TGF-ß1 activates neutrophil signaling and gene expression but not migration.

Tumor-associated neutrophils are found in many types of cancer and are often reported to contribute to negative outcomes. The presence of transforming growth factor-beta (TGF-ß) in the tumor microenvironment reportedly contributes to the skewing of neutrophils to a more pro-tumor phenotype. The effects of TGF-ß on neutrophil signaling and migration are, however, unclear. We sought to characterize TGF-ß signaling in both primary human neutrophils and the neutrophil-like cell line HL-60 and determine whether it directly induces neutrophil migration. We found that TGF-ß1 does not induce neutrophil chemotaxis in transwell or underagarose migration assays. TGF-ß1 does activate canonical signaling through SMAD3 and noncanonical signaling through ERK1/2 in neutrophils in a time- and dose-dependent manner. Additionally, TGF-ß1 present in the tumor-conditioned media (TCM) of invasive breast cancer cells results in SMAD3 activation. We discovered that TCM induces neutrophils to secrete leukotriene B4 (LTB4), which is a lipid mediator important for amplifying the range of neutrophil recruitment. However, TGF-ß1 alone does not induce secretion of LTB4. RNA-sequencing revealed that TGF-ß1 and TCM alter gene expression in HL-60 cells, including the mRNA levels of the pro-tumor oncostatin M (OSM) and vascular endothelial growth factor A (VEGFA). These new insights into the role and impact of TGF-ß1 on neutrophil signaling, migration, and gene expression have significant implications in the understanding of the changes in neutrophils that occur in the tumor microenvironment.

TGF-ß1 activates neutrophil signaling and gene expression but not migration.

Tumor-associated neutrophils are found in many types of cancer and are often reported to contribute to negative outcomes. The presence of transforming growth factor-beta (TGF-ß) in the tumor microenvironment reportedly contributes to the skewing of neutrophils to a more pro-tumor phenotype. The effects of TGF-ß on neutrophil signaling and migration are, however, unclear. We sought to characterize TGF-ß signaling in both primary human neutrophils and the neutrophil-like cell line HL-60 and determine whether it directly induces neutrophil migration. We found that TGF-ß1 does not induce neutrophil chemotaxis in transwell or underagarose migration assays. TGF-ß1 does activate canonical signaling through SMAD3 and noncanonical signaling through ERK1/2 in neutrophils in a time-and dose-dependent manner. Additionally, TGF-ß1 present in the tumor-conditioned media (TCM) of invasive breast cancer cells results in SMAD3 activation. We discovered that TCM induces neutrophils to secrete leukotriene B 4 (LTB 4 ), which is a lipid mediator important for amplifying the range of neutrophil recruitment. However, TGF-ß1 alone does not induce secretion of LTB 4 . RNA-sequencing revealed that TGF-ß1 and TCM alter gene expression in HL-60 cells, including the mRNA levels of the pro-tumor oncostatin M ( OSM ) and vascular endothelial growth factor A ( VEGFA ). These new insights into the role and impact of TGF-ß1 on neutrophil signaling, migration, and gene expression have significant implications in the understanding of the changes in neutrophils that occur in the tumor microenvironment.

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

Neutrophils sense and migrate towards chemotactic factors released at sites of infection/inflammation and contain the affected area using a variety of effector mechanisms. Aside from these established immune defense functions, neutrophils are emerging as one of the key tumor-infiltrating immune cells that influence cancer progression and metastasis. Neutrophil recruitment to the tumor microenvironment (TME) is mediated by multiple mediators including cytokines, chemokines, lipids, and growth factors that are secreted from cancer cells and cancer-associated stromal cells. However, the molecular mechanisms that underlie the expression and secretion of the different mediators from cancer cells and how neutrophils integrate these signals to reach and invade tumors remain unclear. Here, we discuss the possible role of the epithelial to mesenchymal transition (EMT) program, which is a well-established promoter of malignant potential in cancer, in regulating the expression and secretion of these key mediators. We also summarize and review our current understanding of the machineries that potentially control the secretion of the mediators from cancer cells, including the exocytic trafficking pathways, secretory autophagy, and extracellular vesicle-mediated secretion. We further reflect on possible mechanisms by which different mediators collaborate by integrating their signaling network, and particularly focus on TGF-ß, a cytokine that is highly expressed in invasive tumors, and CXCR2 ligands, which are crucial neutrophil recruiting chemokines. Finally, we highlight gaps in the field and the need to expand current knowledge of the secretory machineries and cross-talks among mediators to develop novel neutrophil targeting strategies as effective therapeutic options in the treatment of cancer.

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

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-ß cytokines compared to ER+ cell-derived TCM. TCM activity was inhibited by simultaneously blocking receptors specific to GRO chemokines and TGF-ß, 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-ß and GRO chemokines responsible for guiding neutrophil trafficking to the breast tumor.