title: Intravital Förster resonance energy transfer imaging reveals osteopontin-mediated polymorphonuclear leukocyte activation by tumor cell emboli.
abstract: Myeloid-derived suppressor cells (MDSCs) cause paraneoplastic leukemoid reactions and facilitate tumor cell metastasis. However, the interaction of MDSCs with tumor cells in live tissue has not been adequately visualized. To accomplish this task, we developed an intravital imaging protocol to observe metastasized tumor cells in mouse lungs. For visualization of the activation of MDSCs, bone marrow cells derived from transgenic mice expressing a Förster resonance energy transfer biosensor for ERK were implanted into host mice. Under a two-photon excitation microscope, numerous polymorphonuclear cells (PMNs) were found to infiltrate the lungs of tumor-bearing mice in which 4T1 mammary tumor cells were implanted into the footpads. By Förster resonance energy transfer imaging, we found ERK activation in PMNs around the 4T1 tumor emboli in the lungs. Because antibody array analysis implied the involvement of osteopontin (OPN) in the metastasis of 4T1 cells, we further analyzed the effect of OPN knockdown. The OPN knockdown in 4T1 cells did not affect the cell growth, but markedly suppressed lung metastasis of 4T1 cells and ERK activation in PMNs in the lung. Intravenous injection of recombinant OPN restored the lung metastasis of OPN-deficient 4T1 cells, suggesting that OPN functioned in a paracrine manner. It has been reported that ERK activation of neutrophils causes NETosis and that PMNs promote metastasis of tumor cells by NETosis. In agreement with previous reports, the NETosis inhibitor DNase I inhibited lung metastasis of 4T1 cells. These observations suggest that OPN promotes metastasis of 4T1 cells by activating PMNs and inducing NETosis.
authors.affiliation: Department of Pathology and Biology of Diseases, Graduate School of Medicine, Kyoto University, Kyoto, Japan.|Department of Molecular Genetics, Institute of Biomedical Science, Kansai Medical University, Hirakata, Japan.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Kamioka
authors.affiliation: Imaging Platform for Spatio-Temporal Regulation, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
authors.forename: Kanako
authors.initials: K
authors.lastname: Takakura
authors.affiliation: Laboratory for Mouse Genetic Engineering, Quantitative Biology Center, RIKEN, Osaka, Japan.
authors.forename: Kenta
authors.initials: K
authors.lastname: Sumiyama
authors.affiliation: Department of Pathology and Biology of Diseases, Graduate School of Medicine, Kyoto University, Kyoto, Japan.|Laboratory of Bioimaging and Cell Signaling, Graduate School of Biostudies, Kyoto University, Kyoto, Japan.
authors.forename: Michiyuki
authors.initials: M
authors.lastname: Matsuda
mesh_terms: D000818:Animals; D015374:Biosensing Techniques / Q000379:methods; D016026:Bone Marrow Transplantation; D002465:Cell Movement; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D055785:Gene Knockdown Techniques; D015151:Immunoblotting; D008175:Lung Neoplasms / Q000097:blood / Q000517:prevention & control / Q000556:secondary*; D015674:Mammary Neoplasms, Animal / Q000473:pathology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008822:Mice, Transgenic; D008853:Microscopy / Q000379:methods; D009360:Neoplastic Cells, Circulating; D009504:Neutrophils / Q000502:physiology*; D053495:Osteopontin / Q000097:blood / Q000235:genetics / Q000502:physiology*; D018874:Spheroids, Cellular; D014407:Tumor Cells, Cultured; D059016:Tumor Microenvironment / Q000502:physiology
chemical_list: D053495:Osteopontin; D048049:Extracellular Signal-Regulated MAP Kinases
keywords: Cancer imaging; immune system imaging; immunosurveillance; tumor immunology; two-photon excitation microscopy