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. 2015 Jan;17(1):81-94.
doi: 10.1093/neuonc/nou144. Epub 2014 Aug 1.

Adult mouse subventricular zones stimulate glioblastoma stem cells specific invasion through CXCL12/CXCR4 signaling

Nicolas Goffart  1 , Jérôme Kroonen  1 , Emmanuel Di Valentin  1 , Matthias Dedobbeleer  1 , Alexandre Denne  1 , Philippe Martinive  1 , Bernard Rogister  1
Affiliations

Adult mouse subventricular zones stimulate glioblastoma stem cells specific invasion through CXCL12/CXCR4 signaling

Nicolas Goffart et al. Neuro Oncol. 2015 Jan.

Abstract

Background: Patients with glioblastoma multiforme (GBM) have an overall median survival of 15 months. This catastrophic survival rate is the consequence of systematic relapses that could arise from remaining glioblastoma stem cells (GSCs) left behind after surgery. We previously demonstrated that GSCs are able to escape the tumor mass and specifically colonize the adult subventricular zones (SVZs) after transplantation. This specific localization, away from the initial injection site, therefore represents a high-quality model of a clinical obstacle to therapy and relapses because GSCs notably retain the ability to form secondary tumors.

Method: In this work, we questioned the role of the CXCL12/CXCR4 signaling in the GSC-specific invasion of the SVZs.

Results: We demonstrated that both receptor and ligand are respectively expressed by different GBM cell populations and by the SVZ itself. In vitro migration bio-assays highlighted that human U87MG GSCs isolated from the SVZs (U87MG-SVZ) display stronger migratory abilities in response to recombinant CXCL12 and/or SVZ-conditioned medium (SVZ-CM) compared with cancer cells isolated from the tumor mass (U87MG-TM). Moreover, in vitro inhibition of the CXCR4 signaling significantly decreased the U87MG-SVZ cell migration in response to the SVZ-CM. Very interestingly, treating U87MG-xenografted mice with daily doses of AMD3100, a specific CXCR4 antagonist, prevented the specific invasion of the SVZ. Another in vivo experiment, using CXCR4-invalidated GBM cells, displayed similar results.

Conclusion: Taken together, these data demonstrate the significant role of the CXCL12/CXCR4 signaling in this original model of brain cancer invasion.

Keywords: CXCL12; cancer stem cells; invasion; stem cell microenvironment.

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Figures

Fig. 1.
Fig. 1.
Invasion of the subventricular zone (SVZ) by U87MG cells and GB138 primary cells after intrastriatal implantation. (A) Human GB138 cells specifically colonized the SVZ environment after migration through the corpus callosum (CC). (B) Human U87MG cells invaded the largest part of the right striatum 3 weeks after the graft. At this point, U87MG cells had already escaped the tumor mass (TM) and began to migrate along the CC (white arrows). (C) U87MG cells finally invaded the SVZ environment 4 weeks after the injection, with some of them still being in a proliferation state (white arrows, magnified square). (D) U87MG and GB138 cells isolated from the SVZ displayed stronger abilities to form gliomaspheres than U87MG or GB138 cells isolated from the TM. Human U87MG cells were detected using an antihuman nuclei antibody (Hu. Nuclei - red). GB138 cells were engineered to express the green fluorescent protein (eGFP - green). Cell nuclei were counterstained with DAPI (blue). Scale bars = 500 µm for B, 40 µm for A and C. ** P < .01, ***P < .001.
Fig. 2.
Fig. 2.
Expression of CXCL12 by the subventricular zone (SVZ) environment. (A) RT-qPCR screening notably displayed a high expression level of CXCL12 in the SVZ environment (yellow rim of the graph). (B) This observation was validated by RT-PCR using mesenchymal stem cells (MSC) as a positive control. (C–E) The expression of CXCL12 was next demonstrated on brain coronal sections. This expression was consistent with the presence of U87MG cells (Hu. Nuclei) or GB138 primary cells (eGFP) in the SVZ (white arrows). (F) CXCL12 acquisitions were processed as binary images. The mean intensity, with foreground 255 and background 0, in predefined areas of the SVZ environment (A, B, and C) was calculated. A constant decrease of the CXCL12 expression was observed starting from area A to end at area C, suggesting that CXCL12 is secreted along a decreasing concentration gradient. (G) CXCL12 levels were evaluated by ELISA in conditioned media from SVZ, cerebellum, and olfactory bulb (OB) whole mounts for 24 or 60 hours. (H) CXCL12 was expressed by astrocytes and endothelial cells within the adult SVZ. Immunostaining on organotypic whole mounts showed a closely related expression of CXCL12 (green) with the vasculature and astrocytes (red). SVZ blood vessels and astrocytes were respectively stained using a FITC-coupled lectin or a specific anti-GFAP antibody (red). Cell nuclei were counterstained with DAPI (blue). Scale bars = 20 µm for C and D and 10 µm for E and H. Caption indicates where pictures and materials were taken.
Fig. 3.
Fig. 3.
Expression of CXCR4 by various GBM cell types. (A) CXCR4 mRNA expression was found in 3 human GBM cell lines (U87MG, U373, and LN18) and 2 human primary cultures (GBM 1 and 2). (B and C) Western blot analyses displayed a strong CXCR4 expression pattern in GBM cell lines and primary cultures as well as in U87MG cells isolated from the tumor mass (U87MG-TM) or the SVZ (U87MG-SVZ) and U87MG floating gliomaspheres (U87MG NS). (D) Human U87MG NS showed a combined expression of CXCR4 (red) with GFAP, Sox2, Nestin, and βIII-tubulin proteins (green). E) U87MG-TM and U87MG-SVZ gliomaspheres specifically expressed CXCR4 (red) as well as GSC markers such as CD133 (green), Nestin (green), and Integrin α6 (ITGA6) (pink). Nuclei were counterstained with DAPI (blue). Scale bars = 15 µm for D and E.
Fig. 4.
Fig. 4.
In vitro migration of GBM cells in response to recombinant CXCL12 and subventricular zone-conditioned medium (SVZ-CM). (A and B) Recombinant CXCL12 triggered migration and chemotaxis of human U87MG cells. (C and D) SVZ-CM triggered migration and chemotaxis of human U87MG cells. (E) The migration of U87MG cells and human GBM cells in primary culture (GBM2) in response to SVZ-CM was significantly reduced by using AMD3100, a specific CXCR4 antagonist. (F) AMD3100 disrupted chemotaxis of U87MG cells in response to SVZ-CM. (G and H) AMD3100 did not impact parameters such as the mean accumulated distance (µm) and velocity (µm/min) of U87MG cells. (I) U87MG-SVZ cells showed greater migration abilities in response to recombinant CXCL12 and SVZ-CM compared with U87MG cells or U87MG-TM cells. Moreover, AMD3100 clearly inhibited the migration of U87MG-SVZ cells in response to SVZ-CM. Graphs are mean values ± SEM and are representative of 3 independent experiments. *P < .05; **P < .01; ***P < .001; ns, not significant.
Fig. 5.
Fig. 5.
Inhibition of the SVZ invasion by CXCR4-depleted U87MG cells. (A and B) Western blot and cytological analyses of the CXCR4 expression in U87MGeGFP-Ires-Luc cells infected with lentiviruses encoding either a scrambled shRNA (U87MG-EIL-sc) or 2 short hairpins-RNA directed against CXCR4 (U87MG-EIL-shCXCR4* and U87MG-EIL-shCXCR4**). CXCR4 expression was declined up to 41% in the U87MG-EIL-shCXCR4* cells and up to 64% in the U87MG-EIL-shCXCR4** cells. (C) CXCR4 knockdown also decreased the U87MG cells in vitro migration in response to recombinant CXCL12 (1000nM) compared with the absence of CXCL12 (U87MG-EIL-sc CTL). (D and E) Animals were injected with either control U87MG cells (U87MG-EIL-sc, n = 5 mice) or with CXCR4-depleted U87MG cells (U87MG-EIL-shCXCR4* and U87MG-EIL-shCXCR4**, n = 5 mice per group). Human U87MG cells invaded the corpus callosum (CC) and the SVZ of mice grafted with U87MG-EIL-sc. No eGFP signal could be detected in the CC or the SVZ of both shRNA conditions. Scale bars = 10 µm and 100 µm, respectively, for B and D-E. ***P < .001.
Fig. 6.
Fig. 6.
AMD3100 inhibited the U87MG GSC invasion of the subventricular zone (SVZ). (A) AMD3100-treated animals showed significantly smaller amounts of relative bioluminescence compared with the control group (P < .01) at the end of the treatment period. (B and C) Only a few U87MG cells were found in the corpus callosum (CC) of AMD3100 treated-mice, whereas U87MG cells fully invaded the CC of PBS-treated mice (control). U87MG cells could not be found in the SVZ area in AMD3100-treated animals, whereas U87MG cells from the control group invaded the SVZ as expected. U87MG cells are labeled with a specific antihuman nuclei antibody. Scale bar = 100 µm for C. Graphs are mean values ± SEM. **P < .01, ***P < .001.
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