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 3

Reduction of macrophage infiltration and enhanced T cell effector activity in the tumor microenvironment is due to OPN loss.

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Reduction of macrophage infiltration and enhanced T cell effector activi...
Either WT or Opn–/– background mice were intracranially implanted with GL261 cells that were transfected with an NT control shRNA or shRNA specific to OPN (OPN shRNA) (50,000 cells per mouse, 8 mice/group, 4 groups). All mice were euthanized on day 14, and the tumor brain tissue, spleen, and blood were collected for ex vivo flow cytometry and immunohistochemical staining. (A) Percentages of CD206+CD11b+ macrophages in the spleen and F4/80+CD11b+ macrophages in the brain tumor were diminished when either OPN in the tumor or in the host background was knocked out. (B) Representative immunohistochemistry showing the frequency of Iba-1+ macrophages in the tumor; the graph summarizes the quantitative results. Original magnification, ×100 (scale bars: 100 μm). (C) Percentage of IFN-γ+CD4+ T cells and IFN-γ+CD8+ effector T cells in the spleen (left), PBMCs (middle), and tumor (right). (D) Percentage of IL-2+CD4+ T cells (left) and IL-2+CD8+ effector T cells (right) in the tumor. (E) Percentage of CD4+FoxP3+ Tregs in PBMCs. Data represent mean ± SD of 4 different animals in 1 experiment. P values were calculated based on 2-tailed 2-sample t test with Bonferroni’s correction. *P < 0.05, **P < 0.01, ***P < 0.001.