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Summary A simple method is described for the separation of cells derived from effusions of patients with adenocarcinomas in discontinuous density gradients of Percoll. After separation, cells from different fractions were analyzed by morphologic, histochemical and immunologic criteria. Total cell recovery from 27 experiments was 67+4%. Macrophages (82%) were recovered in the intermediate density fraction (1.056-1.067gml-1) with a purity of 90%. Recovered lymphocytes (98%) were found in the high density fraction (1.067-1.077gml-1) with a purity of 92%. The majority of the lymphocytes recovered were T cells. Malignant adenocarcinoma cells (90%) were recovered in the lowest density fractions (up to 1.056gml-1) with a purity of 79%. Use of effective cell separation procedures should facilitate the analysis of the functional capacities of both normal and neoplastic cells derived from human malignant effusions.

We examined the ability of primary human tumour cells to secrete diffusible factors capable of stimulating anchorage independent growth of normal rat kidney fibroblast (NRK) cells. Conditioned media (CM) prepared from cells derived from 31/43 patients with adenocarcinoma of the breast, colon, ovary or lung were found to induce growth of NRK cells in soft agar. The ability of the CM to induce anchorage independent growth was enhanced in 25/35 cases by the presence of epidermal growth factor (EGF). The CM did not compete with EGF for binding to the EGF receptor site. CM from cells derived from nonmalignant effusions also supported the growth of NRK cells in soft agar. There was no significant difference in the ability of the CM derived from malignant or normal cells to support NRK colony growth. The ability of primary human tumour cells to clone in soft agar was compared to the ability of these cells to produce diffusible colony stimulating factors for NRK cells. No correlation was observed between the ability of the primary human tumour cells to clone in soft agar and their ability to induce anchorage independent growth of NRK cells. The secretion of substances with TGF like activity may be a property of many types of primary human cells.

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A human epithelial-derived cell line, SW-13, releases a soluble substance that functions as an autocrine growth factor. SW-13 cells, derived from a human adenocarcinoma of the adrenal cortex, form a few small colonies when suspended in soft agar at low densities. The number of colonies increased significantly when either viable SW-13 cells or serum-free medium conditioned by SW-13 cells (CM) was added to agar underlayers. CM increased colony formation in a dose-dependent fashion. Clonal growth at low cell densities was dependent on the presence of both horse serum and SW-13 CM. Neither activity alone was capable of sustaining growth. Even when cells were plated at high densities CM could not substitute for serum, but could reduce the threshold serum concentration. The results suggest that autocrine and serum-derived factors act in concert to maintain clonal growth of epithelial tumor cells in soft agar.