Extracellular Matrix as a Key Mediator of Mammary Tumor Cell Normalization

Some epithelial cancers can be induced to revert to quiescent differentiated tissues when combined with embryonic mesenchyme; however, the mechanism of this induction is unknown. This dissertation is based on the hypothesis that because extracellular matrix (ECM) plays a critical role during organ development in the embryo, it also may mediate the differentiation-inducing effects of embryonic mesenchyme on cancer cells. To test this hypothesis, I first optimized methods to isolate ECMs from whole tissues or cultured cells, and to repopulate them with cultured cells, using embryonic tooth as a model system. In Chapter 2, I describe these studies and use them to demonstrate that embryonic ECM is sufficient to regulate odontogenic signaling, cell fate decisions and histodifferentiation during normal tooth development. In Chapter 3, I adapt these methods to show that culture of breast cancer cells with ECM derived from embryonic mammary mesenchyme decreases tumor cell proliferation, and stimulates differentiation, including formation of hollow acini and ducts as well as enhanced expression of estrogen receptor-alpha and decreased migration. Further, when the inductive ECMs were injected into fast-growing breast tumors in mice, they significantly inhibited cancer expansion. Critically, the differentiation observed with ECM was the same as that observed in co-culture with mammary mesenchyme cells, showing that ECM is playing a dominant role in tumor cell normalization. In Chapter 4, I then set out to determine the mechanism by which embryonic ECM normalizes tumor cells, I analyzed the contributions of bound cytokines, ECM composition and mechanics. Western blot analysis revealed several bound growth factors, which remained following decellularization; however, removal of these growth factors using high salt washes had no effect on ECM-mediated normalization of tumors. Further, using proteomics analysis I identified eleven ECM proteins present only within inductive ECMs and by testing these proteins in 3D culture, I found three proteins -- collagen III, biglycan and SPARC -- that increased lumen formation to a similar extent as embryonic ECM. These data confirm that mesenchyme-induced tumor cell normalization is mediated by the insoluble ECM, and reveal the identity of some of the inductive molecules responsible for these effects.