Osteopontin mediates glioblastoma-associated macrophage infiltration and is a potential therapeutic target
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
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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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 6

OPN is expressed in GIMs.

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OPN is expressed in GIMs. (A) Heatmap demonstrating the preferential upr...
(A) Heatmap demonstrating the preferential upregulation of genes in GIMs relative to matched patient peripheral blood monocytes. (B) In newly diagnosed GBM patients, quantitative PCR was used to evaluate the relative expression of OPN in matched specimens of GBM CD14+ GIMs and monocytes (n = 10). Ct values were normalized to GAPDH. Data are presented as mean ± SEM and are representative of 3 independent experiments. (C) Immunofluorescence staining of OPN (green) and macrophages (Iba-1, red) in an ex vivo GBM, including a merged (nuclei, DAPI, blue) and enlarged example. Original magnification, ×400 (scale bars: 10 μm) and ×600 (scale bars: 2 μm). Graph: Iba-1+OPN+ macrophages in the GBM tissue. Data are shown as mean ± SD and represent 3 independent experiments. (D) Western blots demonstrating that ITGαvβ5 is preferentially expressed in the GIMs. (E) Unifying schema for the role of OPN in macrophages within the GBM tumor microenvironment. GBM tumor cells including GSCs elaborate OPN, which acts as a chemokine for M0 and M2 ITGαvβ5-expressing cells. GIMs are mostly in a phenotypic continuum of M0 to M2 and express ITGαvβ5. The GIMs also express OPN, which further amplifies the recruitment of additional M0- and M2-polarized cells into the tumor microenvironment.