Abstract Cancer cells in an aggressive primary tumor are adept at exploiting their local tissue microenvironment. In contrast, when metastatic cells leave these favorable surroundings, they must possess or acquire traits that will allow them to survive and colonize foreign, potentially hostile tissue environments. The obstacles that metastasizing tumor cells encounter vary from organ to organ, and are highly influenced by non-cancerous stromal cells of the tumor microenvironment. For example, the blood-brain barrier, composed of endothelial cells, astrocytes and pericytes, presents a far more formidable structure for tumor cells to penetrate, compared to the fenestrated capillaries in the bone marrow. While the primary tumor microenvironment has emerged as an important regulator of cancer progression, it is less well understood how different tissue environments influence metastatic processes. We used a dual species-specific microarray platform to uncover tumor-stroma interactions that modulate organ tropism of brain, bone and lung metastasis in animal models of cancer. Among the differentially regulated tumor- and stroma-specific genes, we identified cathepsin S as a novel regulator of breast-to-brain metastasis. In breast cancer patients, high cathepsin S expression at the primary site correlated with decreased brain metastasis-free survival. Both macrophages and tumor cells produce cathepsin S, and only the combined depletion significantly reduced brain metastasis in experimental models in vivo. We show that cathepsin S specifically mediates blood-brain barrier transmigration via proteolytic processing of the junctional adhesion molecule (JAM)-B. Pharmacological inhibition of cathepsin S significantly reduced experimental brain metastasis, supporting its consideration as a therapeutic target for this disease. Citation Format: Lisa Sevenich, Robert Bowman, Steve Mason, Daniela Quail, Franck Rapaport, Benelita Elie, Edi Brogi, Priscilla Brastianos, William Hahn, Leslie Holsinger, Joan Massagué, Christina Leslie, Johanna A. Joyce. A brain metastasis-promoting role for cathepsin S identified from analysis of tumor- and stroma-supplied proteolytic networks. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr IA18. doi:10.1158/1538-7445.CHTME14-IA18