Inducing a normal phenotype in breast epithelial cells using a three-dimensional basement membrane extract culture system: A study on the reversion of cancer

Experimentally, traditional developmental models and transgenic animals consistently underscore the importance of studying cell behavior in the correct tissue context. However, live animal experimentation is inherently complex, and systematic assessment of the effects of individual variables, such as cell shape and matrix compliance on cell behavior, is extremely difficult at best. Two-dimensional monolayer culture of key individual cell types has provided abundant, fundamental information on cell response, but cannot be used to show the normal phenotype of breast epithelial cells. Furthermore, their results often fail to translate into in vivo and clinical studies. It has been previously established that normal human breast epithelial cells can form their original phenotype as seen in vivo when embedded in or layered on reconstituted basement membrane extract. This phenotype is characterized as a single or double layer of polarized cells in acinar form with a lumen devoid of cells. Most malignant cell lines cultured under the same conditions exhibit severe morphological deformities, including colony overgrowth, luminal filling (hyperplasia), and resistance to apoptosis. It was hypothesized that malignant breast cells can be reverted to a normal phenotype through the manipulation of two factors: control of the environment via extra-cellular matrix proteins, and control of cellular pathways via signaling inhibitors. It was observed that high levels of epidermal growth factor resulted in disrupted multi-acinar formations. Inhibition of the protein complex known as mammalian target of rapamycin is currently being investigated as a potential method for cancer treatment. Exposure of rapamycin, mammalian target of rapamycin's primary inhibitor, led to decreased proliferation and increased caspase activity. Through the exposure to rapamycin in three-dimensional cultures, proliferation was reduced in malignant cells, while normal cells were not significantly affected. Unfixed fluorescent staining with ethidium bromide indicated the presence of luminal cell death. Increased structural organization was observed by immunofluorescent staining of F-actin and beta-catenin. Through RT-PCR analysis, increased expression of a number of genes related to polarity and structural organization was detected in malignant cells exposed to rapamycin. Further study will be required to better characterize the reversion effects of rapamycin and its derivatives.