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Pharmacological inhibition of BACE1 suppresses glioblastoma growth by stimulating macrophage phagocytosis of tumor cells

Nature Cancer volume 2, pages 1136–1151 (2021)Cite this article

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Abstract

Glioblastoma (GBM) contains abundant tumor-associated macrophages (TAMs). The majority of TAMs are tumor-promoting macrophages (pTAMs), while tumor-suppressive macrophages (sTAMs) are the minority. Thus, reprogramming pTAMs into sTAMs represents an attractive therapeutic strategy. By screening a collection of small-molecule compounds, we find that inhibiting β-site amyloid precursor protein-cleaving enzyme 1 (BACE1) with MK-8931 potently reprograms pTAMs into sTAMs and promotes macrophage phagocytosis of glioma cells; moreover, low-dose radiation markedly enhances TAM infiltration and synergizes with MK-8931 treatment to suppress malignant growth. BACE1 is preferentially expressed by pTAMs in human GBMs and is required to maintain pTAM polarization through trans-interleukin 6 (IL-6)–soluble IL-6 receptor (sIL-6R)–signal transducer and activator of transcription 3 (STAT3) signaling. Because MK-8931 and other BACE1 inhibitors have been developed for Alzheimer’s disease and have been shown to be safe for humans in clinical trials, these inhibitors could potentially be streamlined for cancer therapy. Collectively, this study offers a promising therapeutic approach to enhance macrophage-based therapy for malignant tumors.

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Fig. 1: Identification of the BACE1 inhibitor MK-8931 as a potent activator of macrophage phagocytosis of glioma cells.
Fig. 2: Targeting BACE1 with MK-8931 potently inhibits tumor growth and extends survival of animals bearing intracranial GBM xenografts.
Fig. 3: Inhibiting BACE1 with MK-8931 converts pTAMs into sTAMs.
Fig. 4: Disrupting BACE1 in U937-derived macrophages suppresses tumor growth in GBM xenografts derived from GSCs cotransplanted with macrophages.
Fig. 5: Depletion of monocyte-derived TAMs by clodronate treatment attenuates antitumor effects of MK-8931 treatment in GBM xenografts.
Fig. 6: Low doses of IR synergize with MK-8931 treatment to suppress GBM growth and prolong survival of tumor-bearing mice.
Fig. 7: BACE1 maintains pTAMs by cleaving IL-6R to activate trans-IL-6–sIL-6R–STAT3 signaling.
Fig. 8: BACE1 is preferentially expressed by pTAMs in human primary GBMs.

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Data availability

Any additional data that support the findings of this study are available from the corresponding author upon reasonable request. All RNA-seq data were deposited in the Gene Expression Omnibus under accession codes GSE181649 and GSE181650. The Cancer Genome Atlas dataset was downloaded from GlioVis (http://gliovis.bioinfo.cnio.es/). GSC lines will be provided upon request to S.B. through a material-transfer agreement. Source data are provided with this paper.

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Acknowledgements

We thank the Brain Tumor and Neuro-Oncology Centers at the Cleveland Clinic for providing GBM surgical specimens for this study. We greatly appreciate the help provided by M. McGraw from the Brain Tumor Bank at the Cleveland Clinic. We thank the Flow Cytometry Core, the Imaging Core and Central Cell Services at Cleveland Clinic Lerner Research Institute for their assistance. This work was supported by the Cleveland Clinic Foundation and NIH R01 grants NS091080 and NS099175 (to S.B.). This work made use of an IVIS system (Spectrum CT) that was purchased with NIH SIG grant S10OD018205.

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Authors and Affiliations

  1. Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA

    Kui Zhai, Zhi Huang, Qian Huang, Weiwei Tao, Xiaoguang Fang, Aili Zhang, George R. Stark & Shideng Bao

  2. Department of Inflammation and Immunity, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA

    Xiaoxia Li & Thomas A. Hamilton

  3. Case Comprehensive Cancer Center, Case Western Reserve University School of Medicine, Cleveland, OH, USA

    Xiaoxia Li, George R. Stark & Shideng Bao

  4. Center for Cancer Stem Cell Research, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA

    Shideng Bao

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Contributions

S.B. developed the working hypothesis and scientific concept, designed experimental approaches, oversaw the study and prepared the manuscript. K.Z. designed experiments, performed most experiments, analyzed and prepared data, and wrote the initial draft of the manuscript. Z.H. generated iPSC-derived monocytes and macrophages and established GBM xenograft models. Q.H. and K.Z. performed flow cytometric analyses of macrophage phagocytosis and analyzed data. X.F., W.T., Q.H. and A.Z. prepared human GBM samples and isolated GSCs. X.L., T.A.H. and G.R.S. provided scientific input for the paper. G.R.S. also edited the manuscript.

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Correspondence to Shideng Bao.

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Competing interests

S.B. and K.Z. are listed as inventors in a pending patent application related to this study. The other authors declare no competing interests.

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Peer review information Nature Cancer thanks Samuel Cheshier, Michele De Palma and Benjamin Purow for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Derivation of Macrophages from Human iPS Cells (iPSCs).

a, A brief protocol for generating monocytes and macrophages from human iPS cells (iPSCs) expressing GFP. EB: Embryonic body; PMA: Phorbol 12-myristate 13-acetate. b, A brief protocol for the generation of M2-like macrophages (iPSC-M2) from iPSC-derived monocytes expressing GFP. c, Immunofluorescent analysis of the pan macrophage markers (CCR2, CD11b and IBA1; in red), the M2 macrophage makers (ARG1 and FIZZ1; in red), BACE1 (in red), or the microglia-specific markers (TMEM119 and CX3CR1; in red) in iPSCs-M2 macrophages. Representative immunofluorescent images show expression of BACE1, the pan macrophage markers, and the M2 macrophage markers in iPSC-M2 macrophages. d,e, Flow cytometric analyses of macrophage phagocytosis of human glioma cells in vitro. Representative flow cytometric plots (d) show the gating strategy for identifying macrophage phagocytosis of glioma cells (CCF-3264, tdTomato+) in iPSC-derived macrophages (GFP+) that were pre-treated with MK-8931 (50 μg/mL) or vehicle control for 48 h. Quantification (e) of the flow cytometric analyses shows fractions of macrophage phagocytosis of glioma cells (GFP+/tdTomato+). Data are shown as mean ± SEM. n = 10 independent phagocytosis assays per group. Statistical significance was determined by two-tailed Student’s t-test; P < 0.0001.

Source data

Extended Data Fig. 2 BACE1 Inhibition Promotes Macrophage Phagocytosis of Glioma Cells in vitro and in vivo.

a, Immunofluorescent analyses of pan TAM markers (IBA1 or CD11b), M2 macrophage makers (CD163, ARG1, or FIZZ1), and BACE1 expression in bone marrow-derived M2-like macrophages (BMDMs-M2). Representative images show expression of BACE1 and M2 macrophage markers in BMDMs-M2 macrophages. b, Immunofluorescent analysis of BACE1 expression in BMDMs-M2 macrophages transduced with shBACE1 or shNT (control). c,d, Representative fluorescent images (c) show phagocytosis of BMDMs (in green) against GSCs (CCF-3264, in red) after BACE1 disruption in BMDMs. Quantification (d) shows fractions of BMDMs containing engulfed glioma cells. Data are shown as mean ± SEM. n = 3 independent experiments (about 300 BMDMs/group/experiment). Statistical significance was determined by two-tailed Student’s t-test; P = 0.0337. e, In vitro analyses of macrophage phagocytosis of glioma cells by confocal microscopy and Z-stack reconstruction to confirm the engulfment of GSCs (in red) by BMDMs (in green) after BACE1 disruption. Representative 2D and 3D images are shown. f,g, Representative images (f) and quantification (g) show MK-8931-activated TAM (IBA1+, in green) phagocytosis of glioma cells (TRA-1–85+, in red) in GBM xenografts derived from CCF-DI315 GSCs. Data are shown as mean ± SEM. n = 6 tumor samples (each sample includes about 80 TAMs). Statistical significance was determined by two-tailed Student’s t-test; P = 0.0014. Ctl: Control; MK: MK-8931. h,i, Representative flow cytometric plots (h) show gating strategy for identifying TAM (CD45+/Gr1-/CD11b+/DAPI-) phagocytosis of tumor cells (TRA-1–85+) from GBM xenografts treated with MK-8931 or vehicle control. Quantifications (i) of TAM phagocytosis show fractions of macrophage phagocytosis of glioma cells (CD11b+/TRA-1–85+) in the treated and control xenografts. Data are shown as mean ± SEM. n = 5 tumor samples per group. Statistical significance was determined by two-tailed Student’s t-test; P < 0.0001.

Source data

Extended Data Fig. 3 BACE1 Is Highly Expressed by pTAMs in GBM Xenografts.

a, b, Immunofluorescent analyses of BACE1 and the pan TAM marker IBA1 or CD11b in GBM xenografts derived from GSCs (CCF-3264 or CCF-DI315). Representative immunofluorescent images (a) show the distribution and co-localization of BACE1 (in green) with the pan TAM marker (IBA1 or CD11b; in red) in GBM xenografts. Quantifications (b) show the fractions of BACE1-positive TAMs (BACE1+/IBA1+ or BACE1+/CD11b+ cells) in total TAMs (IBA1+ or CD11b+ cells) in GBM xenografts. Data are shown as mean ± SEM. n = 6 GBM xenografts per group. c,d, Immunofluorescent analyses of BACE1 and the pTAM marker CD163 or FIZZ1 in GBM xenografts derived from GSCs (CCF-3264 or CCF-DI315). Representative immunofluorescent images (c) show the distribution and co-localization of BACE1 (in green) with pTAM markers (CD163 and FIZZ1; in red) in GBM xenografts. Quantification (d) shows the fractions of BACE1-positive pTAMs (BACE1+/CD163+ or BACE1+/FIZZ1+ cells) in total pTAMs (CD163+ or FIZZ1+ cells) in GBM xenografts. Data are shown as mean ± SEM. n = 6 GBM xenografts per group. e,f, Immunofluorescent analyses of BACE1 and the sTAM marker HLA-DR or CD11c in GBM xenografts derived from GSCs (CCF-3264 or CCF-DI315). Representative immunofluorescent images (e) show staining of BACE1 (in green) and the sTAM markers (HLA-DR or CD11c; in red) in GBM xenografts. Quantification (f) shows the fractions of BACE1+ sTAMs (BACE1+/HLA-DR+ or BACE1+/CD11c+ cells) in total sTAMs (HLA-DR+ or CD11c+ cells) in GBM xenografts. Data are shown as mean ± SEM. n = 6 GBM xenografts per group.

Source data

Extended Data Fig. 4 The Effects of MK-8931 Treatment on Apoptosis, Proliferation, Vessel density and GSC population in GBM Xenografts.

a,b, Representative immunofluorescent images (a) of cleaved caspase-3 staining to detect apoptosis in MK-8931-treated or control GBM xenografts derived from GSCs (CCF-3264 or CCF-DI315). Quantifications (b) show fractions of cleaved caspase-3+ cells in the MK-8931-treated or control tumors. CCF-3264 xenografts: n = 10 (MK-8931-treated) or 8 (control) tumors; CCF-DI315 xenografts: n = 5 tumors per group. c,d, Representative immunofluorescent images (c) of CD31 staining to examine vessel density in MK-8931-treated or control GBM xenografts derived from GSCs (CCF-3264 or CCF-DI315). Quantifications (d) show relative vessel density (CD31+ cells) in the MK-8931-treated or control tumors. CCF-3264 xenografts: n = 6 (MK-8931-treated) or 7 (control) tumors; CCF-DI315 xenografts: n = 5 tumors per group. e,f, Representative immunofluorescent images (e) of Ki-67 staining to detect cell proliferation in MK-8931-treated or control GBM xenografts derived from GSCs (CCF-3264 or CCF-DI315). Quantifications (f) show that fractions of proliferative cells (Ki-67+) in the MK-8931-treated or control tumors. CCF-3264 xenografts: n = 10 (MK-8931-treated) or 8 (control) tumors; CCF-DI315 xenografts: n = 5 tumors per group. g,h, Representative immunofluorescent images (g) of SOX2 staining to detect GSC populations in MK-8931-treated or control GBM xenografts derived from GSCs (CCF-3264 or CCF-DI315). Quantifications (h) show the fractions of GSCs (SOX2+ cells) in the MK-8931-treated or control tumors. CCF-3264 xenografts: n = 6 (MK-8931-treated) or 7 (control) tumors; CCF-DI315 xenografts: n = 5 tumors per group. Data are shown as mean ± SEM; statistical significance was determined by two-tailed Student’s t-test; P values are indicated on the figures (b,d,f,h).

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Extended Data Fig. 5 MK-8931 Treatment Shows Little Effect on Glioma Cells in Vitro.

a,b, Representative images (a) of tumorspheres derived from CCF-3264 GSCs treated with MK-8931 (50 μg/mL) or vehicle control. Quantifications (b) show the number and size of tumorspheres treated with MK-8931 or control. Data are shown as mean ± SEM. Statistical significance was determined by two-tailed Student’s t-test. n = 3 independent experiments; ns, not significant. c, Immunoblot analysis of SOX2 expression in GSCs (CCF-3264 and CCF-DI315) treated with MK-8931 (50 μg/mL) or the vehicle control for three days. GAPDH was blotted as control. Similar results were confirmed in 3 independent experiments. d, Immunoblot analyses of cleaved PARP and cleaved caspase 3 to detect apoptosis in GSCs (CCF-3264 and CCF-DI315) treated with MK-8931 (50 μg/mL), etoposide (Etop, 1 μM), or the vehicle control for indicated times. GAPDH was blotted as control. Ctl: Control; Etop: Etoposide. Similar results were confirmed in 3 independent experiments. e, Cell viability assay of GSCs (CCF-3264 and CCF-DI315) treated with MK-8931 (10 or 50 μg/mL) or the vehicle control for three days to examine the effect of MK-8931 treatment on glioma cells. Data are shown as mean ± SEM. Significance was determined by one-way ANOVA analysis. n = 4 independent experiments; ns, not significant. f,g, RNA-seq analyses of GSCs treated with MK-8931 (50 μg/mL) or the vehicle control. The volcano plot (f) shows differentially and non-differentially expressed genes. Green dots represent the up-regulated genes (40); red dots represent the down-regulated genes (57); and blue dots represent the unaffected genes (24289) by MK-8931 treatment in GSCs. Gene ontology analysis (g) predicts differentially expressed genes enriched in the indicated cellular processes, but none of them was significantly affected by MK-8931 treatment in GSCs.

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Extended Data Fig. 6 BACE1 Is Required for the Maintenance of M2-like Macrophages Derived from U937.

a, A protocol for derivation of M2-like macrophages from U937 monocytes (U937-M2) in vitro. Briefly, U937 cells were primed by phorbol 12-myristate 13-acetate (PMA, 5 nM) for two days to generate M0 macrophages and then incubated with IL-4 (20 ng/mL), IL-10 (20 ng/mL), and TGF-β (20 ng/mL) for three days to produce U937-M2 macrophages. b, qPCR analyses of BACE1 mRNA expression in U937 monocytes, U937-derived M0 macrophages, and U937-derived M2-like macrophages. Data are shown as mean ± SEM. n = 3 independent experiments. Statistical significance was determined by one-way ANOVA analysis; U937 vs M0: P = 0.0060; U937 vs M2: P = 0.0002; M0 vs M2: P = 0.0064. c, Immunoblot analyses of BACE1 and the M2 markers (CD163 and FIZZ1) in U937 monocytes, U937-derived M0 macrophages, and U937-derived M2-like macrophages. GAPDH was blotted as the loading control. Similar results were obtained in 3 independent experiments. d, Immunoblot analyses of BACE1, the M2 markers (CD163 and ARG1), and the pan macrophage marker IBA1 in U937-M2 macrophages expressing shBACE1 or non-targeting sequence (shNT). GAPDH was blotted as the loading control. Similar results were obtained in 3 independent experiments. e, Immunoblot analyses of the M2 markers (CD163 and ARG1) and the total macrophage marker IBA1 in U937-M2 macrophages treated with MK-8931 (50 μg/mL) or the vehicle control for three days. GAPDH was blotted as the loading control. Similar results were obtained in 3 independent experiments.

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Extended Data Fig. 7 RNA-seq Analyses of Isolated TAMs from GBM Xenografts Treated with MK-8931 or the Vehicle Control in vivo.

a, The volcano plot of differentially and non-differentially expressed genes revealed by RNA-seq analyses in the total TAMs (CD45+/Gr1-/CD11b+/DAPI-) sorted from CCF-3264 GSCs-derived GBM xenografts treated with MK-8931 or vehicle control. The log2 fold change is plotted on the x-axis and the negative log10-adjusted P-value is plotted on the y-axis. Green dots represent the genes up-regulated by MK-8931 treatment in TAMs; red dots represent the genes down-regulated by MK-8931 treatment in TAMs; and blue dots represent the genes that were not affected by MK-8931 treatment in TAMs. b, Heat map showing differential expression of genes in TAMs (CD45+/Gr1-/CD11b+/DAPI-) isolated from CCF-3264 GSCs-derived GBM xenografts treated with MK-8931 or vehicle control. Expressions of 336 genes (242 genes upregulated, and 94 genes downregulated) were significantly altered by MK-8931 treatment in TMAs sorted from GBM xenografts. Ctl: Control; MK: MK-8931. c, Gene ontology enrichment analyses showing potential association between differentially expressed genes affected by MK-8931 treatment in TAMs sorted from CCF-3264 GSCs-derived GBM xenografts and the cellular activities involved in immune cell migration, activation, and adhesion, and inflammatory response. d, Gene expression analyses of the M1 (sTAM) and M2 (pTAM) macrophage-related genes in TAMs (CD45+/Gr1-/CD11b+/DAPI-) sorted from MK-8931-treated GBM xenografts (from CCF-3264 GSCs) relative to that from the control tumors. Nearly all the M1 macrophage (sTAM)-related genes including iNOS (mouse gene: Nos2) and MHC II (mouse genes: H2ab1, H2eb1, H2aa) were upregulated by MK-8931 treatment, while the majority of the M2 macrophage (pTAM)-related genes including CD163 (mouse gene: Cd163) and FIZZ1 (mouse gene: Retnla) were downregulated in TAMs by MK-8931 in vivo.

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Extended Data Fig. 8 BACE1 Inhibition by MK-8931 Redirects pTAMs into sTAMs in GBM Xenografts.

a, b, Analysis of relative density of pTAMs by immunofluorescent staining of pTAM markers (CD163 and FIZZ1) and the pan TAM marker IBA1 in GBM xenografts derived from CCF-DI315 GSCs and treated with MK-8931 (30 mg/kg/daily) or a vehicle control for two weeks. Representative images (a) show immunofluorescent staining of pTAMs (CD163+ or FIZZ1+, in red) and total TAMs (IBA1+, in green) in the MK-8931-treated or control GBM xenografts. Quantifications (b) show relative density of pTAMs (CD163+/IBA1+ or FIZZ1+/IBA1+) to total TAMs (IBA1+) in GBM xenografts treated with MK-8931 or the vehicle control. Data were shown as mean ± SEM. n = 6 GBM samples per group. Statistical significance was determined by two-tailed Student’s t-test; P < 0.0001. Ctl: Control; MK: MK-8931. c, d, Analysis of relative density of sTAMs by immunofluorescent staining of sTAM markers (HLA-DR and CD11c) and the total TAM marker IBA1 in GBM xenografts derived CCF-DI315 GSCs and treated with MK-8931 (30 mg/kg/daily) or a vehicle control for two weeks. Representative images (c) show immunofluorescent staining of sTAMs (HLA-DR+ or CD11c+, in red) and total TAMs (IBA1+ , in green) in MK-8931-treated or the control GBM xenografts. Quantifications (d) show relative density of sTAMs (HLA-DR+/IBA1+ or CD11c+/IBA1+) to total TAMs (IBA1+) in GBM xenografts treated with MK-8931 or the vehicle control. Data are shown as mean ± SEM. n = 6 GBM tumors. Statistical significance was determined by two-tailed Student’s t-test; P values are indicated on the figure. Ctl: Control; MK: MK-8931.

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Extended Data Fig. 9 The Effect of the Combined Low Doses of Irradiation and MK-8931 Treatment on TAM Polarization in GBM Xenografts.

a–c, Analyses of relative density of pTAMs (a) or sTAMs (b) by double immunofluorescent staining of the pan TAM marker IBA1 (in green) and the pTAM marker CD163 (in red) or the sTAM marker HLA-DR (in red) in CCF3264 GSC-derived GBM xenografts treated with MK-8931, Irradiation (IR, 2×2 Gy), IR plus MK-8931, or a vehicle control. Representative images show immunofluorescent staining of IBA1 and the pTAM marker CD163 (a) or IBA1 and the sTAM marker HLA-DR (b) in GBM xenografts from the four groups. Quantifications (c) show fractions of total TAMs (IBA1+ cells), pTAMs (IBA1+/CD163+ cells) and sTAMs (IBA1+/HLA-DR+ cells) in GBM xenografts from the four groups. Data are shown as mean ± SEM. n = 5 GBM tumors per group. Statistical significance was determined by one-way ANOVA analysis; p values are indicated on the figure. d, e, In vivo analysis of cell apoptosis by immunofluorescent staining of cleaved caspase-3 in CCF3264 GSC-derived GBM xenografts treated with MK-8931, IR (2×2 Gy), IR plus MK-8931, or a vehicle control. Representative images (d) show immunofluorescent staining of cleaved caspase-3 in GBM xenografts from the four groups. Quantifications (e) show the fractions of cleaved caspase-3+ (CC-3+) cells in GBM xenografts from the four groups. Data are shown as mean ± SEM. n = 6–8 GBM tumors per group. Statistical significance was determined by one-way ANOVA analysis; P values are indicated on the figure. Ctl: Control; MK: MK-8931; IR: Irradiation.

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Extended Data Fig. 10 BACE1 Expression Predicts Poor Survival of GBM Patients.

a–d, Kaplan-Meier survival analyses showing an inverse correlation between BACE1 expression and overall survival of GBM patients in the databases of TCGA-GBM (a), Rembrandt (b), Gravendeel (c), and LeeY (d). Two-tailed log-rank test was used for the analyses. a, n = 130 (BACE1high) or 134 (BACE1low) GBM patients; P = 0.0202; b, n = 76 (BACE1high) or 76 (BACE1low) GBM patients; P = 0.0098; c, n = 76 (BACE1high) or 77 (BACE1low) GBM patients; P = 0.0049; d, n = 46 (BACE1high) or 48 (BACE1low) GBM patients; P = 0.0053. e, A schematic illustration showing that BACE1 inhibition by MK-8931 reprograms pTAMs into sTAMs to promote TAM phagocytosis of cancer cells and thus suppresses tumor growth. Most malignant tumors including GBM contain abundant tumor-promoting TAMs (pTAMs). BACE1-mediated STAT3 activation is required for maintaining pTAMs. Targeting BACE1 by its inhibitor MK-8931 redirects pTAMs into sTAMs to engulf glioma cells and re-modulate the tumor microenvironment. Thus, BACE1 inhibition by MK-8931 potently suppresses malignant growth of GBM, highlighting the promising therapeutic potential of the macrophage-based therapy through BACE1 inhibition with MK-8931 to improve survival of cancer patients.

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Zhai, K., Huang, Z., Huang, Q. et al. Pharmacological inhibition of BACE1 suppresses glioblastoma growth by stimulating macrophage phagocytosis of tumor cells. Nat Cancer 2, 1136–1151 (2021). https://doi.org/10.1038/s43018-021-00267-9

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