Fig 1: CSF2 knockdown reduces microglia-dependent invasion of human glioblastoma cells.a–c Cell invasion was determined using matrigel invasion test. Same number of shNeg or shCSF2 LN18 and U87 glioma cells were plated on matrigel-covered inserts with or without BV2 microglia cells in the lower compartments. Cells migrating through the matrigel were stained and counted with laser scanning cytometry (representative histograms are shown in a) or quantified manually in five fields (b, c). d Invasion of shNeg or shCSF2 glioma cells in the absence or presence of human SV40 immortalised microglial cells (HMSV40) was determined. Data are presented as means ± s.d.; statistical significance was analysed by ANOVA and marked as *shNeg versus shCSF2, *p < 0.05; # in the absence versus presence of microglia, #p < 0.05, ##p < 0.01). e Schematic representation of tested conditions. f The effect of antibodies against CSF2 or CSF2Ra on the invasion of U87 glioma cells co-cultured without or with HMSV40 or BV2 microglia in the lower compartment.
Fig 2: CSF2 expression in gliomas.a Boxplots represent CSF2 FPKM values in normal brain samples (NB), low-grade gliomas (WHO grade II and III) and GBM (glioblastoma, WHO grade IV) TCGA datasets. b Boxplots represent CSF2 FPKM values in GBM samples with division into molecular subtypes.31 c Genes overexpressed in CSF2 expressing tumours (FDR p-value < 0.05 and FC > 3) were an input for GO enrichment. Dotplot shows the most enriched GO groups in CSF2 expressing GBMs, i.e. related to leukocyte activation, immune and defence responses. d Quantification of CSF2 expression using qPCR in healthy brain samples, Jurkat cells and glioma cell lines. e CSF2 protein levels in cells culture supernatants from normal human astrocytes and human glioma cell lines were determined using ELISA. Data are presented as means± from three independent experiments, performed in triplicates.
Fig 3: Development and characterisation of CSF2-depleted glioma cells.a–d Quantification of CSF2 mRNA (a, c) and protein (b, d) levels in parental LN18 glioma cells (a, b) and U87 glioma cells (c, d), and two clones of each shNeg and shCSF2 cells; normal human astrocytes and normal brain samples were used as a reference. Bars show means ± s.d., n = 3. e–h CSF2 knockdown did not affect the proliferation (e, g) and viability (f, h) of CSF2-depleted LN18 and U87 glioma cells, when compared to control shNeg cells. All results are expressed as the values relative to those obtained for control cells (shNeg1 = 100%) and are presented as the means ± s.d., n = 3.
Fig 4: Apoptotic microglia/macrophages in CSF2-depleted gliomas.a Microglia/macrophages were visualized using Iba1 staining (green); cell nuclei were counterstained with DAPI (blue). DNA fragmentation was detected by TUNEL staining. Scale bar 100 µm. A right panel shows confocal microscopy images with z-stack projections and orthogonal views along the x and y axes, which demonstrate TUNEL+ nuclei in Iba1+ cells. b Quantification of TUNEL+Iba1+ cells in experimental gliomas (8 mice/shCSF2 group, 6 mice/shNeg group). The statistical significance was determined using Student’s t test, *p<0.05, **p<0.01.
Fig 5: Myeloid infiltrates and tumour growth in control and CSF2-depleted gliomas.a ShNeg or shCSF2 LN18 glioma cells were implanted into the striata of athymic mice and the tumours kept growing for 15 days. Human cells were visualized in brain sections with anti-human PCNA staining; scale bar 100 µm. Inset shows cells at higher magnification. b Quantification of tumour volume of shNeg or CSF2-depleted gliomas. Each dot represents an individual animal, and the bold line depicts the mean (8 mice/shCSF2 group, 6 mice/shNeg group); statistical analysis with U Mann-Whitney test; p = 0.01. c Survival of mice with implanted shCSF2 gliomas (green) compared with mice with control gliomas (red) (p = 0.04). d Microglia/macrophages were visualized using Iba1 staining (red); cell nuclei were counterstained with DAPI (blue). e Quantification of Iba1+ cells in experimental gliomas (8 mice/shCSF2 group, 6 mice/shNeg group). The statistical significance was determined using Student’s t test, *p<0.05, **p<0.01, ***p<0.001.
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