The MCF-10A progression series grown in three dimensional rBM cultures can be a powerful system for studying human mammary tumorigenesis. All three cell lines derive from a common ancestor and the manipulations that have made them increasingly transformed are well defined. While much can be learned by studying these cells in traditional two dimensional tissue culture, real malignant transformation takes place within a three dimensional tissue. The three dimensional rBM system we have utilized to characterize these cells recapitulates much more of the microenvironment of a tissue and allows for a more detailed investigation of the dynamic and reciprocal crosstalk between the extracellular matrix and nuclear gene expression that may play an important role in real breast tumorigenesis [1, 3, 5, 17, 18]. In this study, we assessed cellular parameters of tissue formation in cells of the MCF-10A progression series and documented them as a faithful model of mammary malignant progression.
Variants of differing malignancy have been developed from the first normal human mammary epithelial cell line, HMT-3522 , by spontaneous transformation with selection for EGF-independent growth [21–23]). These variants, cultured in rBM, have offered significant insights into tumor progression [24–27]. However, the molecular mechanisms driving these cells toward malignancy were not defined in advance, although some information about the process might be inferred from gene expression studies of the established lines .
MCF-10A cells were manipulated by the forced expression of a mutated H-ras-1, which is the human analog of the Harvey sarcoma virus oncogene. This constitutively active Ras variant, which was originally isolated from the bladder carcinoma cell line T24, has a single base mutation causing an amino acid change at residue 12 (Gly to Val) . When injected into nude mice, the resulting MCF-10AT cells persisted as xenografts and ultimately developed into carcinomas 25% of the time [12, 13]. In tissue culture studies, expression of this constitutively activated Ras induced transformation of immortalized NIH3T3 cells and of primary rat and hamster cells from multiple tissues, whereas the normal Ras did not [29, 30]. While few established breast cancer cell lines have Ras mutations, a majority have Ras overexpression via amplification of the gene [31–33]. More recently, activating mutations in H-ras, K-ras, or N-ras were observed in 8 out of 40 human breast cancer cell lines, with another 4 lines having activating mutations in B-Raf, which is downstream in the Ras pathway . Although only 5% of breast tumors have activating Ras mutations , it was shown that more than half had a 2- to 6-fold increase in Ras expression and a more than 5-fold activation in downstream MAP Kinase activity . Thus, Ras signaling may be more commonly activated in breast tumor cells by mechanisms other than mutation. In MCF-10A cells, ErbB1-ErbB2 heterodimerization disrupts acinar tissue structure in three dimensional basement membrane cultures and generates a more invasive phenotype . The invasive phenotype was reverted by inhibition of the Ras/MAP Kinase pathway, supporting a role for the Ras pathway in the malignant progression of ErbB2 expressing tumors. For these reasons, the ectopic expression of activated Ras in MCF-10A cells is likely relevant to the mechanisms of patient breast tumor progression.
While MCF-10AT cells and normal MCF-10A cells grown in three-dimensional reconstituted basement membrane culture both exhibited growth arrest, this arrest appeared to be delayed in the MCF-10AT cells. These cells also demonstrated a nearly complete lack of apoptosis, resulting in the absence of notable lumen formation. A recently published report indicated that in MCF-10AT cells grown in three-dimensional culture, apoptosis was decreased, but not absent, leading to reduced lumen formation . MCF-10AT cells were normal or nearly normal in their ability to deposit basement membrane and to form cell:cell junctions. Thus the activation of Ras predominantly affects the proliferation state of the cells in three-dimensional culture, consistent with the idea that these cells recapitulate aspects of breast tissue hyperplasia in vivo .
In contrast, the malignant MCF-10CA1a cells never exhibited growth arrest and never formed normal acinar structures, consistent with reports comparing primary mammary epithelial cells, primary breast tumor cells, and transformed breast cancer cell lines . The MCF-10CA1a cells exhibited unchecked proliferation in three-dimensional culture despite the continuous occurrence of apoptosis, suggesting the loss of normal growth controls. In addition, the MCF-10CA1a cells showed an incomplete deposition of basement membrane proteins and abnormal cell:cell contacts. Interestingly, the expression levels of the markers assayed in these experiments were not significantly altered. Instead, the proper structural organization of the proteins within the cells was compromised. These phenotypes are consistent with more advanced stages of tumorigenesis, at which the dynamic relationships between cell:cell interaction and cell:ECM contact are altered [1, 39].
These progressively malignant cell lines were engineered in the same cell background by a defined series of manipulations. They recapitulate tumor phenotypes in a three dimensional culture system, and this will facilitate future studies of malignant progression. Compared to in vivo studies, organotypic culture systems are easier and less expensive to manipulate, facilitate fluorescence based microscopy imaging, have a reduced complexity of cell types in a simplified microenvironment, and are influenced by a reduced number of uncontrolled factors. This simplicity can be a disadvantage, ignoring features not included in the tissue culture, but it can also be a great advantage for mechanistic studies of malignant changes. Of particular interest is the potential use of these cell lines in three dimensional culture to dissect the signal transduction pathways, and especially the ras pathway, that drive these cells to increasing malignancy.