PROJECT SUMMARY Metastasis causes a majority of breast cancer (BCa)-related deaths. Among all types of BCas, triple-negative breast cancers (TNBCs) are more aggressive and have a poorer prognosis. To metastasize, cancer cells must enter into the circulatory system. These circulating tumor cells (CTCs) are considered the seeds of metastasis. Recently, both our preliminary data and other studies have shown that neutrophils can interact with a subpopulation of CTCs (named CTC-neutrophil clusters) to facilitate metastasis. However, the features of neutrophil-associated CTCs, and how CTC-associated neutrophils facilitate metastasis has yet to be determined. The overall objective of this proposal is to identify and characterize neutrophil-associated CTCs, and how the interaction promotes metastasis, with the hope of developing a novel therapeutic strategy to inhibit metastasis by blocking CTC-neutrophil cluster formation. We found that the intercellular adhesion molecule 1 (ICAM-1), which is well-known to bind to Mac-1 on neutrophils, is overexpressed in lung metastases, and knockdown of ICAM-1 inhibits TNBC metastasis. In addition, ICAM-1+ TNBC cells produce high level of soluble urokinase plasminogen activator receptor (uPAR). Since uPAR is a potent chemoattractant for recruitment of neutrophils, and facilitates Mac-1-mediated adhesion, we hypothesize that ICAM-1+ CTCs are prone to associate with neutrophil by increasing suPAR production to enhance their metastatic ability. Furthermore, our preliminary findings indicate that Mac-1 expression is upregulated in circulating neutrophils from tumor-bearing mice. Since both phospholipase A2 and arachidonate 5-lipoxygenase, the key enzymes in arachidonic acid (AA) metabolism, can upregulate Mac-1 expression, we also hypothesize that enhanced AA metabolism in circulating neutrophils is required for their interaction with CTCs to promote metastasis. We propose 2 Specific Aims: 1) to determine the molecular mechanisms by which ICAM-1 promotes BCa metastasis, particularly whether uPAR facilitates ICAM-1 and Mac-1 mediated CTC and neutrophil interaction using both mouse and human TNBC models; 2) to dissect the role of AA metabolism in the pro-metastatic activity of circulating neutrophils using blood samples from both TNBC mouse models and BCa patients. The results from this proposal will not only reveal new mechanisms of metastasis, but also have significant implications for understanding how AA metabolism regulates pro-metastatic function of neutrophils, which will pave the way for the design of next-generation neutrophil-based immunotherapy.