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.
Highlights
Neutrophils are increasingly recognized as critical tumorinfiltrating immune cells [1]
CXCL members CXCL8 and CXCL1/2 have been implicated in driving neutrophil recruitment in breast cancer [49,50,51,52], and overexpression of these chemokines is associated with breast tumor samples and breast cancer cell lines that have negative estrogen receptor expression status and higher aggressiveness [50, 53,54,55]
Out of 12 CXCL chemokines, we found that the expression of CXCL8, CXCL1, and CXCL2 transcripts was significantly elevated in Estrogen receptor (ER)- breast cancer specimens compared to ER+ samples in seven, four and three independent studies, respectively (Figure S1C)
Summary
Neutrophils are increasingly recognized as critical tumorinfiltrating immune cells [1]. Upon arrival to the infected/injured sites, the primary functions of neutrophils are to eliminate invading pathogens and mount an inflammatory response using diverse mechanisms, ranging from phagocytosis and respiratory burst, to the release of toxic granule contents and DNA-containing neutrophil extracellular traps (NETs) [2]. Apart from these defense functions, neutrophils have gained attention for their newly discovered role at either promoting [3,4,5] or hindering [6,7,8] tumor progression, depending on the types of cancer. Cancer-associated guidance cues that drive neutrophil migration to the breast tumor niche are not well defined, partly because of the molecular heterogeneity among different breast cancer subtypes [18]
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