Glioma cancer stem cells induce immunosuppressive macrophages/microglia

Adam Wu

Jun Wei

Ling Kong

Yongtao Wang

Supplementary Material

Supplementary Data:

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Department of Neurosurgery (A.W., J.W., L.-Y.K., Y.W., R.S., A.B.H.), Department of Experimental Therapeutics (W.P.), Department of Biostatistics (W.Q.), The University of Texas MD Anderson Cancer Center, Houston, Texas

Corresponding Author: Amy B. Heimberger, MD, Department of Neurosurgery, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd., Unit 442, Houston, TX 77030 (gro.nosrednadm@rebmieha).

Received 2010 Feb 18; Accepted 2010 Jun 15.

Copyright © The Author(s) 2010. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org.

Abstract

Macrophages (MΦs)/microglia that constitute the dominant tumor-infiltrating immune cells in glioblastoma are recruited by tumor-secreted factors and are induced to become immunosuppressive and tumor supportive (M2). Glioma cancer stem cells (gCSCs) have been shown to suppress adaptive immunity, but their role in innate immunity with respect to the recruitment and polarization of MΦs/microglia is unknown. The innate immunosuppressive properties of the gCSCs were characterized based on elaborated MΦ inhibitory cytokine-1 (MIC-1), transforming growth factor (TGF-β1), soluble colony-stimulating factor (sCSF), recruitment of monocytes, inhibition of MΦ/microglia phagocytosis, induction of MΦ/microglia cytokine secretion, and the inhibition of T-cell proliferation. The role of the signal transducer and activator of transcription 3 (STAT3) in mediating innate immune suppression was evaluated in the context of the functional assays. The gCSCs produced sCSF-1, TGF-β1, and MIC-1, cytokines known to recruit and polarize the MΦs/microglia to become immunosuppressive. The gCSC-conditioned medium polarized the MΦ/microglia to an M2 phenotype, inhibited MΦ/microglia phagocytosis, induced the secretion of the immunosuppressive cytokines interleukin-10 (IL-10) and TGF-β1 by the MΦs/microglia, and enhanced the capacity of MΦs/microglia to inhibit T-cell proliferation. The inhibition of phagocytosis and the secretion of IL-10 were reversed when the STAT3 pathway was blocked in the gCSCs. The gCSCs modulate innate immunity in glioblastoma by inducing immunosuppressive MΦs/microglia, and this capacity can be reversed by inhibiting phosphorylated STAT3.

Keywords: cancer stem cells, innate immunity, macrophages, microglia, immune suppression, signal transducer and activator of transcription 3

Abstract

Glioblastoma multiforme (GBM) is a lethal cancer that responds poorly to radiotherapy and chemotherapy. Macrophages (MΦs) and microglia constitute the dominant tumor-infiltrating immune cells. MΦs/microglia that are capable of phagocytosis,¹ antigen processing and presentation,² cytotoxicity, and the promotion of inflammation³ are designated M1. However, upon recruitment into the tumor microenvironment by chemoattractants such as CC chemokine ligand 2 (CCL2)⁴,⁵ and soluble colony-stimulating factor 1 (sCSF-1),⁶,⁷ MΦs/microglia become immunosuppressive⁸–¹⁰ as designated as M2 and are unable to produce proinflammatory cytokines, induce effector T-cell anergy, demonstrate impairments in cytotoxicity²,¹¹–¹³, and induce Tregs.¹⁴

Tumor-associated MΦs are related to myeloid-derived suppressor cells, which have known immunosuppressive properties¹⁵; the tumor-associated MΦ probably represents a more mature subset of this cell population.¹⁶ Tumor-associated M2 MΦs have been shown to promote cancer by secreting proangiogenic factors and enhancing invasion¹⁰,¹⁷ mediated by the production of soluble factors such as transforming growth factor-β (TGF-β),¹⁸ interleukin (IL)-1,¹⁹ vascular endothelial growth factor, and matrix metallopeptidase-9.²⁰ The presence of MΦs correlates with poor prognosis in a variety of malignancies,²¹ and polymorphism in the gene CX3CR1, which plays a role in MΦ and microglial mobilization and migration, was found to potentially be associated with differential survival in patients with GBM.²² Thus, it appears that malignant gliomas actively recruit microglia and MΦs to the tumor site and induce them to adopt tumor-supportive phenotypes capable of mediating immunosuppression and promoting invasion. The mechanisms and participating cell populations that are mediating immunosuppressive MΦs/microglia have not been fully established.

Malignant gliomas contain glioma cancer stem cells (gCSCs) that are a heterogeneous population of multipotent undifferentiated cells with the capacity for self-renewal and are able to form neurospheres that are nonadherent in vitro, are highly tumorigeneic,²³ and can recapitulate the characteristics of malignant gliomas.²⁴ We have recently shown that gCSCs are powerful mediators of immunosuppression of the adaptive immune system, specifically T-cell responses.²⁵ These processes are dependent on the phosphorylated signal transducer and activator of transcription 3 (p-STAT3) pathway,²⁶ which has been previously been demonstrated to be a key pathway in cancer-mediated immune suppression²⁷ and is overexpressed in most cancers including gliomas.²⁸

STAT3 induces a variety of transcriptional factors that propagate tumorigenesis²⁹ and upregulate tumor-mediated immunosuppressive factors.²⁷,³⁰ The STAT3-regulated tumor-secreted factors then activate STAT3 in diverse immune cells, including both innate immune cells such as MΦs and T cells,³¹ resulting in global immune suppression.³¹–³⁴ When p-STAT3 is blocked in the gCSCs, cell proliferation is inhibited, neurosphere formation is impaired, the CD133-positive (CD133+) cells are depleted,³⁵ and the gCSC-mediated immunosuppression is blocked.²⁶ Additionally, using cytokine microarray analysis, we found that the gCSCs²⁵ were making MΦ inhibitory cytokine-1 (MIC-1), a member of the TGF-β super family,³⁶ which has been shown to be produced by glioblastoma cells³⁷ and found to be elevated in the cerebrospinal fluid of GBM patients, with higher levels associated with poorer outcome.³⁸ We have therefore hypothesized that the gCSCs can mediate innate immune suppression, possibly secondary to MIC-1, which can be reversed with STAT3 blockade. In this manuscript, we demonstrate that gCSCs induce MΦs derived from peripheral blood monocytes, the myeloid precursors to normal and tumor-infiltrating microglia/MΦs,³⁹ to an immunosuppressive phenotype that is partially reversed with STAT3 blockade.

MIC-1, Macrophage inhibitory cytokine-1; CSF-1, Colony-stimulating factor-1; TGF-β1, Transforming growth factor-β1; SD, Standard deviation.

^{Detection limit for ELISA is 0.015 ng/mL (0.6 pg/mL/10}^{cells/24 h for 5 × 10}^{cells cultured for 5 days).}

MΦ, macrophages; IL, interleukin; TGF-β1, transforming growth factor-β1; MIC-1, macrophage inhibitory cytokine-1.

^{Number of times (fold) the level seen in the control.}

^{Control macrophages produced no IL-23.}

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Acknowledgments

We thank Lamonne Crutcher for assistance in obtaining tissue specimens and David M. Wildrick, PhD, for editorial assistance.

Conflict of interest statement. W.P. and A.B.H. hold patents on WP1066.

Acknowledgments

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