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. Nf1 optic glioma associated microglia from mice were flow sorted. Upregulated genes of glioma associated microglia were verified and further examined.

实体瘤发生于支持性微环境中，该微环境通过分泌有丝分裂原与趋化因子，促进肿瘤形成并维持其持续生长。在这类肿瘤中，单核细胞（包括巨噬细胞与小胶质细胞）是这类基质因子的重要来源。依托1型神经纤维瘤病（NF1）低级别脑肿瘤（视神经胶质瘤）的基因工程小鼠模型，既往研究已证实小胶质细胞对胶质瘤的发生与维持具有关键作用。为筛选可支持胶质瘤生长的肿瘤相关小胶质细胞因子（胶质瘤促生因子，gliomagens），我们采用优化的高级RNA测序技术开展了大规模系统性筛选研究。我们优先筛选候选胶质瘤促生因子，以识别潜在的分泌型或膜结合型蛋白；随后通过体内米诺环素介导的小胶质细胞失活模型，采用定量RT-PCR与RNA荧光原位杂交（RNA FISH）对候选因子进行验证。基于上述筛选标准，我们在基因工程Nf1小鼠与人视神经胶质瘤中均鉴定出高表达的趋化因子Ccl5。作为候选胶质瘤促生因子，重组Ccl5在体外可促进Nf1缺陷型视神经星形胶质细胞的增殖。尤为重要的是，其在维持肿瘤生长中发挥关键作用，使用中和抗体抑制Ccl5可在体内降低Nf1小鼠视神经胶质瘤的生长速度。综上，本研究证实Ccl5是低级别胶质瘤维持过程中的关键基质生长因子，可为未来脑肿瘤的小胶质细胞靶向治疗提供潜在参考。本研究对小鼠来源的Nf1型视神经胶质瘤相关小胶质细胞进行了流式分选，并对胶质瘤相关小胶质细胞的差异上调基因进行了验证与深入分析。