Triple-Negative Breast Cancer Cells Recruit Neutrophils by Secreting TGF-β and CXCR2 Ligands

Abstract

Tumor associated neutrophils (TANs) are frequently detected in triple-negative breast cancer (TNBC). Recent studies also reveal the importance of neutrophils in promoting tumor progression and metastasis during breast cancer. However, the mechanisms regulating neutrophil trafficking to breast tumors are less clear. We sought to determine whether neutrophil trafficking to breast tumors is determined directly by the malignant potential of cancer cells. We found that tumor conditioned media (TCM) harvested from highly aggressive, metastatic TNBC cells induced a polarized morphology and robust neutrophil migration, while TCM derived from poorly aggressive estrogen receptor positive (ER+) breast cancer cells had no activity. In a three-dimensional (3D) type-I collagen matrix, neutrophils migrated toward TCM from aggressive breast cancer cells with increased velocity and directionality. Moreover, in a neutrophil-tumor spheroid co-culture system, neutrophils migrated with increased directionality towards spheroids generated from TNBC cells compared to ER+ cells. Based on these findings, we next sought to characterize the active factors secreted by TNBC cell lines. We found that TCM-induced neutrophil migration is dependent on tumor-derived chemokines, and screening TCM elution fractions based on their ability to induce polarized neutrophil morphology revealed the molecular weight of the active factors to be around 12 kDa. TCM from TNBC cell lines contained copious amounts of GRO (CXCL1/2/3) chemokines and TGF-β cytokines compared to ER+ cell-derived TCM. TCM activity was inhibited by simultaneously blocking receptors specific to GRO chemokines and TGF-β, while the activity remained intact in the presence of either single receptor inhibitor. Together, our findings establish a direct link between the malignant potential of breast cancer cells and their ability to induce neutrophil migration. Our study also uncovers a novel coordinated function of TGF-β and GRO chemokines responsible for guiding neutrophil trafficking to the breast tumor.

Prognostic Relevance of Neutrophil to Lymphocyte Ratio (NLR) in Luminal Breast Cancer: A Retrospective Analysis in the Neoadjuvant Setting

“…The evidence that increased levels of neutrophils (both peripheral and in primary tumors) in triple-negative and HER2-positive tumors are associated with tumor progression and dismal prognosis is indicative of a prevalent N2 phenotype in these tumors. Consistently, overexpression of N2-promoting cytokines, TGF-β and G-CSF, as well as NET formation, have been frequently observed in TN breast cancer [69][70][71]. Due to the lack of information on neutrophil characterization in breast cancer subtypes in previous publications, we can try to explain the association between low neutrophils and poor prognosis in patients with luminal breast cancer assuming that the tumor microenvironment of luminal tumors may induce neutrophil polarization toward the pro-immunogenic N1 phenotype.…”

The Recruitment of Neutrophils to the Tumor Microenvironment Is Regulated by Multiple Mediators

“…G-CSF and GM-CSF are important growth factors commonly upregulated in cancer, and their primary functions are to regulate the release of mature neutrophils from the bone marrow into the blood and to extend the survival of neutrophils (38,39). In addition, cytokines, including interleukins (IL-17A, IL-6) (10,12,25,40), interferons (IFN-b) (9), TNF-a (25), and TGF-b (5,8,24,28), have been linked to neutrophil recruitment and extended neutrophil survival, as well as regulating neutrophil function. While a growing number of studies report the effect of individual mediators on neutrophil recruitment and function, it is likely that antagonistic, additive, or synergistic effects of different classes of mediators are crucial for neutrophil recruitment and function in the context of cancer.…”

“…Neutrophil navigation to the tumor niche could therefore be orchestrated by the interplay of different mediators. We recently reported that CXCR2 ligands, potentially growth-related oncogene (GRO) members (CXCL1/2/3), and TGF-b1, which are abundantly secreted by triple-negative breast cancer cells, concertedly induce robust neutrophil migration (24). TGF-b ligands belong to the TGF-b subfamily, with three known mammalian isoforms: TGF-b1, TGF-b2 and TGF-b3, of which TGF-b1 is the most commonly expressed.…”

CXCL1: Gene, Promoter, Regulation of Expression, mRNA Stability, Regulation of Activity in the Intercellular Space

“…Additionally, it causes the mobilization of these cells from the bone marrow [ 198 ]. These processes lead to the recruitment of TAN [ 199 , 200 ] and granulocytic, myeloid-derived suppressor cells (G-MDSC) [ 195 , 201 , 202 ] into the tumor niche. In doing so, this chemokine also increases the expansion of monocytic, myeloid-derived suppressor cells (Mo-MDSCs) in the bone marrow, which increases the number of these cells in the body, and thus in the tumor after the recruitment of these cells by other chemokines [ 203 ].…”