No abstract

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 approximately 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.

The reproducible establishment of representative stages in early breast cancer progression from the MCF10 model offers a new opportunity to analyze critical events of carcinogenesis and progression in breast cancer.

A 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 10(5) 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: they formed domes and exhibited renewal inhibition, they produced ductlike formations in collagen gels, they contained epithelium-specific keratin filaments, and they were diploid.

We have previously described the mortal human breast epithelial culture MCF‐10M, that was derived from fibrocystic breast tissue, was cultivated in medium with low calcium content for over 2 years, and spontaneously gave rise to the immortal MCF‐10 cell line. The emergence of immortalized cells, characterized by growth in conventional calcium levels, from mortal cells has proven to be a reproducible event. Here we report the establishment of a second immortal line from MCF‐10M, designated MCF‐10‐2, and establishment of the MCF‐12 immortal line after long‐term cultivation of MCF‐12M mortal cells from reduction mammoplasty tissue. DNA fingerprinting demonstrated the independent, human origin and lineage of the MCF‐10‐2 and MCF‐12 cell lines. Both lines require cortisol and EGF for maximal growth. The expression in these cultures of in vivo breast epithelial phenotypes was analyzed using 2‐dimensional gel Western blots and immunoper‐ oxidase staining with antibodies to cytokeratins and polymorphic epithelial mucin. MCF‐10M and MCF‐12M retain the cytokeratin profile of the luminal cell (7, 8, 18, 19), and also express cytokeratin 14, found predominantly in basal cells. The immortal lines express a similar profile, except that cytokeratin 19, a component of the fully differentiated luminal cell, is not expressed in the more uniform population seen in MCF‐10 and MCF‐12, but is retained in the morphologically mixed, less‐ selected population of MCF‐10‐2. Epitopes on the polymorphic epithelial mucin, recognized by antibodies HMFG 1, HMFG 2 and SM‐3, were detected in the mortal cultures and in the immortal lines, indicating the occurrence of both normal and abnormal mucin processing. MCF‐10, MCF‐10‐2 and MCF‐12 cells do not form tumors in nude mice, but appear to organize as duct‐like structures before regressing in the 5th week post injection.