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 8

OPN expression can be exploited for therapeutic targeting.

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OPN expression can be exploited for therapeutic targeting. (A) Transwell...
(A) Transwell migration assays of M0- and M2-skewed macrophages exposed to OPN antibody or antagonistic OPN aptamers (OPN-R3) at 48 hours. Data are shown as mean ± SD and are representative of 3 independent experiments. (B) Treatment schema and graph of the Kaplan-Meier survival estimate demonstrating improved survival with the 4-1BB–OPN aptamer conjugate (37 days) relative to the untreated control (22 days), 4-1BB aptamer (27 days), OPN aptamer (26 days), or a mixture of the 4-1BB and OPN aptamers (23 days) (n = 5 mice/group) (P < 0.05). Data represent 2 independent experiments. i.c., intracerebrally. (C) Localization of the 4-1BB–OPN aptamer in the brain tumor microenvironment visualized with an antisense 4-1BB RNA probe using ISH. A control aptamer that does not target the glioma, 4-1BB–PSMA, was not detected in the brain tumor using the 4-1BB RNA probe. Right panels: Colocalizing H&E staining of the tumor within the brain. Original magnification, ×100 (scale bars: 200 μm). (D) Summary graph of the treatment groups from B showing quantification of CD3+ T cells within the intracerebral glioma (4-1BB–OPN aptamer conjugate relative to other groups; P < 0.05). Data indicate mean ± SD from 5 different animals per group in a single experiment. P values were calculated based on the 2-tailed 2-sample t test.