RNA-sequencing of tumor-associated microglia reveals Ccl5 as a stromal chemokine critical for neurofibromatosis-1 glioma growth

Solid cancers develop within a supportive microenvironment that promotes tumor formation and continued 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), previous studies have demonstrated that microglia are important for glioma formation and maintenance. To identify the tumor-associated microglial factors that support glioma growth (gliomagens), we employed a comprehensive large scale discovery effort using optimized advanced RNA-sequencing methods. Candidate gliomagens were prioritized to identify potential secreted or membrane-bound proteins, which were next validated by quantitative RT-PCR and RNA FISH following minocycline-mediated microglial inactivation in vivo. Using these selection criteria, Ccl5 was identified as a highly expressed chemokine in both genetically engineered Nf1 mouse and human optic gliomas. 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, Ccl5 inhibition with neutralizing antibodies reduced Nf1 mouse optic glioma growth in vivo. Collectively, these findings establish Ccl5 as critical stromal growth factor in low-grade glioma maintenance relevant to future microglia-targeted therapies for brain tumors. Overall design: Nf1 optic glioma associated microglia from mice were flow sorted. Upregulated genes of glioma associated microglia were verified and further examined.

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Solga AC, Pong WW, Kim KY, Cimino PJ, Toonen JA, Walker J, Wylie T, Magrini V, Griffith M, Griffith OL, Ly A, Ellisman MH, Mardis ER, Gutmann DH