Breast cancer is one of the most commonly diagnosed cancers in women, accounting for 25.1% of all cancers and resulting in more than 500’000 deaths per year worldwide (http://www.who.int). The leading cause of death for breast cancer patients is not the primary tumor itself, but the development of metastasis. Metastasis-related mortality in breast cancer reflects our limited understanding of the complex processes that drive the spread of cancer and points to the inability of the currently used preclinical models to fully recapitulate the clinical setting. Currently, most of these preclinical models rely on cancer cell lines that have been adapted to long-term culture in two-dimensional plastic dishes, and do not share many of the properties of metastatic cancer cells, including their mutational profile.Using blood specimens from cancer patients, we have recently improved our understanding on the processes driving the establishment of metastasis. For example, we discovered that circulating tumor cells (CTCs) can enter the bloodstream as single cells or clusters of cells (CTC-clusters), with the latter being associated to higher propensity to seed metastasis. Further, we have shown that CTCs isolated from breast cancer patients can also be propagated in culture under specific conditions, retaining a clustered 3D structure. These CTC-derived cell lines represent a unique preclinical model for discovery of new therapies, particularly because they can be used as three-dimensional drug screening platforms, they share the mutational profile of metastatic cancers, are tumorigenic, and spontaneously metastasize in mice reflecting the metastatic pattern of the patient of origin.In parallel to the development of new preclinical models, small molecules have been instrumental in several breakthrough studies to investigate the biology of cancer and to identify biological targets along with existing genetic and molecular biology technologies. Small molecules as a chemical tool offer a unique opportunity to investigate cancer vulnerabilities and unknown complex biological mechanisms that define cancer biology, leading to the identification of new targets and potential therapeutic agents. Along these lines, there are extraordinary examples of how small molecules helped to understand various biological pathways in the recent past, including cytoskeleton biology through colchicine and paclitaxel, mTOR signaling through Rapamycin, MG132 in proteasome function, and recently CRBN-CUL4 E3 by thalidomide analogs and JQ1 for bromodomain biology and pharmacology. In this proposal, we aim to combine for the first time two very powerful tools, i.e., state-of-the-art human CTC-derived models that recapitulate metastatic cells and a large natural- and synthetic-origin compound library (~80.000 compounds) to identify novel inhibitors of metastasis. Specifically, this collaborative project will be divided into two main aims. First, we will screen libraries of small molecules capable of selectively inducing cell death in CTCs, or impede their self-renewal potential that is needed for metastasis formation. This approach will be complemented by RNA sequencing experiments to identify affected pathways, and synthetic lethal screens to identify relationships between mutations and drug sensitivity. Second, we will perform medicinal chemistry to improve structure-activity relationships on selected lead compounds, and test them in preclinical (mouse) cancer models.All together, our project brings together the unique tools provided by each of the project partners to unravel vulnerabilities of circulating tumor cells and to identify novel inhibitors of metastasis. In the long-term, our project should represent the first step towards the development of novel metastasis-tailored compounds for the treatment of cancer.