Targeting CXCR4 by a selective peptide antagonist modulates tumor microenvironment and microglia reactivity in a human glioblastoma model.

Laura Mercurio,Stefania Scala,Serena Cecchetti,Giuseppina Bozzuto,Maria Antonietta Ajmone-Cat,Luisa Minghetti,Isabella Manni,Giulia Carpinelli,Agnese Molinari,Bianca Pollo,Alessandro Ricci

doi:10.1186/s13046-016-0326-y

Abstract

BackgroundThe CXCL12/CXCR4 pathway regulates tumor cell proliferation, metastasis, angiogenesis and the tumor-microenvironment cross-talk in several solid tumors, including glioblastoma (GBM), the most common and fatal brain cancer. In the present study, we evaluated the effects of peptide R, a new specific CXCR4 antagonist that we recently developed by a ligand-based approach, in an in vitro and in vivo model of GBM. The well-characterized CXCR4 antagonist Plerixafor was also included in the study.MethodsThe effects of peptide R on CXCR4 expression, cell survival and migration were assessed on the human glioblastoma cell line U87MG exposed to CXCL12, by immunofluorescence and western blotting, MTT assay, flow cytometry and transwell chamber migration assay. Peptide R was then tested in vivo, by using U87MG intracranial xenografts in CD1 nude mice. Peptide R was administered for 23 days since cell implantation and tumor volume was assessed by magnetic resonance imaging (MRI) at 4.7 T. Glioma associated microglia/macrophage (GAMs) polarization (anti-tumor M1 versus pro-tumor M2 phenotypes) and expressions of vascular endothelial growth factor (VEGF) and CD31 were assessed by immunohistochemistry and immunofluorescence.ResultsWe found that peptide R impairs the metabolic activity and cell proliferation of human U87MG cells and stably reduces CXCR4 expression and cell migration in response to CXCL12 in vitro. In the orthotopic U87MG model, peptide R reduced tumor cellularity, promoted M1 features of GAMs and astrogliosis, and hindered intra-tumor vasculature.ConclusionsOur findings suggest that targeting CXCR4 by peptide R might represent a novel therapeutic approach against GBM, and contribute to the rationale to further explore in more complex pre-clinical settings the therapeutic potential of peptide R, alone or in combination with standard therapies of GBM.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-016-0326-y) contains supplementary material, which is available to authorized users.

Highlights

The CXCL12/CXCR4 pathway regulates tumor cell proliferation, metastasis, angiogenesis and the tumor-microenvironment cross-talk in several solid tumors, including glioblastoma (GBM), the most common and fatal brain cancer
Gliomas are surrounded by resident non-tumor cells, including infiltrating lymphocytes and a large number of glioma-associated microglia/macrophages (GAMs), which are correlated with the malignancy grade [3, 4] and can support tumor structure, angiogenesis, growth and invasiveness in response to autocrine and paracrine molecular regulators secreted by tumor cells [5, 6]
After 72 h, CXCR4 down-modulation on cell membrane of peptide R-treated cells was maintained, while in Plerixafor-treated cells the chemokine receptor was re-expressed at levels comparable to unstimulated (-CXCL12) or CXCL12-stimulated cells (+CXCL12)

Summary

Introduction

The CXCL12/CXCR4 pathway regulates tumor cell proliferation, metastasis, angiogenesis and the tumor-microenvironment cross-talk in several solid tumors, including glioblastoma (GBM), the most common and fatal brain cancer. The binding of its ligand, the chemokine stromal cell-derived factor 1-α (SDF1-α, or CXCL12), triggers different downstream signaling pathways in tumor cells and in cells of the surrounding microenvironment, which result in a variety of cellular responses including angiogenesis, metastasis, proliferation and survival [18,19,20]. Multiple preclinical studies in animal models of glioma have demonstrated that disrupting the CXCL12/CXCR4 pathway by specific antagonists affects tumor growth, vasculogenesis and post-radiation recurrence, suggesting that this approach represents a promising strategy for glioblastoma therapy [25,26,27,28]. Cardiotoxicity and other adverse events were reported following long-term usage of Plerixafor [34], prompting the search for new safer and selective CXCR4 inhibitors suitable as anti-GBM agents

Methods

Results

Discussion

Conclusion