Breast cancer is a priority in U.S. research and resulting clinical advances include better detection techniques and targeted therapies. Most breast cancer in the U.S. is now detected at clinical stage I. Breast cancer research, however, has long been dominated by a small set of well-established cell lines derived from stage IV tumors. Using techniques from mouse embryonic stem cell culture, we have developed a tissue engineering system that allows us to reliably establish proliferative cell cultures of normal breast epithelial cells as well as breast tumors of all stages, 0-IV. Microarray analysis of these unique resources reveals that the traditional late-stage cell lines are poor models for early stage tumors. We therefore propose to prepare a detailed molecular profile of two cell lines derived from our unique panel of sporadic, stage I breast tumors, using complete sequencing of coding and regulatory regions for mutations, in comparison to adjacent non-tumor tissue, with the results interpreted in the context of complete gene expression microarrays. The combination of polymorphic, mutant and epigenetically altered genes should explain the altered gene expression profile found in the tumor itself. These data will then be used to determine what subset of alterations found in late stage tumors are also seen in early stage tumors; to determine mechanisms of mutation in early stage tumors, be they environmental exposure or genomic instability; to identify established and potential new alterations suitable for targeted therapy; and to understand the underlying basis of transformation in breast epithelial cells. These explant cultures were established in the early 1990s and continue to grow in our culture system today; they may be considered as a novel set of cell lines that can be utilized experimentally to specifically address questions related to early stage breast cancer. From this perspective the complete characterization of these cultures will provide a rich molecular context for future investigators.