Metastatic disease is still largely unexplored, poorly understood and incurable. Accumulating evidence indicates that cells and mediators of the immune system can facilitate metastasis. Neutrophil accumulation in cancer patients has been associated with metastasis formation. In mouse tumor models, neutrophils have been reported to be pro- or anti- metastatic, but the underlying mechanisms involved in either function remain largely elusive. This proposal outlines a research program aimed at resolving the pro-metastatic role of neutrophils in breast cancer, as our preliminary data indicate that neutrophils proactively mediate breast cancer metastasis. Using a state-of-the art spontaneous breast cancer metastasis mouse model, we will mechanistically study how neutrophils facilitate metastasis formation and how mammary tumors provoke the metastasis-facilitating function of neutrophils. Building upon my previous studies and our current data, we will focus on the unexplored crosstalk between the adaptive immune system and neutrophils in facilitating spontaneous metastatic disease. These crucial questions will be addressed by undertaking a multidisciplinary approach, involving sophisticated mouse models for metastatic breast cancer, RNA sequencing on tumor-associated neutrophil populations, state-of-the-art mouse engineering, intravital imaging and in vivo neutrophil manipulations. Moreover, we will validate our findings from the mouse metastasis model in human breast cancer samples. We will determine the metastasis predicting power of the identified murine pro-metastatic neutrophil-specific pathways by immunohistochemistry and multi-parameter immunofluorescence on breast cancer samples and blood of untreated patients of which clinical follow-up is available. Thus, we will identify novel molecular pathways that can be targeted to selectively inhibit the pro-metastatic activity of the immune system.