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. 2010 Nov;12(11):1113-25.
doi: 10.1093/neuonc/noq082. Epub 2010 Jul 28.

Glioma cancer stem cells induce immunosuppressive macrophages/microglia

Adam Wu  1 , Jun Wei, Ling-Yuan Kong, Yongtao Wang, Waldemar Priebe, Wei Qiao, Raymond Sawaya, Amy B Heimberger
Affiliations

Glioma cancer stem cells induce immunosuppressive macrophages/microglia

Adam Wu et al. Neuro Oncol. 2010 Nov.

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.

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Figures

Fig. 1.
Fig. 1.
Attraction and phenotype alteration of monocytes by gCSCs. Monocytes upon exposure to the gCSC-conditioned medium (n = 4) demonstrated (A) increased migration in a cell-migration chemotaxis assay compared with both control neurosphere medium alone and supernatants from bulk and CD133+ depleted primary GBM and (B) an increase in p-STAT3 expression, as shown by flow cytometry, relative to control monocytes exposed to neurosphere medium alone. (C) Western blot showing that p-STAT1 expression was decreased within monocytes and MΦs by gCSC-conditioned medium and MIC-1, and that this was partially reversed by p-STAT3 blockade with WP1066 (WP = 2 µM WP1066). C is control neurosphere medium.
Fig. 2.
Fig. 2.
Functional inhibition of MΦs/microglia by gCSCs. (A) Representative microscope (×40) images showing inhibition of phagocytosis of fluorescent microbeads (red) in MΦs exposed to (i) positive control (neurosphere medium) compared with (ii) gCSC-conditioned medium. In contrast, (iii) bulk primary GBM supernatant and (iv) CD133+ depleted primary GBM supernatant did not inhibit phagocytosis. Furthermore, representative microscopy demonstrates that phagocytosis is inhibited in MΦs exposed to (v) 10 ng/mL MIC-1. MΦ nuclei were stained with DAPI (blue). A similar inhibition of phagocytosis by the gCSC-conditioned medium was seen in normal microglia (Supplementary Fig. 1). (B) T-cell proliferation was inhibited by MΦs treated with gCSC-conditioned medium (n = 4) compared with MΦs treated with neurosphere medium alone. When MΦs were pulsed with gCSC-conditioned medium (n = 4), inhibition of T-cell proliferation was maintained even after gCSC-conditioned medium was removed and replaced with a fresh growth medium. Inhibition of T-cell proliferation is expressed as a percent positive control neurosphere medium alone (*significantly different from MΦs alone ; P < .05). (C) Representative CFSE histograms comparing the inhibition of T-cell proliferation between MΦs alone and MΦs pulsed with the gCSC-conditioned medium.
Fig. 3.
Fig. 3.
STAT3 blockade diminishes gCSC-mediated modulation of MΦ immunosuppressive function. (A) Representative microscope (×40) images showing phagocytosis in MΦs exposed to (i) control medium and (ii) gCSC-conditioned medium (67.7% ± 4.3% of control level), with reversal of phagocytosis inhibition after inhibition of STAT3 in gCSCs by (iii) 2 µM WP1066 medium (110.6% ± 15.8% of control level), and (iv) STAT3 siRNA (100.2% ± 5.8% of control level), but not by 2 different control siRNAs, (v) 57.8% ± 3.5% of control level and (vi) 66.8% ± 1.3% of control level, respectively. MΦ nuclei stained with DAPI. (B) STAT3 blockade reversed the induction of MΦ IL-10 production by gCSCs. The increase in MΦ IL-10 production by the gCSC-conditioned medium was reduced by treatment of gCSCs with WP1066 and STAT3 siRNA to levels not significantly different from that of controls treated with the neurosphere medium, whereas MΦ IL-10 production induced by gCSCs treated with 2 different control siRNAs remained significantly increased relative to controls treated with the neurosphere medium.
Fig. 4.
Fig. 4.
Theoretical schema showing relationship between gCSCs and MΦs. The gCSCs recruit circulating monocytes and Tregs via CSF-1 and CCL2 respectively and mediate differentiation and polarization of the monocytes into M2 tumor-associated MΦs in part by elaboration of soluble MIC-1, increasing expression of p-STAT3 and decreasing expression of p-STAT1 in the monocytes/MΦs. M2 tumor-associated MΦs possess an immunosuppressive phenotype characterized by reduced phagocytosis, production of immunosuppressive cytokines including IL-10, TGF-β1, and/or IL-23, and the ability to inhibit T-cell proliferation. M2 tumor-associated MΦs also contribute to other tumor-supportive functions, including angiogenesis and tissue remodeling allowing for invasion. The gCSCs also directly inhibit adaptive immunity by inducing Tregs. These immunosuppressive functions of gCSCs are mediated in part by STAT3.
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