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As a model for the effect of hormones and growth factors on three-dimensional growth of mammalian cells, we have analyzed the effect of insulin on the three-dimensional growth and morphology of Chinese hamster ovary (CHO) colonies grown on the surface of agar. Sequential photographs in dark-field illumination of growing colonies have been analyzed with computer-assisted techniques. In this analysis the entire shape of each colony in a sizeable population (up to 10 5 colonies per experiment) can be measured and distributions of parameters derived from these measurements can be studied. In fetal calf serum (FCS), insulin has a dose-related stimulatory effect on cell growth that is most pronounced when growth has slowed down. In 10% FCS, insulin has a similar but diminished effect. When CHO cells are grown conventionally on plastic substrata or in suspension, insulin has little effect on cell growth at 4% serum concentration. Computer analysis of changes in the distribution of colony morphology proved to be a sensitive, dose-dependent, and reproducible assay of a hormonal effect. As little as 5 ng of insulin per ml added to 10% FCS causes a shift in the distribution of colony morphologies. In 4% FCS, 50 ng of insulin per ml is required to produce a detectable change in the colony morphology distribution. Computer analysis of cells grown three-dimensionally on agar provides a powerful approach to studying the effects of hormones and provides observations not available when cells are grown on plastic substrata.

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Attempts were made to initiate hormone-dependent tumor (HDT) transformation in ductal phase mammary outgrowths. These outgrowths, when carried in virgin GR/A mice, were not tumorous and usually resembled the host's mammary glands. After 2-3 months' initial growth, the following hormone treatments were initiated: 1) An isologous pituitary was implanted into each fat pad having previously received an HDT transplant. Some neoplastic transformation of the pregnancy-dependent tumor outgrowth occurred after 1-2 months; all 10 mammary glands were well stimulated and had many hyperplastic alveolar nodules. 2) In experiments involving exogenous hormone administration, estrogen or progesterone administered alone caused no neoplastic changes in the 2- to 3-month-old ductal phase outgrowths. However, 1 mg progesterone plus 1 mug estrogen daily given sc for 3 weeks to both ovariectomized and intact mice with HDT transplants caused neoplastic changes in the outgrowths and induced small ductal hyperplasias in the host glands. Termination of hormone treatment resulted in a return to the normal ductal structure. The addition of 100 mug prolactin and 100 mug growth hormone to the progesterone and estrogen mixture did not further alter transplants but increased stimulation to the host glands.