COX-2 blockade suppresses gliomagenesis by inhibiting myeloid-derived suppressor cells

Abstract

Epidemiologic studies have highlighted associations between the regular use of nonsteroidal anti-inflammatory drugs (NSAID) and reduced glioma risks in humans. Most NSAIDs function as COX-2 inhibitors that prevent production of prostaglandin E₂ (PGE₂). Because PGE₂ induces expansion of myeloid-derived suppressor cells (MDSC), we hypothesized that COX-2 blockade would suppress gliomagenesis by inhibiting MDSC development and accumulation in the tumor microenvironment (TME). In mouse models of glioma, treatment with the COX-2 inhibitors acetylsalicylic acid (ASA) or celecoxib inhibited systemic PGE₂ production and delayed glioma development. ASA treatment also reduced the MDSC-attracting chemokine CCL2 (C-C motif ligand 2) in the TME along with numbers of CD11b(+)Ly6G(hi)Ly6C(lo) granulocytic MDSCs in both the bone marrow and the TME. In support of this evidence that COX-2 blockade blocked systemic development of MDSCs and their CCL2-mediated accumulation in the TME, there were defects in these processes in glioma-bearing Cox2-deficient and Ccl2-deficient mice. Conversely, these mice or ASA-treated wild-type mice displayed enhanced expression of CXCL10 (C-X-C motif chemokine 10) and infiltration of cytotoxic T lymphocytes (CTL) in the TME, consistent with a relief of MDSC-mediated immunosuppression. Antibody-mediated depletion of MDSCs delayed glioma growth in association with an increase in CXCL10 and CTLs in the TME, underscoring a critical role for MDSCs in glioma development. Finally, Cxcl10-deficient mice exhibited reduced CTL infiltration of tumors, establishing that CXCL10 limited this pathway of immunosuppression. Taken together, our findings show that the COX-2 pathway promotes gliomagenesis by directly supporting systemic development of MDSCs and their accumulation in the TME, where they limit CTL infiltration.

Figure 1. Effects of NSAIDs on glioma development

Gliomas were induced in neonatal C57BL/6 mice by intraventricular transfection of the following plasmids: pT2/C-Luc//PGK-SB1.3 (0.2 μg), pT/CAG-NRas (0.4 μg), and pT/shp53 (0.4 μg). A, glioma cell lines were established from WT mice and treated in vitro with the following NSAIDs at indicated concentrations: ASA (left), celecoxib (center), and dimethylcelecoxib (right). The cells were incubated for 24 h, and WST-1 assay was performed for cell viability. Untreated cells were used as a control. B, supernatants were collected from A to perform ELISA for PGE₂ levels. C, mice with developing tumors received daily treatment of ASA (left and center) or celecoxib (right) with indicated doses by oral gavage or through drinking water initiated on indicated days. Symptom-free survival was monitored. P-values are based on log-rank test. “Med” in the tables stands for median survival. D, plasma samples were collected from these mice at days 50 to 60 to perform ELISA for systemic PGE₂ levels. Numbers in the “ASA” section represent the days the treatment was initiated. A, B, and D, lines within boxes denote means; box upper and lower bounds indicate SD; whiskers indicate minimum and maximum values. P-values are based on Holm’s post hoc test.

Figure 2. Effects of ASA on glioma microenvironment

Gliomas were induced and treated with ASA or control PBS initiated on day 0 in WT mice as described in Fig. 1. The mice were sacrificed when similar tumor size was observed by BLI at days around 50 to 60. A, indicated organs were collected from each mouse (3 mice/group), and total RNA was extracted to perform quantitative RT-PCR for mRNA expression levels of Ccl2 (top) and Cxcl10 (bottom). B, leukocytes were isolated from each organ to perform flow cytometry for Ly6G^hiLy6C^lo granulocytic MDSCs (gMDSCs) and Ly6G⁻Ly6C^hi monocytic MDSCs (mMDSCs). Representative flow data (left) and cumulative enumerations from multiple experiments (right) are shown. Numbers in the left panels indicate percentage of gated subpopulations in leukocyte-gated populations. C, both MDSC subpopulations were sorted, and total RNA was extracted to perform quantitative RT-PCR for mRNA expression levels of Nos2. D, the brain-infiltrating leukocytes (BILs) isolated in B were analyzed by flow cytometry for CD8⁺CD107a⁺ cells. Representative flow data (left) and cumulative enumerations for CD8⁺ cells from multiple experiments (right) are shown. Numbers in dot plots indicate percentage of gated subpopulations in leukocyte-gated populations and MFI of CD107a on CD8-gated subpopulation.

Figure 3. Deletion of Cox-2 alleles leads to similar effects to those by the ASA treatment

Gliomas were induced in C57BL/6-background Cox-2^−/−, Cox-2^+/−, and Cox-2^+/+ mice. A, symptom-free survival was monitored. B, plasma samples were collected as described in Fig. 1 to perform ELISA for systemic PGE₂ levels. C, total RNA was extracted from the mouse brains as described in Fig. 2 to perform quantitative RT-PCR for mRNA expression levels of Ccl2 (left) and Cxcl10 (right). D, BILs were isolated to perform flow cytometry for subpopulations of CD11b⁺Gr-1⁺ (upper) and CD8⁺CD107a⁺ (lower) cells. Representative flow data (left) and cumulative enumerations from multiple experiments (right) are shown. Numbers in dot plots indicate percentage of gated subpopulations in leukocyte-gated populations; MFI of CD107a on CD8-gated subpopulation is also indicated in the bottom panels.

Figure 4. Endogenous CCL2 promotes glioma development and tumor-infiltration of MDSCs

Gliomas were induced in C57BL/6-background Ccl2-deficient or WT mice. A, symptom-free survival was monitored. B, total RNA was extracted from the mouse brains with or without gliomas to perform quantitative RT-PCR for Cxcl10 mRNA expression levels. C, BILs were isolated to perform flow cytometry for subpopulations of CD11b⁺Gr-1⁺ (upper) and CD8⁺CD107a⁺ (lower) cells. Representative flow data (left) and cumulative enumerations from multiple experiments (right) are shown. Numbers in dot plots indicate percentage of gated subpopulations in leukocyte-gated populations; MFI of CD107a on CD8-gated subpopulation is also indicated in the bottom panels.

Figure 5. mAb-mediated depletion of Gr-1⁺ cells inhibits the glioma development

Gliomas were induced in C57BL/6 WT mice. The mice with developing gliomas received i.p. injections of anti-Gr-1 mAb (RB6-8C5; 0.25 mg/dose) or control IgG on days 21, 23, 25, and 27 after tumor induction. A, symptom-free survival was monitored. B, total RNA was extracted from the mouse brains to perform quantitative RT-PCR for Cxcl10 mRNA expression levels. C, BILs were isolated to perform flow cytometry for subpopulations of CD11b⁺Gr-1⁺ (upper) and CD8⁺ (lower) cells. Representative flow data (left) and cumulative enumerations from multiple experiments (right) are shown. Numbers in dot plots indicate percentage of gated subpopulations in leukocyte-gated populations; MFI of CD107a on CD8-gated subpopulation is indicated in the bottom panels.

Figure 6. Endogenous CXCL10 inhibits glioma development and promotes tumor-infiltration of CD8⁺ T-cells

Gliomas were induced in C57BL/6-background Cxcl10-deficient or WT mice. A, symptom-free survival was monitored. B and C, BILs were isolated to perform flow cytometric evaluation of the following molecules on CD8⁺ subpopulations: CXCR3 (B) and CD107a (C). C, representative flow data (left) and cumulative enumerations from multiple experiments (right) are shown. The left panels also indicate percentage of gated subpopulations in leukocyte-gated populations and MFI of CD107a on CD8-gated subpopulation.