1 A novel 3D in vitro metastasis model elucidates differential invasive strategies during and after breaching basement membrane Asja Guzman, Víctor Sánchez Alemany, Yen Nguyen, Catherine Ruiqi Zhang, Laura J. Kaufman Columbia University, Department of Chemistry, New York, NY 10027 Abstract: Invasive breast cancer and other tumors of epithelial origin must breach a layer of basement membrane (BM) that surrounds the primary tumor before invading into the surrounding extracellular matrix. To analyze invasive strategies of breast cancer cells during BM breaching and subsequent invasion into a collagen I-rich extracellular matrix (ECM), we developed a physiologically relevant 3D in vitro model that recreates the architecture of a solid tumor with an intact, degradable, cell-assembled BM layer embedded in a collagen I environment. Using this model we demonstrate that while the BM layer fully prevents dissemination of non-malignant cells, cancer cells are capable of breaching it and invading into the surrounding collagen, indicating that the developed system recreates a hallmark of invasive disease. We demonstrate that cancer cells exhibiting individual invasion in collagen matrices preferentially adopt a specific mode of collective invasion when transmigrating a cell-assembled BM that is not observed in any other tested fibrillar, non-fibrillar, or composite ECM. Matrix-degrading enzymes are found to be crucial during BM breaching but not during subsequent invasion in the collagen matrix. It is further shown that multicellular transmigration of the BM is less susceptible to pharmacological MMP inhibition than multicellular invasion in composite collagen/basement membrane extract matrices. The newly developed in vitro model of metastasis allows 3D cancer cell invasion to be studied not only as a function of a particular tumor’s genetics but also as a function of its heterogeneous environment and the different stages of invasion. As such, this model is a valuable new tool with which to dissect basic mechanisms of invasion and metastasis and develop new therapeutic approaches in a physiologically relevant, yet inexpensive and highly tunable, in vitro setting. keywords: metastasis, spheroid, collagen, invasion, model system, cancer © 2016. This manuscript version is made available under the Elsevier user license http://www.elsevier.com/open-access/userlicense/1.0/