Targeting legumain-mediated cell-cell interaction sensitizes glioblastoma to immunotherapy in preclinical models
Lizhi Pang, … , Justin D. Lathia, Peiwen Chen
Lizhi Pang, … , Justin D. Lathia, Peiwen Chen
Published March 25, 2025
Citation Information: J Clin Invest. 2025;135(10):e186034. https://doi.org/10.1172/JCI186034.
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Research Article Immunology Oncology

Targeting legumain-mediated cell-cell interaction sensitizes glioblastoma to immunotherapy in preclinical models

Abstract

Tumor-associated macrophages (TAMs) are the most prominent immune cell population in the glioblastoma (GBM) tumor microenvironment and play critical roles in promoting tumor progression and immunosuppression. Here we identified that TAM-derived legumain (LGMN) exhibited a dual role in regulating the biology of TAMs and GBM cells. LGMN promoted macrophage infiltration in a cell-autonomous manner by activating the GSK3β/STAT3 pathway. Moreover, TAM-derived LGMN activated integrin αv/AKT/p65 signaling to drive GBM cell proliferation and survival. Targeting of LGMN-directed macrophage (inhibiting GSK3β and STAT3) and GBM cell (inhibiting integrin αv) mechanisms resulted in an antitumor effect in immunocompetent GBM mouse models that was further enhanced by combination with anti–PD-1 therapy. Our study reveals a paracrine and autocrine mechanism of TAM-derived LGMN that promotes GBM progression and immunosuppression, providing effective therapeutic targets to improve immunotherapy in GBM.

Authors

Lizhi Pang, Songlin Guo, Yuyun Huang, Fatima Khan, Yang Liu, Fei Zhou, Justin D. Lathia, Peiwen Chen

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Figure 3

Macrophage-derived LGMN regulates GBM cell proliferation and apoptosis.

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Macrophage-derived LGMN regulates GBM cell proliferation and apoptosis. ...
(A) GSEA shows enrichment of proliferation signature in tumors from the LGMN-low compared with the LGMN-high macrophage group. Normalized enrichment score (NES) and FDR q values are shown. (B–D) Proliferation curves of SF763 (B), LN229 (C), and U87 (D) cells treated with CM from THP1 macrophages expressing shRNA control (shC) and LGMN shRNAs (shLGMN). GBM cell proliferation was recorded and analyzed using the Incucyte imaging system for 72 hours. n = 6 independent samples. (E) Proliferation curves of CT2A cells treated with CM from Raw264.7 macrophages expressing shC and shLgmn. CT2A GBM cell proliferation was recorded and analyzed using the Incucyte imaging system for 48 hours. n = 6 independent samples. (F–I) Colony formation assay and quantifications showing proliferation of SF763 (F), LN229 (G), U87 (H), and CT2A (I) cells treated with CM from THP1 or Raw264.7 macrophages expressing shC and shLGMN. n = 4 independent samples. (J) GSEA shows enrichment of apoptosis signature in the LGMN-low compared with the LGMN-high macrophage group. NES and FDR q values are shown. (K and L) Representative images and quantification of Apotracker and propidium iodide (PI) staining showing apoptosis of SF763 (K) and CT2A (L) cells treated with CM from THP1 or Raw264.7 macrophages expressing shC and shLGMN. n = 4 independent samples. (M) Diagram showing procedures of coinjection of CT2A cells and CT2A CM–educated Raw264.7 macrophages harboring shC or shLgmn into brains of C57BL/6 mice. (N) Representative images and quantification of IF for relative expression of Ki67 and cleaved caspase-3 (CC3) in size-matched tumors from C57BL/6 mice implanted with CT2A cells and CT2A CM–polarized Raw264.7 macrophages expressing shC or shLgmn. Scale bars: 25 μm. n = 3 independent samples. (O) Representative images and quantification of IF for relative expression of Ki67 and CC3 in size-matched tumors from control and LGMN–macrophage-specific knockdown (LGMN-mKD) mice implanted with CT2A cells. Scale bars: 25 μm. n = 3 independent samples. Two-way ANOVA test (B–E); 1-way ANOVA test (F–I, K, L, N, and O). *P < 0.05, **P < 0.01, ***P < 0.001.