Targeting Prometastatic Neutrophils to Inhibit Breast Cancer Metastasis

Background: Triple negative breast cancer (TNBC) accounts for 10-15% of all breast cancer and have a high mortality rate owing to early recurrence and metastasis and a lack of effective therapeutic options. Our preliminary studies suggest that neutrophils counts are increased in tumor-bearing mice, and neutrophils frequently interacted with circulating tumor cells (CTCs). However, how neutrophils interact with CTCs and the consequences of their interaction are largely unknown. In addition, the intercellular adhesion molecule 1 (ICAM1), which is the receptor for Mac-1, are highly expressed on CTCs. Previous studies have shown that neutrophils can promote metastasis via arachidonate 5-lipoxygenase (Alox5)-dependent leukotriene synthesis, and induce immunosuppression by expressing PD-L1. However, whether Alox5 inhibition could be a novel therapeutic strategy to block metastasis and the mechanisms to induce PD-L1 expression on neutrophils are yet to be determined. We hypothesize that neutrophils promote metastasis by activating Alox5-Leukotriene pathway to (1) promote ICAM-1 and Mac-1 mediated CTCs and neutrophils interaction, and (2) induce immunosuppression by expressing PD-L1. We also hypothesize that combination of Alox5 inhibitors with anti-PD-1 Ab could have synergistic effects. Specific Aims: (1) determine whether interaction of neutrophils with CTCs promotes metastasis; (2) determine whether neutrophils induce immunosuppression through Alox5-leukotriene-IFN gamma-PD-L1 pathway; (3) determine whether Alox5 inhibitors can synergize anti-PD-1 treatment. Study designs: We will use patient-derived-xenograft (PDX) breast cancer models as well as the syngeneic 4T1 and E0771 mouse breast cancer models, all of which spontaneously develop lung metastasis. In order to monitor tumor growth and metastasis by bioluminescence imaging, the cells will be transduced with dual-reporter vector which expresses both Luciferase 2 and tdTomato (L2T). The effects of Alox5 inhibitors and combination with anti-PD-1 antibody on metastasis will be determined in mouse models. The CD11b deficient mice and ICAM1 knockout tumor cells will be used for functional analysis of interaction between CTCs and neutrophils. In vitro, the ICAM-1 and Mac-1 mediated interaction between neutrophils and CTCs will be detected by Imagestream. Alox5 activity, LTB4 and IFN gamma production, PD-L1 expression and T cell immunity will be analyzed to determine the mechanisms of neutrophils-induced immunosuppression. The clinical correlation of ICAM-1+ CTCs with patient’s outcomes and correlation between PD-L1+ neutrophils and Alox5 activity will be determined in metastatic TNBC patients. Impact: This proposal will explore the novel strategies of targeting prometastatic activity of neutrophils by Alox5 inhibitors or combination with immunotherapy to inhibit metastasis. We expect to develop more effective and less toxic immunotherapy combinations to block breast cancer metastasis.