Stem-like signatures in human meningioma cells are under the control of CXCL11/CXCL12 chemokine activity

Abstract

Background: Meningiomas are mainly benign brain tumors, although about 20% of histologically benign cases are clinically aggressive and recur after resection. We hypothesize that meningioma brain invasiveness and recurrence may be related to the presence of cancer stem cells and their high responsiveness to the CXCL12-CXCR4/CXCR7 chemokine axis. The aim of this study was to isolate meningioma stem cells from human samples, characterize them for biological features related to malignant behavior, and to identify the role of CXCR4/CXCR7 in these processes.

Methods: Meningioma stem cells were isolated from patient-derived primary cultures in stem cell-permissive conditions, and characterized for phenotype, self-renewal, proliferation and migration rates, vasculogenic mimicry (VM), and in vivo tumorigenesis, in comparison with differentiated meningioma cells and stem-like cells isolated from normal meninges. These cell populations were challenged with CXCL12 and CXCL11 and receptor antagonists to define the chemokine role in stem cell-related functions.

Results: Stem-like cells isolated from meningioma cultures display higher proliferation and migration rates, and VM, as compared to meningioma non-stem cells or cells isolated from normal meninges and were the only tumorigenic population in vivo. In meningioma cells, these stem-like functions were under the control of the CXCR4/CXCR7 chemokine axis.

Conclusions: We report a role for CXCL11 and CXCL12 in the control of malignant features in stem-like cells isolated from human meningioma, providing a possible basis for the aggressive clinical behavior observed in subsets of these tumors. CXCR4/CXCR7 antagonists might represent a useful approach for meningioma at high risk of recurrence and malignant progression.

Keywords: CXCL11/CXCL12; meningioma; stem cells; tumorigenesis.

Figure 1.

Isolation and characterization of patient-derived stem-like and differentiated cells from meningiomas. (A) Representative images of meningioma primary culture morphology (a-b-g) after selection in stem-permissive medium (STEM, c-d-h) or maintained in serum-containing medium (DIFF, e-f-i). Scale bars: 150 μm (a-c-e-g-h-i), 50 μm (b-d-f). (B)Uniform Manifold Approximation and Projection representation of single cell RNA-seq profiles of PD-Mg-S (stem) and PD-Mg-D (diff) isolated from M69 and M71. Dots are colorized accordingly the PCA clustering of their profile. A small number (about 1.2%) of monocytes and CD8 T lymphocytes were also detected, representing residual cells persisting since few passages from the primary culture were performed (not reported in the graph for sake of clarity). (C) Flow-cytometry analysis of mesenchymal markers (CD73, CD90, and CD105) and negative indicators (CD14, CD19, CD31, CD34, CD45, and HLA-DR). p0 cultures display a mesenchymal stem cell profile in a mixed population, while p1, grown in the STEM medium (PD-Mg-S), showed homogeneous CD73⁺/CD90⁺/CD105⁺ phenotype and depletion of hematopoietic and endothelial contaminants. (D) Flow-cytometry analysis comparing CD73⁺/CD90⁺/CD105⁺ in PD-Mg-S (STEM), PD-Mg-D (DIFF) (N = 6) and PD-NMe-S (NORM), at p2. Scatter dot plots depict percentage of marker positive cells from individual cultures; lines represent mean ± SD. (E) Representative immunoblots of CD90 and CD105 expression in PD-Mg-S and PD-Mg-D matching cultures (N = 4, tumor ID#: M28-M30-M33-M34). β-actin was used as reference for protein loading. Densitometric analysis is reported in Supplementary Figure 4. (F) Stacked bar chart shows the number of cultures able to differentiate into osteogenic and adipogenic lineages (white) on the total of PD-Mg-S (N = 6) and PD-NMe-S (N = 2) examined. (G) Growth curves of cultures demonstrating the sustained proliferation of PD-Mg-S as compared to matched PD-Mg-D (N = 6), and PD-NMe-S (N = 1). Each point represents mean ± SD of the cumulative time-course expressed as percentage of respective time 0. (H) Analysis of sphere-formation from low density meningioma cultures. Pie charts show that all PD-Mg-S (N = 9) generate meningospheres with high or low efficiency, while only 15% sphere-forming cultures originated from the corresponding PD-Mg-D. Phase-contrast images of 10 days–cultures depict representative tumor spheroids in stem (scale bar: 100 μm) and differentiating (scale bar: 50 μm) conditions.

Figure 2.

Pattern of expression of markers in meningioma stem-like cells and differentiated counterpart. (A) Representative IF images of PD-Mg-S, PD-Mg-D, and PD-NMe-S showing markers’ expression within cell monolayers. Scale bars: 100 μm. (B) Stacked bar graph depicting the IF results of Figure 2A, as the number of positive (white) and negative (black) cultures. (C) Quantitative RT-PCR analysis of SSTR2 mRNA levels in PD-MG-S and PD-Mg-D cultures (N = 6). Histogram shows the mean ± SD of tested cultures. (D) SSTR2 expression in PD-Mg-S and PD-Mg-D cells. Representative IF images are reported. Scale bars: 100 μm. (E) Representative immunoblots for Sox2 and Oct4 in PD-Mg-S and PD-Mg-D matching cultures (N = 4, tumor ID# M28-M30-M33-M343). β-actin was used as reference for protein loading. (F) Histogram reports the densitometric analysis of normalized protein levels.

Figure 3.

Meningioma stem-like cells show migratory and vasculogenic potential in vitro and tumorigenesis in vivo. (A) Representative photomicrographs of transwell migration assays of green fluorescent-labeled PD-Mg-S (N = 11) and PD-Mg-D (N = 8). Scatter dot plot depicts the quantification of transmigrated cells toward FBS in each culture. Migration was assessed after 16 hours to avoid interference of cell proliferation. Lines represent mean ± SD *P < .05, **P < .01 versus respective control (CTR); °P < .05 versus FBS chemoattractant. Scale bar: 200 μm. (B) Representative photomicrographs of tube formation in the presence of VEGF. Capillary-like networks were more efficiently formed by PD-Mg-S (N = 6) as compared to PD-Mg-D (N = 4). Scatter dot plot shows the statistical results of the quantification of each culture. Lines represent mean ± SD. ***P < .001 versus respective control (CTR); °°P < .01 versus VEGF stimulation. Scale bar: 50 μm. (C)WT embryonic mouse brain supports PD-Mg-S growth. a) Method for cell labeling using CM-DiI or lentivirus-expressing dsRED or GFP. b) Cell were transplantated within second ventricle (V) of E12.5 embryos, brains were analyzed post-transplantation at E18.5-P0. c) Dorsal view of a mouse head at E18.5; arrow: dsRED⁺ xenograft; dashed lines: Brain boundaries. d) IF image of PD-Mg-S mass in the mice meninges, coronal cryosections through embryonic brains; red: dsRED⁺ tumor (T) cells; dashed lines: Meninge (M) boundaries; NCx: Neocortex. e) IF images of PD-Mg-S in the secondV of embryonic brains; red: CM-DiI⁺PD-Mg-S; green: HuNu⁺; dashed lines: V boundary; dotted line: T mass. f) images of PD-MC in the secondV of embryonic brains; red: dsRED⁺ PD-Mg-S; green: Epithelial membrane antigen (EMA). Dashed lines: V boundary; dotted line: T mass; arrow: EMA⁺ cells invading the host parenchyma. Nuclei are counterstained with DAPI (blue). Scale bars: c) 2 mm; d) 100 μm; e) and f) 200 μm. (D) Patient-derived xenografts (PDX) are only detected upon transplantation of PD-Mg-S in WT embryonic mouse brain. IF image of cells in mice secondV analyzed at E18.5-P0. Blue: Nuclei (DAPI), red: CM-DiI⁺ PD-Mg-S (STEM) and PD-Mg-D (DIFF), or GFP⁺ PD-NMe-S (NORM); dashed lines: V boundary; dotted line: tumor (T). Neocortex (NCx); mouse meninges (m). Scale bar: 200 μm.

Figure 4.

Differential expression of CXCR4 and CXCR7 and their ligands in meningioma-derived cultures and xenografts. (A) Chemokine ligands and receptors mRNA levels in PD-Mg-S and PD-Mg-D (N = 6) and PD-NMe-S (N = 2). Scatter dot plot reports individual cultures. Lines represent mean ± SD. *P < .05. (B) Fold changes of gene expression for CXCR4 and CXCL11 and CXCR7 and CXCL12 in PD-Mg-S in comparison to PD-Mg-D. (C) Representative IF images showing CXCR4 homogenously expressed in PD-Mg-S cells and CXCR7 is restricted to cell subsets. Blue: Nuclei (DAPI). Scale bars: 100 μm. (D) Xenotransplants of PD-Mg-S express CXCR4 and CXCR7. IF image of cells transplanted in the mouse embryo second V analyzed at E18.5-P0. a, b) Cells expressing CXCR4 (arrow). Note the expression of CXCR4 in host murine meninges (m). c, d) Cells expressing CXCR7 (arrowheads). Blue: nuclei (DAPI); red: CM-DiI⁺; green: CXCR4; gray: E-Cadherin (a, b); green: CXCR7 (c, d). Dashed lines: tissue boundary; dotted line: tumor (T) boundary. NCx: Neocortex. Scale bars: a, c) 100 μm; b) 35 μm; d) 50 μm.

Figure 5.

CXCL11-12/CXCR4-7 system supports proliferation, migration, and neo-angiogenesis in meningioma stem-like cells. (A) In vitro proliferation of PD-Mg-S (N = 11), PD-Mg-D (N = 6), and PD-NMe-S treated with CXCL12 or CXCL11 for 24 hours. Scatter dot plot depicts the percentage of cell proliferation versus vehicle-treated controls (CTR, 100%); lines represent mean ± SD. Each point represents one individual tumor. *P < .05 versus respective CTR. (B) Representative photomicrographs of transwell migration of green-fluorescent PD-Mg-S (N = 14) and PD-Mg-D (N = 7) toward CXCL11 or CXCL12 (25 nM). Scale bar: 200 μm. Scatter dot plot depicts the quantification of transmigrated cells in each culture. Each points represents one individual tumor. Lines represent mean ± SD. **P < .01 and ***P < .001, versus respective control (CTR, 100%), °°°P < .001 versus chemokine stimulation. Scale bar: 150 μm. (C) Representative photomicrographs of tube networks formed by PD-Mg-S (N = 10) or PD-Mg-D (N = 4), after exposure to CXCL11 or CXCL12 (25 nM). Scatter dot plot shows the quantification of tube formation. Each points represents one individual tumor. Lines represent mean ± SD. *P < .05 versus respective control (CTR, 100%). (D) PD-Mg-S-derived PDX in mouse embryo contain CD31⁺ cells. Images depict two independent tumors: Panels a-d refer to a mass developed in the host brain parenchyma in proximity of the meninges; panels e-h depict a tumor mass developed inside the second ventricle. Panel i is an enlargement of panel e. Injected PD-Mg-S were stained with Ds Red (red), nuclei counterstained with Hoechst (blue), and CD31 staining is evidenced in cyan (Alexa Fluor®647). Scale bar: 20 µm.

Figure 6.

Pharmacologic inhibition of CXCL11 and CXCL12 impairs meningioma stem-like cell activity. (A) (Upper left panel) Antiproliferative effects of CXCR4 (AMD3100) and CXCR7 (CCX771) inhibitors in PD-Mg-S (N = 7) stimulated with CXCL11 or CXCL12 (25 nM). Bars represent mean ± SD of the percentage of vehicle-treated controls (CTR). **P < .01 and ***P < .001 versus respective CTR; °P < .05 versus CXCL11; ^§P < .05 versus CXCL12. (Upper right panel) Migrastatic effects of AMD3100 and CCX771 in CXCL11 or CXCL12 (25nM) -stimulated PD-Mg-S (N = 3). *P < .05 and **P < .01 versus respective CTR; °P < .05 versus CXCL11; ^§P < .05 versus CXCL12. (Lower left panel) Inhibition of CXCL11- and CXCL12-mediated vessel-like formation by AMD3100 or CCX771 in PD-Mg-S (N = 2). **P < .01 and ***P < .001 versus respective CTR; °P < .05 versus CXCL11; ^§P < .05 and ^§§P < .01 versus CXCL12. (B) Inhibition of ERK1/2 activation induced by CXCL12 and CXCL11 by treatment with AMD3100 and CCX771. Left panel: Representative WB using phospho-ERK1/2 (upper panel) and α-tubulin (lower panel). Right panel: Densitometric analysis showing the pERK1/2-α/tubulin ratio. (C) Schematic diagram showing the ability of CXCR4 and CXCR7 antagonists (AMD3100 and CCX771), individually added, to completely prevent meningioma stem-like cell proliferation, migration and tube formation, induced by both CXCL11 and CXCL12.