Microenvironmental Cues Determine Tumor Cell Susceptibility to Neutrophil Cytotoxicity

Introduction: In cancer, neutrophils are not homogenous population and they feature both pro and anti-tumor properties. We have recently shown that neutrophil anti-tumor cytotoxicity is Ca²⁺-dependent and is mediated by TRPM2, an H₂O₂-dependent Ca²⁺ channel. Although tumor cells at the primary site express TRPM2 they are not targeted by cytotoxic neutrophils. In contrast, disseminated tumor cells are efficiently targeted and eliminated by neutrophils, which limit metastatic seeding. This has led us to question whether disseminated tumor cells are more susceptible to neutrophil cytotoxicity than cells at the primary site and whether Epithelial-to-Mesenchymal Transition (EMT) and Mesenchymal-to-Epithelial Transition (MET), which may be critical for primary tumor cells dissemination, seeding and metastasis formation, play a role in this process. Material and methods: using in vitro killing assay and iv vivo circulating tumor cells seeding assay, we determine the influence of EMT and MET on the fate of tumor cells susceptibility to neutrophils cytotoxicity and seeding capacity. Results and discussion: To examine our hypothesis we compared the expression levels of TRPM2 in primary and disseminated circulating 4T1 tumor cells (CTC). We found that CTC have higher levels of TRPM2, which accompanies their more mesenchymal gene expression profile. In line with this result, we found that TRPM2 expression on tumors cells is modulated by EMT or MET driving factors. Specifically, expression of EMT diving factors such as TGFβ, TWIST and Snail upregulate TRPM2 levels. High levels of TRPM2 exposed tumor cells to neutrophil cytotoxicity, decrease survivability and inhibits seeding capacity in the pre-metastatic tissue. Vice versa, cells undergoing MET using factors such as BMP7 and Klf4, express reduced levels of TRPM2, rendering them resistant to neutrophils cytotoxicity. Cells expressing reduced levels of TRPM2 are protected from neutrophil and seed more efficiently in the premetastatic lung. The reduced susceptibility to neutrophils in cells expressing reduced levels of TRPM2 is reflected by a higher metastatic potential. This might be explained by the important anti-metastatic role neutrophils play in the pre-metastatic lung. Conclusion: These data identify TRPM2 as the link between environmental cues at the primary or the pre-metastatic tumor sites, tumor cell susceptibility to neutrophil cytotoxicity and disease progression. Furthermore, these data identify EMT as a process enhancing tumor-cell immune susceptibility and in contrast, MET as a novel mode of immune evasion.