopulations of cell to test for tumorigenicity in vivo.