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Kunitz-type protease inhibitor TFPI2 remodels stemness and immunosuppressive tumor microenvironment in glioblastoma

Nature Immunology volume 24, pages 1654–1670 (2023)Cite this article

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Abstract

Glioblastoma (GBM) tumors consist of multiple cell populations, including self-renewing glioblastoma stem cells (GSCs) and immunosuppressive microglia. Here we identified Kunitz-type protease inhibitor TFPI2 as a critical factor connecting these cell populations and their associated GBM hallmarks of stemness and immunosuppression. TFPI2 promotes GSC self-renewal and tumor growth via activation of the c-Jun N-terminal kinase–signal transducer and activator of transcription (STAT)3 pathway. Secreted TFPI2 interacts with its functional receptor CD51 on microglia to trigger the infiltration and immunosuppressive polarization of microglia through activation of STAT6 signaling. Inhibition of the TFPI2–CD51–STAT6 signaling axis activates T cells and synergizes with anti-PD1 therapy in GBM mouse models. In human GBM, TFPI2 correlates positively with stemness, microglia abundance, immunosuppression and poor prognosis. Our study identifies a function for TFPI2 and supports therapeutic targeting of TFPI2 as an effective strategy for GBM.

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Fig. 1: TFPI2 overexpression promotes GSC self-renewal.
Fig. 2: TFPI2 depletion impairs stemness and extends survival of GSC-bearing mice.
Fig. 3: TFPI2 promotes stemness and tumor growth by activating JNK–STAT3 signaling.
Fig. 4: TFPI2 activates STAT6 to promote microglia infiltration.
Fig. 5: TFPI2–STAT6 signaling axis triggers microglia immunosuppressive polarization.
Fig. 6: CD51 is a membrane receptor for TFPI2 on microglia.
Fig. 7: TFPI2–CD51–STAT6 axis inhibition activates antitumor immunity and synergizes with anti-PD1 therapy.
Fig. 8: TFPI2 is a prognostic biomarker correlating with GSC stemness and microglia abundance in GBM.

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Data availability

Proteomic data generated by IP–MS have been deposited in the Mass spectrometry Interactive Virtual Environment under dataset accession number of MSV000090624 (https://massive.ucsd.edu/ProteoSAFe/dataset.jsp?accession = MSV000090624). RNA-seq data have been desposited in the Gene Expression Omnibus under the accession code is GSE232486 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE232486). The TCGA GBM datasets, TCGA GBMLGG merged datasets, Brain Tumor Immune Micro Environment dataset (https://joycelab.shinyapps.io/braintime/) and published GEO datasets EGAS00001004422 (https://ega-archive.org/datasets/EGAD00001006206) and GSE131928 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE131928) were also accessed. All other data are available within the article and supplementary information. Source data are provided with this paper.

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Acknowledgements

We thank S. D. Rabkin, F. Lang, J. Hu and H. Okada for providing 005 GSCs, patient-derived GSCs, QPP7 GSC and SB28, respectively. This work was supported in part by NIH R00 CA240896 (P.C.), NIH R01 NS124594 (P.C.), DoD Career Development Award W81XWH-21-1-0380 (P.C.), Cancer Research Foundation Young Investigator Award (P.C.), Lynn Sage Scholar Award (P.C.), American Cancer Society Institutional Research Grant IRG-21-144-27 (P.C.), NIH P50 CA221747 (to P.C., Brain Cancer SPORE CEP Award), philanthropic donation from Mindy Jacobson and the Bill Bass Foundation (P.C.), Northwestern University start-up funds (P.C.), the Northwestern Medicine Malnati Brain Tumor Insitute of the Robert H. Lurie Comprehensive Cancer Center (P.C.), NIH R24 NS104160 (C.G.) and NIH P30 AG072977 (C.G.). W.H.H. is funded by the CPRIT Training Program (RP210028). Imaging work was performed at the Northwestern University Center for Advanced Microscopy generously supported by NCI CCSG P30 CA060553. Proteomics experiments were performed at the Northwestern Proteomics Core Facility, which is generously supported by NCI CCSG P30 CA060553, instrumentation award (S10OD025194) from NIH Office of Director, and P41 GM108569. Incucyte analysis was performed in the Analytical bioNanoTechnology Core (ANTEC) Facility supported by the Soft and Hybrid Nanotechnology Experimental (SHyNE) Resource (NSF ECCS-2025633).

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Author notes

  1. These authors contributed equally: Lizhi Pang, Madeline Dunterman.

Authors and Affiliations

  1. Department of Neurological Surgery, Lou and Jean Malnati Brain Tumor Institute, Robert H Lurie Comprehensive Cancer Center, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA

    Lizhi Pang, Madeline Dunterman, Songlin Guo, Fatima Khan, Yang Liu, Craig Horbinski, Charles David James & Peiwen Chen

  2. Mesulam Center for Cognitive Neurology and Alzheimer’s Disease; Department of Cell and Developmental Biology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA

    Erfan Taefi, Atousa Bahrami & Changiz Geula

  3. Department of Cancer Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

    Wen-Hao Hsu

  4. Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA

    Craig Horbinski

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Contributions

L.P. performed most of the experiments. M.D. performed some in vitro and in vivo studies. S.G. provided help for IHC staining. F.K. performed the kinase array profiling and provided assistance for tumor implantation. Y.L. provided help for migration assay and IP. E.T., A.B. and C.G. isolated and cultured human primary microglia. W.-H.H. provided help for scRNA-seq data analysis. C.H. and C.D.J. provided help with human patient samples and commented the study. P.C. designed the project and analyzed data. P.C., L.P. and M.D. wrote the paper. All authors participated in editing the paper.

Corresponding author

Correspondence to Peiwen Chen.

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Competing interests

L.P. and P.C. are listed as inventors on a patent related to targeting the TFPI2–CD51–STAT6 signaling axis combining with or without anti-PD1 therapy. All other authors declare no competing interests.

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Nature Immunology thanks the anonymous reviewers for their contribution to the peer review of this work. N. Bernard was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 TFPI2 promotes GSC self-renewal.

a, Correlation between TFPI2 or LOXL2 expression and IDH1-WT GBM survival in TCGA datasets. P values are shown. b, c, Correlation between stemness (b) or GSC signature (c) and TFPI2 or LOXL2 expression in TCGA GBM dataset. R and P values were determined by Pearson correlation. d, e, Expression of TFPI2 (d) and LOXL2 (e) in CD45− cells and immune cells of non-tumor tissues and GBM tumors. n = 5 and 17 for CD45− cells, 8 and 15 for microglia (MG), 7 and 13 for macrophages (MDM), 6 and 13 for neutrophils, 6 and 11 for CD4+ T cells, as well as 7 and 6 for CD8+ T cells of non-tumor tissues and GBM, respectively. f, TFPI2 copy number correlates with TFPI2 in HG-U133A TCGA GBM tumors. n = 6, 88, 405 and 9 for Hetloss, Diploid, Gain and Amp, respectively. g, TFPI2 expression in glioma tumors at different stages. n = 256, 244, and 150 for stage II, III and IV, respectively. h, TFPI2 expression in different types of brain tumors. n = 191, 130, 194 and 152 for oligodendroglioma (OG), oligoastrocytoma (OA), astrocytoma and GBM, respectively. i, TFPI2 expression in different subtypes of GBM tumors. n = 182, 156 and 151 for classical, mesenchymal and proneural, respectively. j, k, High-resolution UMAP dimensional reduction of all cells (j) and malignant cells (k) from GBM patient tumors. l, m, UMAP dimensional reduction of the basis of the expression pattern of hallmark stemness signature (l) and TFPI2 (m) in malignant cells. n, Quantification of hallmark stemness signature in TFPI2-low and TFPI2-high tumors. o, Immunoblots for CD133 and SOX2 in cell lysates from GSC20 treated with TFPI2 protein at indicated concentrations. p–r, Representative (p) and quantification (q,r) of flow cytometry analysis of apoptosis in GSC262 and GSC23. n = 3 biological replicates. Error bars indicate mean ± SD (f–i) or SEM (q, r). One-way ANOVA test (f–i, q, r) and two-tailed Student’s t-test (n).

Source data

Extended Data Fig. 2 TFPI2 regulates GSC self-renewal, proliferation, and apoptosis.

a, b, Representative of tumorspheres of QPP7 (a) and CT2A (b) cells expressing control shRNA (shC) and Tfpi2 shRNAs (shTfpi2). Scale bar, 200 µm. c, d, Representative of proliferation in shC and shTfpi2 QPP7 (c) and CT2A (d) cells. e–j, Representative and quantification of flow cytometry for apoptosis in GSC272 (e, f), QPP7 GSCs (g, h) and CT2A cells (i, j) expressing shC and shTFPI2. k, Immunoblots for cleaved caspase 3 (CC3) in cell lysates from GSC272, QPP7, and CT2A cells expressing shC and shTFPI2. l, Immunoblots for CD133 and SOX2 in cell lysates from QPP7 GSCs expressing shTfpi2 with or without reexpression of shRNA-resistant TFPI2 cDNA (OE). m, Representative of tumorspheres in QPP7 GSCs expressing shTfpi2 with or without TFPI2 OE. Scale bar, 400 µm. n, Immunoblots for TFPI2 in cell lysates from TFPI2 wild-type (WT) and different clones of CRISPR knockout (KO) GSC272. o, Immunoblots for CD133 and SOX2 in cell lysates from TFPI2-WT and TFPI2-GSC272. p, Representative of tumorspheres in TFPI2-WT and TFPI2-KO GSC272. Scale bar, 400 µm. q, r, Representative (q) and quantification (r) of immunofluorescence staining of SOX2 in size-matched tumors from C57BL/6 mice intracranially implanted with shC and shTfpi2 CT2A cells. Scale bar, 50 µm. s, t, Representative (s) and quantification (t) of immunochemistry staining of CD133 in tumors from C57BL/6 mice intracranially implanted with shC and shTfpi2 CT2A cells. Scale bar, 200 µm. u–x, Representative and quantification of immunofluorescence staining of Ki67 (u,v) and CC3 (w,x) in tumors from C57BL/6 mice intracranially implanted with shC and shTfpi2 CT2A cells. Scale bar, 50 µm. In (f, h, j, r, t, v, x), n = 3 biological replicates. Error bars indicate mean ± SEM. One-way ANOVA test.

Source data

Extended Data Fig. 3 TFPI2 regulates GSC proliferation and apoptosis via JNK and STAT3 pathways.

a, b, Representative (a) and quantification (b) of proliferation in GSC23 expressing control and TFPI2 overexpression (OE) plasmid treated with or without JNK inhibitor JNK-IN-8 (10 nM). n = 3 biological replicates. c, d, Representative (c) and quantification (d) of proliferation in GSC262 expressing control and TFPI2 OE plasmid treated with or without JNK-IN-8 (10 nM). n = 3 biological replicates. e, f, Representative (e) and quantification (f) of apoptosis in GSC23 expressing control and TFPI2 OE plasmid treated with or without JNK-IN-8 (10 nM). n = 3 biological replicates. g, h, Representative (g) and quantification (h) of apoptosis in GSC262 expressing control and TFPI2 OE plasmid treated with or without JNK-IN-8 (10 nM). n = 3 biological replicates. i, j, Representative (i) and quantification (j) of proliferation in GSC23 expressing control and TFPI2 OE plasmid treated with or without STAT3 inhibitor WP1066 (20 nM). n = 3 biological replicates. k, l, Representative (k) and quantification (l) of proliferation in GSC262 expressing control and TFPI2 OE plasmid treated with or without WP1066 (20 nM). n = 3 biological replicates. m, n, Representative (m) and quantification (n) of apoptosis in GSC23 expressing control and TFPI2 OE plasmid treated with or without WP1066 (20 nM). n = 3 biological replicates. o, p, Representative (o) and quantification (p) of apoptosis in GSC262 expressing control and TFPI2 OE plasmid treated with or without WP1066 (20 nM). n = 3 biological replicates. Error bars indicate mean ± SEM. One-way ANOVA test.

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Extended Data Fig. 4 TFPI2 promotes microglia migration via activating STAT6 signaling.

a, Amplification pattern of TFPI2 and CLOCK in GBM and low-grade glioma (LGG) patient tumors. b, GSEA analysis shows the top 10 enriched hallmark pathways in TFPI2-high GBM patient tumors compared to TFPI2-low tumors. Blue highlighted pathways relate to immune response. c, Representative of transwell migration of PrhMG following stimulation with conditioned media (CM) from GSC262 or GSC23 expressing control and TFPI2 overexpression (OE) plasmid. Scale bar, 400 µm. d, Representative of transwell migration of PrhMG, HMC3 and SIM-A9 microglia following stimulation with CM from GSC272 (for PrhMG and HMC3) or QPP7 cells (for SIM-A9) expressing control shRNA (shC) and TFPI2 shRNAs (shTFPI2). Scale bars, 400 µm for PrhMG; 200 µm for HMC3 and SIM-A9. e, f, Representative (e) and quantification (f) of flow cytometry for the percentage of CD45lowCD11b+ and CD45highCD11b+ cells in size matched tumors from C57BL/6 mice intracranially implanted with shC and shTfpi2 CT2A cells. n = 4 and 3 biological replicates for shC and shTfpi2, respectively. g, h, Representative (g) and quantification (h) of flow cytometry for the percentage of CD45highCD11b+CD68+ macrophages in size matched tumors from C57BL/6 mice intracranially implanted with shC and shTfpi2 CT2A cells. n = 4 and 3 biological replicates for shC and shTfpi2, respectively. i, Correlation between microglia signature and STAT6, AKT1, AKT2 or AKT3 in TCGA GBM dataset. R and P values were determined by Pearson correlation. j, Representative of transwell migration of mouse primary microglia isolated from wild-type (WT) and STAT6 knockout (KO) mice treated with or without TFPI2 recombinant protein (20 ng/ml). Scale bar, 200 µm. k, Representative of transwell migration of PrhMG, HMC3 and SIM-A9 microglia following stimulation with TFPI2 protein (20 ng/ml) in the presence or absence of STAT6 inhibitor AS1517499 at indicated concentrations. Scale bars, 400 µm for PrhMG; 200 µm for HMC3 and SIM-A9. Error bars indicate mean ± SEM. One-way ANOVA test.

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Extended Data Fig. 5 TFPI2-STAT6 axis regulates microglia polarization.

a, Representative of flow cytometry for CD206 in PrhMG treated with conditioned media (CM) from GSC23 expressing control and TFPI2 overexpression (OE) plasmid. b, Representative of flow cytometry for CD206 in HMC3 microglia treated with CM from GSC272 expressing shRNA control (shC) and TFPI2 shRNAs (shTFPI2). c, Representative of flow cytometry for CD206 in SIM-A9 microglia treated with CM from QPP7 GSCs expressing shC and shTfpi2 with or without reexpression of shRNA-resistant TFPI2 cDNA (OE). d, e, Representative (d) and quantification (e) of flow cytometry for the percentage of CD45hiCD11b+CD68+CD206+ macrophages in size matched tumors from C57BL/6 mice intracranially implanted with shC and shTfpi2 CT2A cells. n = 4 and 3 for shC and shTfpi2, respectively. f–i, Immunofluorescence and quantification of CX3CR1+CD206+ (f,g) and CX3CR1+CD163+ (h,i) cells in tumors from C57BL/6 mice intracranially implanted with shC and shTfpi2 CT2A cells. Scale bar, 25 μm. n = 3 (g) or 6 (i) biological replicates. j–m, Immunofluorescence and quantification of CX3CR1+CD206+ (j,k) and CX3CR1+CD163+ (l,m) cells in tumors from nude mice intracranially implanted with shC and shTFPI2 GSC272. Scale bar, 25 μm. n = 6 biological replicates. n, Representative of CD206 positive microglia isolated from wild-type (WT) and STAT6 knockout (KO) mice and treated with or without TFPI2 recombinant protein (20 ng/ml). o, p, Representative of flow cytometry for CD206 (o) and ARG1 (p) in SIM-A9 microglia treated with or without TFPI2 protein (20 ng/ml) and STAT6 inhibitor (STAT6i) AS1517499 (25 nM). q, r, Representative of flow cytometry for CD206 (q) and ARG1 (r) in HMC3 microglia treated with or without TFPI2 protein (20 ng/ml) and AS1517499 (25 nM). s, Relative mRNA expression of IL6, TNFA, LFNG, and HLA-DR in HMC3 microglia treated with or without TFPI2 protein (20 ng/ml) and AS1517499 (25 nM). n = 3 biological replicates. Error bars indicate mean ± SEM. A one-way ANOVA followed by a Tukey test was performed to compare more than two groups.

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Extended Data Fig. 6 CD51 is a receptor for TFPI2 activities on microglia.

a, CD51 expression level in human GBM samples from the TIME dataset. n = 17, 15, 13, 13, 11, and 6 for CD45− cells, microglia (MG), macrophages (MDM), neutrophils, CD4+ T cells and CD8+ T cells, respectively. b, CD51 expression in different cell populations in mouse CT2A tumor tissues. c, d, Immunoprecipitation with TFPI2 antibody (TFPI2 Ab) and immunoblotting for the interaction between TFPI2 and CD51 in SIM-A9 (c) and HMC3 (d) microglia. e, f, Calcium mobilization triggered by TFPI2 protein in HMC3 microglia (e) and PrhMG (f) treated with or without CD51 inhibitor MK-0429 at indicated concentrations. g, Immunoblots for CD51 in cell lysates of SIM-A9 microglia expressing shRNA control (shC) and Itgav shRNAs (shItgav). h, Immunoblots for P-PKCζ and PKCζ in cell lysates of shC and shItgav SIM-A9 microglia treated with or without TFPI2 protein. i, Immunoblots for P-STAT6 and STAT6 in cell lysates from SIM-A9 microglia and PrhMG treated with TFPI2 protein and MK-0429. j, Representative of transwell migration of PrhMG human HMC3, and mouse SIM-A9 microglia treated with or without TFPI2 protein and MK-0429 at indicated concentrations. Scale bars, 200 µm for HMC3 and SIM-A9, and 400 µm for PrhMG. k–n, Representative and quantification of flow cytometry for the percentage of CD206+ (k,l) and ARG1+ (m,n) SIM-A9 microglia treated with or without TFPI2 protein and MK-0429. n = 3 biological replicates. o–r, Representative (o) and quantification (p) of flow cytometry for the percentage of CD206+ (o,p) and ARG1+ (q,r) HMC3 microglia treated with or without TFPI2 recombinant protein and MK-0429 (50 nM). n = 3 biological replicates. s, t, Representative of flow cytometry for the percentage of CD206+ (s) and ARG1+ (t) PrhMG treated with or without TFPI2 protein and MK-0429. TFPI2 protein at 20 ng/ml and MK-0429 at 50 nM were used unless indicated. Error bars indicate mean ± SEM. One-way ANOVA test.

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Extended Data Fig. 7 TFPI2-STAT6 signaling mediates microglia-induced T cell suppression.

a, b, Survival curves of nude mice implanted with QPP7 cells (2x104 cells; a) or CT2A cells (2x104 cells; b) expressing control shRNA (shC) and Tfpi2 shRNAs (shTfpi2). n = 8 biological replicates. c, d, Immunofluorescence (c) and quantification (d) of CD8 in size matched tumors from C57BL/6 mice intracranially implanted with shC and shTfpi2 CT2A cells expressing. Scale bar, 50 μm. n = 3 biological replicates. e, f, Immunofluorescence (e) and quantification (f) of CD4 in size matched tumors from C57BL/6 mice intracranially implanted with shC and shTfpi2 QPP7 GSCs. Scale bar, 50 μm. n = 3 biological replicates. g, Representative of flow cytometry analysis for CD45+CD3+CD8+PD1high and CD45+CD3+CD4+PD1high T cells in the spleens of C57BL/6 mice bearing size matched shC and shTfpi2 CT2A tumors. h, Representative of flow cytometry for IFNγ+CD8+ T cells cocultured with SIM-A9 mouse microglia treated with or without TFPI2 recombinant protein (20 ng/ml) and STAT6 inhibitor (STAT6i) AS1517499 (25 nM). i, Representative of flow cytometry for fixable viability dye-labeled JURKAT T cells cocultured with PrhMG treated with or without TFPI2 protein (20 ng/ml) and AS1517499 (25 nM). Percentage of live cells and dead cells are indicated. j, k, Representative (j) and quantification (k) of flow cytometry for the percentage of HLA-DR+ cells out of total JURKAT T cells cocultured with PrhMG treated with TFPI2 protein (20 ng/ml) in the presence or absence of AS1517499 (25 nM). n = 3 biological replicates. l, Representative of flow cytometry for CD69+ JURKAT T cells cocultured with PrhMG treated with or without TFPI2 protein (20 ng/ml) and AS1517499 (25 nM). m, Representative of flow cytometry analysis for IFNγ+ JURKAT T cells cocultured with PrhMG treated with TFPI2 protein (20 ng/ml) in the presence or absence of AS1517499 (25 nM). Error bars indicate mean ± SEM. One-way ANOVA test. In (a,b), log-rank test.

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Extended Data Fig. 8 TFPI2-CD51-STAT6 signaling mediates microglia-induced T cell suppression.

a, b, CT2A cancer cell cytotoxicity induced by activated mouse primary CD8+ T cells cocultured with SIM-A9 microglia treated with TFPI2 recombinant protein (20 ng/ml) in the presence or absence of STAT6 inhibitor (STAT6i) AS1517499 (25 nM). The ratios between CD8+ T cells and CT2A cells are indicated. n = 10 biological replicates. c, GSC272 cytotoxicity induced by activated JURKAT T cells cocultured with PrhMG treated with TFPI2 protein (20 ng/ml) in the presence or absence of AS1517499 (25 nM). The ratios between CD8+ T cells and CT2A cells are indicated. n = 10 biological replicates. d, Correlation between CD274 (PD-L1) and TFPI2, STAT6, or ITGAV (CD51) in TCGA GBM dataset. e, Correlation between PDCD1LG2 (PD-L2) and TFPI2, STAT6, or ITGAV (CD51) in TCGA GBM dataset. In (d, e), R and P values were determined by Pearson correlation. Error bars indicate mean ± SEM. One-way ANOVA test.

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Extended Data Fig. 9 TFPI2 correlates with proliferation and apoptosis markers in GBM patient tumors.

a, Representative images show low, medium, and high expression of TFPI2, Ki67, and cleaved caspase 3 (CC3) in human GBM tumor samples based on immunohistochemistry staining. Scale bar, 100 µm. b,c, Quantification of immunohistochemistry staining showing strong positive correlation between TFPI2 and Ki67 (b) and negative correlation between TFPI2 and CC3 (c) in human GBM tumor samples (n = 24). R and P values were determined by Pearson correlation. d-f, The plasma TFPI2 level in newly diagnosed (n = 38) and recurrent (n = 17) (d), female (n = 26) and male (n = 29) (e), as well as old (>65) (n = 29) and young (<65) (n = 26) GBM patients (f). Error bars indicate mean ± SEM. Student’s t-test.

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Extended Data Fig. 10 Working model.

a, Illustration shows a proposed mechanism for the TFPI2 and its associated signaling axis in regulating self-renewal of glioma stem cells (GSCs) and GSC-microglia symbiosis in GBM. TFPI2 is amplified in GBM and essential for tumor progression. Upon secreting from GSCs, TFPI2 promotes GSC self-renewal by activating the JNK-STAT3 signaling pathway. Secreted TFPI2 binds to CD51 on microglia to promote microglia infiltration and immunosuppressive polarization via activation of STAT6 signaling. Consequently, these activated microglia inhibit the infiltration and activation of T cells and damage their tumor killing potential. b, Inhibition of TFPI2-CD51-STAT6 signaling axis impairs tumor progression by reducing immunosuppressive microglia and activating T cell-mediated antitumor immunity and synergizes with anti-PD1 therapy in GBM. This image was created with BioRender.com.

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Pang, L., Dunterman, M., Guo, S. et al. Kunitz-type protease inhibitor TFPI2 remodels stemness and immunosuppressive tumor microenvironment in glioblastoma. Nat Immunol 24, 1654–1670 (2023). https://doi.org/10.1038/s41590-023-01605-y

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