Project Summary The daunting heterogeneity of breast cancer is apparent even within a tumor in an individual patient. The clinical consequence is that a targeted therapeutic e ective against some cancer cells remains impotent against counterparts that have evolved alternative pathways to accomplish disease-driving functions. Thus, there is an urgent need for single-cell approaches to discover molecular targets that span the complementary pathways by which heterogeneous cancer cells metastasize. An early step in metastasis involves cancer cells invading through the primary tumor microenvironment to reach nearby vascular and lymphatic networks through which they disseminate to secondary sites. A common mode of breast cancer invasion in the primary tumor microenvironment involves cancer cell migration along collagen bers. The proteins that regulate brillar migration are poorly understood, and the diversity of pathways by which breast cancer subtypes accomplish this invasive mode of migration remains to be elucidated. In the proposed work, we seek to develop, optimize and apply single-cell migration and proteomics (scMAP), an innovative method to quantify both the migration properties and the proteome of the same individual cell. Thus, scMAP enables the collection of a novel data set that directly links cell migration and the abundance of thousands of proteins at the single-cell level. We will apply scMAP to discover proteins whose abundance is associated with enhanced brillar migration of a panel of heterogenous breast cancer cell lines. Analysis of this novel data set by total least squares regression and mutual information will reveal proteins that exhibit quantitative linear and non-linear relationships to brillar migration properties. These results will identify distinct sets of proteins that explain the disparate brillar migration properties of heterogeneous breast cancer cells. The identi ed proteins feed a pipeline for future functional testing in complex 3d brillar matrices and subsequent potential therapeutic application to curb brillar invasive migration. In addition, scMAP opens the door to proteomic-scale, single-cell resolution insights into cell migration in other contexts, including other modes of motility in metastasis.