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. 2020 Jun 12;21(12):4199.
doi: 10.3390/ijms21124199.

CCR5-Mediated Signaling Is Involved in Invasion of Glioblastoma Cells in Its Microenvironment

Metka Novak  1 , Miha Koprivnikar Krajnc  1 , Barbara Hrastar  1 , Barbara Breznik  1 , Bernarda Majc  1   2 , Mateja Mlinar  1 , Ana Rotter  1 , Andrej Porčnik  3 , Jernej Mlakar  4 , Katja Stare  5 , Richard G Pestell  6 , Tamara Lah Turnšek  1   7
Affiliations

CCR5-Mediated Signaling Is Involved in Invasion of Glioblastoma Cells in Its Microenvironment

Metka Novak et al. Int J Mol Sci. .

Abstract

The chemokine CCL5/RANTES is a versatile inflammatory mediator, which interacts with the receptor CCR5, promoting cancer cell interactions within the tumor microenvironment. Glioblastoma is a highly invasive tumor, in which CCL5 expression correlates with shorter patient survival. Using immunohistochemistry, we identified CCL5 and CCR5 in a series of glioblastoma samples and cells, including glioblastoma stem cells. CCL5 and CCR5 gene expression were significantly higher in a cohort of 38 glioblastoma samples, compared to low-grade glioma and non-cancerous tissues. The in vitro invasion of patients-derived primary glioblastoma cells and glioblastoma stem cells was dependent on CCL5-induced CCR5 signaling and is strongly inhibited by the small molecule CCR5 antagonist maraviroc. Invasion of these cells, which was enhanced when co-cultured with mesenchymal stem cells (MSCs), was inhibited by maraviroc, suggesting that MSCs release CCR5 ligands. In support of this model, we detected CCL5 and CCR5 in MSC monocultures and glioblastoma-associated MSC in tissue sections. We also found CCR5 expressing macrophages were in close proximity to glioblastoma cells. In conclusion, autocrine and paracrine cross-talk in glioblastoma and, in particular, glioblastoma stem cells with its stromal microenvironment, involves CCR5 and CCL5, contributing to glioblastoma invasion, suggesting the CCL5/CCR5 axis as a potential therapeutic target that can be targeted with repositioned drug maraviroc.

Keywords: CCL5; CCR5; chemokines; glioblastoma; invasion; maraviroc; mesenchymal stem cells.

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Conflict of interest statement

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Figures

Figure 1
Figure 1
Brain tissue sections immunolabeling for CCL5 and CCR5. Immunohistochemical localization of CCL5 and CCR5 in glioblastoma and non-cancerous tissue (NB1 and NB2) sections was performed as described in Materials and Methods. Cell nuclei were counterstained by hematoxylin (blue). CCR5 epitope blocking peptide (P) was used (in CCR5+P images) as a control for specific binding of the primary antibody. Scale bar represents 100 µm. Black arrows indicate examples of CCL5 and CCR5 positive cells. Microscopy was carried out at 20× objective magnification.
Figure 2
Figure 2
Immunocytochemical localization of CCL5 and CCR5 in primary glioblastoma cells. ICC localization of CCL5 and CCR5 in primary glioblastoma cells isolated from patients’ tumors and the glioblastoma cell line U373 as performed as described in Materials and Methods. Cell nuclei were counterstained by hematoxylin (blue). CCR5 epitope blocking peptide (P) was used (in CCR5 + P images) as a control. Negative control staining was performed in the absence of the primary antibody. Scale bar represents 50 µm. Microscopy was carried out at 20× objective magnification.
Figure 3
Figure 3
Primary glioblastoma stem cells express CCR5. Immunofluorescence labeling was performed as described in Materials and Methods on GSC spheroids, established from the patient’s Nb.4 tissue and established GSC lines NCH644 and NCH421k. Nuclei were stained with DAPI (blue), CCR5 expression is shown as a green and CCL5 as red fluorescence. The last panel presents merged channels. Confocal microscopy was carried out at 20× objective magnification. Scale bar represents 100 µm.
Figure 4
Figure 4
Mesenchymal stem cells in glioblastoma tissues express CCL5. Fluorescence immunohistochemical staining of CCL5 antigen was performed on glioblastoma sections of 3 patients, Nb. 8, Nb. 12 and Nb. 5. MSCs were immunolabeled using the antibody against their specific marker CD105. Nuclei were stained with DAPI (blue), CCL5 with Alexa Fluor 546 (red), and CD105 with Alexa Fluor 488 (green) dye. Merged images represent colocalization (violet color) of CD105 and CCL5. Microscopy was carried out at 20× objective magnification. Scale bar represents 100 µm.
Figure 5
Figure 5
Glioblastoma-associated macrophages express CCR5. Fluorescence immunohistochemical staining of CCR5 antigen was performed on glioblastoma sections of 3 patients, Nb. 8, Nb. 12 and Nb. 5. Macrophages were immunolabeled, using an antibody against the specific marker CD68. Nuclei were stained with DAPI (blue), CD68 with Alexa Fluor 546 (red), and CCR5 with Alexa Fluor 488 (green) dye. Merged images represent colocalization (yellow color) of CD68 and CCR5. Microscopy was carried out at 20× objective magnification. Scale bar represents 100 µm.
Figure 6
Figure 6
Effects of CCL5 and MSC on the invasion of primary glioblastoma cells and glioblastoma stem cells. Primary glioblastoma cells from patient Nb. 2 (GB Nb.2) (10,000 cells/insert) and GSC cells (NCH644) (80,000 cells/insert), were seeded in the upper compartment alone or in combination with maraviroc (MRV) (final concentration 10 µM) which was coated with 0.5 mg/mL Matrigel in serum-free medium. (A,C) Recombinant CCL5 (final concentration 300 ng/mL) was added to the lower chamber (B,D) MSCs (20,000/insert) were added to the lower chamber. The cells that invaded the matrigel after 48 h, were stained with 0.1% crystal violet and counted using an inverted microscope. Each value represents mean ± SD (n = 3). ★ p < 0.05, ★★ p < 0.01, ★★★ p < 0.001 vs. control group (t-test).
Figure 7
Figure 7
CCL5 and CCR5 gene expression in glioma tissues and primary glioblastoma cells. The expression of CCL5 (A) and CCR5 (B) at mRNA was determined in glioma, non-cancerous brain tissues and glioblastoma cells analyzed by RT-qPCR. mRNA values were normalized to housekeeping genes HPRT1 and GAPDH and analyzed with quantGenius software [40] as described in Materials and Methods. n-number of samples; N-non-cancerous brain tissues; glioma I-II- low-grade gliomas: pilocytic astrocytoma, astrocytoma, oligodendroglioma; glioma III-anaplastic astrocytoma, anaplastic oligodendroglioma, and anaplastic mixed oligoastrocytoma; GB-glioblastoma; GB rec- recurrent glioblastoma; GB cells-primary glioblastoma cells; GSC-glioblastoma stem cells isolated from patient tumor samples. ★★ p < 0.01, ★★★ p < 0.001 versus the non-cancerous brain tissues.
Figure 8
Figure 8
CCL5 and CCR5 gene expression in glioblastoma subtypes. mRNA expression of CCL5 (A) and CCR5 (B) in glioblastoma tissues and glioblastoma cells analyzed by RT-qPCR. mRNA values were normalized to housekeeping genes HPRT1 and GAPDH and analyzed with quantGenius software as described in Materials and Methods. n-number of samples; CL-classical, MES-mesenchymal, PN-proneural, and MIX subtypes. ★ p < 0.05, ★★ p < 0.01, ★★★ p < 0.001 versus the non-cancerous brain tissues.
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