More than 90% of cancer-related death is caused by spreading of tumor cells from the primary sites to distant organs (metastasis). Triple-negative breast cancers (TNBCs) are more aggressive and metastatic than other types of breast cancers. To metastasize, tumor cells have to enter into the blood vessels (circulation). It has been recognized that these circulating tumor cells (CTCs) contain a rare subset of metastasis-initiating cells with the ability to generate metastatic tumors. However, the properties of these metastasis-initiating CTCs are largely unknown. Both my preliminary data and other studies have shown that neutrophils can interact with CTCs (CTC-neutrophil clusters) to facilitate metastasis, but little is known about how they interact. By single-cell RNA sequencing, I found that ICAM-1 was highly expressed in lung metastatic cells compared to the primary tumor cells in TNBCs. In addition, knockdown of ICAM-1 inhibited self-renewal ability of tumor cells in vitro, uPAR secretion, and TNBC metastasis in vivo. It has been known that neutrophil-endothelial cell interactions are mediated by Mac-1 and ICAM-1, and uPAR pariicipates to the recruitment of neutrophils, and facilitate Mac-1-mediated adhesion. Based on these findings, I hypothesize that ICAM-1+ CTCs contain metastasis-initiating cells which secrete uPAR to recruit neutrophils, and then associate with them through ICAM-1-Mac-1 interaction to promote metastasis. I propose to test this hypothesis in Aim1. Accumulating evidence indicate that neutrophils have both antimetastatic and pro-metastatic activities (plasticity). Inhibition of AloxS, one of the key enzymes in the arachidonic acid (AA)-Alox5 metabolic pathway, abrogates pro-metastatic activity of neutrophils and consequently reduces metastasis. In addition, AA-cyclooxygenase (COX) metabolic product prostaglandin E2 (PGE2) is also involved in pro-tumor function of neutrophils. Interestingly, both PLA2 (the initial enzyme of the AA metabolism) and Alox5 can increase Mac-1 expression, and I have found that Mac-1 on circulating neutrophils from tumor bearing mice is upregulated. Based on these findings, I hypothesize that AA metabolism regulates pro-metastatic activity of neutrophils, and facilitates CTC-neutrophil interaction by upregulating Mac-1 expression. I will test this hypothesis in Aim2. The results from this proposal will not only reveal new mechanisms of metastasis, but also have significant implication for understanding how AA metabolism regulates pro-metastatic function of neutrophils, which will facilitate the development of the novel cancer immunotherapeutic strategies by targeting pro-metastatic neutrophils.
