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

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

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