Crosstalk between β-Catenin and CCL2 Drives Migration of Monocytes towards Glioblastoma Cells

Abstract

Isocitrate dehydrogenase (IDH)-wildtype glioblastoma (GBM) is a fast growing and highly heterogeneous tumor, often characterized by the presence of glioblastoma stem cells (GSCs). The plasticity of GSCs results in therapy resistance and impairs anti-tumor immune response by influencing immune cells in the tumor microenvironment (TME). Previously, β-catenin was associated with stemness in GBM as well as with immune escape mechanisms. Here, we investigated the effect of β-catenin on attracting monocytes towards GBM cells. In addition, we evaluated whether CCL2 is involved in β-catenin crosstalk between monocytes and tumor cells. Our analysis revealed that shRNA targeting β-catenin in GBMs reduces monocytes attraction and impacts CCL2 secretion. The addition of recombinant CCL2 restores peripheral blood mononuclear cells (PBMC) migration towards medium (TCM) conditioned by shβ-catenin GBM cells. CCL2 knockdown in GBM cells shows similar effects and reduces monocyte migration to a similar extent as β-catenin knockdown. When investigating the effect of CCL2 on β-catenin activity, we found that CCL2 modulates components of the Wnt/β-catenin pathway and alters the clonogenicity of GBM cells. In addition, the pharmacological β-catenin inhibitor MSAB reduces active β-catenin, downregulates the expression of associated genes and alters CCL2 secretion. Taken together, we showed that β-catenin plays an important role in attracting monocytes towards GBM cells in vitro. We hypothesize that the interactions between β-catenin and CCL2 contribute to maintenance of GSCs via modulating immune cell interaction and promoting GBM growth and recurrence.

Keywords: CCL2; GSCs; MSAB; Wnt; glioblastoma; immune evasion; monocytes; β-catenin.

Figure 1

β-catenin knockdown in GBM cells reduces migration of CD14⁺ monocytes in vitro: (A) GBM cell lines (GBM1, JHH520, SF188) were transduced with lentiviral particles containing shβ-catenin plasmids and knockdown efficiency (relative mRNA expression) was confirmed using RT-qPCR and (B) Western blotting. (C) CD14⁺ monocyte migration towards TCM of β-catenin knockdown cells was decreased compared to migration towards TCM of control (pLKO.1) cells. (D) CCL2 levels in TCM of shβ-catenin GBM cells were measured after 24 h incubation by ELISA and compared to control cells (pLKO.1). The relative CCL2 secretion data are presented as mean ± SD (n = 3). Statistical significance was calculated with unpaired t-test. * p ≤ 0.05 *** p ≤ 0.001.

Figure 2

Recombinant CCL2 restored PBMC migration in shβ-catenin TCM and CCL2 knockdown reduced monocyte migration: (A) Recombinant CCL2 (100 ng/mL) was added to the TCM of β-catenin knockdown cells and restored PBMC-attracting ability. GBM cell lines were transduced with lentiviral particles containing shCCL2 plasmids and knockdown efficiency (relative mRNA expression and relative CCL2 secretion) was confirmed using (B) RT-qPCR and (C) ELISA, respectively. (D) CD14⁺-monocyte migration was decreased after treatment with TCM of shCCL2 knockdown cells compared to treatment with TCM of control (pLKO.1) cells. Data are presented as mean ± SD (n = 3). Statistical significance was calculated with unpaired t-test. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Figure 3

CCL2 knockdown alters expression of β-catenin target and related genes as well as clonogenicity of GBM cells: (A) β-catenin, the β-catenin target genes Axin2, CCND1 and c-Myc and the β-catenin-associated genes ZEB1, SNAI1 and SNAI2 relative mRNA expression levels were analyzed by RT-qPCR in shCCL2 cells and compared to control cells (pLKO.1) (B) Non-phospho-(active)-β-catenin protein levels were detected using immunoblotting in shCCL2 and control cells (pLKO.1). (C) CCL2 suppression led to decreased clonogenicity of GBM1 and JHH520 while increasing clonogenicity of SF188 as detected by using a soft agar assay. Representative pictures of NBT stained colonies are shown. Abbreviations: NBT, 4-Nitro blue tetrazolium chloride. Data are presented as mean ± SD (n = 3). Statistical significance was calculated with unpaired t-test. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Figure 4

MSAB treatment reduces viability, active β-catenin protein levels and clonogenicity of GBM cells: (A) Cell viability was decreased by MSAB treatment in a dose-dependent manner. (B) Pharmacological β-catenin inhibition with MSAB led to suppression of non-phospho-(active) β-catenin in a dose-dependent manner as assessed by immunoblotting. Cells were treated with shown concentrations for 24 h. GAPDH was used as loading control. (C) MSAB treatment decreased clonogenicity of GBM cells in soft agar assay. Representative pictures of NBT stained colonies are shown. (D) The relative mRNA expression levels of β-catenin target genes (Axin2, c-Myc), -associated genes (SNAI1, SNAI2), neural stem cell marker SOX2 and chemokine CCL2 were measured by RT-qPCR in MSAB-treated cells compared to control cells (DMSO). Data are presented as mean ± SD (n = 3). Statistical significance was calculated with unpaired t-test. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Figure 5

MSAB reduces Wnt-activity, induces apoptosis and modulates CCL2 secretion in GBM cells: (A) 24 h MSAB treatment (10 µM) reduced Wnt-activity in glioblastoma cell lines as assessed by Luciferase Reporter Assay. The relative luciferase activity data from three cell lines are shown. (B) 24 h treatment with MSAB induced apoptosis in GBM cell lines in a dose-dependent manner. Apoptosis was assessed with Muse Annexin V and Dead Cell Kit in three cell lines. (C) Altered CCL2 protein levels (relative CCL2 secretion) in the conditioned medium measured after 24 h incubation by ELISA. Data are presented as the mean ± SD (n = 3). Statistical significance was calculated with unpaired t-test. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.