Characteristics of the Alternative Phenotype of Microglia/Macrophages and its Modulation in Experimental Gliomas

Microglia (brain resident macrophages) accumulate in malignant gliomas and instead of initiating the anti-tumor response, they switch to a pro-invasive phenotype, support tumor growth, invasion, angiogenesis and immunosuppression by release of cytokines/chemokines and extracellular matrix proteases. Using immunofluorescence and flow cytometry, we demonstrate an early accumulation of activated microglia followed by accumulation of macrophages in experimental murine EGFP-GL261 gliomas. Those cells acquire the alternative phenotype, as evidenced by evaluation of the production of ten pro/anti-inflammatory cytokines and expression profiling of 28 genes in magnetically-sorted CD11b+ cells from tumor tissues. Furthermore, we show that infiltration of implanted gliomas by amoeboid, Iba1-positive cells can be reduced by a systematically injected cyclosporine A (CsA) two or eight days after cell inoculation. The up-regulated levels of IL-10 and GM-CSF, increased expression of genes characteristic for the alternative and pro-invasive phenotype (arg-1, mt1-mmp, cxcl14) in glioma-derived CD11b+ cells as well as enhanced angiogenesis and tumor growth were reduced in CsA-treated mice. Our findings define for the first time kinetics and biochemical characteristics of glioma-infiltrating microglia/macrophages. Inhibition of the alternative activation of tumor-infiltrating macrophages significantly reduced tumor growth. Thus, blockade of microglia/macrophage infiltration and their pro-invasive functions could be a novel therapeutic strategy in malignant gliomas

Quantification of selected M1/M2 phenotype-associated gene expression in CD11b⁺ cells isolated from naïve and tumor-bearing mice.

<p>Gene expression was analyzed by real-time PCR and the results are presented as fold changes of CD11b⁺ cells isolated from tumor brains versus those from naïve brain tissue. Numbers corresponding to the significantly changed genes (t-test generated p-value<0.05) are marked in bold; NA - not available.</p

Influx of microglia/macrophages into the tumor is blocked by CsA.

<p>A. Representative confocal images of Iba1 staining in intact brain tissue, tumor-bearing brain slices from mice treated with PBS or CsA. Scale bar = 20 µm. B–C. Quantification of microglia and blood-derived macrophages in naïve, tumor-bearing and CsA-treated mice (4 per group). Each bar represents the mean ± SEM. ^***<i>p</i><0.001, ^**<i>p</i><0.01 tumor-bearing versus naïve mice; ^##<i>p</i><0.01, CsA-treated versus PBS-treated tumor-bearing mice.</p

Accumulation and activation of microglia/macrophages in experimental glioma.

<p>A. Representative confocal images of brain sections 15 days after implantation of pEGFP-N1 GL261 cells into the striatum of C57BL/6 mice. Note the infiltration and morphological transformation of glioma-infiltrating Iba1⁺ cells. Scale bar: left image – 1000 µm, right image – 20 µm. B. Contralateral and ipsilateral hemisphere from tumor-bearing brain 15 days after injection of pEGFP-N1 GL261 cells. Images showed merged Iba1 and EGFP fluorescence. Scale bar = 750 µm. C. Microglia/macrophages were separated using a magnetic-bead-conjugated anti-CD11b antibody and stained with CD45 PerCP-Cy5.5 and CD11b PE prior to FACS acquisition. Propidium iodide staining was performed to eliminate necrotic/apoptotic cells (Gate R3, R4) and viable cells were gated (Gate R1; <b>B1</b>, Gate R2; <b>B2</b>). D. Efficiency of CD11b-positive cells separation in the negative fraction (CD11b-negative cells) from each sample was controlled. E. Representative dot plots for microglia (Gate R4, CD11b⁺/CD45^low) and macrophages (Gate R5, CD11b⁺/CD45^high) from tumor-bearing hemispheres. F. Kinetics of microglia/macrophage influx into tumor tissue. CD11b⁺ cells separated from the brains of naïve, sham operated and tumor-bearing mice at day 3, 8 or 15 after implantation (n = 4/group) were sorted according to CD45 expression. Each bar represents the mean ± SEM; ^***<i>p</i><0.001, ^*<i>p</i><0.05 tumor-bearing mice at 8th day versus naïve mice; ^##<i>p</i><0.01 tumor-bearing mice at day 15 versus day 8.</p

Cytokine profile in glioma-bearing brains.

<p>A. The levels of ten pro/anti-inflammatory cytokines were determined by flow cytometry in protein extracts isolated from hemispheres of naïve, LPS-injected and tumor-bearing mice. The results show the means ± SEM (n = 5 per group); ^#<i>p</i><0.05 significant change between LPS-treated v. naïve mice; ^*<i>p</i><0.05 significant difference between tumor-bearing v. naïve mice. B. Elevated levels of IL-10 and GM-CSF detected in tumors are reduced by CsA treatment. Each dot represents an individual animal; a horizontal line represents a mean of each group; ^*<i>p</i><0.05; ^**<i>p</i><0.01. C. Expression of IL-10 in glioma-infiltrating microglia and macrophages. Left panel: expression of IL-10 on sorted CD11b⁺ cells (50,000 cells) determined by flow cytometry with the anti-IL-10 antibody conjugated to Alexa Fluor647. Representative histograms of IL-10 detection in microglia (light grey) and macrophages (dark grey) cells from naïve (a) and tumor-bearing (b) brains. Right panel: quantification of microglia/macrophages expressing IL-10 in naïve and tumor-bearing brains (means ± SEM, 3 animals per group); significant increase of IL-10-positive microglia (** <i>p</i><0.01) and macrophages (^###<i>p</i><0.001) in tumor-bearing brains. D. Quantification of <i>gm-csf</i> and <i>m-csf</i> expression in GL261 glioma cells and non-transformed astrocytes (means ± SD from 3 experiments). E. Quantitative evaluation of <i>gm-csf</i> mRNA expression in GL261 glioma cells exposed to 0.1 and 1 µM CsA for 24 hours (means ± SD from two experiments with three or four replicates per condition) compared to untreated control cells. Statistical analysis was done by Student <i>t</i> test, ** <i>p</i><0.01.</p

Alterations of gene expression in infiltrating microglia/macrophages and intracranial gliomas are modulated by CsA.

<p>A. Gene expression in magnetically sorted CD11b⁺ cells from tumor-bearing and naïve brains was determined by qPCR. Expression of five genes was significantly altered in CD11b⁺ cells: <i>arg-1 (p = 0.000003)</i>; <i>cxcl14 (p = 0.0001)</i>; <i>ifn-β1 (p = 0.0002)</i>; <i>cox-2 (p = 0.000002)</i>; <i>mt1-mmp (p = 0.00002)</i>; n = 5 animals per group; ^*<i>p</i><0.05, ^**<i>p</i><0.01. The middle line represents the median value. Lower ΔC_T are consistent with higher gene expression. B. Quantification of arginase activity in brain tissue extracts from naïve and tumor-bearing mice treated either with PBS or CsA. Results represent the mean ± SEM of 4–5 mice; ^*<i>p</i><0.05, tumor-bearing versus tumor-free hemispheres; ^#<i>p</i><0.05, CsA (10 mg/kg, 8th) versus PBS-treated, tumor-bearing mice. C. MMP-2 activity in proteins extracts from the brains of naïve (N1–5) and tumor-bearing mice (T1–5) determined by gel zymography. Active MMP-2 detected as a prominent band at 62 kDa. D. Quantification of MMP-2 activity using the cleavage of fluorescent DQ-gelatin substrate; means ± SEM of 4–6 mice; ^**<i>p</i><0.01, tumor-bearing versus naïve brain extracts; ^###<i>p</i><0.001, ^#<i>p</i><0.05, CsA- versus PBS-treated tumor-bearing mice.</p