Fluorescent Detection of Peroxynitrite Produced by Myeloid-Derived Suppressor Cells in Cancer and Inhibition by Dasatinib

Abstract

Myeloid-derived suppressor cells (MDSCs) are immature myeloid cells that expand dramatically in many cancer patients. This expansion contributes to immunosuppression in cancer and reduces the efficacy of immune-based cancer therapies. One mechanism of immunosuppression mediated by MDSCs involves production of the reactive nitrogen species peroxynitrite (PNT), where this strong oxidant inactivates immune effector cells through destructive nitration of tyrosine residues in immune signal transduction pathways. As an alternative to analysis of nitrotyrosines indirectly generated by PNT, we used an endoplasmic reticulum (ER)-targeted fluorescent sensor termed PS3 that allows direct detection of PNT produced by MDSCs. When the MDSC-like cell line MSC2 and primary MDSCs from mice and humans were treated with PS3 and antibody-opsonized TentaGel microspheres, phagocytosis of these beads led to production of PNT and generation of a highly fluorescent product. Using this method, we show that splenocytes from a EMT6 mouse model of cancer, but not normal control mice, produce high levels of PNT due to elevated numbers of granulocytic (PMN) MDSCs. Similarly, peripheral blood mononuclear cells (PBMCs) isolated from blood of human melanoma patients produced substantially higher levels of PNT than healthy human volunteers, coincident with higher peripheral MDSC levels. The kinase inhibitor dasatinib was found to potently block the production of PNT both by inhibiting phagocytosis in vitro and by reducing the number of granulocytic MDSCs in mice in vivo, providing a chemical tool to modulate the production of this reactive nitrogen species (RNS) in the tumor microenvironment.