Figure 1.
Accumulation and activation of microglia/macrophages in experimental glioma.
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; B1, Gate R2; B2). 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+/CD45low) and macrophages (Gate R5, CD11b+/CD45high) 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; ***p<0.001, *p<0.05 tumor-bearing mice at 8th day versus naïve mice; ##p<0.01 tumor-bearing mice at day 15 versus day 8.
Figure 2.
Influx of microglia/macrophages into the tumor is blocked by CsA.
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. ***p<0.001, **p<0.01 tumor-bearing versus naïve mice; ##p<0.01, CsA-treated versus PBS-treated tumor-bearing mice.
Figure 3.
CsA induces cell death within tumor-associated microglia/macrophages.
A. Quantification of the number of TUNEL-positive cells per 1 mm2 of tumor area in saline and CsA-treated mice was counted using Image Pro-Plus software. The results show the median of 5–6 mice in each group. A statistical significance was determined by U-Mann-Whitney test; **p<0.01. B. Detection of DNA fragmentation in situ in EGFP-N1 GL261 tumour bearing brain section visualised with TUNEL staining (red fluorescence) by confocal microscopy. Scale bar = 20 µm. C. Identification of TUNEL-positive cells (red) in tumour-bearing slices. Sections were subjected to TUNEL reaction and stained for Iba1 (microglia/macrophages). Images were acquired using confocal microscopy and Z-stack projection was performed. Representative Z-stack images (1–4) of saline- and CsA-treated mice (10 mg/kg, postponed treatment) demonstrate a co-localization of TUNEL-positive cells (red) and Iba1-positive cells (blue). Scale bar = 20 µm. D. Quantification of Iba+/TUNEL+ cells among TUNEL-positive cells. The results show the mean ± SD (4–6 mice per group); **p<0.01 significant change between postponed (10 mg/kg) and early treated mice (10 mg/kg).
Figure 4.
Cytokine profile in glioma-bearing brains.
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); #p<0.05 significant change between LPS-treated v. naïve mice; *p<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; *p<0.05; **p<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 (** p<0.01) and macrophages (### p<0.001) in tumor-bearing brains. D. Quantification of gm-csf and m-csf expression in GL261 glioma cells and non-transformed astrocytes (means ± SD from 3 experiments). E. Quantitative evaluation of gm-csf 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 t test, ** p<0.01.
Figure 5.
Alterations of gene expression in infiltrating microglia/macrophages and intracranial gliomas are modulated by CsA.
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: arg-1 (p = 0.000003); cxcl14 (p = 0.0001); ifn-β1 (p = 0.0002); cox-2 (p = 0.000002); mt1-mmp (p = 0.00002); n = 5 animals per group; *p<0.05, **p<0.01. The middle line represents the median value. Lower ΔCT 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; *p<0.05, tumor-bearing versus tumor-free hemispheres; #p<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; **p<0.01, tumor-bearing versus naïve brain extracts; ###p<0.001, #p<0.05, CsA- versus PBS-treated tumor-bearing mice.
Table 1.
Quantification of selected M1/M2 phenotype-associated gene expression in CD11b+ cells isolated from naïve and tumor-bearing mice.
Figure 6.
Reduction of tumor growth in CsA-treated animals.
A. Scheme of experiment. B. Representative images of EGFP-GL261 tumors in mice treated with PBS or 10 mg/kg CsA, 2nd; scale bar = 1 mm. C–D. Quantification of tumor volumes in PBS, CsA-treated or FK506-treated mice after early (C) or postponed (D) treatment. Results for individual animals are presented; the bold line represents the median of 5–10 mice in each group. **p<0.01, ***p<0.001, tumor volumes in drug-treated versus control mice.