Abstract 5322: Glioma cancer stem cells induce immune suppressive macrophages

Abstract

Abstract Introduction: The tumor microenvironment can induce monocytes to become immune suppressive macrophages (M2) via the phosphorylation of the signal transducer activator of transcription 3 (STAT3). The M2 cells assume a phenotype with impaired anti-tumor activity, support tumor progression, angiogenesis, and invasion and portend a poor prognosis. Gliomas contain cancer stem cells (gCSCs) that are self-renewing, multipotent, recapitulate the phenotype of the original tumor, and mediate resistance to radiation and chemotherapy. The gCSCs inhibit adaptive immune responses by inhibiting T cell proliferation and effector responses, inducing T cell apoptosis and inducing Tregs secondary to phosphorylated STAT3 (p-STAT3) regulated processes such as secreted TGF-β1 and Galectin-3 and the expression of cell surface molecules such as B7-H1. We therefore hypothesized that gCSCs also exert immune suppression of the innate immune system by inducing M2 cells. Methods: The gCSCs (n=4) were isolated from surgical glioblastoma specimens. Normal microglia were isolated from brain specimens obtained during resections of extra-axial lesions. Peripheral blood monocytes were isolated from healthy donors and differentiated into macrophages (MDMs), which were then treated with gCSC supernatants and recombinant cytokines. Cell surface markers were assessed by flow cytometry, secreted cytokines measured using ELISA, T cell proliferation by CFSE, Treg induction by flow cytometry and phagocytosis by fluorescent bead uptake. Western blot assays were used to assess activation of the p-STAT1 and p-STAT3 pathways. The p-STAT3 pathway was inhibited with the small molecule WP1066 and siRNA. Results: The gCSCs produce macrophage inhibitory cytokine (MIC-1) (22.3 - 1056.2 pg/106 cells/24 hours), macrophage inhibitory factor (MIF) (69.1 - 1463.3 pg/106 cells/24 hours), and TGF-β1 (24 - 73.8 pg/106 cells/24 hours) but not IL-23. The MDMs exposed to the gCSC supernatants increased expression of CD80 and B7-H1, and had significantly potentiated secretion of TGF-β1 (106.3 - 3001.8 pg/106 cells/24 hours) and IL-23 (176.8 - 5650.6 pg/106 cells/24 hours). The induced TGF-β1 and IL-23 correlated with inhibition of T cell proliferation and the induction of Tregs. MDM phagocytosis was markedly inhibited by the gCSC supernatants, and physiological amounts of recombinant MIC-1 and MIF. Similar findings were obtained with the human microglia. The gCSC supernatants up-regulated p-STAT3 in the MDMs; however the M2 phenotype could be reversed when p-STAT3 was blocked in either the gCSCs or the MDMs. Conclusions: The gCSCs secrete factors such as MIC-1 and MIF that can induce immune suppression in macrophages characterized by altered expression of cell surface markers, increased secretion of TGF-β1 and IL-23 with the associated ability to inhibit T cell proliferation and induce Tregs, and impaired phagocytosis. The M2 phenotype is reversed with p-STAT3 blockade. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5322.