Tumor-activated Neutrophils Promote Metastasis in Breast Cancer via the G-CSF-RLN2-MMP-9 Axis

Immune component of tumor microenvironment is essential for the regulation of cancer progression. Tumor mass is frequently infiltrated by neutrophils (tumor-associated neutrophils, TANs) in breast cancer (BC) patients. However, the role of TANs and the mechanisms of their action in BC are still unclear. Using quantitative IHC, ROC and Cox analysis, we demonstrate here that high density of TANs infiltrating tumor parenchyma is predictive for poor prognosis and for the decreased progression-free survival of patients with BC that underwent surgical tumor removal without previous neoadjuvant chemotherapy in all 3 cohorts (training cohort, n = 170; validation cohorts, n = 130, independent cohorts, n = 95). Conditioned medium of human breast cancer cell lines prolonged life span of healthy donor neutrophils ex vivo. Neutrophils, activated by supernatants of breast cancer lines, demonstrated the increased ability to stimulate proliferation, migration and invasive activity of breast cancer cells. Cytokines involved in this process have been identified by antibody array. Relationships between these cytokines and density of TANs were validated by ELISA and IHC in fresh breast cancer surgical samples. Identified by antibody arrays, tumor-derived G-CSF significantly prolonged the lifespan and increased the metastasis-promoting activities of neutrophils via PI3K-AKT and NFκB pathway. Simultaneously, TAN-derived RLN2 promoted the migratory abilities of MCF-7 cells via PI3K-AKT-MMP-9. Analysis of tumor tissues from 20 BC patients and RNA-seq from TCGA BC patients identified a positive correlation of density of TANs with the activation of G-CSF-RLN2-MMP-9 axis. Our data indicate that TANs in human breast cancer have detrimental activity supporting malignant cells invasion and migration.