CXCL12 mediates glioblastoma resistance to radiotherapy in the subventricular zone

CXCL12 mediates glioblastoma resistance to radiotherapy in the subventricular zone Abstract Background. Patients with glioblastoma (GBM) have an overall median survival of 15 months despite multimodal therapy. These catastrophic survival rates are to be correlated to systematic relapses that might arise from remaining glioblastoma stem cells (GSCs) left behind after surgery. In this line, it has recently been demonstrated that GSCs are able to escape the tumor mass and preferentially colonize the adult subventricular zone (SVZ). At a distance from the initial tumor site, these GSCs might therefore represent a high-quality model of clinical resilience to therapy and cancer relapses as they specifically retain tumor-initiating abilities. Method. While relying on recent findings that have validated the existence of GSCs in the human SVZ, we questioned the role of the SVZ niche as a potential GSC reservoir involved in therapeutic failure. Results. Our results demonstrate that (i) GSCs located in the SVZ are specifically resistant to radiation in vivo, (ii) these cells display enhanced mesenchymal roots that are known to be associated with cancer radioresistance, (iii) these mesenchymal traits are specifically upregulated by CXCL12 (stromal cell-derived factor-1) both in vitro and in the SVZ environment, (iv) the amount of SVZ-released CXCL12 mediates GBM resistance to radiation in vitro, and (v) interferes with the CXCL12/CXCR4 signalling system, allowing weakening of the tumor mesenchymal roots and radiosensitizing SVZ-nested GBM cells. Conclusion. Together, these data provide evidence on how the adult SVZ environment, through the release of CXCL12, supports GBM therapeutic failure and potential tumor relapse. CXCL12, glioblastoma, mesenchymal activation, radioresistance, subventricular zone Primary brain tumors are considered as one of the nastiest scourges faced in oncology. Their most aggressive form, glioblastoma (GBM, WHO grade IV), is also regarded as the most common and lethal subtype.1 Patients' poor survival rates are typically correlated with unsatisfactory therapeutic strategies leading to systematic GBM relapses.2 Trying to better understand the origin of those relapses might therefore be of clinical benefit for improving both outcomes and patients' survival. In this line, recent studies have reported that a fraction of GBM cells, further characterized as GBM stem cells (GSCs), show a specific tropism for the adult subventricular zone (SVZ).3–5 This body of experimental evidence has recently been brought into sharp focus following the identification of GSCs within the SVZ of GBM patients.6 Because they are away from their initial tumor site and reside in a permissive microenvironment, these SVZ-nested GSCs might therefore represent a reliable example of clinical resilience to therapy. Additionally, a second core group of clinical reports shows a correlation between doses of radiotherapy delivered to the SVZ and increased progression-free survival (PFS) as well as overall survival (OS) in newly diagnosed GBM patients.7–9 Together, these findings might be explained by the persistence of GSCs within the SVZ environment and advocate the need for better characterization of the communication lines between GSCs and the SVZ environment. In this work, we evaluated the influence of SVZ-related factors in GBM resistance to radiotherapy. More specifically, we questioned the role of SVZ-released CXCL12 in the extrinsic resistance of GSCs to irradiation (IR). As a matter of fact, the CXCL12/CXCR4 signaling axis has been recently identified as a key mediator of GBM mesenchymal activation in vitro,10 a process described as sustaining cancer radioresistance.11,12 Additionally, CXCR4 has further been identified as a potential biomarker for radioresistant cancer stem cells.13 Here, we demonstrate that SVZ-nested GSCs are specifically resistant to IR in vivo. These cells also display an enhanced mesenchymal signature in the SVZ compared with their counterparts from the tumor mass. Our results also demonstrate that these mesenchymal traits are specifically upregulated by CXCL12 both in vitro and in vivo. Interestingly, the use of a CXCL12/CXCR4 antagonist (AMD3100) allowed us to radiosensitize and disrupt the mesenchymal signature of SVZ-nested GBM cells simultaneously. Similarly, the in-vitro blockade of CXCL12 within the soluble environment of the SVZ revealed a significant reduction in the expression of GBM mesenchymal markers, together with a significant increase in the radiosensitivity of GBM cells. Together, these data emphasize the critical role of CXCL12 in mediating GBM resistance to radiotherapy and identify the adult SVZ environment as a potential niche involved in GBM recurrence. Methods Study Approval Patients gave informed consent for the use of their GBM specimens. Regarding human brain tissues, we obtained the patients’ written consent and the approval by the ethical committee of the University of Liège for experimental purposes. Cell Culture Human GBM primary cultures (GBM1, GBM2, and GB138) were established from consenting patients and cultivated as previously described.14 Additional details can be found in the Supplementary Data section. Animals Adult P40 immunodeficient nude mice, Crl:NU-Foxn1nu (obtained from Charles River Laboratories) were used for xenograft purposes. Athymic nude mice were housed in sterilized filter-topped cages and were processed with the consent of the ethical committee of the University of Liège. All animals were cared for in compliance with the guidelines of the Belgium Ministry of Agriculture and in accord with the ethical committee's laboratory animal care and use regulation (86/609/CEE, CE of J n_L358, December 18, 1986). Intracranial Transplantation and AMD3100 Treatment Intracranial xenografts were performed as described previously.4 AMD3100, a specific CXCL12/CXCR4 bicyclam antagonist, was injected twice a day for 10 consecutive days (1,25 mg/kg–Sigma). The injections started at week 10 post implantation. In Vivo Radiotherapy Protocol A dose of 30 Gy in 5 consecutive days was delivered in the whole brain with a dedicated small animal radiotherapy device (SmART Irradiator from Precision X-Ray Inc). Radiation was delivered using a photon beam (maximum energy of 225 kV and 13 mA), which provided a dose rate of 3 Gy/min. Additional details are provided in the Supplementary Data section. Whole-mount Dissection The lateral walls of the murine ventricles were dissected from the caudal aspect of the telencephalon as previously described.15 When dissecting the human SVZ, the brain was separated into 2 hemispheres, and the lateral walls of the ventricles were dissected from the caudal aspect of the telencephalon towards the cephalic region of the SVZ. This procedure was started within 15 minutes after the patient's death. The strips of SVZ tissue were separated into 3 parts (cephalic, median, and caudal) and cultured for 60 hours in DMEM/F12 (Life Technologies) supplemented with recombinant EGF (20 ng/mL, Preprotech) and recombinant FGF-2 (10 ng/mL, Preprotech) to prepare the human SVZ-conditioned medium. Gene-expression Profiling PCR arrays were performed using RT2 profiler PCR array (SABiosciences, Epithelial to Mesenchymal Transition PCR Array, PAHS-090A) on a LightCycler 480 qPCR system (Roche) according to the manufacturer's protocol. Additional details are provided in the Supplementary Data section. Western Blot Analysis Western blots were performed as previously described.5 Reagents, buffers, and antibodies are provided in the Supplementary Data section. Proteome Profiler Array Screening for the human SVZ-CM components was performed using the proteome profiler method (human chemokine array kit, R&D Systems) according to the manufacturer's protocol. Dot blots (standardized for loading control) were imaged with the ImageQuant 350 scanning system (cooled-CCD camera, GE Healthcare). Immunostaining Immunostainings were performed as previously described.5 Reagents, buffers, and antibodies are provided in the Supplementary Data section. Statistical Analysis Quantitative data are expressed as mean ± SEM. Two-way ANOVA was performed, followed by Tukey posttest, and a P value <.05 was considered statistically significant. Each experiment was run at least 3 times independently. Student t tests were performed for group comparison. All statistics were computed using Statistica 10.0 software. Results The Adult Subventricular Zone Acts as a Radioprotective Niche for Glioblastoma Cells To investigate the radioprotective role of the SVZ niche, we grafted RFP-positive GB138 primary cells into the right striatum of immunocompromised mice. Ten weeks after the implantation, 8 mice were submitted to brain-restricted doses of radiation (6 Gy) for 5 days. By the end of the 11th week, animals from both control and irradiated groups were euthanized. The efficacy of IR was assessed by histological examination of RFP-positive cells in the brain. As expected, control animals displayed massive infiltration of the corpus callosum (CC) and SVZ (Fig. 1 B and C).4 The number of GB138 primary cells dropped by 68% in the tumor mass (TM) (P = .027), 65% in the CC (P = .057), and 73% in the SVZ (P = .029) after IR (Fig. 1 A–D). These results specifically highlight the persistence of GBM cells in the CC and the SVZ environment after radiotherapy. These persisting cells, away from the initial tumor site (TM) might therefore play a key role in GBM recurrence and might corroborate with late periventricular patterns of recurrence observed in GBM patients every so often.16 Fig. 1 View largeDownload slide GB138 Primary cells leave the tumor mass and migrate through the corpus callosum to reach the subventricular zone (SVZ). The number of RFP-positive GB138 primary cells initially found in the striatum (A), corpus callosum (B), and subventricular zone (C) of nonirradiated animals significantly decreased in irradiated animals. A minimum of 5 mice were used in each group for quantification. GB138 primary cells were detected using a specific anti-RFP antibody (red). Cell nuclei were counterstained with DAPI (blue). Captions show where pictures were taken (D). Scale bars = 40 µm for A, B and C. * P < .05. Fig. 1 View largeDownload slide GB138 Primary cells leave the tumor mass and migrate through the corpus callosum to reach the subventricular zone (SVZ). The number of RFP-positive GB138 primary cells initially found in the striatum (A), corpus callosum (B), and subventricular zone (C) of nonirradiated animals significantly decreased in irradiated animals. A minimum of 5 mice were used in each group for quantification. GB138 primary cells were detected using a specific anti-RFP antibody (red). Cell nuclei were counterstained with DAPI (blue). Captions show where pictures were taken (D). Scale bars = 40 µm for A, B and C. * P < .05. Murine and Human SVZ-CM Mediate GBM Resistance to Radiation in Vitro To validate whether the SVZ endorses the role of a radioprotective niche for GBM cells, we focused on its soluble environment. To do so, we grew GBM2 primary cells and U87MG cells for 12 hours in minimal culture media (serum starvation). We then supplemented these GBM cells with murine SVZ-conditioned media (mSVZ-CM) and irradiated them (10 Gy) to assess the γH2AX response. Interestingly, both GBM2 primary cells and U87MG cells supplemented with mSVZ-CM prior to IR displayed a significant decrease in γH2AX reactivity compared with cells in control media (Fig. 2 A). A similar observation was made with GBM1 primary cells (Supplementary material, Fig. S1A). We then conducted a γH2AX kinetic on GBM2 primary cells and U87MG cells to further assess the DNA damage response. Again, we found that mSVZ-CM protected these 2 GBM cell populations from IR all along the different time points of the kinetic (P < .001, Fig. 2B). Similar observations were made with a 53BP1 kinetic to further confirm the radioprotective role of mSVZ-CM (Supplementary material, Fig. S1B). Together, these data show how mSVZ-CM counteracts with the DNA damage response by decreasing the radiosensitivity of GBM cells. Fig. 2 View largeDownload slide Mouse and human SVZ-CM mediate glioblastoma (GBM) resistance to radiation in vitro. Culture media were conditioned with murine SVZ whole-mounts for 60 hours and then added to GBM monolayers prior to irradiation (IR). This significantly decreased the sensitivity of GBM2 primary cells and U87MG cells to IR by reducing the DNA damage response (A). Radiodesensitization was associated with a significant decrease of γH2AX-positive cells. A γH2AX kinetic (t0, t15, t30, and t60) was performed on GBM2 primary cells and U87MG cells after IR. For each time point, both GBM cell populations activated fewer H2AX after IR when supplemented with subventricular zone-conditioned media (SVZ-CM) compared with control media (B). Irradiation of GBM2 primary cells and U87MG cells supplemented with olfactory bulb-conditioned media (OB-CM) or cerebellum-conditioned media (CRBL-CM) did not affect the DNA damage response as similar levels of γH2AX-positive cells were found in comparison with irradiated cells in control media (C). Culture media were conditioned with human SVZ whole-mounts for 60 hours and added to GBM monolayers (GBM2 and U87MG) prior to IR. This significantly decreased the sensitivity of both GBM2 primary cells and U87MG cells to IR by reducing the DNA damage response (D). The latter was specifically assessed by measuring the γH2AX response in these GBM populations following IR. Cell nuclei were counterstained with DAPI (blue). Scale bars = 10 µm for A. * P < .05, ** P < .01, *** P < .001. Fig. 2 View largeDownload slide Mouse and human SVZ-CM mediate glioblastoma (GBM) resistance to radiation in vitro. Culture media were conditioned with murine SVZ whole-mounts for 60 hours and then added to GBM monolayers prior to irradiation (IR). This significantly decreased the sensitivity of GBM2 primary cells and U87MG cells to IR by reducing the DNA damage response (A). Radiodesensitization was associated with a significant decrease of γH2AX-positive cells. A γH2AX kinetic (t0, t15, t30, and t60) was performed on GBM2 primary cells and U87MG cells after IR. For each time point, both GBM cell populations activated fewer H2AX after IR when supplemented with subventricular zone-conditioned media (SVZ-CM) compared with control media (B). Irradiation of GBM2 primary cells and U87MG cells supplemented with olfactory bulb-conditioned media (OB-CM) or cerebellum-conditioned media (CRBL-CM) did not affect the DNA damage response as similar levels of γH2AX-positive cells were found in comparison with irradiated cells in control media (C). Culture media were conditioned with human SVZ whole-mounts for 60 hours and added to GBM monolayers (GBM2 and U87MG) prior to IR. This significantly decreased the sensitivity of both GBM2 primary cells and U87MG cells to IR by reducing the DNA damage response (D). The latter was specifically assessed by measuring the γH2AX response in these GBM populations following IR. Cell nuclei were counterstained with DAPI (blue). Scale bars = 10 µm for A. * P < .05, ** P < .01, *** P < .001. We further verified whether this decline in radiosensitivity was specific to the SVZ environment. To do so, we irradiated GBM2 primary cells and U87MG cells, either supplemented with mSVZ-CM or murine olfactory bulb-conditioned media (mOB-CM) or murine cerebellum-conditioned media (mCRBL-CM). As expected, we showed a significant decrease in γH2AX-positive GBM2 and U87MG cells supplemented with mSVZ-CM following IR (P < .001). Interestingly, mOB-CM and mCRBL-CM did not impact the DNA damage response of GBM2 and U87MG cells following IR (10 Gy) (Fig. 2 C). We also compared the γH2AX response in U87MG cells isolated from the tumor mass (U87MG-TM) and from the SVZ (U87MG-SVZ) in the presence of mSVZ-CM. These GBM subpopulations were isolated according to a well-defined protocol4 and preincubated in mSVZ-CM or control media prior to IR (10 Gy). U87MG-TM cells displayed a γH2AX pattern of expression similar to U87MG cells. Indeed, mSVZ-CM allowed a significant decrease in the number of γH2AX-positive cells in this subpopulation after IR (P < .01). On the other hand, U87MG-SVZ cells significantly fewer γH2AX-positive cells when incubated in mSVZ-CM prior to IR (P < .05), further suggesting different patterns of DNA damage responses in these 2 GBM subpopulations (Supplementray material, Fig. S1C). GBM2 primary cells and U87MG cells were finally cultured for 12 hours in serum-starvation conditions and supplemented with human SVZ-CM (hSVZ-CM) prior to IR (10 Gy). hSVZ-CM allowed a significant decrease in the radiosensitivity of GBM2 primary cells and U87MG cells after IR (Fig. 2 D). Consistent with prior studies in human epithelial cells,17 this radio-desensitization was again correlated with a significant decrease in γH2AX reactivity (P < .001). Similar data were collected by assessing the expression of 53BP1 in response to IR in both GBM2 primary cells and U87MG cells supplemented with hSVZ-CM (P < .001, Supplemetary material, Fig. S1D). These results collectively provide evidence that the soluble environment of the SVZ helps to abrogate the DNA damage response of GBM cells after IR. Identification of SVZ-released CXCL12 as a Key Mediator of GBM Resistance to Irradiation and Survival in Vitro CXCL12 has recently been suggested to regulate the mesenchymal activation of cancer cells in vitro, especially through Snail and N-cadherin regulation.10 In this line, the process has also been reported to enhance GBM radioresistance in a NF-κB-dependent manner.11 Furthermore, we recently highlighted the expression of CXCL12 in mSVZ-CM (approximately 600 pg/mL).5,18 With reliance on these facts, we investigated whether SVZ-released CXCL12 could play a role in GBM radioresistance. To do so, we preincubated mSVZ-CM with specific CXCL12-blocking antibodies and/or nonrelevant immunoglobulins (IgG) for 45 minutes. We then supplemented GBM2 primary cells and U87MG cells with these 2 different mSVZ-CMs prior to IR (10 Gy) and quantified the impact of blocking CXCL12 on the γH2AX response of GBM cells. As expected, the number of γH2AX-positive GBM2 and U87MG cells had decreased significantly in mSVZ-CM and mSVZ-CM/IgG conditions (Fig. 3 A). Interestingly, the blockade of CXCL12 allowed sensitization of these 2 GBM populations to IR (P < .001, Fig. 3 A). However, the inhibition of CXCL12 did not completely restore the number of γH2AX-positive cells encountered without any mSVZ-CM supplementation, further suggesting the role of other SVZ-released components in GBM resistance to IR in vitro. Fig. 3 View largeDownload slide Identification of CXCL12 as a key mediator of glioblastoma (GBM) survival and resistance to radiation. The inhibition of CXCL12 in the mSVZ-CM prior to irradiation (IR) leads to a significant rescue of radiosensitization in human GBM2 primary cells and U87MG cells (A). The number of γH2AX-positive cells was higher after the blockade of CXCL12 in both GBM populations compared with cells supplemented with mSVZ-CM and a nonrelevant immunoglobulin (IgG). Conversely, growing concentrations of recombinant CXCL12 (25/50/100 nM) significantly increased GBM2 primary cells and U87MG cells radioprotection in a dose-dependent manner (B). mSVZ-CM significantly propelled the proliferation of U87MG cells compared with U87MG cells in control media (C–D). U87MG cells supplemented with mSVZ-CM prior to IR displayed stronger proliferation abilities than irradiated U87MG cells in control media (D). U87MG cells supplemented with mSVZ-CM and specific CXCL12 blocking antibodies prior to IR had the same proliferation rate compared with irradiated U87MG cells in control media (D). * P < .05, ** P < .01, *** P < .001. Fig. 3 View largeDownload slide Identification of CXCL12 as a key mediator of glioblastoma (GBM) survival and resistance to radiation. The inhibition of CXCL12 in the mSVZ-CM prior to irradiation (IR) leads to a significant rescue of radiosensitization in human GBM2 primary cells and U87MG cells (A). The number of γH2AX-positive cells was higher after the blockade of CXCL12 in both GBM populations compared with cells supplemented with mSVZ-CM and a nonrelevant immunoglobulin (IgG). Conversely, growing concentrations of recombinant CXCL12 (25/50/100 nM) significantly increased GBM2 primary cells and U87MG cells radioprotection in a dose-dependent manner (B). mSVZ-CM significantly propelled the proliferation of U87MG cells compared with U87MG cells in control media (C–D). U87MG cells supplemented with mSVZ-CM prior to IR displayed stronger proliferation abilities than irradiated U87MG cells in control media (D). U87MG cells supplemented with mSVZ-CM and specific CXCL12 blocking antibodies prior to IR had the same proliferation rate compared with irradiated U87MG cells in control media (D). * P < .05, ** P < .01, *** P < .001. Conversely to the blockade of CXCL12, we conducted rescue experiments to prove that CXCL12 could act as a radioprotective chemokine for GBM cells. We grew GBM2 primary cells and U87MG cells for 12 hours in serum-starvation conditions. We next stimulated these 2 GBM populations with growing concentrations of recombinant CXCL12 (rCXCL12 at 25 nM, 50 nM, and 100 nM) prior to IR (10 Gy) and assessed the γH2AX response within each of the 3 conditions. We noticed a significant decrease of γH2AX-positive GBM2 primary cells starting at 50 nM (P = .02, Fig. 3 B). A similar observation was made in U87MG cells at a concentration of 25 nM (P = .04, Fig. 3 B). This slight but constant radio-desensitization was observed up to 100 nM in both GBM cell populations (P < .001). We finally investigated the impact of mSVZ-CM on GBM cell survival and growth abilities. We seeded 7500 U87MG cells in 6-well plates and serum-starved them for 12 hours. mSVZ-CM was then added to U87MG monolayers prior to IR (4 Gy). Six hours later, U87MG cells were refreshed in DMEM supplemented with fetal bovine serum (10%) and cultured at an exponential growth phase for 7 days (Fig. 3 C). Besides IR, we noticed that mSVZ-CM had a significant positive impact on the proliferation of U87MG cells. These cells proliferated twice as much (2.03/1 ratio) compared with U87MG cells in control media (P < .001) (Fig. 3 D). We then estimated the impact of an IR dose of 4 Gy on the survival of GBM cells. Irradiating U87MG monolayers led to a 43.5% reduction of cell survival (P < .001) (Fig. 3 D). Surprisingly, the addition of mSVZ-CM to U87MG monolayers prior to IR significantly propelled their proliferation as U87MG cells proliferated 2.5 times faster compared with control media (P < .001) (Fig. 3 D). Similar data were collected with GBM2 primary cells (Supplementary material, Fig. S2). We also assessed the role of SVZ-released CXCL12 on GBM cell survival and growth abilities after IR by blocking its expression in mSVZ-CM. Irradiated U87MG monolayers supplemented with a combination of mSVZ-CM and specific CXCL12 blocking antibodies grew at the same rate as irradiated U87MG cells in control media (Fig. 3 D). Collectively, these data demonstrated how mSVZ-CM actively promotes GBM cell proliferation and survival after IR in a CXCL12-dependent manner. Expression of CXCL12 by the Human SVZ and Role in GBM Radioprotection Since we demonstrated that hSVZ-CM specifically protects GBM cells from IR in vitro, we next investigated the hypothetical role of hSVZ-released CXCL12 in GBM radioprotection. To do so, we dissected the SVZ of a fresh human brain in 3 distinct regions (cephalic, median, and caudal, Fig. 4 A) and independently conditioned culture media with each of these regions for 60 hours. ELISA experiments highlighted a significant difference of CXCL12 expression between the cephalic/median hSVZ-CM and its caudal part (P = .004, Fig. 4 A). These findings were further confirmed by Western blots, showing the preferential expression of CXCL12 in the cephalic/median human SVZ compared with the caudal region (Fig. 4 B). Fig. 4 View largeDownload slide Expression of CXCL12 in the human subventricular zone (SVZ) and validation of its role in glioblastoma (GBM) radioprotection. The presence of CXCL12 in the hSVZ-CM was assessed by ELISA (A) and further confirmed by Western-blot analysis (B). The blockade of CXCL12 in hSVZ-CM prior to irradiation (IR) induced a partial loss of radioprotection in GBM2 primary cells and U87MG cells (C). This observation correlated with a significant decrease of γH2AX reactivity in these 2 GBM populations. CXCL12, hSVZ-CM also contains different proteins such as IL-8, MIP-3α and PF4 among others. * P < .05, ** P < .01, *** P < .001. Fig. 4 View largeDownload slide Expression of CXCL12 in the human subventricular zone (SVZ) and validation of its role in glioblastoma (GBM) radioprotection. The presence of CXCL12 in the hSVZ-CM was assessed by ELISA (A) and further confirmed by Western-blot analysis (B). The blockade of CXCL12 in hSVZ-CM prior to irradiation (IR) induced a partial loss of radioprotection in GBM2 primary cells and U87MG cells (C). This observation correlated with a significant decrease of γH2AX reactivity in these 2 GBM populations. CXCL12, hSVZ-CM also contains different proteins such as IL-8, MIP-3α and PF4 among others. * P < .05, ** P < .01, *** P < .001. We then supplemented hSVZ-CM with CXCL12-blocking antibodies and/or nonrelevant IgG for 45 minutes and finally grew GBM2 primary cells and U87MG in these 2 different media prior to IR (10 Gy). Similar amounts of γH2AX-positive cells were found in both hSVZ-CM and hSVZ-CM/IgG conditions (Fig. 4 C). Interestingly, the blockade of CXCL12 allowed radiosensitization of GBM2 primary cells and U87MG cells (P < .001 and P = .02, respectively) (Fig. 4 C). Again, the CXCL12 inhibition did not completely restore the number of γH2AX-positive cells encountered without any hSVZ-CM supplementation (CTL), suggesting the role of other SVZ-released components in GBM resistance to IR in vitro. In this line, we performed a proteome-profiling analysis of hSVZ-CM and specifically highlighted the expression of different proteins already known to play a role in cancer radioresistance including MIP-3α, PF4, and IL-8 among others ( Fig. 4 D). SVZ-released CXCL12 Promotes the Mesenchymal Activation of GBM Cells So far, we have demonstrated that CXCL12 endorses a key role in GBM resistance to IR, but the mechanisms working behind the scene remained unclear. In this context, CXCL12 has recently been suggested to regulate the mesenchymal activation of GBM in vitro,10 a process known to be involved in cancer radioresistance.19 Moreover, the acquisition of a mesenchymal signature corresponds to the most rebellious type of response to therapy in GBM.20 Relying on these findings, we questioned the role of SVZ-released CXCL12 in promoting the mesenchymal activation of GBM cells. In order to identify key mediators involved in the mesenchymal activation of GBM cells, we conducted wide-scale RT-qPCR screening on CXCL12-stimulated GB138 primary cells. This analysis highlighted a significant increase in the expression of 26 genes typically involved in the acquisition of a mesenchymal phenotype (Supplementary material, Fig. S3A and B). Among these genes, we showed the upregulation of 3 crucial mesenchymal markers including CDH2, FN1, and vimentin (Fig. 5 A) as well as key transcription factors including ESR1, FOXC2, SOX10, and ZEB2 (Fig. 5 B). Fig. 5 View largeDownload slide Subventricular (SVZ)-released CXCL12 promotes the mesenchymal activation of glioblastoma (GBM) cells. CXCL12-stimulated GB138 primary cells upregulated 3 major mesenchymal genes including CDH2, FN1 and vimentin and 4 key EMT-related transcription factors including ESR1, FOXC2, SOX10, and ZEB2 (A-B). Both CXCL12-stimulated GB138 primary cells and U87MG cells overexpressed mesenchymal proteins including N-cadherin and vimentin (C). Pretreatment of GB138 primary cells and U87MG cells with AMD3100 prior to CXCL12 treatment disrupted the expression of N-cadherin and vimentin (C). Fig. 5 View largeDownload slide Subventricular (SVZ)-released CXCL12 promotes the mesenchymal activation of glioblastoma (GBM) cells. CXCL12-stimulated GB138 primary cells upregulated 3 major mesenchymal genes including CDH2, FN1 and vimentin and 4 key EMT-related transcription factors including ESR1, FOXC2, SOX10, and ZEB2 (A-B). Both CXCL12-stimulated GB138 primary cells and U87MG cells overexpressed mesenchymal proteins including N-cadherin and vimentin (C). Pretreatment of GB138 primary cells and U87MG cells with AMD3100 prior to CXCL12 treatment disrupted the expression of N-cadherin and vimentin (C). Thereafter, we stimulated both GB138 primary cells and U87MG cells with rCXCL12 (20 nM) and assessed the expression level of mesenchymal proteins. Our results showed an increase in N-cadherin expression in both GBM populations within one hour of stimulation (Fig. 5 C). The expression of vimentin, a type III intermediate filament protein specifically expressed in mesenchymal cells, was also found to be upregulated in U87MG cells within an hour (Fig. 5 C). The vimentin expression started to increase after 4 hours of stimulation in GB138 primary cells, suggesting different posttranslational regulations in U87MG cells and GB138 primary cells (Fig. 5 C). Interestingly, the upregulation of N-cadherin and vimentin in U87MG cells as well as GB138 primary cells was hampered by pretreating these GBM populations with a specific CXCL12/CXCR4 antagonist (AMD310021) prior to the rCXCL12 stimulation (Fig. 5 C). These findings particularly highlighted how CXCL12 mediates the acquisition of an enhanced mesenchymal phenotype in both GB138 primary cells and U87MG cells in vitro. AMD3100 Sensitizes SVZ-nested GBM Cells to Radiation in Vivo Since CXCL12 enhances GBM mesenchymal roots, we then looked for a potential mesenchymal signature that would be an indicator of therapeutic resistance in GBM cells nested in the SVZ environment. Western blot analyses on GB138 primary cells isolated from the TM and SVZ surprisingly revealed a higher expression level of N-cadherin and vimentin in GB138-SVZ cells (Fig. 6 A). This observation suggested a SVZ-dependent enhancement of GBM mesenchymal traits through the release of CXCL12 in the nearby environment. To prove this, xenograft experiments were performed with GB138 primary cells. Half of the cohort was treated twice a day for 10 days with intraperitoneal injections of AMD3100 (1.25 mg/kg), whereas the other group was treated with PBS (control). Histological examination of brain coronal sections confirmed the expression of N-cadherin and vimentin (red) in GB138 primary cells (green) located in the ventricular walls of control animals (Fig. 6 B). Interestingly, the in vivo use of AMD3100 significantly decreased the amount of GB138 primary cells invading both the CC and the SVZ regions (P < .01 and P < .001, respectively; Supplementary material, Fig. S4A) and disrupted the expression of vimentin and N-cadherin in SVZ-nested GB138 primary cells (Fig. 6 C). These results demonstrate how SVZ-released CXCL12 plays a key role in the acquisition of GBM mesenchymal properties, a signature typically associated with therapeutic resistance.20 Fig. 6 View largeDownload slide Inhibition of CXCL12 in the subventricular zone (SVZ) weakens the tumor mesenchymal roots and promotes radiosensitization. GB138 primary cells isolated from the SVZ exhibit a higher expression level of N-cadherin and vimentin than GB138 primary cells isolated from tumor mass (A). Immunohistostaining on brain coronal sections of mice grafted with GB138 primary cells confirmed the expression of N-cadherin and vimentin (red) in SVZ-nested G138 primary cells (green) (B). The use of AMD3100 in vivo disrupted the expression of N-cadherin and vimentin (red) in SVZ-nested GB138 primary cells (green) (C). Combining AMD3100 and irradiation (IR) treatments allowed a significantly decreased number of GB138 primary cells in the corpus callosum and the SVZ compared with irradiated mice only (D). GB138 primary cells were labeled with a specific anti-RFP antibody. Scale bars = 20 µm for B and C and 30 µm for D. * P < .05. Fig. 6 View largeDownload slide Inhibition of CXCL12 in the subventricular zone (SVZ) weakens the tumor mesenchymal roots and promotes radiosensitization. GB138 primary cells isolated from the SVZ exhibit a higher expression level of N-cadherin and vimentin than GB138 primary cells isolated from tumor mass (A). Immunohistostaining on brain coronal sections of mice grafted with GB138 primary cells confirmed the expression of N-cadherin and vimentin (red) in SVZ-nested G138 primary cells (green) (B). The use of AMD3100 in vivo disrupted the expression of N-cadherin and vimentin (red) in SVZ-nested GB138 primary cells (green) (C). Combining AMD3100 and irradiation (IR) treatments allowed a significantly decreased number of GB138 primary cells in the corpus callosum and the SVZ compared with irradiated mice only (D). GB138 primary cells were labeled with a specific anti-RFP antibody. Scale bars = 20 µm for B and C and 30 µm for D. * P < .05. Relying on these data, new xenograft experiments with RFP-positive GB138 primary cells were conducted to assess the efficacy of combining radiotherapy with adjuvant AMD3100 chemotherapy. Half of the cohort was treated twice a day for 10 days with intraperitoneal injections of AMD3100 (1.25 mg/kg), whereas the other group was treated with PBS. Brain-restricted radiotherapy started 5 days after the initial injection of AMD3100 and lasted for 5 days. Histological examination of brain coronal sections highlighted a significant decrease in the number of GB138 primary cells found in the CC and SVZ regions of irradiated/AMD3100-treated animals compared with irradiated animals (P = .015). On the other hand, AMD3100 had no effect on the number of cancer cells found in the TM after IR (Fig. 6 D and Supplementary material, Fig. S4B). Collectively, these data show that interfering with SVZ-released CXCL12 allows sensitization of SVZ-nested GBM cells to radiotherapy. Discussion The present study has demonstrated that a fraction of GBM cells hiding in the SVZ environment are resistant to radiotherapy. Of importance, we previously demonstrated that GBM cells located in the SVZ are enriched in tumor-initiating capacities and were in this way characterized as GSC. 4,5 Interestingly, GSC display intrinsic radioresistant abilities.22 It thus makes sense to find a fraction of tumor cells remaining in the SVZ environment after IR; however, we questioned the role of the SVZ niche as a potential GBM reservoir involved in GSC extrinsic resistance to IR. We specifically assumed that the composition of the niche finely regulates both fate specification and protection of these GSCs. Physiologically, the SVZ acts as a supportive niche, promoting self-renewal of neural stem cells and inhibiting differentiation.23 This “seed-and-soil” relationship has also been adapted to cancer stem cell research as GSCs also rely on specific niches to maintain their stem cell properties and ability to drive tumor growth.24–26 In this line, Piccirillo et al. recently showed that GSCs isolated from the human SVZ are specifically resistant to supramaximal chemotherapy doses along with differential patterns of drug response.6 Together, these findings suggest that the SVZ environment might provide GBM cells with radio/chemoprotective inputs and could play a determinant role in malignant brain tumor recurrence. To further explore the role of the SVZ in GBM resistance to IR, we established an in-vitro protocol of radiotherapy in which we focused on the soluble environment of the SVZ (SVZ-CM). This experimental protocol allowed us to highlight a significant increase of radioprotection in GBM cells stimulated with SVZ-CM. This observation was made with SVZ-CM collected from both mice and human donors. We next identified CXCL12 as a key mediator involved in GBM radioprotection. The blockade of CXCL12 in SVZ-CM allowed sensitizing GBM cells to IR in vitro. Concomitant to our findings, the pharmacological inhibition of CXCL12 in combination with IR was shown to abrogate GBM regrowth in mice by preventing the development of functional tumor blood vessels post-IR.27 Domanska et al. also reported that the inhibition of CXCL12-dependent protective signals from stromal cells renders prostate cancer cells more sensitive to ionizing radiations.28 Collectively, these data advocate for a better characterization of CXCL12 inhibitors in preclinical cancer models. Their potential therapeutic benefits, in combination with IR, might indeed contribute to understanding the role of CXCL12 in GBM resistance to therapy and could facilitate translation of these inhibitors to the clinic. Besides the role of SVZ-released CXCL12 in GBM resistance to IR, the mechanisms underlying these findings were yet to be determined. To address the issue, we focused on the tumor's mesenchymal profile. The acquisition of a mesenchymal phenotype is indeed associated with poor outcomes and corresponds to the most rebellious type of GBM to therapy.20,29 GBMs are known to frequently shift toward a mesenchymal status upon recurrence.29,30 With that in mind, we demonstrated that GBM cells express a basal level of vimentin and N-cadherin that further suggests the mesenchymal origin of our GBM populations. Interestingly, these 2 mesenchymal markers were specifically upregulated upon CXCL12 stimulation at the mRNA and protein levels. The inhibition of the CXCL12/CXCR4 signaling axis in GBM has been recently reported to affect the in-vitro expression of mesenchymal markers including Twist, Snail, and N-cadherin (which supports our findings).31,32 Interestingly, we showed that GBM cells hosted by the SVZ display a higher expression level of N-cadherin and vimentin compared with the rest of the tumor. In this line, the use of AMD3100, a specific CXCL12/CXCR4 antagonist,21 allowed weakening of the tumor's mesenchymal signature within the SVZ and sensitized both CC and SVZ-nested GBM cells. These data demonstrate how SVZ-released CXCL12 undertakes a key role in GBM radioprotection by underlying the acquisition of strong tumor mesenchymal roots. In light of our findings, many studies have established a close link between GBM's mesenchymal activation and its ability to better resist chemotherapy.33 By contrast, very little is known about the involvement of this process in radioresistance. To our knowledge, a single study so far has reported that the mesenchymal activation in GBM promotes resistance to IR in a NF-κB-dependent manner.11 A recent study has also shown that ATM kinase stabilizes ZEB1 in response to DNA damage.12 Interestingly, the expression of ZEB1 is upregulated upon CXCL12 stimulation in GB138 primary cells (Supplementary material, Fig. S2B). ZEB1 then directly interacts with USP7 to enhance its ability to de-ubiquitinate CHK1, thereby promoting homologous recombination-dependent DNA repair and resistance to IR in breast cancer cells.12 Whether Nf-κB and/or ZEB1 are specifically involved in our model of GBM resistance to IR remains to be elucidated. Besides CXCL12, we have also shown the expression of different effectors in the human SVZ-CM that could further undertake a key role in GBM resistance to treatment. Among these proteins, MIP3α and IL8 are known to enhance the epithelial-to-mesenchymal transition of hepatocellular carcinoma and sustain nasopharyngeal carcinoma radioresistance, respectively.34,35 Together these other SVZ components could support CXCL12 in mediating GBM resistance to IR in the SVZ environment. Collectively, our findings provide the very first evidence that the SVZ niche actively participates in GBM resistance to IR. Disrupting the CXCL12/CXCR4 signaling axis has indeed weakened the mesenchymal roots of SVZ-nested GBM cells and sensitized these cells to IR. Supplementary Material Supplementary material is available at Neuro-Oncology Journal online (http://neuro-oncology.oxfordjournals.org/). Funding This work was supported by grants from the National Fund for Scientific Research (F.N.R.S/F.R.I.A/TELEVIE); the Special Funds of the University of Liège; the Anti-Cancer Center near the University of Liège, and the Leon Fredericq Grant. J.K was supported by the Bohnenn Fund for Neuro-Oncology Research. Conflict of interest statement. None declared. Acknowledgments The authors want to thank the GIGA viral vector platform and the GIGA imaging platform for valuable technical support. References 1. Louis DN , Ohgaki H , Wiestler OD, et al.   . The 2007 WHO classification of tumours of the central nervous system. Acta Neuropathol  . 2007; 114 (2): 97– 109. Google Scholar CrossRef Search ADS PubMed  2. Furnari FB , Fenton T , Bachoo RM, et al.   . Malignant astrocytic glioma: genetics, biology, and paths to treatment. Genes Dev  . 2007; 21 (21): 2683– 2710. Google Scholar CrossRef Search ADS PubMed  3. Sadahiro H , Yoshikawa K , Ideguchi M et al.   . Pathological features of highly invasive glioma stem cells in a mouse xenograft model. Brain Tumor Pathol  . 2014; 31 (2): 77– 84. Google Scholar CrossRef Search ADS PubMed  4. Kroonen J , Nassen J , Boulanger YG, et al.   . Human glioblastoma-initiating cells invade specifically the subventricular zones and olfactory bulbs of mice after striatal injection. Int J Cancer  . 2011; 129 (3): 574– 585. Google Scholar CrossRef Search ADS PubMed  5. Goffart N , Kroonen J , Di Valentin E, et al.   . Adult mouse subventricular zones stimulate glioblastoma stem cells specific invasion through CXCL12/CXCR4 signaling. Neuro Oncol  . 2015; 17 (1): 81– 94. Google Scholar CrossRef Search ADS PubMed  6. Piccirillo SG , Spiteri I , Sottoriva A, et al.   . Contributions to drug resistance in glioblastoma derived from malignant cells in the sub-ependymal zone. Cancer Res  . 2015; 75 (1): 194– 202. Google Scholar CrossRef Search ADS PubMed  7. Chen L , Guerrero-Cazares H , Ye X, et al.   . Increased subventricular zone radiation dose correlates with survival in glioblastoma patients after gross total resection. Int J Radiat Oncol Biol Phys  . 2013; 86 (4): 616– 622. Google Scholar CrossRef Search ADS PubMed  8. Evers P , Lee PP , DeMarco J, et al.   . Irradiation of the potential cancer stem cell niches in the adult brain improves progression-free survival of patients with malignant glioma. BMC Cancer  . 2010; 10: 384. Google Scholar CrossRef Search ADS PubMed  9. Lee P , Eppinga W , Lagerwaard F, et al.   . Evaluation of high ipsilateral subventricular zone radiation therapy dose in glioblastoma: a pooled analysis. Int J Radiat Oncol Biol Phys  . 2013; 86 (4): 609– 615. Google Scholar CrossRef Search ADS PubMed  10. Liao A , Shi R , Jiang Y, et al.   . SDF-1/CXCR4 Axis Regulates Cell Cycle Progression and Epithelial-Mesenchymal Transition via Upregulation of Survivin in Glioblastoma. Mol Neurobiol  . 2016; 53 (1): 210– 215. Google Scholar CrossRef Search ADS PubMed  11. Bhat KP , Balasubramaniyan V , Vaillant B, et al.   . Mesenchymal differentiation mediated by NF-κB promotes radiation resistance in glioblastoma. Cancer Cell  . 2013; 24 (3): 331– 346. Google Scholar CrossRef Search ADS PubMed  12. Zhang P , Wei Y , Wang L et al.   . ATM-mediated stabilization of ZEB1 promotes DNA damage response and radioresistance through CHK1. Nat Cell Biol  . 2014; 16 (9): 864– 875. Google Scholar CrossRef Search ADS PubMed  13. Trautmann F , Cojoc M , Kurth I, et al.   . CXCR4 as biomarker for radioresistant cancer stem cells. Int J Radiat Biol  . 2014; 90 (8): 687– 699. Google Scholar CrossRef Search ADS PubMed  14. Seidel S , Garvalov BK , Acker T . Isolation and culture of primary glioblastoma cells from human tumor specimens. Methods Mol Biol  . 2015; 1235: 263– 275. Google Scholar PubMed  15. Mirzadeh Z , Doetsch F , Sawamoto K , Wichterle H , Alvarez-Buylla A . The subventricular zone en-face: wholemount staining and ependymal flow. J Vis Exp  . 2010; 6 39: 1938. 16. Adeberg S , Konig L , Bostel T, et al.   . Glioblastoma recurrence patterns after radiation therapy with regard to the subventricular zone. Int J Radiat Oncol Biol Phys  . 2014; 90 (4): 886– 893. Google Scholar CrossRef Search ADS PubMed  17. Bouquet F , Pal A , Pilones KA, et al.   . TGFbeta1 inhibition increases the radiosensitivity of breast cancer cells in vitro and promotes tumor control by radiation in vivo. Clin Cancer Res  . 2011; 17 (21): 6754– 6765. Google Scholar CrossRef Search ADS PubMed  18. Kokovay E , Goderie S , Wang Y, et al.   . Adult SVZ lineage cells home to and leave the vascular niche via differential responses to SDF1/CXCR4 signaling. Cell Stem Cell  . 2010; 7 (2): 163– 173. Google Scholar CrossRef Search ADS PubMed  19. Marie-Egyptienne DT , Lohse I , Hill RP . Cancer stem cells, the epithelial to mesenchymal transition (EMT) and radioresistance: potential role of hypoxia. Cancer Lett  . 2013; 341 (1): 63– 72. Google Scholar CrossRef Search ADS PubMed  20. Phillips HS , Kharbanda S , Chen R, et al.   . Molecular subclasses of high-grade glioma predict prognosis, delineate a pattern of disease progression, and resemble stages in neurogenesis. Cancer Cell  . 2006; 9 (3): 157– 173. Google Scholar CrossRef Search ADS PubMed  21. De Clercq E . The bicyclam AMD3100 story. Nature reviews. Drug Discov  . 2003; 2 (7): 581– 587. Google Scholar CrossRef Search ADS   22. Bao S , Wu Q , McLendon RE, et al.   . Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature  . 2006; 444 (7120): 756– 760. Google Scholar CrossRef Search ADS PubMed  23. Scadden DT . The stem-cell niche as an entity of action. Nature  . 2006; 441 (7097): 1075– 1079. Google Scholar CrossRef Search ADS PubMed  24. Calabrese C , Poppleton H , Kocak M, et al.   . A perivascular niche for brain tumor stem cells. Cancer Cell  . 2007; 11 (1): 69– 82. Google Scholar CrossRef Search ADS PubMed  25. Chiffer D , Mellai M , Annovazzi L, et al.   . Stem cell niches in glioblastoma: a neuropathological view. BioMed Res. Int  . 2014; 2014: 725921. Google Scholar PubMed  26. Goffart N , Kroonen J , Rogister B . Glioblastoma-initiating cells: relationship with neural stem cells and the micro-environment. Cancers  . 2013; 5 (3): 1049– 1071. Google Scholar CrossRef Search ADS PubMed  27. Kioi M , Vogel H , Schultz G , Hoffman RM , Harsh GR , Brown JM . Inhibition of vasculogenesis, but not angiogenesis, prevents the recurrence of glioblastoma after irradiation in mice. J Clin Invest  . 2010; 120 (3): 694– 705. Google Scholar CrossRef Search ADS PubMed  28. Domanska UM , Boer JC , Timmer-Bosscha H, et al.   . CXCR4 inhibition enhances radiosensitivity, while inducing cancer cell mobilization in a prostate cancer mouse model. Clin Exp Metastasis  . 2014; 31 (7): 829– 839. Google Scholar CrossRef Search ADS PubMed  29. Verhaak RG , Hoadley KA , Purdom E, et al.   . Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1. Cancer Cell  . 2010; 17 (1): 98– 110. Google Scholar CrossRef Search ADS PubMed  30. Lombard A , Goffart N , Rogister B . Glioblastoma Circulating Cells: Reality, Trap or Illusion? Stem Cells Int  . 2015; 2015: 182985. Google Scholar CrossRef Search ADS PubMed  31. Lv B , Yang X , Lv S, et al.   . CXCR4 Signaling Induced Epithelial-Mesenchymal Transition by PI3K/AKT and ERK Pathways in Glioblastoma. Mol Neurobiol  . 2014; 52 (3): 1263– 1268. Google Scholar PubMed  32. Yao C , Li P , Song H, et al.   . CXCL12/CXCR4 Axis Upregulates Twist to Induce EMT in Human Glioblastoma. Mol Neurobiol  . 2015. doi:10.1007/s12035-015-9340-x . 33. Singh A , Settleman J . EMT, cancer stem cells and drug resistance: an emerging axis of evil in the war on cancer. Oncogene  . 2010; 29 (34): 4741– 4751. Google Scholar CrossRef Search ADS PubMed  34. Hou KZ , Fu ZQ , Gong H . Chemokine ligand 20 enhances progression of hepatocellular carcinoma via epithelial-mesenchymal transition. World J Gastroenterol  . 2015; 21 (2): 475– 483. Google Scholar CrossRef Search ADS PubMed  35. Qu JQ , Yi HM , Ye X . MiRNA-203 Reduces Nasopharyngeal Carcinoma Radioresistance by Targeting IL8/AKT Signaling. Mol Cancer Ther  . 2015; 14 (11): 2653– 2664. Google Scholar CrossRef Search ADS PubMed  © The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Neuro-Oncology Oxford University Press

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Abstract

Abstract Background. Patients with glioblastoma (GBM) have an overall median survival of 15 months despite multimodal therapy. These catastrophic survival rates are to be correlated to systematic relapses that might arise from remaining glioblastoma stem cells (GSCs) left behind after surgery. In this line, it has recently been demonstrated that GSCs are able to escape the tumor mass and preferentially colonize the adult subventricular zone (SVZ). At a distance from the initial tumor site, these GSCs might therefore represent a high-quality model of clinical resilience to therapy and cancer relapses as they specifically retain tumor-initiating abilities. Method. While relying on recent findings that have validated the existence of GSCs in the human SVZ, we questioned the role of the SVZ niche as a potential GSC reservoir involved in therapeutic failure. Results. Our results demonstrate that (i) GSCs located in the SVZ are specifically resistant to radiation in vivo, (ii) these cells display enhanced mesenchymal roots that are known to be associated with cancer radioresistance, (iii) these mesenchymal traits are specifically upregulated by CXCL12 (stromal cell-derived factor-1) both in vitro and in the SVZ environment, (iv) the amount of SVZ-released CXCL12 mediates GBM resistance to radiation in vitro, and (v) interferes with the CXCL12/CXCR4 signalling system, allowing weakening of the tumor mesenchymal roots and radiosensitizing SVZ-nested GBM cells. Conclusion. Together, these data provide evidence on how the adult SVZ environment, through the release of CXCL12, supports GBM therapeutic failure and potential tumor relapse. CXCL12, glioblastoma, mesenchymal activation, radioresistance, subventricular zone Primary brain tumors are considered as one of the nastiest scourges faced in oncology. Their most aggressive form, glioblastoma (GBM, WHO grade IV), is also regarded as the most common and lethal subtype.1 Patients' poor survival rates are typically correlated with unsatisfactory therapeutic strategies leading to systematic GBM relapses.2 Trying to better understand the origin of those relapses might therefore be of clinical benefit for improving both outcomes and patients' survival. In this line, recent studies have reported that a fraction of GBM cells, further characterized as GBM stem cells (GSCs), show a specific tropism for the adult subventricular zone (SVZ).3–5 This body of experimental evidence has recently been brought into sharp focus following the identification of GSCs within the SVZ of GBM patients.6 Because they are away from their initial tumor site and reside in a permissive microenvironment, these SVZ-nested GSCs might therefore represent a reliable example of clinical resilience to therapy. Additionally, a second core group of clinical reports shows a correlation between doses of radiotherapy delivered to the SVZ and increased progression-free survival (PFS) as well as overall survival (OS) in newly diagnosed GBM patients.7–9 Together, these findings might be explained by the persistence of GSCs within the SVZ environment and advocate the need for better characterization of the communication lines between GSCs and the SVZ environment. In this work, we evaluated the influence of SVZ-related factors in GBM resistance to radiotherapy. More specifically, we questioned the role of SVZ-released CXCL12 in the extrinsic resistance of GSCs to irradiation (IR). As a matter of fact, the CXCL12/CXCR4 signaling axis has been recently identified as a key mediator of GBM mesenchymal activation in vitro,10 a process described as sustaining cancer radioresistance.11,12 Additionally, CXCR4 has further been identified as a potential biomarker for radioresistant cancer stem cells.13 Here, we demonstrate that SVZ-nested GSCs are specifically resistant to IR in vivo. These cells also display an enhanced mesenchymal signature in the SVZ compared with their counterparts from the tumor mass. Our results also demonstrate that these mesenchymal traits are specifically upregulated by CXCL12 both in vitro and in vivo. Interestingly, the use of a CXCL12/CXCR4 antagonist (AMD3100) allowed us to radiosensitize and disrupt the mesenchymal signature of SVZ-nested GBM cells simultaneously. Similarly, the in-vitro blockade of CXCL12 within the soluble environment of the SVZ revealed a significant reduction in the expression of GBM mesenchymal markers, together with a significant increase in the radiosensitivity of GBM cells. Together, these data emphasize the critical role of CXCL12 in mediating GBM resistance to radiotherapy and identify the adult SVZ environment as a potential niche involved in GBM recurrence. Methods Study Approval Patients gave informed consent for the use of their GBM specimens. Regarding human brain tissues, we obtained the patients’ written consent and the approval by the ethical committee of the University of Liège for experimental purposes. Cell Culture Human GBM primary cultures (GBM1, GBM2, and GB138) were established from consenting patients and cultivated as previously described.14 Additional details can be found in the Supplementary Data section. Animals Adult P40 immunodeficient nude mice, Crl:NU-Foxn1nu (obtained from Charles River Laboratories) were used for xenograft purposes. Athymic nude mice were housed in sterilized filter-topped cages and were processed with the consent of the ethical committee of the University of Liège. All animals were cared for in compliance with the guidelines of the Belgium Ministry of Agriculture and in accord with the ethical committee's laboratory animal care and use regulation (86/609/CEE, CE of J n_L358, December 18, 1986). Intracranial Transplantation and AMD3100 Treatment Intracranial xenografts were performed as described previously.4 AMD3100, a specific CXCL12/CXCR4 bicyclam antagonist, was injected twice a day for 10 consecutive days (1,25 mg/kg–Sigma). The injections started at week 10 post implantation. In Vivo Radiotherapy Protocol A dose of 30 Gy in 5 consecutive days was delivered in the whole brain with a dedicated small animal radiotherapy device (SmART Irradiator from Precision X-Ray Inc). Radiation was delivered using a photon beam (maximum energy of 225 kV and 13 mA), which provided a dose rate of 3 Gy/min. Additional details are provided in the Supplementary Data section. Whole-mount Dissection The lateral walls of the murine ventricles were dissected from the caudal aspect of the telencephalon as previously described.15 When dissecting the human SVZ, the brain was separated into 2 hemispheres, and the lateral walls of the ventricles were dissected from the caudal aspect of the telencephalon towards the cephalic region of the SVZ. This procedure was started within 15 minutes after the patient's death. The strips of SVZ tissue were separated into 3 parts (cephalic, median, and caudal) and cultured for 60 hours in DMEM/F12 (Life Technologies) supplemented with recombinant EGF (20 ng/mL, Preprotech) and recombinant FGF-2 (10 ng/mL, Preprotech) to prepare the human SVZ-conditioned medium. Gene-expression Profiling PCR arrays were performed using RT2 profiler PCR array (SABiosciences, Epithelial to Mesenchymal Transition PCR Array, PAHS-090A) on a LightCycler 480 qPCR system (Roche) according to the manufacturer's protocol. Additional details are provided in the Supplementary Data section. Western Blot Analysis Western blots were performed as previously described.5 Reagents, buffers, and antibodies are provided in the Supplementary Data section. Proteome Profiler Array Screening for the human SVZ-CM components was performed using the proteome profiler method (human chemokine array kit, R&D Systems) according to the manufacturer's protocol. Dot blots (standardized for loading control) were imaged with the ImageQuant 350 scanning system (cooled-CCD camera, GE Healthcare). Immunostaining Immunostainings were performed as previously described.5 Reagents, buffers, and antibodies are provided in the Supplementary Data section. Statistical Analysis Quantitative data are expressed as mean ± SEM. Two-way ANOVA was performed, followed by Tukey posttest, and a P value <.05 was considered statistically significant. Each experiment was run at least 3 times independently. Student t tests were performed for group comparison. All statistics were computed using Statistica 10.0 software. Results The Adult Subventricular Zone Acts as a Radioprotective Niche for Glioblastoma Cells To investigate the radioprotective role of the SVZ niche, we grafted RFP-positive GB138 primary cells into the right striatum of immunocompromised mice. Ten weeks after the implantation, 8 mice were submitted to brain-restricted doses of radiation (6 Gy) for 5 days. By the end of the 11th week, animals from both control and irradiated groups were euthanized. The efficacy of IR was assessed by histological examination of RFP-positive cells in the brain. As expected, control animals displayed massive infiltration of the corpus callosum (CC) and SVZ (Fig. 1 B and C).4 The number of GB138 primary cells dropped by 68% in the tumor mass (TM) (P = .027), 65% in the CC (P = .057), and 73% in the SVZ (P = .029) after IR (Fig. 1 A–D). These results specifically highlight the persistence of GBM cells in the CC and the SVZ environment after radiotherapy. These persisting cells, away from the initial tumor site (TM) might therefore play a key role in GBM recurrence and might corroborate with late periventricular patterns of recurrence observed in GBM patients every so often.16 Fig. 1 View largeDownload slide GB138 Primary cells leave the tumor mass and migrate through the corpus callosum to reach the subventricular zone (SVZ). The number of RFP-positive GB138 primary cells initially found in the striatum (A), corpus callosum (B), and subventricular zone (C) of nonirradiated animals significantly decreased in irradiated animals. A minimum of 5 mice were used in each group for quantification. GB138 primary cells were detected using a specific anti-RFP antibody (red). Cell nuclei were counterstained with DAPI (blue). Captions show where pictures were taken (D). Scale bars = 40 µm for A, B and C. * P < .05. Fig. 1 View largeDownload slide GB138 Primary cells leave the tumor mass and migrate through the corpus callosum to reach the subventricular zone (SVZ). The number of RFP-positive GB138 primary cells initially found in the striatum (A), corpus callosum (B), and subventricular zone (C) of nonirradiated animals significantly decreased in irradiated animals. A minimum of 5 mice were used in each group for quantification. GB138 primary cells were detected using a specific anti-RFP antibody (red). Cell nuclei were counterstained with DAPI (blue). Captions show where pictures were taken (D). Scale bars = 40 µm for A, B and C. * P < .05. Murine and Human SVZ-CM Mediate GBM Resistance to Radiation in Vitro To validate whether the SVZ endorses the role of a radioprotective niche for GBM cells, we focused on its soluble environment. To do so, we grew GBM2 primary cells and U87MG cells for 12 hours in minimal culture media (serum starvation). We then supplemented these GBM cells with murine SVZ-conditioned media (mSVZ-CM) and irradiated them (10 Gy) to assess the γH2AX response. Interestingly, both GBM2 primary cells and U87MG cells supplemented with mSVZ-CM prior to IR displayed a significant decrease in γH2AX reactivity compared with cells in control media (Fig. 2 A). A similar observation was made with GBM1 primary cells (Supplementary material, Fig. S1A). We then conducted a γH2AX kinetic on GBM2 primary cells and U87MG cells to further assess the DNA damage response. Again, we found that mSVZ-CM protected these 2 GBM cell populations from IR all along the different time points of the kinetic (P < .001, Fig. 2B). Similar observations were made with a 53BP1 kinetic to further confirm the radioprotective role of mSVZ-CM (Supplementary material, Fig. S1B). Together, these data show how mSVZ-CM counteracts with the DNA damage response by decreasing the radiosensitivity of GBM cells. Fig. 2 View largeDownload slide Mouse and human SVZ-CM mediate glioblastoma (GBM) resistance to radiation in vitro. Culture media were conditioned with murine SVZ whole-mounts for 60 hours and then added to GBM monolayers prior to irradiation (IR). This significantly decreased the sensitivity of GBM2 primary cells and U87MG cells to IR by reducing the DNA damage response (A). Radiodesensitization was associated with a significant decrease of γH2AX-positive cells. A γH2AX kinetic (t0, t15, t30, and t60) was performed on GBM2 primary cells and U87MG cells after IR. For each time point, both GBM cell populations activated fewer H2AX after IR when supplemented with subventricular zone-conditioned media (SVZ-CM) compared with control media (B). Irradiation of GBM2 primary cells and U87MG cells supplemented with olfactory bulb-conditioned media (OB-CM) or cerebellum-conditioned media (CRBL-CM) did not affect the DNA damage response as similar levels of γH2AX-positive cells were found in comparison with irradiated cells in control media (C). Culture media were conditioned with human SVZ whole-mounts for 60 hours and added to GBM monolayers (GBM2 and U87MG) prior to IR. This significantly decreased the sensitivity of both GBM2 primary cells and U87MG cells to IR by reducing the DNA damage response (D). The latter was specifically assessed by measuring the γH2AX response in these GBM populations following IR. Cell nuclei were counterstained with DAPI (blue). Scale bars = 10 µm for A. * P < .05, ** P < .01, *** P < .001. Fig. 2 View largeDownload slide Mouse and human SVZ-CM mediate glioblastoma (GBM) resistance to radiation in vitro. Culture media were conditioned with murine SVZ whole-mounts for 60 hours and then added to GBM monolayers prior to irradiation (IR). This significantly decreased the sensitivity of GBM2 primary cells and U87MG cells to IR by reducing the DNA damage response (A). Radiodesensitization was associated with a significant decrease of γH2AX-positive cells. A γH2AX kinetic (t0, t15, t30, and t60) was performed on GBM2 primary cells and U87MG cells after IR. For each time point, both GBM cell populations activated fewer H2AX after IR when supplemented with subventricular zone-conditioned media (SVZ-CM) compared with control media (B). Irradiation of GBM2 primary cells and U87MG cells supplemented with olfactory bulb-conditioned media (OB-CM) or cerebellum-conditioned media (CRBL-CM) did not affect the DNA damage response as similar levels of γH2AX-positive cells were found in comparison with irradiated cells in control media (C). Culture media were conditioned with human SVZ whole-mounts for 60 hours and added to GBM monolayers (GBM2 and U87MG) prior to IR. This significantly decreased the sensitivity of both GBM2 primary cells and U87MG cells to IR by reducing the DNA damage response (D). The latter was specifically assessed by measuring the γH2AX response in these GBM populations following IR. Cell nuclei were counterstained with DAPI (blue). Scale bars = 10 µm for A. * P < .05, ** P < .01, *** P < .001. We further verified whether this decline in radiosensitivity was specific to the SVZ environment. To do so, we irradiated GBM2 primary cells and U87MG cells, either supplemented with mSVZ-CM or murine olfactory bulb-conditioned media (mOB-CM) or murine cerebellum-conditioned media (mCRBL-CM). As expected, we showed a significant decrease in γH2AX-positive GBM2 and U87MG cells supplemented with mSVZ-CM following IR (P < .001). Interestingly, mOB-CM and mCRBL-CM did not impact the DNA damage response of GBM2 and U87MG cells following IR (10 Gy) (Fig. 2 C). We also compared the γH2AX response in U87MG cells isolated from the tumor mass (U87MG-TM) and from the SVZ (U87MG-SVZ) in the presence of mSVZ-CM. These GBM subpopulations were isolated according to a well-defined protocol4 and preincubated in mSVZ-CM or control media prior to IR (10 Gy). U87MG-TM cells displayed a γH2AX pattern of expression similar to U87MG cells. Indeed, mSVZ-CM allowed a significant decrease in the number of γH2AX-positive cells in this subpopulation after IR (P < .01). On the other hand, U87MG-SVZ cells significantly fewer γH2AX-positive cells when incubated in mSVZ-CM prior to IR (P < .05), further suggesting different patterns of DNA damage responses in these 2 GBM subpopulations (Supplementray material, Fig. S1C). GBM2 primary cells and U87MG cells were finally cultured for 12 hours in serum-starvation conditions and supplemented with human SVZ-CM (hSVZ-CM) prior to IR (10 Gy). hSVZ-CM allowed a significant decrease in the radiosensitivity of GBM2 primary cells and U87MG cells after IR (Fig. 2 D). Consistent with prior studies in human epithelial cells,17 this radio-desensitization was again correlated with a significant decrease in γH2AX reactivity (P < .001). Similar data were collected by assessing the expression of 53BP1 in response to IR in both GBM2 primary cells and U87MG cells supplemented with hSVZ-CM (P < .001, Supplemetary material, Fig. S1D). These results collectively provide evidence that the soluble environment of the SVZ helps to abrogate the DNA damage response of GBM cells after IR. Identification of SVZ-released CXCL12 as a Key Mediator of GBM Resistance to Irradiation and Survival in Vitro CXCL12 has recently been suggested to regulate the mesenchymal activation of cancer cells in vitro, especially through Snail and N-cadherin regulation.10 In this line, the process has also been reported to enhance GBM radioresistance in a NF-κB-dependent manner.11 Furthermore, we recently highlighted the expression of CXCL12 in mSVZ-CM (approximately 600 pg/mL).5,18 With reliance on these facts, we investigated whether SVZ-released CXCL12 could play a role in GBM radioresistance. To do so, we preincubated mSVZ-CM with specific CXCL12-blocking antibodies and/or nonrelevant immunoglobulins (IgG) for 45 minutes. We then supplemented GBM2 primary cells and U87MG cells with these 2 different mSVZ-CMs prior to IR (10 Gy) and quantified the impact of blocking CXCL12 on the γH2AX response of GBM cells. As expected, the number of γH2AX-positive GBM2 and U87MG cells had decreased significantly in mSVZ-CM and mSVZ-CM/IgG conditions (Fig. 3 A). Interestingly, the blockade of CXCL12 allowed sensitization of these 2 GBM populations to IR (P < .001, Fig. 3 A). However, the inhibition of CXCL12 did not completely restore the number of γH2AX-positive cells encountered without any mSVZ-CM supplementation, further suggesting the role of other SVZ-released components in GBM resistance to IR in vitro. Fig. 3 View largeDownload slide Identification of CXCL12 as a key mediator of glioblastoma (GBM) survival and resistance to radiation. The inhibition of CXCL12 in the mSVZ-CM prior to irradiation (IR) leads to a significant rescue of radiosensitization in human GBM2 primary cells and U87MG cells (A). The number of γH2AX-positive cells was higher after the blockade of CXCL12 in both GBM populations compared with cells supplemented with mSVZ-CM and a nonrelevant immunoglobulin (IgG). Conversely, growing concentrations of recombinant CXCL12 (25/50/100 nM) significantly increased GBM2 primary cells and U87MG cells radioprotection in a dose-dependent manner (B). mSVZ-CM significantly propelled the proliferation of U87MG cells compared with U87MG cells in control media (C–D). U87MG cells supplemented with mSVZ-CM prior to IR displayed stronger proliferation abilities than irradiated U87MG cells in control media (D). U87MG cells supplemented with mSVZ-CM and specific CXCL12 blocking antibodies prior to IR had the same proliferation rate compared with irradiated U87MG cells in control media (D). * P < .05, ** P < .01, *** P < .001. Fig. 3 View largeDownload slide Identification of CXCL12 as a key mediator of glioblastoma (GBM) survival and resistance to radiation. The inhibition of CXCL12 in the mSVZ-CM prior to irradiation (IR) leads to a significant rescue of radiosensitization in human GBM2 primary cells and U87MG cells (A). The number of γH2AX-positive cells was higher after the blockade of CXCL12 in both GBM populations compared with cells supplemented with mSVZ-CM and a nonrelevant immunoglobulin (IgG). Conversely, growing concentrations of recombinant CXCL12 (25/50/100 nM) significantly increased GBM2 primary cells and U87MG cells radioprotection in a dose-dependent manner (B). mSVZ-CM significantly propelled the proliferation of U87MG cells compared with U87MG cells in control media (C–D). U87MG cells supplemented with mSVZ-CM prior to IR displayed stronger proliferation abilities than irradiated U87MG cells in control media (D). U87MG cells supplemented with mSVZ-CM and specific CXCL12 blocking antibodies prior to IR had the same proliferation rate compared with irradiated U87MG cells in control media (D). * P < .05, ** P < .01, *** P < .001. Conversely to the blockade of CXCL12, we conducted rescue experiments to prove that CXCL12 could act as a radioprotective chemokine for GBM cells. We grew GBM2 primary cells and U87MG cells for 12 hours in serum-starvation conditions. We next stimulated these 2 GBM populations with growing concentrations of recombinant CXCL12 (rCXCL12 at 25 nM, 50 nM, and 100 nM) prior to IR (10 Gy) and assessed the γH2AX response within each of the 3 conditions. We noticed a significant decrease of γH2AX-positive GBM2 primary cells starting at 50 nM (P = .02, Fig. 3 B). A similar observation was made in U87MG cells at a concentration of 25 nM (P = .04, Fig. 3 B). This slight but constant radio-desensitization was observed up to 100 nM in both GBM cell populations (P < .001). We finally investigated the impact of mSVZ-CM on GBM cell survival and growth abilities. We seeded 7500 U87MG cells in 6-well plates and serum-starved them for 12 hours. mSVZ-CM was then added to U87MG monolayers prior to IR (4 Gy). Six hours later, U87MG cells were refreshed in DMEM supplemented with fetal bovine serum (10%) and cultured at an exponential growth phase for 7 days (Fig. 3 C). Besides IR, we noticed that mSVZ-CM had a significant positive impact on the proliferation of U87MG cells. These cells proliferated twice as much (2.03/1 ratio) compared with U87MG cells in control media (P < .001) (Fig. 3 D). We then estimated the impact of an IR dose of 4 Gy on the survival of GBM cells. Irradiating U87MG monolayers led to a 43.5% reduction of cell survival (P < .001) (Fig. 3 D). Surprisingly, the addition of mSVZ-CM to U87MG monolayers prior to IR significantly propelled their proliferation as U87MG cells proliferated 2.5 times faster compared with control media (P < .001) (Fig. 3 D). Similar data were collected with GBM2 primary cells (Supplementary material, Fig. S2). We also assessed the role of SVZ-released CXCL12 on GBM cell survival and growth abilities after IR by blocking its expression in mSVZ-CM. Irradiated U87MG monolayers supplemented with a combination of mSVZ-CM and specific CXCL12 blocking antibodies grew at the same rate as irradiated U87MG cells in control media (Fig. 3 D). Collectively, these data demonstrated how mSVZ-CM actively promotes GBM cell proliferation and survival after IR in a CXCL12-dependent manner. Expression of CXCL12 by the Human SVZ and Role in GBM Radioprotection Since we demonstrated that hSVZ-CM specifically protects GBM cells from IR in vitro, we next investigated the hypothetical role of hSVZ-released CXCL12 in GBM radioprotection. To do so, we dissected the SVZ of a fresh human brain in 3 distinct regions (cephalic, median, and caudal, Fig. 4 A) and independently conditioned culture media with each of these regions for 60 hours. ELISA experiments highlighted a significant difference of CXCL12 expression between the cephalic/median hSVZ-CM and its caudal part (P = .004, Fig. 4 A). These findings were further confirmed by Western blots, showing the preferential expression of CXCL12 in the cephalic/median human SVZ compared with the caudal region (Fig. 4 B). Fig. 4 View largeDownload slide Expression of CXCL12 in the human subventricular zone (SVZ) and validation of its role in glioblastoma (GBM) radioprotection. The presence of CXCL12 in the hSVZ-CM was assessed by ELISA (A) and further confirmed by Western-blot analysis (B). The blockade of CXCL12 in hSVZ-CM prior to irradiation (IR) induced a partial loss of radioprotection in GBM2 primary cells and U87MG cells (C). This observation correlated with a significant decrease of γH2AX reactivity in these 2 GBM populations. CXCL12, hSVZ-CM also contains different proteins such as IL-8, MIP-3α and PF4 among others. * P < .05, ** P < .01, *** P < .001. Fig. 4 View largeDownload slide Expression of CXCL12 in the human subventricular zone (SVZ) and validation of its role in glioblastoma (GBM) radioprotection. The presence of CXCL12 in the hSVZ-CM was assessed by ELISA (A) and further confirmed by Western-blot analysis (B). The blockade of CXCL12 in hSVZ-CM prior to irradiation (IR) induced a partial loss of radioprotection in GBM2 primary cells and U87MG cells (C). This observation correlated with a significant decrease of γH2AX reactivity in these 2 GBM populations. CXCL12, hSVZ-CM also contains different proteins such as IL-8, MIP-3α and PF4 among others. * P < .05, ** P < .01, *** P < .001. We then supplemented hSVZ-CM with CXCL12-blocking antibodies and/or nonrelevant IgG for 45 minutes and finally grew GBM2 primary cells and U87MG in these 2 different media prior to IR (10 Gy). Similar amounts of γH2AX-positive cells were found in both hSVZ-CM and hSVZ-CM/IgG conditions (Fig. 4 C). Interestingly, the blockade of CXCL12 allowed radiosensitization of GBM2 primary cells and U87MG cells (P < .001 and P = .02, respectively) (Fig. 4 C). Again, the CXCL12 inhibition did not completely restore the number of γH2AX-positive cells encountered without any hSVZ-CM supplementation (CTL), suggesting the role of other SVZ-released components in GBM resistance to IR in vitro. In this line, we performed a proteome-profiling analysis of hSVZ-CM and specifically highlighted the expression of different proteins already known to play a role in cancer radioresistance including MIP-3α, PF4, and IL-8 among others ( Fig. 4 D). SVZ-released CXCL12 Promotes the Mesenchymal Activation of GBM Cells So far, we have demonstrated that CXCL12 endorses a key role in GBM resistance to IR, but the mechanisms working behind the scene remained unclear. In this context, CXCL12 has recently been suggested to regulate the mesenchymal activation of GBM in vitro,10 a process known to be involved in cancer radioresistance.19 Moreover, the acquisition of a mesenchymal signature corresponds to the most rebellious type of response to therapy in GBM.20 Relying on these findings, we questioned the role of SVZ-released CXCL12 in promoting the mesenchymal activation of GBM cells. In order to identify key mediators involved in the mesenchymal activation of GBM cells, we conducted wide-scale RT-qPCR screening on CXCL12-stimulated GB138 primary cells. This analysis highlighted a significant increase in the expression of 26 genes typically involved in the acquisition of a mesenchymal phenotype (Supplementary material, Fig. S3A and B). Among these genes, we showed the upregulation of 3 crucial mesenchymal markers including CDH2, FN1, and vimentin (Fig. 5 A) as well as key transcription factors including ESR1, FOXC2, SOX10, and ZEB2 (Fig. 5 B). Fig. 5 View largeDownload slide Subventricular (SVZ)-released CXCL12 promotes the mesenchymal activation of glioblastoma (GBM) cells. CXCL12-stimulated GB138 primary cells upregulated 3 major mesenchymal genes including CDH2, FN1 and vimentin and 4 key EMT-related transcription factors including ESR1, FOXC2, SOX10, and ZEB2 (A-B). Both CXCL12-stimulated GB138 primary cells and U87MG cells overexpressed mesenchymal proteins including N-cadherin and vimentin (C). Pretreatment of GB138 primary cells and U87MG cells with AMD3100 prior to CXCL12 treatment disrupted the expression of N-cadherin and vimentin (C). Fig. 5 View largeDownload slide Subventricular (SVZ)-released CXCL12 promotes the mesenchymal activation of glioblastoma (GBM) cells. CXCL12-stimulated GB138 primary cells upregulated 3 major mesenchymal genes including CDH2, FN1 and vimentin and 4 key EMT-related transcription factors including ESR1, FOXC2, SOX10, and ZEB2 (A-B). Both CXCL12-stimulated GB138 primary cells and U87MG cells overexpressed mesenchymal proteins including N-cadherin and vimentin (C). Pretreatment of GB138 primary cells and U87MG cells with AMD3100 prior to CXCL12 treatment disrupted the expression of N-cadherin and vimentin (C). Thereafter, we stimulated both GB138 primary cells and U87MG cells with rCXCL12 (20 nM) and assessed the expression level of mesenchymal proteins. Our results showed an increase in N-cadherin expression in both GBM populations within one hour of stimulation (Fig. 5 C). The expression of vimentin, a type III intermediate filament protein specifically expressed in mesenchymal cells, was also found to be upregulated in U87MG cells within an hour (Fig. 5 C). The vimentin expression started to increase after 4 hours of stimulation in GB138 primary cells, suggesting different posttranslational regulations in U87MG cells and GB138 primary cells (Fig. 5 C). Interestingly, the upregulation of N-cadherin and vimentin in U87MG cells as well as GB138 primary cells was hampered by pretreating these GBM populations with a specific CXCL12/CXCR4 antagonist (AMD310021) prior to the rCXCL12 stimulation (Fig. 5 C). These findings particularly highlighted how CXCL12 mediates the acquisition of an enhanced mesenchymal phenotype in both GB138 primary cells and U87MG cells in vitro. AMD3100 Sensitizes SVZ-nested GBM Cells to Radiation in Vivo Since CXCL12 enhances GBM mesenchymal roots, we then looked for a potential mesenchymal signature that would be an indicator of therapeutic resistance in GBM cells nested in the SVZ environment. Western blot analyses on GB138 primary cells isolated from the TM and SVZ surprisingly revealed a higher expression level of N-cadherin and vimentin in GB138-SVZ cells (Fig. 6 A). This observation suggested a SVZ-dependent enhancement of GBM mesenchymal traits through the release of CXCL12 in the nearby environment. To prove this, xenograft experiments were performed with GB138 primary cells. Half of the cohort was treated twice a day for 10 days with intraperitoneal injections of AMD3100 (1.25 mg/kg), whereas the other group was treated with PBS (control). Histological examination of brain coronal sections confirmed the expression of N-cadherin and vimentin (red) in GB138 primary cells (green) located in the ventricular walls of control animals (Fig. 6 B). Interestingly, the in vivo use of AMD3100 significantly decreased the amount of GB138 primary cells invading both the CC and the SVZ regions (P < .01 and P < .001, respectively; Supplementary material, Fig. S4A) and disrupted the expression of vimentin and N-cadherin in SVZ-nested GB138 primary cells (Fig. 6 C). These results demonstrate how SVZ-released CXCL12 plays a key role in the acquisition of GBM mesenchymal properties, a signature typically associated with therapeutic resistance.20 Fig. 6 View largeDownload slide Inhibition of CXCL12 in the subventricular zone (SVZ) weakens the tumor mesenchymal roots and promotes radiosensitization. GB138 primary cells isolated from the SVZ exhibit a higher expression level of N-cadherin and vimentin than GB138 primary cells isolated from tumor mass (A). Immunohistostaining on brain coronal sections of mice grafted with GB138 primary cells confirmed the expression of N-cadherin and vimentin (red) in SVZ-nested G138 primary cells (green) (B). The use of AMD3100 in vivo disrupted the expression of N-cadherin and vimentin (red) in SVZ-nested GB138 primary cells (green) (C). Combining AMD3100 and irradiation (IR) treatments allowed a significantly decreased number of GB138 primary cells in the corpus callosum and the SVZ compared with irradiated mice only (D). GB138 primary cells were labeled with a specific anti-RFP antibody. Scale bars = 20 µm for B and C and 30 µm for D. * P < .05. Fig. 6 View largeDownload slide Inhibition of CXCL12 in the subventricular zone (SVZ) weakens the tumor mesenchymal roots and promotes radiosensitization. GB138 primary cells isolated from the SVZ exhibit a higher expression level of N-cadherin and vimentin than GB138 primary cells isolated from tumor mass (A). Immunohistostaining on brain coronal sections of mice grafted with GB138 primary cells confirmed the expression of N-cadherin and vimentin (red) in SVZ-nested G138 primary cells (green) (B). The use of AMD3100 in vivo disrupted the expression of N-cadherin and vimentin (red) in SVZ-nested GB138 primary cells (green) (C). Combining AMD3100 and irradiation (IR) treatments allowed a significantly decreased number of GB138 primary cells in the corpus callosum and the SVZ compared with irradiated mice only (D). GB138 primary cells were labeled with a specific anti-RFP antibody. Scale bars = 20 µm for B and C and 30 µm for D. * P < .05. Relying on these data, new xenograft experiments with RFP-positive GB138 primary cells were conducted to assess the efficacy of combining radiotherapy with adjuvant AMD3100 chemotherapy. Half of the cohort was treated twice a day for 10 days with intraperitoneal injections of AMD3100 (1.25 mg/kg), whereas the other group was treated with PBS. Brain-restricted radiotherapy started 5 days after the initial injection of AMD3100 and lasted for 5 days. Histological examination of brain coronal sections highlighted a significant decrease in the number of GB138 primary cells found in the CC and SVZ regions of irradiated/AMD3100-treated animals compared with irradiated animals (P = .015). On the other hand, AMD3100 had no effect on the number of cancer cells found in the TM after IR (Fig. 6 D and Supplementary material, Fig. S4B). Collectively, these data show that interfering with SVZ-released CXCL12 allows sensitization of SVZ-nested GBM cells to radiotherapy. Discussion The present study has demonstrated that a fraction of GBM cells hiding in the SVZ environment are resistant to radiotherapy. Of importance, we previously demonstrated that GBM cells located in the SVZ are enriched in tumor-initiating capacities and were in this way characterized as GSC. 4,5 Interestingly, GSC display intrinsic radioresistant abilities.22 It thus makes sense to find a fraction of tumor cells remaining in the SVZ environment after IR; however, we questioned the role of the SVZ niche as a potential GBM reservoir involved in GSC extrinsic resistance to IR. We specifically assumed that the composition of the niche finely regulates both fate specification and protection of these GSCs. Physiologically, the SVZ acts as a supportive niche, promoting self-renewal of neural stem cells and inhibiting differentiation.23 This “seed-and-soil” relationship has also been adapted to cancer stem cell research as GSCs also rely on specific niches to maintain their stem cell properties and ability to drive tumor growth.24–26 In this line, Piccirillo et al. recently showed that GSCs isolated from the human SVZ are specifically resistant to supramaximal chemotherapy doses along with differential patterns of drug response.6 Together, these findings suggest that the SVZ environment might provide GBM cells with radio/chemoprotective inputs and could play a determinant role in malignant brain tumor recurrence. To further explore the role of the SVZ in GBM resistance to IR, we established an in-vitro protocol of radiotherapy in which we focused on the soluble environment of the SVZ (SVZ-CM). This experimental protocol allowed us to highlight a significant increase of radioprotection in GBM cells stimulated with SVZ-CM. This observation was made with SVZ-CM collected from both mice and human donors. We next identified CXCL12 as a key mediator involved in GBM radioprotection. The blockade of CXCL12 in SVZ-CM allowed sensitizing GBM cells to IR in vitro. Concomitant to our findings, the pharmacological inhibition of CXCL12 in combination with IR was shown to abrogate GBM regrowth in mice by preventing the development of functional tumor blood vessels post-IR.27 Domanska et al. also reported that the inhibition of CXCL12-dependent protective signals from stromal cells renders prostate cancer cells more sensitive to ionizing radiations.28 Collectively, these data advocate for a better characterization of CXCL12 inhibitors in preclinical cancer models. Their potential therapeutic benefits, in combination with IR, might indeed contribute to understanding the role of CXCL12 in GBM resistance to therapy and could facilitate translation of these inhibitors to the clinic. Besides the role of SVZ-released CXCL12 in GBM resistance to IR, the mechanisms underlying these findings were yet to be determined. To address the issue, we focused on the tumor's mesenchymal profile. The acquisition of a mesenchymal phenotype is indeed associated with poor outcomes and corresponds to the most rebellious type of GBM to therapy.20,29 GBMs are known to frequently shift toward a mesenchymal status upon recurrence.29,30 With that in mind, we demonstrated that GBM cells express a basal level of vimentin and N-cadherin that further suggests the mesenchymal origin of our GBM populations. Interestingly, these 2 mesenchymal markers were specifically upregulated upon CXCL12 stimulation at the mRNA and protein levels. The inhibition of the CXCL12/CXCR4 signaling axis in GBM has been recently reported to affect the in-vitro expression of mesenchymal markers including Twist, Snail, and N-cadherin (which supports our findings).31,32 Interestingly, we showed that GBM cells hosted by the SVZ display a higher expression level of N-cadherin and vimentin compared with the rest of the tumor. In this line, the use of AMD3100, a specific CXCL12/CXCR4 antagonist,21 allowed weakening of the tumor's mesenchymal signature within the SVZ and sensitized both CC and SVZ-nested GBM cells. These data demonstrate how SVZ-released CXCL12 undertakes a key role in GBM radioprotection by underlying the acquisition of strong tumor mesenchymal roots. In light of our findings, many studies have established a close link between GBM's mesenchymal activation and its ability to better resist chemotherapy.33 By contrast, very little is known about the involvement of this process in radioresistance. To our knowledge, a single study so far has reported that the mesenchymal activation in GBM promotes resistance to IR in a NF-κB-dependent manner.11 A recent study has also shown that ATM kinase stabilizes ZEB1 in response to DNA damage.12 Interestingly, the expression of ZEB1 is upregulated upon CXCL12 stimulation in GB138 primary cells (Supplementary material, Fig. S2B). ZEB1 then directly interacts with USP7 to enhance its ability to de-ubiquitinate CHK1, thereby promoting homologous recombination-dependent DNA repair and resistance to IR in breast cancer cells.12 Whether Nf-κB and/or ZEB1 are specifically involved in our model of GBM resistance to IR remains to be elucidated. Besides CXCL12, we have also shown the expression of different effectors in the human SVZ-CM that could further undertake a key role in GBM resistance to treatment. Among these proteins, MIP3α and IL8 are known to enhance the epithelial-to-mesenchymal transition of hepatocellular carcinoma and sustain nasopharyngeal carcinoma radioresistance, respectively.34,35 Together these other SVZ components could support CXCL12 in mediating GBM resistance to IR in the SVZ environment. Collectively, our findings provide the very first evidence that the SVZ niche actively participates in GBM resistance to IR. Disrupting the CXCL12/CXCR4 signaling axis has indeed weakened the mesenchymal roots of SVZ-nested GBM cells and sensitized these cells to IR. Supplementary Material Supplementary material is available at Neuro-Oncology Journal online (http://neuro-oncology.oxfordjournals.org/). Funding This work was supported by grants from the National Fund for Scientific Research (F.N.R.S/F.R.I.A/TELEVIE); the Special Funds of the University of Liège; the Anti-Cancer Center near the University of Liège, and the Leon Fredericq Grant. J.K was supported by the Bohnenn Fund for Neuro-Oncology Research. Conflict of interest statement. None declared. Acknowledgments The authors want to thank the GIGA viral vector platform and the GIGA imaging platform for valuable technical support. References 1. Louis DN , Ohgaki H , Wiestler OD, et al.   . The 2007 WHO classification of tumours of the central nervous system. Acta Neuropathol  . 2007; 114 (2): 97– 109. Google Scholar CrossRef Search ADS PubMed  2. Furnari FB , Fenton T , Bachoo RM, et al.   . Malignant astrocytic glioma: genetics, biology, and paths to treatment. Genes Dev  . 2007; 21 (21): 2683– 2710. Google Scholar CrossRef Search ADS PubMed  3. Sadahiro H , Yoshikawa K , Ideguchi M et al.   . Pathological features of highly invasive glioma stem cells in a mouse xenograft model. Brain Tumor Pathol  . 2014; 31 (2): 77– 84. Google Scholar CrossRef Search ADS PubMed  4. Kroonen J , Nassen J , Boulanger YG, et al.   . 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Neuro-OncologyOxford University Press

Published: Jul 1, 2016

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