Osteopontin mediates glioblastoma-associated macrophage infiltration and is a potential therapeutic target
Jun Wei, … , Shulin Li, Amy B. Heimberger
Jun Wei, … , Shulin Li, Amy B. Heimberger
Published October 11, 2018
Citation Information: J Clin Invest. 2019;129(1):137-149. https://doi.org/10.1172/JCI121266.
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Research Article Immunology

Osteopontin mediates glioblastoma-associated macrophage infiltration and is a potential therapeutic target

Abstract

Glioblastoma is highly enriched with macrophages, and osteopontin (OPN) expression levels correlate with glioma grade and the degree of macrophage infiltration; thus, we studied whether OPN plays a crucial role in immune modulation. Quantitative PCR, immunoblotting, and ELISA were used to determine OPN expression. Knockdown of OPN was achieved using complementary siRNA, shRNA, and CRISPR/Cas9 techniques, followed by a series of in vitro functional migration and immunological assays. OPN gene–deficient mice were used to examine the roles of non-tumor-derived OPN on survival of mice harboring intracranial gliomas. Patients with mesenchymal glioblastoma multiforme (GBM) show high OPN expression, a negative survival prognosticator. OPN is a potent chemokine for macrophages, and its blockade significantly impaired the ability of glioma cells to recruit macrophages. Integrin αvβ5 (ITGαvβ5) is highly expressed on glioblastoma-infiltrating macrophages and constitutes a major OPN receptor. OPN maintains the M2 macrophage gene signature and phenotype. Both tumor-derived and host-derived OPN were critical for glioma development. OPN deficiency in either innate immune or glioma cells resulted in a marked reduction in M2 macrophages and elevated T cell effector activity infiltrating the glioma. Furthermore, OPN deficiency in the glioma cells sensitized them to direct CD8+ T cell cytotoxicity. Systemic administration in mice of 4-1BB–OPN bispecific aptamers was efficacious, increasing median survival time by 68% (P < 0.05). OPN is thus an important chemokine for recruiting macrophages to glioblastoma, mediates crosstalk between tumor cells and the innate immune system, and has the potential to be exploited as a therapeutic target.

Authors

Jun Wei, Anantha Marisetty, Brett Schrand, Konrad Gabrusiewicz, Yuuri Hashimoto, Martina Ott, Zacharia Grami, Ling-Yuan Kong, Xiaoyang Ling, Hillary Caruso, Shouhao Zhou, Y. Alan Wang, Gregory N. Fuller, Jason Huse, Eli Gilboa, Nannan Kang, Xingxu Huang, Roel Verhaak, Shulin Li, Amy B. Heimberger

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Figure 7

OPN is required for M2 polarization maintenance.

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OPN is required for M2 polarization maintenance. (A) CD14+ monocytes wer...
(A) CD14+ monocytes were polarized, and the M1 and M2 cells were harvested on day 7. Typical phenotypic characteristics of polarized macrophages are shown in the top row. Original magnification, ×20. Flow cytometry data demonstrate that OPN is most abundant in the M2-polarized macrophage (bottom row). Isotype is the shaded blue curve, and OPN staining is shaded red. Representative FACS data from 3 independent experiments are shown. (B) Quantitative PCR to measure OPN mRNA levels in M2 macrophages 2 days after transfection of OPN or NT siRNAs. Data are shown as mean ± SD from 6 different donors. (C) Significantly downregulated genes in OPN siRNA–transfected M2 macrophages, as determined by transforming the associated NanoString data to fit the normal distribution and analyzing the fold changes for the genes with P values less than 0.05 by Wilcoxon’s signed-rank test. Data are from 6 different donors (a–f). (D) M2 phagocytic activity based on the uptake of pHrodo Red BioParticles conjugate in triplicate assays. Data are shown as mean ± SD and are representative of 3 independent experiments. P < 0.05 by 2-tailed 2-sample t test.