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Periostin secreted by glioblastoma stem cells recruits M2 tumour-associated macrophages and promotes malignant growth

Nature Cell Biology volume 17, pages 170–182 (2015)Cite this article

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Abstract

Tumour-associated macrophages (TAMs) are enriched in glioblastoma multiformes (GBMs) that contain glioma stem cells (GSCs) at the apex of their cellular hierarchy. The correlation between TAM density and glioma grade suggests a supportive role for TAMs in tumour progression. Here we interrogated the molecular link between GSCs and TAM recruitment in GBMs and demonstrated that GSCs secrete periostin (POSTN) to recruit TAMs. TAM density correlates with POSTN levels in human GBMs. Silencing POSTN in GSCs markedly reduced TAM density, inhibited tumour growth, and increased survival of mice bearing GSC-derived xenografts. We found that TAMs in GBMs are not brain-resident microglia, but mainly monocyte-derived macrophages from peripheral blood. Disrupting POSTN specifically attenuated the tumour-supportive M2 type of TAMs in xenografts. POSTN recruits TAMs through the integrin αvβ3 as blocking this signalling by an RGD peptide inhibited TAM recruitment. Our findings highlight the possibility of improving GBM treatment by targeting POSTN-mediated TAM recruitment.

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Figure 1: POSTN is preferentially secreted by GSCs and its levels correlate with TAM density in GBMs.
Figure 2: Disrupting POSTN by shRNA markedly inhibited tumour growth and reduced TAM density in GSC-derived xenografts.
Figure 3: TAMs in human primary GBMs and xenografts are monocyte-derived macrophages from peripheral blood.
Figure 4: POSTN-recruited TAMs are maintained as the M2 subtype in GSC-derived xenografts.
Figure 5: Silencing POSTN in GSCs increased the relative fraction of M1 TAMs in GSC-derived xenografts.
Figure 6: GSC-secreted POSTN is a chemoattractant to macrophages and monocytes.
Figure 7: POSTN recruits macrophages through αvβ3 integrin signalling.
Figure 8: M2 TAMs accelerated GSC tumour growth in mouse brains.

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Acknowledgements

We thank the Brain Tumor and Neuro-Oncology Centers at Cleveland Clinic and University Hospitals Case Medical Center for providing GBM surgical specimens for this study. We are grateful to members in J.N.R.’s laboratory for their assistance and scientific discussion. We also thank C. Shemo and S. O’Bryant of the Flow Cytometry Core, J. Drazba and L. Vargo of the Imaging Core at Cleveland Clinic Lerner Research Institute, and D. Schumick of the Center for Medical Art and Photography for their assistance. This work was supported by the Cleveland Clinic Foundation and an NIH R01 grant (NS070315) to S.B.

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Authors and Affiliations

  1. Department of Stem Cell Biology and Regenerative Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195, USA

    Wenchao Zhou, Susan Q. Ke, Zhi Huang, William Flavahan, Xiaoguang Fang, Jeremy Paul, Jeremy N. Rich & Shideng Bao

  2. Department of Immunology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195, USA

    Ling Wu & Xiaoxia Li

  3. Departments of Neurological Surgery & Pathology, University Hospitals, Case Medical Center & Case Comprehensive Cancer Center, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA

    Andrew E. Sloan

  4. Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710, USA

    Roger E. McLendon

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Contributions

W.Z. and S.B. designed the experiments, analysed data and prepared the manuscript. W.Z., S.Q.K., Z.H., X.F., J.P. and L.W. performed the experiments. W.F. performed database analysis. A.E.S. and R.E.M. provided GBM surgical specimens. R.E.M. performed pathological analyses. J.N.R. and X.L. provided scientific input and helped to edit the manuscript.

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Correspondence to Shideng Bao.

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Integrated supplementary information

Supplementary Figure 2 Identification of POSTN as a dominant chemoattractant to macrophages, and the correlation between POSTN expression and GBM prognosis, GSC population, or TAM density.

(a,b) Graphical analyses of migration (a) and invasion (b) assays showing that POSTN is a dominant chemoattractant in recruiting PMA-primed U937 macrophage-like cells. Migration was allowed for 24 h and invasion was allowed for 36 h. ∗∗∗, P < 0.001; ∗, P < 0.05; ns, P > 0.05. (n = 5 fields; mean ± s.e.m.; two-tailed unpaired t-test; experiments were repeated 3 times). (c,d) Bioinformatic analyses of the relationship between POSTN expression and overall survival (c) or progression-free survival (d) of GBM patients in TCGA dataset (The Cancer Genome Atlas) from Oncomine (http://www.oncomine.com). The threshold for high/low POSTN expression is determined by the average of POSTN expression levels in 525 GBM patients. The Kaplan–Meier survival curves demonstrate a significant invert correlation between POSTN levels and overall survival (c) (n = 525 patients; POSTN low, n = 115 patients; POSTN high, n = 400 patients; two-tailed log-rank test) or progression-free survival (d) (n = 337 patients; POSTN low, n = 164 patients; POSTN high, n = 173 patients; two-tailed log-rank test) of GBM patients. (e) Immunofluorescent staining of POSTN (green) and the GSC marker SOX2 (red) in primary GBMs. POSTN was preferentially expressed in GSCs and distributed in areas near GSCs in GBMs. In CW690 GBM with less GSCs, POSTN showed little diffusion staining and strictly co-localized with SOX2+ cells. Scale bar, 40 μm. (f) Immunofluorescent analysis of POSTN (red) and the TAM marker Iba1 (green). Three sets of representative staining from 3 tumours (B7, C4 and D8) in a tissue microarray slide (GL803a, US Biomax) were presented to show enrichment of TAMs in POSTN abundant regions in GBMs. Areas indicated with squares were enlarged and shown on right side of each picture. Scale bar, 80 μm. (g) Graphical analysis of (f) showing the enrichment of TAMs in POSTN-high areas. Average levels of POSTN and Iba1 signals were determined by ImageJ. Iba1+ TAM densities from five random POSTN-high and POSTN-low areas in each GBM were normalized to total TAMs. ∗∗, P < 0.01; ∗, P < 0.05. (n = 5 fields; mean ± s.e.m.; two tailed unpaired t-test).

Supplementary Figure 3 Silencing POSTN by shRNA reduced TAM density and extended survival of mice bearing the GSC-derived xenografts.

(a) Immunoblot analysis of POSTN in GSCs expressing non-targeting shRNA (shNT) or POSTN shRNA (shPOSTN, O55). Disrupting POSTN by the shRNA clone O55 through lentiviral infection reduced POSTN expression by >90% in GSCs. (b,c) Kaplan–Meier survival curves of mice implanted with GSCs expressing shPOSTN or shNT (control). GSCs (T3691 or T4121) were transduced with shPOSTN (O55, O56, or O57) or shNT through lentiviral infection and then transplanted intracranially into athymic/nude mice (20,000 cells per mouse). Mice bearing GSC-derived tumours expressing shPOSTN showed a significant survival extension relative to the control group. (n = 5 mice for each group; P = 0.0477 for T3691-O55 versus shNT; P = 0.0026 for T4121-O56 versus shNT; P = 0.0027 for T4121-O57 versus shNT; two-tailed log-rank test). (d) Immunofluorescent analysis of the TAM marker Iba1 (green) in GBM tumours derived from GSCs expressing shPOSTN (O55) or shNT. Frozen sections of GBM tumours derived from GSCs (T3691) expressing shPOSTN (O55) or shNT (NT) were immunostained with antibodies against Iba1 and counterstained with DAPI (blue). A dramatic reduction of TAMs was detected in the GSC-derived tumours expressing shPOSTN. Scale bar, 40 μm. (e) Graphical analysis of (d) showing a significant reduction of TAM density by ∼70% in the GSC-derived tumours expressing shPOSTN. POSTN intensity and TAM density were analysed with ImageJ. ∗∗, P < 0.01 (n = 5 tumours; mean ± s.e.m.; two tailed unpaired t-test).

Supplementary Figure 4 TAMs in GSC-derived tumours were CD11b+ and Iba1+ macrophages that were reduced after POSTN disruption.

(a) Immunofluorescent analysis of POSTN (red) and the TAM marker CD11b (green) in GBM tumours derived from GSCs expressing POSTN shRNA (shPOSTN) or NT shRNA (shNT). Frozen sections of GBM tumours derived from GSCs with shPOSTN (O56 and O57) or shNT were immunostained with antibodies against POSTN and Iba1 and counterstained with DAPI (blue). Reductions of both POSTN and TAMs were detected in GSC-derived tumours expressing shPOSTN. Scale bar, 80 μm. (b) Graphical analysis of (a) showing a decrease of POSTN signal intensity by ∼80% in the GSC-derived tumours expressing POSTN shRNA. ∗∗, P < 0.01 (n = 5 tumours; mean ± s.e.m; two tailed unpaired t-test). (c) Graphical analysis of (a) showing a significant reduction of TAM density by ∼60% in the GSC-derived tumours expressing shPOSTN (O56 or O57). POSTN intensity and TAM density were analysed with ImageJ. ∗∗∗, P < 0.001 (n = 5 tumours; mean ± s.e.m; two tailed unpaired t-test). (d) Co-immunofluorescent staining of two TAM markers Iba1 (red) and CD11b (green) in sections of GSC-derived orthotopic xenografts. Frozen sections of GBM xenografts derived from GSCs (T387) expressing shNT or shPOSTN (O57) were immunostained with antibodies against Iba1 and CD11b and counterstained with DAPI (blue). Overlapping staining of Iba1 and CD11b was detected in the majority of TAMs in GSC-derived tumours. Scale bar, 80 μm. (e) Co-immunofluorescent staining of two TAM markers Iba1 (red) and CD11b (green) in TAMs isolated from subcutaneous xenografts. Subcutaneous tumour derived from GSCs (CW0322) were dissociated and cultured in complete Neurobasal media for 24 h, and the attached, macrophage-like cells were trypsinized and transferred to RPMI/1640 media supplemented with 10% FBS. The attached cells were immunostained with the pan macrophage marker CD11b (green) along with Iba1 (red). All CD11b+ TAMs are also Iba1+, indicating that Iba1 is a universal TAM marker. Scale bar, 80 μm.

Supplementary Figure 5 TAMs in human GBMs are mainly monocyte-derived M2 macrophages that correlate with the tumour prognosis.

(a) Immunohistochemical (IHC) staining of monocyte-derived TAM marker (CCR2) or resident microglia marker (CX3CR1) in human primary GBM tumours. Two consecutive human GBM tissue microarray slides (GL806b, US Biomax) were immunostained with antibodies against CCR2 or CX3CR1, respectively. CCR2 or CX3CR1 staining in representative tumour cases (A4, B4, and B7) were presented. The majority of TAMs in GBMs were CCR2+/CX3CR1− (monocyte-derived macrophages). Representative image of the IHC staining in normal brain (H2) were presented to show CCR2−/CX3CR1+ microglia (indicated by arrays) in normal brain tissue. Scale bar, 40 μm. (b–d) Immunofluorescent staining of the pan macrophage marker Iba1 (red) and M2 TAM markers CD163 (b) and Fizz1 (c), or M1 TAM marker HLA-DR (d) and in primary GBM tumours. Frozen sections of GBM tumours were immunostained with the indicated antibodies and counterstained with DAPI (blue). Scale bar, 40 μm. (e–g) Graphical analyses of (b), (c), (d) showing fractions of CD163+/Fizz1+ TAMs (M2 subtype) and HLA-DR+ TAMs (M1 subtype) in primary GBM tumours (n = 5 human GBMs; mean ± s.e.m.). (h) Bioinformatic analysis of the relationship between the M2 TAM marker CD163 expression and GBM patient survival in TCGA dataset (The Cancer Genome Atlas) from Oncomine (http://www.oncomine.com). The threshold for high/low CD163 expression is determined by the average of CD163 expression levels in 525 GBM patients. The Kaplan–Meier survival curves demonstrate a significant invert correlation between CD163 levels and overall survival (n = 525 patients; CD163 low, n = 244 patients; CD163 high, n = 271 patients; two-tailed log-rank test; P = 0.0013) of GBM patients. (i) Bioinformatic analysis of the relationship between the M1 TAM marker MHCII expression and GBM patient survival in TCGA dataset (The Cancer Genome Atlas) from Oncomine (http://www.oncomine.com). The threshold for high/low MHCII expression is determined by the average of MHCII expression levels in 525 GBM patients. The Kaplan–Meier survival curves demonstrate a significant positive correlation between MHCII levels and overall survival of GBM patients. (n = 525 patients; MHCII low, n = 280 patients; MHCII high, n = 235 patients; two-tailed log-rank test; P = 0.0007).

Supplementary Figure 6 The M2 subtype TAMs are enriched in POSTN abundant areas in GBMs and POSTN promotes M2 TAM marker expression.

(a) Immunofluorescent analysis of POSTN (red) and the M2 subtype TAM marker CD206 (green) in a GBM tissue microarray (GL806c, US Biomax). Two sets of representative staining were presented to show the enrichment of TAMs in POSTN abundant regions in GBMs. Scale bar, 80 μm. (b) Immunohistochemical staining of POSTN and the M2 subtype TAM marker CD163 in two consecutive tissue microarray slides, respectively (GL806c, US Biomax). Representative staining images were presented to show that the GBM (GL806c-F1) with higher POSTN levels contains more CD163+ cells (M2 TAMs) and the GBM (GL806c-E3) with lower POSTN levels has less CD163+ (M2 TAMs). Scale bar, 40 μm. (c) Graphical analysis of POSTN and CD163 staining in the tissue microarray slides. 63.2% of GBM cases showed POSTNHigh/CD163High staining, and 22.1% of GBM cases showed POSTNLow/CD163Low staining. Only 11.8% of GBM cases showed POSTNHigh/CD163Low staining, and 2.94% of GBM cases showed POSTNLow/CD163High staining. The majority (85.3%) of GBM cases showed that M2 TAM density correlates with POSTN protein levels (data from 68 tumours). (d) RT-qPCR analyses showing up-regulation of the M2 TAM markers induced by POSTN in the isolated mouse peritoneal macrophages. The peritoneal macrophages were stimulated with rPOSTN (0.5 μg ml−1) in the RPMI media supplemented with 0.1% BSA for 24 h. RT-qPCR analysis indicates upregulation of the M2 markers STAB1 and Lyve1, along with the downregulation of the M1 markers CCL17 and IL1b. ∗∗, P < 0.01; ∗∗∗, P < 0.001. (n = 3 biologically independent samples per group; mean ± s.e.m; two tailed unpaired t-test; experiment was repeated three times). (e) RT-qPCR analyses showing downregulation of M1 TAM markers induced by POSTN in PMA-primed macrophage-like U937 cells. PMA-primed U937 cells were treated with recombinant POSTN (0.5 μg ml−1) in RPMI media supplemented with 0.1% BSA for 24 h. RT-qPCR analysis indicated downregulation of the M1 TAM markers including CCL17, IL-12a, and iNOS. ∗∗, P < 0.01; ∗∗∗, P < 0.001; ns, P > 0.05. (n = 3 biologically independent samples per group; mean ± s.e.m; two tailed unpaired t-test; experiment was repeated three times).

Supplementary Figure 7 POSTN disruption shows negligible impact on GSC growth in vitro and differentiated glioma cells rarely express POSTN in GBMs.

(a) Cell titre assay showing proliferation of GSCs expressing shPOSTN or shNT. GSCs were attached to human embryonic stem cell matrix and then transduced with shNT (NT) or shPOSTN (O56 or O57) through lentiviral infection. Cell proliferation was monitored with the luminescent cell viability assay kit at indicated time points. No significant difference was detected in cell proliferation between GSC populations expressing shPOSTN and shNT. (n = 3 biologically independent samples per group; mean ± s.e.m.; one representative experiment shown, and the experiment was repeated 3 times). (b) Immunoblot analysis showing knock-down efficiency of shPOSTN in GSCs through lentiviral infection on Day 2. An 85 ∼ 90% decrease of endogenous POSTN was detected in GSCs transduced with shPOSTN. (c) Representative images of attached GSCs expressing shNT or shPOSTN. Scale bar, 40 μm. Only mild or negligible effect of POSTN disruption on GSC morphology was detected. (d,e) Immunofluorescent staining of POSTN (green) and the neuron marker TUJ1 (red) in human GBM samples. Scale bar, 40 μm. Statistical analysis (e) showing that less than 5% of TUJ1+ cells overlapped with POSTN staining (n = 3 human GBMs; mean ± s.e.m.). (f,g) Immunofluorescent staining of POSTN (green) and the astrocyte marker GFAP (red) in human GBM samples. Scale bar, 40 μm. Statistical analysis (g) showing that approximately 5% of GFAP+ cells are positive POSTN staining. (n = 3 human GBMs; mean ± s.e.m.). (h,i) Immunofluorescent staining of POSTN (green) and the oligodendrocyte marker Galc (red) in human GBM samples. Scale bar, 40 μm. Statistical analysis (i) showing that less than 6% of Galc+ cells overlapped with POSTN staining (n = 3 human GBMs; mean ± s.e.m.).

Supplementary Figure 8 Disrupting POSTN or its signalling showed very mild effect on tumour vascularization.

(a) Immunofluorescent analysis of blood vessels marked by Glut1 (red) and TAMs labelled by Iba1 (green) in GBM xenografts derived from GSCs expressing shPOSTN or shNT. Frozen sections of GBM tumours derived from GSCs (T387) expressing shPOSTN (O56 and O57) or shNT were immunostained with antibodies against Glut1 and Iba1 and counterstained with DAPI (blue). A dramatic reduction of TAMs in the GSC-derived tumours expressing shPOSTN was confirmed, while the blood vessels were not affected by POSTN disruption. Scale bar, 100 μm. (b) Graphical analysis of (a) showing a significant reduction of TAM density by ∼70% in the GSC-derived tumours expressing shPOSTN. ∗∗∗, P < 0.001 (n = 5 tumours; mean ± s.e.m; two tailed unpaired t-test). (c) Graphical analysis of (a) showing no significant difference in vessel intensity between control tumours (shNT) and the tumours expressing shPOSTN. ns, P > 0.05. Vessel density and TAM density were analysed with ImageJ. (n = 5 tumours; mean ± s.e.m.; two tailed unpaired t-test). (d) Immunofluorescent staining of the vessel marker Glut1 (red) in GBM xenografts treated with RGD inhibitory or control peptide. Mice bearing GBM xenografts were treated with the RGD inhibitory or control peptide at 25 mg kg−1 for 5 days through intraperitoneal delivery. Frozen tumour sections were immunostained with antibodies against Glut1 (red) and counterstained with DAPI (blue). Scale bar, 100 μm. (e) Graphical analysis of (d) showing no significant difference of vessel density between the GBM xenografts treated with the RGD inhibitory peptide and control peptide. ns, P > 0.05 (n = 5 tumours; mean ± s.e.m.; two tailed unpaired t-test).

Supplementary Figure 9 POSTN disruption did not affect GSC population and cell proliferation but induced apoptosis in GSC-derived xenografts.

(a) Immunofluorescent staining of SOX2 in GBM xenografts derived from GSCs expressing shPOSTN or shNT to determine the effect of POSTN knockdown on GSC populations. Frozen sections of GBM xenografts derived from GSCs (T387) expressing shNT or shPOSTN were immunostained with the antibody against SOX2 (red) and counterstained with DAPI (blue). Scale bar, 40 μm. (b) Graphical analysis of (a) showing no significant difference in SOX2+ populations between tumours expressing shPOSTN and shNT (control). Scale bar, 40 μm. P > 0.05 (n = 5 tumours; mean ± s.e.m.; two tailed unpaired t-test). (c) Immunohistochemical (IHC) staining of Ki67 (brown) in GBM xenografts derived from GSCs expressing shPOSTN (O56 or O57) or shNT to determine the effect of POSTN down-regulation on cell proliferation. Scale bar, 40 μm. (d) Graphical analysis of (c) showing no significant difference in Ki67 staining between the tumours expressing shPOSTN and shNT. Scale bar, 40 μm. P > 0.05 (n = 5 tumours; mean ± s.e.m.; two tailed unpaired t-test). (e) TUNEL assay detecting cell apoptosis in GSC-derived tumours expressing shPOSTN or shNT. More apoptotic cell deaths were detected in the tumours with POSTN down-regulation (shPOSTN, O56 or O57) than the control tumours (shNT). Scale bar, 20 μm. (f) Graphical analysis of (e) showing a significant increase (250–300%) in TUNEL positive cells in tumours derived from GSCs expressing shPOSTN relative to the control tumours (shNT). ∗∗∗, P < 0.001 (n = 5 tumours; mean ± s.e.m.; two tailed unpaired t-test). (g) qPCR analysis of multiple M1 and M2 macrophage markers in CD11b+ populations from normal mouse brains or GBM xenografts. M2 macrophage markers STAB1 and Lyve1 were markedly up-regulated in CD11b+ population isolated from GBM xenografts, whereas M1 marker IL1b was significantly down-regulated. ∗∗∗, P < 0.001; ns, P > 0.05. (mean ± s.e.m.; n = 3 biologically independent samples per group, one representative experiment shown, and the experiment was repeated 3 times).

Supplementary Figure 10

Scans of uncropped blots.

Supplementary Table 1 A list of the secreted proteins differentially expressed by GSCs relative to matched non-stem tumour cells (NSTCs) from primary GBMs.
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Zhou, W., Ke, S., Huang, Z. et al. Periostin secreted by glioblastoma stem cells recruits M2 tumour-associated macrophages and promotes malignant growth. Nat Cell Biol 17, 170–182 (2015). https://doi.org/10.1038/ncb3090

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