TGF-ß1 activates neutrophil signaling and transcription but not migration
Tumor-associated neutrophils are found in many types of cancer and are often reported to contribute to negative outcomes. Several studies have shown that the presence of TGF-ß in the tumor microenvironment contributes to the skewing of neutrophils to have a more pro-tumor phenotype. However, the direct effects of TGF-ß on neutrophil signaling and migration are unclear. We sought to characterize TGF-ß signaling in both primary human neutrophils and the neutrophil-like cell line HL-60 and determine whether TGF-ß directly induces neutrophil migration. We found that TGF-ß1 does not induce neutrophil migration in either a transwell or an underagarose migration assay. However, TGF-ß1 does activate signals canonically through SMAD3 and noncanonically through ERK1/2 in neutrophils in a time and dose-dependent manner. Additionally, TGF-ß1 present in the tumor-conditioned media (TCM) is responsible for SMAD3 activation. Moreover, we discovered that TCM from aggressive breast cancer cells induces neutrophils to secrete leukotriene B4 (LTB4), which is a lipid mediator important for amplifying neutrophil recruitment. However, we found that TGF-ß1 alone does not induce secretion of LTB4. We next performed RNA-sequencing to evaluate the effects of TGF-ß1 and TCM on the neutrophil transcriptome. We found that TGF-ß1 and TCM result in changes in gene transcription in HL-60 cells, specifically of two pro-tumor genes OSM and VEGFA. Together, our findings characterize the effects of TGF-ß1 on neutrophil signaling, migration, and gene expression that can be applied to understanding the changes in neutrophils that occur in the tumor microenvironment. Overall design: We performed RNA-seq analysis using 2 cell lines, 2 time points, and 2 treatments each with their own media control