title: RNA Sequencing of Tumor-Associated Microglia Reveals Ccl5 as a Stromal Chemokine Critical for Neurofibromatosis-1 Glioma Growth.
abstract: Solid cancers develop within a supportive microenvironment that promotes tumor formation and growth through the elaboration of mitogens and chemokines. Within these tumors, monocytes (macrophages and microglia) represent rich sources of these stromal factors. Leveraging a genetically engineered mouse model of neurofibromatosis type 1 (NF1) low-grade brain tumor (optic glioma), we have previously demonstrated that microglia are essential for glioma formation and maintenance. To identify potential tumor-associated microglial factors that support glioma growth (gliomagens), we initiated a comprehensive large-scale discovery effort using optimized RNA-sequencing methods focused specifically on glioma-associated microglia. Candidate microglial gliomagens were prioritized to identify potential secreted or membrane-bound proteins, which were next validated by quantitative real-time polymerase chain reaction as well as by RNA fluorescence in situ hybridization following minocycline-mediated microglial inactivation in vivo. Using these selection criteria, chemokine (C-C motif) ligand 5 (Ccl5) was identified as a chemokine highly expressed in genetically engineered Nf1 mouse optic gliomas relative to nonneoplastic optic nerves. As a candidate gliomagen, recombinant Ccl5 increased Nf1-deficient optic nerve astrocyte growth in vitro. Importantly, consistent with its critical role in maintaining tumor growth, treatment with Ccl5 neutralizing antibodies reduced Nf1 mouse optic glioma growth and improved retinal dysfunction in vivo. Collectively, these findings establish Ccl5 as an important microglial growth factor for low-grade glioma maintenance relevant to the development of future stroma-targeted brain tumor therapies.
authors.affiliation: Department of Neurology, Washington University School of Medicine, St. Louis, MO.
authors.forename: Anne C
authors.initials: AC
authors.lastname: Solga
authors.affiliation: Department of Neurology, Washington University School of Medicine, St. Louis, MO.
authors.forename: Winnie W
authors.initials: WW
authors.lastname: Pong
authors.affiliation: National Center for Microscopy and Imaging Research, University of California, San Diego, CA.
authors.forename: Keun-Young
authors.initials: KY
authors.lastname: Kim
authors.affiliation: Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO.
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Cimino
authors.affiliation: Department of Neurology, Washington University School of Medicine, St. Louis, MO.
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Toonen
authors.affiliation: The Genome Institute, Washington University School of Medicine, St. Louis, MO.
authors.forename: Jason
authors.initials: J
authors.lastname: Walker
authors.affiliation: The Genome Institute, Washington University School of Medicine, St. Louis, MO.
authors.forename: Todd
authors.initials: T
authors.lastname: Wylie
authors.affiliation: The Genome Institute, Washington University School of Medicine, St. Louis, MO.
authors.forename: Vincent
authors.initials: V
authors.lastname: Magrini
authors.affiliation: The Genome Institute, Washington University School of Medicine, St. Louis, MO.
authors.forename: Malachi
authors.initials: M
authors.lastname: Griffith
authors.affiliation: The Genome Institute, Washington University School of Medicine, St. Louis, MO.
authors.forename: Obi L
authors.initials: OL
authors.lastname: Griffith
authors.affiliation: The Genome Institute, Washington University School of Medicine, St. Louis, MO.
authors.forename: Amy
authors.initials: A
authors.lastname: Ly
authors.affiliation: National Center for Microscopy and Imaging Research, University of California, San Diego, CA.
authors.forename: Mark H
authors.initials: MH
authors.lastname: Ellisman
authors.affiliation: The Genome Institute, Washington University School of Medicine, St. Louis, MO.
authors.forename: Elaine R
authors.initials: ER
authors.lastname: Mardis
authors.affiliation: Department of Neurology, Washington University School of Medicine, St. Louis, MO. Electronic address: gutmannd@neuro.wustl.edu.
authors.forename: David H
authors.initials: DH
authors.lastname: Gutmann
mesh_terms: D000293:Adolescent; D000818:Animals; D001253:Astrocytes / Q000378:metabolism / Q000473:pathology*; D015153:Blotting, Western; D001932:Brain Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D018946:Chemokine CCL5 / Q000235:genetics / Q000378:metabolism*; D002648:Child; D002675:Child, Preschool; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism; D005260:Female; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D007124:Immunoenzyme Techniques; D017404:In Situ Hybridization, Fluorescence; D007223:Infant; D007231:Infant, Newborn; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D017628:Microglia / Q000378:metabolism / Q000473:pathology*; D009456:Neurofibromatosis 1 / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D020339:Optic Nerve Glioma / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017154:Stromal Cells / Q000378:metabolism / Q000473:pathology*; D014407:Tumor Cells, Cultured
chemical_list: C483024:CCL5 protein, human; D018946:Chemokine CCL5; D012333:RNA, Messenger