Background: The members of the slit homolog (SLIT) and roundabout homolog (ROBO) families have emerged as important signaling molecules in tumor metastasis. This study analyzed their role in regulating breast cancer (BC) cell motility and chemotaxis and assessed expression of ROBO1 in brain metastases (BMs) of breast, lung, and colon cancer, and in peritoneal metastases (PMs) of ovarian cancer. Materials and Methods: The BC cell line MDA-MB231 was subjected to scratch, motility, and chemotaxis assays using heparin and a purified recombinant N-terminal SLIT2 fragment. Protein expression was assessed in primary tumors and metastases by immunohistochemistry. Results: Exposure to SLIT2 induced MDA-MB231 cell motility, but no significant chemotaxis without the presence of heparin. ROBO1 was expressed in 4/5 primary BC and in 18/21 BC-derived BM samples; 7/9 BM primary lung cancer samples also stained positive. In contrast, BMs from colorectal cancer were negative for ROBO1. Primary ovarian cancer and ovarian PM showed ROBO1 expression in 0/6 and in only 2/6 samples, respectively, whereas SLIT2 was observed in 1/6 primary cancer and in 6/6 PMs samples. Conclusion: SLIT2 can induce BC cell motility and chemotaxis, but the latter requires the presence of heparin. BM expression of ROBO1 is a common feature of some, but not all cancer types. SLIT2 expression appears to be a general feature of ovarian cancer-derived PMs.Metastasis is a process that involves multiple steps, starting with the mobilization and release of tumor cells from the primary tumor, which are then transported through the lymphatic system and blood vessels to distant sites, where they form new tumors after extravasation and tissue invasion. This process is not random, as it has been established that certain primary cancer types metastasize preferentially to distinct target organs (1). Within the sites of metastasis, distinct molecules, such as cytokines and chemokines play an important role in attracting tumor cells which express the corresponding cell surface receptors (2, 3). C-X-C motif chemokine 12 (CXCL12) with its receptor C-X-C chemokine receptor type 4 (CXCR4), originally identified as a major inducer and regulator of leukocyte chemotaxis (4), is an example of this process. Specifically, CXCR4 is expressed by malignant and pre-malignant breast tumors, such as those at the stage of atypical ductal hyperplasia (5). CXCR4-expressing tumor cells mediate targeted metastasis to bone and other tissues expressing the CXCR4 ligand CXCL12. A new candidate for a modulatory role in CXCR4-/CXCL12-mediated metastasis is the neuronal repellent slit homolog (SLIT) 2 protein. The SLIT proteins (SLIT1, -2, and -3) are a family of large, extracellular-matrix secreted glycoproteins that act as ligands to the Roundabout receptor family (ROBO1, -2, -3, and -4). They have been linked to neuronal development, myogenesis, leukocyte chemotaxis, and cancer (6, 7). SLIT2 (acting through its receptor ROBO1) was found to inhibit CXCL12-induced leukocyte chemotaxis (8). In pre...