Infiltration of Cancer-Related Circulating Neutrophil Subsets Into Tumor Tissues.

Introduction: The role of neutrophils in cancer biology has been recently a matter of increasing interest. Neutrophils account for a significant portion of the immune infiltrate in cancer, but their function in tumor progression and metastatic development remains controversial. We have recently identified a heterogeneous subset of circulating low-density neutrophils (LDN) that accumulate with cancer progression and display immunosuppressive properties compared to the normal high-density neutrophils (HDN). Which of these circulating subsets are the source of intratumoral neutrophils and the mechanisms driving this infiltration are still unkown. We aimed to assess in animal models of lung and breast cancer the ability of circulating neutrophils to infiltrate primary tumors and the chemotactic forces driving it.

Material and methods: The percentage of circulating LDN was evaluated in lung cancer and COPD patients. Blood was collected from mice bearing AB12 (mesothelioma), LLC (lung carcinoma) or 4T1 (breast) tumors, and circulating neutrophils were isolated using a density gradient centrifugation, followed by Neutrophil Enrichment EasySep isolation kit. HDN and LDN stained with CFSE or CellTrace Violet were incubated separately or together, with isolated whole tumors, or injected intraperitonally to mice. The presence of each subset in the blood, lung, spleen and tumors was assessed after 3 hours. The chemokines forcing their infiltration were evaluated.

Results and discussion: LDN was found only in advanced lung cancer patients, but not in early stage or COPD patients, and negatively correlated with survival. In murine models, both HDN and LDN infiltrated the tumor in ex-vivo settings. LDN entered tumor tissues 215% more than HDN (p<0.05). This was maintained when the two subsets were co-incubated with tumor tissues – 67% of infiltrating cells were LDN (p<0.05). Consistently, IP injection of both HDN and LDN in tumor-bearing mice resulted in higher infiltration of LDN in lung tissues and tumor (71% of infiltrated cells). Interestingly, injected HDN and LDN could both be retrieved in both fractions, suggesting these cells retain a certain level of plasticity and are able to acquire new properties in the circulation. The contribution of various chemokine receptors expressed in each subset was further evaluated.

Conclusion: LDN correlated with advanced cancer and poor prognosis. They preferably enter to tumors, though plasticity of circulating neutrophils is maintained. Proper understanding of the ways neutrophils can support or fight cancer and affect tumor progression will help us develop new strategies to direct the immune system, and more specifically neutrophils, against the tumor.