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Recently developed techniques of culturing human tissues permit the in vitro recovery of progenitor cells and the study of factors that regulate the growth of these cells. We applied this technique to benign and malignant breast tissues to determine how their ability to grow in agar changes as transformation occurs and as tumours progress. We also measured the responsiveness of the clonogenic cells from these tissues to a combination of four growth factors. We found that there was a continuing progression of increasing proliferation in agar from benign through primary carcinoma to metastases. We also found that the clonogenic cells of tissues from all stages retained their responsiveness to the tested growth factors and that the degree of their hormonal responsiveness remained similar to that of the parent tissue. In common with their parent tissues, tumours are composed of stem cells and of their progenitor cells. Unlike the parent tissues, however, the proliferative activity of tumour progenitor cells is regulated by growth factors unique to tumours (Todaro et al., 1980; Dickson et al., 1986). A contributory role of the growth regulators of the parent tissue is not certain. Techniques to recover the clonogenic tumour cell populations (the in vitro counterparts of stem cell progenitors) have recently been developed (Hamburger & Salmon, 1977a, b; Courtenay et al., 1978; Kinball et al., 1978). Since the culturing of these cells provides a tool to study tissue growth regulation, we used the technique to define the colony forming ability and the proliferative response to some selected hormones, of benign and malignant breast tissues. Normal breast tissue was obtained from 9 women, 7 of whom underwent a reduction mammoplasty and 2 a modified radical mastectomy. Four of these women had in the past been treated for stages I or II breast carcinoma. The remaining 5 women underwent reduction mammoplasty for cosmetic reasons. Malignant breast tissue was obtained from 21 patients with primary breast carcinoma and from 63 patients with metastatic breast carcinoma (33 solid tumour tissues, and 30 malignant effusions). Oestrogen receptors (ER) were measured by the dextrancoated charcoal method. Values for .10 fmolmg1 protein were considered positive, and values <10 fmol mg 1 protein were considered negative. Forty-one tumours (9 primary and 32 metastatic) were ER positive, and 35 tumours (12 primary and 23 metastatic) were ER negative. Solid tissues were collected in 50ml culture medium and sliced into 1 mm cubes. Effusions were centrifuged at 40g for 10min, and cells resuspended in Ham's F12 medium (F12; GIBCO, Grand Island, NY), supplemented with 10% heatinactivated foetal bovine serum (FBS; KC Biological, Lenexa, KS). Cell aggregates were then incubated in a mixture of 1.0% Worthington type III collagenase (Worthington Biochemical Corporation, Freehold, NJ),

Clonogenic growth and hormone sensitivity of benign and malignant breast tissues