76P Circadian control of neutrophil extracellular trap formation temporally regulates metastatic lung cancer progression

Neutrophils are the first responders of the innate immune system and have been implicated in lung cancer progression and metastasis to secondary organs. Neutrophil trafficking and effector functions are temporally controlled by the chemokine receptor CXCR2 and the circadian rhythm. Due to their diurnal regulation, it remains elusive whether neutrophils can be effectively targeted in lung cancer. To study the role of tumor-derived CXCR2 ligands in neutrophil chemoattraction in vitro, we designed a migration assay using microfluidic chips. We developed orthotopic and liver metastasis models of lung cancer using intrathoracic and intrasplenic injections of syngeneic lung adenocarcinoma cells. We combined spectral flow cytometry, immunofluorescence and imaging mass cytometry to assess changes to the immune response. To study the role of the CXCR2 axis in our cancer models, we generated a neutrophil-specific CXCR2 knock-out mouse model or blocked CXCR2 signaling using a small molecule inhibitor. We show that neutrophil maturation programs, including CXCR2 expression and neutrophil extracellular traps (NETs) formation, accurately predict adverse outcomes in lung adenocarcinoma patients. Using primary and metastatic lung cancers models, we found that tumor-supplied CXCR2 ligands dictate neutrophil recruitment and activation during disease progression. Interestingly, we discovered that lung cancer liver metastases followed a diurnal pattern that coincided with circadian oscillations in circulating neutrophils and NETs. Moreover, quantitative systems modelling identified optimal posological schedules to curb diurnal surges in neutrophil trafficking using CXCR2 inhibitors, which were functionally validated in vivo. Finally, highly multiplexed histological analyses of human lung adenocarcinoma by imaging mass cytometry revealed a robust time-dependent oscillation in neutrophil infiltration, confirming that circadian control of the tumor microenvironment is also present in patients. Our findings suggest that NET-mediated metastases are time-dependent and support the need for the temporal optimization of immune-based cancer treatments targeting neutrophils.