Herbert D. Soule

Malignant MCF10CA1 cell lines derived from premalignant human breast epithelial MCF10AT cells

The MCF10 series of cell lines was derived from benign breast tissue from a woman with fibrocystic disease. The MCF10 human breast epithelial model system consists of mortal MCF10M and MCF10MS (mortal cells grown in serum-free and serum-containing media, respectively), immortalized but otherwise normal MCF10F and MCF10A lines (free-floating versus growth as attached cells), transformed MCF10AneoT cells transfected with T24 Ha-ras, and premalignant MCF10AT cells with potential for neoplastic progression. The MCF10AT, derived from xenograft-passaged MCF10-AneoT cells, generates carcinomas in ∼25% of xenografts. We now report the derivation of fully malignant MCF10CA1 lines that complete the spectrum of progression from relatively normal breast epithelial cells to breast cancer cells capable of metastasis. MCF10CA1 lines display histologic variations ranging from undifferentiated carcinomas, sometimes with focal squamous differentiation, to well-differentiated adenocarcinomas. At least two metastasize to the lung following injection of cells into the tail vein; one line grows very rapidly in the lung, with animals moribund within 4 weeks, whereas the other requires 15 weeks to reach the same endpoint. In addition to variations in efficiency of tumor production, the MCF10CA1 lines show differences in morphology in culture, anchorage-independent growth, karyotype, and immunocytochemistry profiles. The MCF10 model provides a unique tool for the investigation of molecular changes during progression of human breast neoplasia and the generation of tumor heterogeneity on a common genetic background.

Estrogen responsive proliferation of clonal human breast carcinoma cells in athymic mice

A clone of MCF-7 (MCF-7ED), a cell in continuous in vitro cultivation derived from an estrogen-responsive human breast carcinoma, requires estrogen supplementation for progressive exponential (double time: 50--85 h) tumor growth in the mammary fat of athymic mice. The plasma concentration of estradiol which stimulated exponential growth of MCF-7ED corresponded to physiologic premenopausal levels in women. The tumors were carcinomas with murine and MCF-7ED cells intermixed. MCF-7ED cells could be repurified in subcultures of mixed tumors. Comparative studies of breast and non-breast cell lines showed concordance between the presence of estradiol receptor, sensitivity to the anti-estrogen tamoxifen for growth in vitro, and estradiol dependence for tumorigenic growth in athymic mice. Progesterone alone did not stimulate MCF-7ED growth, but acted synergistically with estrogen. Progesterones action was to decrease tumor latent period, not to increase final tumor incidence or growth rate. Under estrogen-deficient conditions, condsitions approximating postmenopausal status in women, (10(-10) M in plasma), a dormant state was established between MCF-7ED cells and murine mammary stroma which could be maintained several months. The dormant state could be broken by introduction of estradiol, but not progesterone. This system should be useful for defining host and cancer cell determinants in estrogen-responsive breast cancer growth.

A simplified method for passage and long-term growth of human mammary epithelial cells.

SummaryA method is described for culturing human mammary epithelial cells in primary culture and allowing more than 50 generations and a 1000-fold increase from starting inocula without need of enzymatic transfers. Organoids dissociated from breast tissue are plated in medium containing 1.05 mM Ca++ to effect attachment and growth to monolayer density. Medium is then switched to one containing 0.06 mM Ca++ to overcome “renewal inhibition” and to stimulate growth. In low Ca++ media, primary cultures become a long-term, continuous source of free-floating viable cells free of fibroblasts. A fundamental requirement for extended growth in primary culture is maintaining calcium levels at approximately 0.06 mM. Above 0.06 mM Ca++, cells divide only 3 to 4 times in primary cultures before terminal differentiation occurs. At 0.06 mM Ca++, cells continue to divide for periods of time determined partly by feeding schedule, but up to 6 mo. and 50 generations of (linear) growth. Cells released from monolayer were greater than 90% viable and yielded 105 cells/cm2 of attached cells every 72 h. Free-floating single cells readily replated and cloned, when transferred, without need of trypsin for dissociation. Long-term free-floating cells were typical mammary epithelium: (a) they formed domes and exhibited renewal inhibition, (b) they produced ductlike formations in collagen gels, (c) they contained epithelium-specific keratin filaments, and (d) they were diploid.

Calcium regulation of normal human mammary epithelial cell growth in culture

SummaryThe concentration of Ca++ in culture media profoundly affected the growth and differentiation properties of normal human mammary epithelial cells in short-term culture. In media where Ca++ was above 0.06 mM, longevity was limited to an average of three to four cell divisions. The extended growth fraction (those cells able, to divide more than once) was only approximately 50% and diminished to zero quickly with time. Stationary cells inhibited from dividing appeared differentiated in the formation of lipid vacuoles and accumulation of α-lactalbumin. Growth of stationary cultures could be reinstituted in about half the cells, either by disruption and transfer or by a reduction in Ca++ to less than 0.08 mM.The reduction of Ca++ to levels below 0.08 mM extended the longevity of normal cells to eight to nine divisions. The extended growth fraction was 100%. Under these conditions, cells did not differentiate. The effects of Ca++ on growth and differentiation were specific (Mg++ and Mn++ variations were without effect) and reversible and in many respects resembled Ca++ effects on epidermal cells. One major difference is that the dual pathways of growth and differentiation in mammary cells were controlled by glucocorticoid and insulin. Based on the kinetics of the reversible Ca++-induced coupling and uncoupling of proliferation and the program of differentiation, we propose that Ca++ may be an essential trigger for cell divisions that commit a mammary cell to differentiate progressively in a permissive hormonal milieu.

The etiology of breast cancer.

A dominant focus of the research programs at our own and other laboratories remains the identification and characterization of the populations at high risk to breast cancer. The goal: the early detection of the disease and, ultimately, its biological control.

Expression of transforming growth factor-β receptor type II and tumorigenicity in human breast adenocarcinoma MCF-7 cells

To analyze transforming growth factor‐β (TGF‐β) response during MCF‐7 cell progression, early passage (MCF‐7E, <200 passage) and late passage (MCF‐7L, > 500 passage) cells were compared. MCF‐7E cells showed an IC50 of ∼10 ng/ml of TGF‐β1, whereas MCF‐7L cells were insensitive. MCF‐7E cells contained approximately threefold higher levels of TGF‐β receptor type II (TβRII) mRNA than MCF‐7L, but their TβRI levels were similar. MCF‐7E parental cells showed higher TβRII promoter activity than MCF‐7L cells, which could be attributed to changes in Sp1 nuclear protein levels. Receptor cross‐linking studies indicated that the cell surface receptor levels parallel mRNA levels in both cell lines. Limiting dilution clones of MCF‐7E cells were established to determine the heterogeneity of TβRII expression in this cell line, and they showed varying degrees of TβRII expression. Fibronectin was induced at higher levels in cells expressing higher TβRII levels. All three TGF‐β isoforms were detected in limiting dilution clones and parental cells, but TGF‐β1 was more abundant relative to TGF‐β2 or 3, and no correlation between TGF‐β isoform profile with TGF‐β sensitivity was found. MCF‐7L cells were tumorigenic and formed xenografts rapidly and progressively, whereas MCF‐7E parental and limiting dilution clonal cells showed transient tumor formation followed by regression. These results indicate that decreased TβRII transcription in breast cancer cells leads to a loss of TβRII expression, resulting in cellular resistance to TGF‐β which contributes to escape from negative growth regulation and tumor progression. J. Cell. Physiol. 176:424–434, 1998.

Preservation of Human Epithelial-Like and Fibroblast-Like Cell Strains at Low Temperatures.

Summary Studies of cell preservation by slow freezing in 5% glycerol medium, storage at −70°C. and rapid thawing were carried out on 17 epithelial-like and 2 fibroblast-like human cell strains (Detroit). Following this procedure, each of the strains could be quickly recovered as actively proliferating cell strains with no changes in growth rate or strain characteristics.

Fine structure of a murine mammary carcinoma cell line

SummaryA fine structural study was made of cells from the epithelioid MCF-8/5-2A cell line derived from a MuMTV-free, D2 transplantable hyperplastic outgrowth. Electron microscopy shows the cells to be truly epithelial with many cell-to-cell junctions and microvilli. The cells are similar in many respects to normal mouse mammary gland and some of the conventional mammary tumor derived cell lines. This study supports previous observations of the absence of MuMTV in MCF-8 within the limits of morphological detection, and demonstrates the presence of numerous virus particles within, or budding into, cisternae of the endoplasmic reticulum and nuclear envelope. These intracisternal A particles have not been previously described in such abundance in mammary tumor tissue culture cells.