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Normal mammary epithelial cells from BALB/cfC3H middpregnant mice were freed from stromal cell types by Percoll density gradient centrifugation after collagenase digestion and were then embedded within collagen gels. Sustained growth leading to an increase in cell number was accomplished in response to cholera toxin and high concentrations of horse serum. The extent of growth was found to be dependent on the horse serum concentration, the maximum growth being attained at 50%. A serum concentration of 12.5% horse serum and 2.5% fetal calf serum, along with cholera toxin at 0.01 Lg/ml, allowed maintenance but failed to cause any significant increase in cell number during the experimental period of 2 weeks. This same maintenance medium was used to determine the effects of various exogenously added steroids, protein hormones, and organ extracts on the proliferation of mammary epithelial cells in culture. Hormones failed to elicit any proliferative response, but extracts of kidney, brain, uterus, and spleen produced proliferative responses equal to or greater than the response obtained with 50% horse serum and cholera toxin. Kidney extracts prepared from midpregnant mice, virgin mice, and virgin mice given pituitary isografts all showed comparable activities, suggesting that the concentration of stimulatory factor(s) was not influenced by the hormonal status of the donor. Normal mammary epithelial cells that had undergone a 10-to 15-fold increase in cell number over initial values during 2-3 weeks in culture were passaged to secondary gel cultures. Outgrowths similar to those seen in primary culture were seen again in secondary culture. The present system provides a method for sustaining growth in culture of primary mammary epithelial cells from normal tissues.Classical endocrinology involving organ ablation and hormone therapy has developed the concept that pituitary, ovarian, and adrenocortical hormones are involved in mammary gland development (1, 2). Detailed analysis, however, requires an in vitro system in which the direct mitogenic effect of hormones can be assessed on appropriate target cells for prolonged periods. Early studies using organ and fragment cultures have shown insulin and growth factors to be the major factors responsible for mammary cell proliferation, but not the in vivo mammogenic hormones, an observation presenting a paradox with the in vivo situation (1, 2). Even insulin and the growth factors have been shown to cause only one or two rounds of DNA synthesis in vitro (3-6) and never a sustained growth.Recent studies indicate that, while estrogens have a direct mitogenic effect on a human mammary tumor cell line (MCF-7) (7) as assessed by an increase in cell number, estrogen mitogenicity cannot be demonstrated on the MTW9/PL mammary cell line (8). It has been proposed for the latter case that estrogen acts indirectly on the target cells through the production of intermediary growth factors that in turn promote growth of the mammary cell line (18). An inherent limitation of the use of e...

Sustained growth of normal mouse mammary epithelial cells in primary culture, leading to an increase in cell number, in response to growth factors [epidermal growth factor (EGF) and fibroblast growth factor (FGF)] or cholera toxin has been achieved by embedding the cells inside collagen cells. Inclusion of agents known to increase the level of cellular cAMP have been found to be favorable for mammary epithelial cell proliferation. Cholera toxin is by far the best of all of the agents tested (prostaglandins E1 and E2, isoproterenol, theophylline, and dibutyryl cAMP). When growth factors (EGF or FGF) are added with cholera toxin, a synergistic effect resulting in a response much greater than with either of them alone is seen. This synergism was best seen in normal mammary epithelial cells from nonpregnant mice. The extent of this synergistic effect was found to be less in normal cells from pregnant mice, suggesting that these cells may be less responsive to EGF during pregnancy. Tumor cells were found to be rather inconsistent in their responses to EGF and cholera toxin, ranging from a minimal response, similar to that of normal cells from pregnant animals, to a maximal response, similar to that of normal cells from nonpregnant animals.

SUMMARY: Mammary glands are enzymatically dissociated and the resulting tissue digest enriched for epithelial cells by isopycnic banding on a density gradient of Percoll. The cells are embedded within a rat tail collagen gel matrix and fed with the appropriate medium. Growth and differentiation are superior in such a system when compared to culture on plastic, using identical media.

In vitro and in vivo experimental studies have demonstrated the role of lysophosphatidic acid (LPA) signaling in tumor proliferation, invasiveness, and metastasis. Among LPA receptors, the overexpression of LPA receptor 3 (LPAR3) in transgenic mice has resulted in the highest rate of breast cancer metastasis. Our goal is to evaluate the LPA-producing enzyme autotaxin and LPAR3 as potential therapeutic targets in breast cancer patients. The expression of autotaxin and LPAR3 was examined by immunohistochemical analysis of 87 invasive human breast carcinomas. Carcinomas were more frequently positive for autotaxin and LPAR3 (24.4 and 43 %, respectively) compared to adjacent normal breast tissue (6.1 and 2.9 %, respectively). Increased stromal autotaxin expression was found in 16.3 % of the tumors. LPAR3 overexpression was associated with less differentiated tumors, human epidermal growth factor receptor 2 expression, and absence of progesterone receptors. The luminal type A carcinomas showed the lowest frequency of autotaxin and LPAR3 expression. Strong desmoplastic stromal reaction was more frequent among the carcinomas with autotaxin-positive tumor cells or autotaxin-positive stroma. Patients with carcinomas overexpressing LPAR3 in epithelial cells or autotaxin in stromal cells were more likely to have larger tumors, nodal involvement, and higher stage disease. Autotaxin overexpression in tumor cells also correlated with tumor size and clinical stage. Our data indicate that the increased expression of LPAR3 and autotaxin in human breast cancer is associated with tumor aggressiveness. They also suggest that LPA mediates tumor metastatic ability and peritumoral desmoplastic reaction through autocrine-paracrine mechanisms. A substantial portion of breast cancer patients might benefit from autotoxin/LPA receptor-targeted therapies.

A serum-free primary cell culture system was used to examine the direct effects and interactions of mammogenic hormones and epidermal growth factor (EGF) on the growth of mouse mammary epithelial cells. Epithelial cells were isolated by collagenase dissociation followed by Percoll gradient centrifugation and cultured within collagen gels in a mixture of Ham's F-12-Dulbecco's Minimum Essential Medium (1:1) containing insulin (10 micrograms/ml), crude soybean lecithin, trace elements, trypsin inhibitor, and antioxidants. Progesterone (P; 10(-6) - 10(-8) M) or ovine PRL (1 microgram/ml), in the absence of EGF, stimulated the growth of cells from mature virgin mice 2- to 4-fold over that of controls cultured in basal medium only. P and PRL synergized in stimulating growth 3- to 17-fold. 17 beta-Estradiol (10(-7) - 10(-10) M) alone did not stimulate growth or synergize with P and/or PRL. This lack of growth stimulation by 17 beta-estradiol was also observed in medium containing a low concentration of insulin (0.1 microgram/ml). EGF (10 ng/ml) alone stimulated growth to the same extent as the combination of P and PRL. EGF at 1, but not 10, ng/ml when combined with P and PRL could additively stimulate growth. Cells from midpregnant mice were less responsive than cells from virgin mice to the growth-stimulating effects of the combination of P and PRL (2-fold stimulation at most), but not to EGF (3- to 6-fold stimulation). Corticosterone, deoxycorticosterone, and aldosterone, but not cortisol, could synergize with PRL in stimulating the growth of cells from mature virgin mice. However, only deoxycorticosterone could stimulate growth in the absence of PRL. These results suggest that PRL, P, and adrenal corticoids may directly stimulate the growth of mouse mammary epithelial cells. The physiologically relevent adrenal corticoids, corticosterone and aldosterone, only potentiate the stimulatory effect of PRL. The hormonal stimulation of growth in vitro can be obscured by an optimum concentration (10 ng/ml) of EGF. The relative growth responses to mammogenic hormones and EGF may depend on the degree of differentiation of the cells.

Freshly isolated normal and tumor mouse mammary epithelial cells embedded within a collagen gel matrix undergo sustained growth when cultured for as long as 3 wk in a serum-free medium composed of a 1:1 (vol/vol) mixture ofHepesbuffered Ham's F12 and Dulbecco's modified Eagle's medium supplemented with insulin, epidermal growth factor (EGF), transferrin, bovine serum albumin fraction V, and cholera toxin. Of these additives, only insulin, EGF, and albumin are required for the growth ofmost normal cells. Albumin is not always an absolute requirement for growth but greatly enhances it. Lithium has been found to stimulate the growth of normal cells and can replace EGF. The collagen matrix culture system allows sustained growth of primary cultures of both normal and neoplastic mammary epithelium in serum-free conditions. This serum-free system will be useful in identifying and investigating the role of hormones, growth factors, and nutritional factors in regulating the growth of mammary epithelial cells.The hormonal control of mammary gland growth and development has long been the subject ofintense study. Both in vivo and in vitro organ culture studies have revealed apparent requirements for hypophyseal polypeptide and steroid hormones for mammary gland growth and development (1,2). However, the results from in vivo studies, with their inherent complexity, and from organ culture studies, which are often characterized by a limited growth response with obligatory hormonal priming of the donor animal, do not permit a clear distinction between direct and indirect effects of hormones upon the growth of the mammary epithelium.Recently, a collagen gel culture system was developed in which mammary epithelial cells (mouse and human) embedded within the gel undergo sustained (i.e., prolonged) multifold growth in primary culture (3-5). The importance of sustained growth in reducing the impact of endogenous factors with long half-lives, such as steroid hormones carried into culture by the cells (6, 7), should not be overlooked. The collagen gel system has permitted assessment of the ability of hormones such as prolactin, estradiol, and progesterone, known to stimulate growth in vivo and in organ culture, to stimulate directly the growth of mammary epithelial cells. In contrast to the in vivo findings, prolactin and ovarian steroids did not stimulate the growth ofmouse mammary epithelial cells when cultured in this system in medium containing serum (3). These results are compatible with the hypothesis that classical in vivo mammogenic hormones may not be direct mitogens for the mammary epithelium (8, 9). However, a limitation of these studies was the use of medium containing serum which may mask, inhibit, or modulate the growth-promoting effects of exogenous hormones. To circumvent this problem, the development of a serum-free culture medium would be ofthe utmost importance.Serum-free media have been established for the propagation of cell lines derived from human mammary carcinomas (10, 11) and various primary cell cul...