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: Anne C Solga, Winnie W Pong, Keun-Young Kim, Patrick J Cimino, Joseph A Toonen, Jason Walker, Todd Wylie, Vincent Magrini, Malachi Griffith, Obi L Griffith, Amy Ly, Mark H Ellisman, Elaine R Mardis, David H Gutmann
• Journal: Neoplasia (New York, N.Y.) (Neoplasia) Vol. 17 Issue 10 Pg. 776-88 (Oct 2015) ISSN: 1476-5586 [Electronic] United States
• PMID: 26585233 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Chemical References

• CCL5 protein, human
• Chemokine CCL5
• RNA, Messenger

Topics

• Adolescent
• Animals
• Astrocytes (metabolism, pathology)
• Blotting, Western
• Brain Neoplasms (genetics, metabolism, pathology)
• Chemokine CCL5 (genetics, metabolism)
• Child
• Child, Preschool
• Embryo, Mammalian (cytology, 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, pathology)
• Neurofibromatosis 1 (genetics, metabolism, pathology)
• Optic Nerve Glioma (genetics, metabolism, pathology)
• RNA, Messenger (genetics)
• Real-Time Polymerase Chain Reaction
• Reverse Transcriptase Polymerase Chain Reaction
• Stromal Cells (metabolism, pathology)
• Tumor Cells, Cultured