Remodeling of the stromal extracellular matrix and elevated expression of specific proto-oncogenes within the adjacent epithelium represent cardinal features of breast cancer, yet how these events become integrated is not fully understood. To address this question, we focused on tenascin-C (TN-C), a stromal extracellular matrix glycoprotein whose expression increases with disease severity. Initially, nonmalignant human mammary epithelial cells (MCF-10A) were cultured within a reconstituted basement membrane (BM) where they formed three-dimensional (3-D) polarized, growth-attenuated, multicellular acini, enveloped by a continuous endogenous BM. In the presence of TN-C, however, acini failed to generate a normal BM, and net epithelial cell proliferation increased. To quantify how TN-C alters 3-D tissue architecture and function, we developed a computational image analysis algorithm, which showed that although TN-C disrupted acinar surface structure, it had no effect on their volume. Thus, TN-C promoted epithelial cell proliferation leading to luminal filling, a process that we hypothesized involved c-met, a proto-oncogene amplified in breast tumors that promotes intraluminal filling. Indeed, TN-C increased epithelial c-met expression and promoted luminal filling, whereas blockade of c-met function reversed this phenotype, resulting in normal BM deposition, proper lumen formation, and decreased cell proliferation. Collectively, these studies, combining a novel quantitative image analysis tool with 3-D organotypic cultures, demonstrate that stromal changes associated with breast cancer can control proto-oncogene function.