Growth of MTW9/PL2 Estrogen-responsive Rat Mammary Tumor Cells in Hormonally Defined Serum-free Media

Growth of the estrogen-responsive MTW9/PL2 rat mammary tumor cells was demonstrated in serum-free defined medium (designated DDM-1) formulated with F12-DME (1:1 vol/vol) supplemented with 15 mM HEPES pH 7.4, insulin 10 micrograms/ml, transferrin 10 micrograms/ml, sodium selenite 10 ng/ml, triiodo-L-thyronine 0.3 nM, phosphoethanolamine 5 microM, epidermal growth factor (20 ng/ml), 17 beta-estradiol 2 nM, and bovine serum albumin 20 micrograms/ml. In DDM-1, the growth rate was about one-half that seen in serum-containing medium. When ethanolamine (50 microM), glutathione (20 micrograms/ml), and linoleic acid/bovine serum albumin (150 micrograms/ml) were added (formulation DDM-2), the growth rate was 80% of serum-containing medium and not seed-density dependent. Deletion of estradiol from DDM-1 or DDM-2 had no effect on growth rate. Also, cells grown in steroid hormone deficient medium for 4 mo. continued to form estrogen-responsive tumors in rats as did cells cultured for the same period in 2 nM estradiol. To investigate autocrine growth factor secretion, a third medium (DDM-3) was prepared by deleting insulin, epidermal growth factor, phosphoethanolamine, estradiol, and both forms of bovine serum albumin from DDM-2. Growth in mitogen-free medium equaled 86% of the serum-stimulated rate and was seed-density dependent; phenol red deletion from DDM-3 had no effect on growth rate. Evidence presented suggests that autocrine factors stimulate growth of the MTW9/PL2 cells in DDM-3, and that this secretion may support the growth of estrogen-responsive cells in culture in the absence of steroid hormone.