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 1

OPN expression is prognostic in human glioblastoma and associates with the mesenchymal subtype.

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OPN expression is prognostic in human glioblastoma and associates with t...
(A) OPN expression is higher in GBM (n = 528) than in non-tumor brain tissue (n = 10, P = 0.000000067) based on TCGA data. (B) Kaplan-Meier survival estimates of glioblastoma patients in relation to expression levels of OPN in the tumors based on TCGA data sets. Median survival time: OPN high–expressing (50%) group, 318 days versus low-expressing (50%) group, 422 days (n = 403); OPN high–expressing (25%) group, 447 days versus low-expressing (25%) group, 296 days (n = 203). (C) OPN expression preferentially associates with the mesenchymal (M; n = 51) versus the classical (C; n = 59) or proneural (P; n = 46) GBM subtype (total n = 156; M vs. C: P = 7 × 10–6; M vs. P: P = 0.0043). A similar preferential association was found in IDH-WT GBM (n = 139): M (n = 48) vs. C (n = 56): P = 6 × 10–6; M vs. P (n = 35): P = 0.0459. In all GBMs (left), C: minimum (min) 10.68, 25% percentile 14.17, median 15.08, 75% percentile 15.95, maximum (max) 18.92; M: min 9.927, 25% percentile 15.46, median 16.54, 75% percentile 17.59, max 18.92; P: min 12.37, 25% percentile 14.71, median 15.64, 75% percentile 16.54, max 18.25. In IDH-WT GBMs (right), C: min 10.68, 25% percentile 14.13, median 15.02, 75% percentile 15.92, max 17.77; M: min 9.927, 25% percentile 15.45, median 16.59, 75% percentile 17.62, max 18.92; P: min 12.49, 25% percentile 14.88, median 15.81, 75% percentile 16.65, max 18.25. RPKM, reads per kilobase million. (D) Ex vivo human GBM immunofluorescently stained for expression of OPN (green) and glioma stem cells, denoted by SOX2 expression (red). Nuclei were stained with DAPI (blue) and the images merged, illustrating the prominent expression of OPN in the GBM tumor microenvironment. Original magnification, ×400 (scale bars: 10 μm). (E) Human GSCs, glioma cell lines, and macrophages were analyzed for production of OPN at 48 hours by ELISA (cells were seeded at 0.5 × 106/ml as a starting culture density). P values were calculated using the 2-tailed 2-sample t test. Data indicate mean ± SD and are representative of 3 independent experiments. ***P < 0.001, **P < 0.001, *P < 0.05.