RNA Sequencing of Tumor-Associated Microglia Reveals Ccl5 as a Stromal Chemokine Critical for Neurofibromatosis-1 Glioma Growth

• Published in: Neoplasia (New York, N.Y.) N.Y.), 2015-10, Vol.17 (10), p.776-788
• Main Authors: Solga, Anne C, Pong, Winnie W, Kim, Keun-Young, Cimino, Patrick J, Toonen, Joseph A, Walker, Jason, Wylie, Todd, Magrini, Vincent, Griffith, Malachi, Griffith, Obi L, Ly, Amy, Ellisman, Mark H, Mardis, Elaine R, Gutmann, David H
• Format: Article
• Language: English
• Subjects: Adolescent; Animals; Astrocytes - metabolism; Astrocytes - pathology; Blotting, Western; Brain Neoplasms - genetics; Brain Neoplasms - metabolism; Brain Neoplasms - pathology; Chemokine CCL5 - genetics; Chemokine CCL5 - metabolism; Child; Child, Preschool; Embryo, Mammalian - cytology; Embryo, Mammalian - metabolism; Female; Flow Cytometry; Fluorescent Antibody Technique; High-Throughput Nucleotide Sequencing - methods; Humans; Immunoenzyme Techniques; In Situ Hybridization, Fluorescence; Infant; Infant, Newborn; Male; Mice; Mice, Inbred C57BL; Microglia - metabolism; Microglia - pathology; Neurofibromatosis 1 - genetics; Neurofibromatosis 1 - metabolism; Neurofibromatosis 1 - pathology; Oncology; Optic Nerve Glioma - genetics; Optic Nerve Glioma - metabolism; Optic Nerve Glioma - pathology; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger - genetics; Stromal Cells - metabolism; Stromal Cells - pathology; Tumor Cells, Cultured
• ISSN: 1476-5586; 1522-8002; 1476-5586; 1522-8002
• DOI: 10.1016/j.neo.2015.10.002

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.