(A) GL261 cells were orthotopically implanted into the right hemisphere of syngeneic immunocompetent C57BL/6 mice. At 1 week post-intracranial (i.c.) injection whole brains were homogenized followed by Percoll gradient separation of leukocytes and non-leukocytes. qRT-PCR was performed for CCL2 in mice intracranially-injected with GL261 cells (n = 4) or PBS-injected controls (n = 3). (B) Detection of CCL2 protein in both brain hemispheres at 1 week-i.c. GL261 (n = 3). (C) Immunoreactivity for CCL2 was detected at 1 week post-GL261 injection. 20Ã- and 63Ã- magnification. Scale bar = 50 microm. (D) qRT-PCR for CCL1, CCL28, CCL2, CCL22, CCL17, and CCL20 from non-leukocytes (NL) and leukocytes (L) as in (A). Images in C are representative of 3 independent replicates. Data are representative of at least 2 independent experiments. Data are represented as mean {plus minus} SEM; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by one-way ANOVA with Tukey's multiple comparisons test (A, D) or Student's t test (B).
ARTICLE ABSTRACT
In many aggressive cancers, such as glioblastoma multiforme, progression is enabled by local immunosuppression driven by the accumulation of regulatory T cells (Treg) and myeloid-derived suppressor cells (MDSC). However, the mechanistic details of how Tregs and MDSCs are recruited in various tumors are not yet well understood. Here we report that macrophages and microglia within the glioma microenvironment produce CCL2, a chemokine that is critical for recruiting both CCR4+ Treg and CCR2+Ly-6C+ monocytic MDSCs in this disease setting. In murine gliomas, we established novel roles for tumor-derived CCL20 and osteoprotegerin in inducing CCL2 production from macrophages and microglia. Tumors grown in CCL2-deficient mice failed to maximally accrue Tregs and monocytic MDSCs. In mixed-bone marrow chimera assays, we found that CCR4-deficient Treg and CCR2-deficient monocytic MDSCs were defective in glioma accumulation. Furthermore, administration of a small-molecule antagonist of CCR4 improved median survival in the model. In clinical specimens of glioblastoma multiforme, elevated levels of CCL2 expression correlated with reduced overall survival of patients. Finally, we found that CD163-positive infiltrating macrophages were a major source of CCL2 in glioblastoma multiforme patients. Collectively, our findings show how glioma cells influence the tumor microenvironment to recruit potent effectors of immunosuppression that drive progression. Cancer Res; 76(19); 5671–82. ©2016 AACR.
