Abstract A11: Cancer cell-secreted CXCL1 chemokine acts on neutrophils to support metastasis

Innate immune cells such as macrophages, monocytes and neutrophils can be recruited to tumors by chemokines, which act on chemokines receptors on these cells. Macrophages and monocytes have well-established roles in promotion of metastasis. However, neutrophils have been reported to have both pro- and anti-metastatic effects. In this study, we used the metastatic 4T1 and non-metastatic 4T07 murine breast carcinoma cells lines (originating from the same tumor) to study effects of neutrophils in breast metastasis. Primary tumors from 4T1 cells, orthotopically injected into syngeneic mice, had more infiltrating neutrophils than did primary tumors from 4T07 cells. The increased numbers of neutrophils, which expressed the chemokine receptor CXCR2, was associated with significantly higher levels of cancer cell-secreted CXCL1, a chemokine ligand for CXCR2. To test whether cancer cell-secreted CXCL1 acted on neutrophils to regulate metastasis, we used shRNAs to reduce CXCL1 expression in the 4T1 cells. Consistent with the lack of CXCR2 expression by the cancer cells, this did not influence cancer cell proliferation or migration in vitro. However, in vivo, tumor infiltration of neutrophils and lung metastasis were both reduced. Moreover, when parental 4T1 cells were orthotopically injected into syngeneic Cxcr2 -/- mice, neutrophil infiltration and lung metastasis were significantly decreased as compared to tumors in wild type littermate. These results suggest that paracrine CXCL1 by cancer cells facilitates neutrophil infiltration via CXCR2 and that neutrophils in turn promote lung metastasis. To examine the mechanisms by which interactions between cancer cells and neutrophils might promote lung metastasis, we performed co-culture experiments with cancer cells and neutrophils. We found that neutrophils directly stimulated 4T1 cell invasion through matrigel. Interestingly, cancer-released CXCL1 also promoted the formation of neutrophil extraceullar traps (NETs), which is an expulsion of DNA and proteases from the neutrophils. Both invasion and NET formation were blocked by inhibition of phagocyte NADPH oxidase. Moreover, when mice with 4T1 tumors were treated with a phagocyte NADPH oxidase inhibitor, tumor growth and lung metastasis was reduced. Finally, decreased levels of the phosphorylated form of p47phox, a cytosolic component of phagocyte NAPDH oxidase that is activated by phosphorylation, was decreased in the tumor lysates from 4T1 injected Cxcr2 -/- mice as compared to Cxcr2 +/- mice. In conclusion, our study revealed that cancer cell-secreted CXCL1 regulates neutrophil infiltration and extracellular trap formation, and that phagocyte NADPH oxidase activity in these neutrophils promoted cancer cell invasion and metastasis. Citation Format: Juwon Park, Jing Qiu, Elizabeth S. Nakasone, Mikala Egeblad. Cancer cell-secreted CXCL1 chemokine acts on neutrophils to support metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A11.