Neutrophils comprise a crucial component of the innate immune system and rely on chemotaxis (directed migration) to track down, engulf, and destroy pathogens. A variety of tumor-produced chemokines also recruit neutrophils to sites of tumor growth where, through mechanisms that remain unclear, they exhibit both beneficial and detrimental effects on tumor clearance. Effective chemotaxis requires that neutrophils restrict activity of the GTPase Rac to a single location to properly form a leading edge. However, multiple GEFs function redundantly to activate Rac and it has been difficult to determine where within the signaling cascade each specific GEF is required. Here, we develop an optogenetic tool to drive PIP3 production, which itself is sufficient to activate Rac in human neutrophil-like HL-60 cells. In cells where we used CRISPR-mediated editing to knockout the Rac GEF PREX1, we find that optogenetic stimulation of PIP3 production fails to activate Rac. In contrast, when these PREX1null neutrophils are instead treated with chemoattractant, other GEFs that do not explicitly rely on PIP3 production can still activate Rac. While signaling cascades often feature multiple redundant pathways functioning in parallel, our optogenetics-based approach reveals the location within such a network where a particular factor is necessary for the transmission of information. A more nuanced understanding of chemotactic signaling in neutrophils should lead to the development of treatments that can more precisely modulate the interactions between neutrophils and the tumor microenvironment.
Citation Format: Brian R. Graziano, Delquin Gong, Karen E. Anderson, Orion D. Weiner. Optogenetics-based dissection of chemotactic signaling in neutrophils [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B096.
- ©2016 American Association for Cancer Research.