CX3CR1/CX3CL1 axis negatively controls glioma cell invasion and is modulated by transforming growth factor-β1

Abstract

The chemokine CX3CL1 is constitutively expressed in the central nervous system by neurons and astrocytes controlling neuronal survival and neurotransmission. In this work, we analyzed the expression and function of the chemokine CX3CL1 and its receptor, CX3CR1, by human glioma cells. We show that both molecules are expressed on the tumor cell plasma membrane and that soluble CX3CL1 accumulates in the culture supernatants, indicating that the chemokine is constitutively released. We found that CX3CR1 is functional, as all the cell lines adhered to immobilized recombinant CX3CL1 and migrated in response to the soluble form of this chemokine. In addition, the blockade of endogenous CX3CL1 function by means of a neutralizing monoclonal antibody markedly delayed tumor cell aggregation and increased their invasiveness. We also show that CX3CL1 expression is potently modulated by the transforming growth factor-beta1 (TGF-beta1), a key regulator of glioma cell invasiveness. Indeed, both the treatment of glioma cells with recombinant TGF-beta1 and the inhibition of its endogenous expression by siRNA showed that TGF-beta1 decreases CX3CL1 mRNA and protein expression. Overall, our results indicate that endogenously expressed CX3CL1 negatively regulates glioma invasion likely by promoting tumor cell aggregation, and that TGF-beta1 inhibition of CX3CL1 expression might contribute to glioma cell invasive properties.

Figures

Fig. 1.

CX3CL1 is expressed by human glioma cells. (A) CX3CL1 mRNA was analyzed by RT–PCR on 3 glioma cell lines (upper panel) and 3 primary glioma cell cultures (lower panel). Data are expressed as CX3CL1/beta-actin mRNA ratio, and represent the mean ± SD of 3 independent experiments. (B) Cell surface expression of membrane-bound CX3CL1 on glioma cell lines was revealed by staining with a specific anti-CX3CL1 mAb. Shown are the histogram plot overlays of anti-CX3CL1 mAb staining (empty histogram) against IC mAb (grey histogram). Numbers in the histograms indicate the average ± SD of the geometric mean fluorescence intensity (MFI) of at least 3 independent experiments performed. MFI of control staining was always

Fig. 2.

CX3CR1 is functionally expressed on glioma cell lines. (A) CX3CR1 cell surface expression on glioma cell lines was revealed by staining with a specific PE-labeled anti-CX3CR1 mAb. Shown are the histogram plot overlays of anti-CX3CR1 mAb staining (empty histogram) against IC mAb (grey histogram). Numbers in the histograms indicate the average ± SD of the MFI of at least 3 independent experiments performed. MFI of control staining was always

Fig. 3.

CX3CL1 inhibits glioma cell invasion. In vitro invasion assay was performed in 24 matrigel-coated wells. Anti-CX3CL1 mAb (black columns) or IC mAb (white columns) was added together with the cells in the upper chamber. Cell invasion was evaluated at 18 hours for U87MG and U251, and at 36 hours for T98G glioma cells. Invasion was quantified as the average number of cells counted in 10 randomly selected fields at 400× magnification. Experiments were performed in duplicate. Results presented are the mean ± SD of 3 independent experiments and are expressed as fold increase in the invasion capacity of anti-CX3CL1 Ab-treated cells vs that of IC-treated cells. Student's t-test was performed by comparing invasion of IC-treated cells vs anti-CX3CL1 mAb-treated cells. *P < .05. Invasion in the absence or presence of IC mAb was similar.

Fig. 4.

CX3CL1 is involved in glioma cell aggregation. (A) Slow aggregation assay on agar substrate was performed in the presence of 40 µg/mL of anti-CX3CL1 or IC mAb in 96-well plates, at 37°C, 5% CO2 as described in material and methods. Images shown were collected at 40× magnification and indicate a representative experiment of 3 independent experiments performed. Scale bar: 100 µm (B) Aggregation of T98G cells after 1 hour incubation in presence of 40 µg/mL of anti-CX3CL1 or IC mAb. Shown is a representative field (25 000 pixel2) obtained using Image J software. Data were expressed as the percentage of the mean ± SD of the number of single cells or aggregates containing 2 or more cells relative to a total of 500 cells, counted in several central fields.

Fig. 5.

TGF-beta1 down-modulates CX3CL1 expression in glioma cell lines. (A) TGF-beta1 levels were evaluated in the supernatants of glioma cells after 18 hours culture by ELISA assay. Histograms represent the mean ± SD of at least 5 independent experiments performed. (B) CX3CL1 mRNA was analyzed by RT–PCR on 3 glioma cell lines following 18 hours culture with or without (–) recombinant human TGF-beta1 (10 ng/mL) at 37°C, 5% CO2. Data, expressed as the mean ± SD of arbitrary units and derived from 3 independent experiments, were normalized with beta-actin, and referred as percent of untreated cells considered as calibrator. (C) Soluble CX3CL1 (sCX3CL1) levels were evaluated in the supernatants of glioma cells following 18 hours of stimulation with TGF-beta1 (10 ng/mL) by ELISA assay. Histograms represent the percentage of the mean ± SD of CX3CL1 concentration relative to untreated control (–) from at least 4 independent experiments performed. (D) Expression of cell surface-associated CX3CL1 in glioma cell lines following TGFbeta-1 (10 ng/mL) stimulation for 18 hours at 37°C, 5% CO2 detected by immunofluorescence and FACS analysis using a specific anti-CX3CL1 mAb. Histograms represent the percentage of the mean ± SD of CX3CL1 MFI relative to untreated control (–) from at least 4 independent experiments performed. In B, C, and D, Student's t-test was performed by comparing TGF-beta1 treated cell values with untreated cell values. *P < .05.

Fig. 6.

TGF-beta1 down-modulates CX3CL1 expression in primary glioma cell cultures. (A) TGF-beta1 levels were evaluated in the supernatants of glioma cells after 18 hours culture by ELISA assay. Histograms represent the mean ± SD of at least 3 independent experiments performed. (B) CX3CL1 mRNA was analyzed by RT–PCR on 3 primary glioma cells following 18 hours culture with or without (–) recombinant human TGF-beta1 (10 ng/mL) at 37°C, 5% CO2. Data are expressed as in Fig. 5B.

Fig. 7.

Increased expression of CX3CL1 and reduced invasiveness in TGFbeta-1 siRNA glioma cells. The release of TGF-beta1 (A) and sCX3CL1 (B, left panel) in supernatants of pSUPER-puro-TGF-beta1 stably transfected U87MG, T98G cells (TGF-beta1 siRNA) or mock transfectants (control) was monitored by ELISA assay. CX3CL1 mRNA was analyzed by RT-PCR in control and TGF-beta1 siRNA T98G cells (B, right panel). The mean ± SD of the concentration (TGF-beta1 and CX3CL1 protein) or arbitrary units (CX3CL1 mRNA) derived from 3 independent experiments and normalized with beta-actin was calculated and results were expressed as percentage of untreated cells. (C, left panel) Control or TGF-beta1 siRNA cells were assayed in migration assay against a gradient of FBS, performed using the 48-well microchamber (C, right panel). Control or TGF-beta1 siRNA cells treated with anti-CX3CL1 or IC mAb were assayed in invasion assay across a matrigel barrier (right panel) as described in material and methods. Student's t-test was performed by comparing control cell values versus TGF-beta1 siRNA cell values except in C, right panel in which IC- vs anti-CX3CL1 Ab-treated cell invasion values were compared. *P < .05.