A zinc transporter drives glioblastoma progression via extracellular vesicles-reprogrammed microglial plasticity

Abstract

Glioblastoma (GBM) is the most aggressive form of brain cancer, with limited therapeutic options. While microglia contribute to GBM progression, the mechanisms by which they foster a protumorigenic immune environment remain poorly understood. We identify the zinc transporter Zrt- And Irt-Like Protein 4 (ZIP4) as a pivotal regulator of the GBM immune landscape. In orthotopic mouse models, ZIP4 drives tumor growth and behavioral changes. Mechanistically, ZIP4 modulates microglial plasticity through tumor-derived extracellular vesicles carrying triggering receptor expressed on myeloid cells-1 (TREM1), a process regulated by the zinc-dependent transcription factor Zinc Finger E-box Binding Homeobox 1 in GBM cells. TREM1 enhances microglial plasticity through the spleen associated tyrosine kinase-Pyruvate dehydrogenase kinase-signal transducer and activator of transcription 3 (SYK-PDK-STAT3) signaling axis, ultimately promoting an immune environment favorable to tumor progression. ZIP4 depletion or TREM1 inhibition attenuates tumor growth and behavioral effects in vivo by disrupting the tumor-microglia interaction. These findings establish ZIP4 as a key modulator of the GBM immune landscape and suggest a promising therapeutic target to counteract microglia-mediated tumor progression.

Keywords: TREM1; exosome; glioblastoma; microglia.

Fig. 1.

ZIP4 expression is associated with GBM progression and microglial activation. (A and B) Representative images and statistical analysis of ZIP4 expression in four histologic types of gliomas from Cohort I. Mean ± SEM. (C and D) Representative images and statistical analysis of ZIP4 expression in four histologic types of gliomas from Cohort II. Mean ± SEM. (E and F) Kaplan–Meier survival analysis based on ZIP4 expression in Cohort I and Cohort II. Log-rank test applied. (G) Representative images of IHC staining of ZIP4, CD68, and p-ERK1/2 in GBM tumor tissue. The scale bars represent 200 µm at 10× magnification and 50 µm at 40× magnification.

Fig. 2.

ZIP4 is associated with GBM progression in patient-derived models. (A) Spheroids were constructed using three glioma patient–derived cells. H&E staining of GPDC spheroids. n = 3/group; Mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by t test. (B) Migration and invasion assay were performed using these GPDCs. (C) Cell proliferation of these GPDCs was evaluated by Cell Counting Kit-8 (CCK-8) assay. n = 3/group; Mean ± SEM; ****P < 0.0001 by two-way ANOVA test. (D) MRI T2 imaging monitoring GPDC progression in intracranial models (days 8, 15, and 30 postsurgery).

Fig. 3.

ZIP4 drives tumor growth and metastasis of GBM In Vivo. (A) Luminescence imaging of intracranial tumor models (ZIP4 VCT/ZIP4 and koVCT/koZIP4 groups, n = 10; scale: ROI 300 to 8,000). (B) Kaplan–Meier survival analysis of intracranial tumor models of mice bearing U251 and LN229 cell lines. (C) Overview and H&E staining of intracranial U251 VCT/ZIP4 tumors. (D) IHC staining for ZIP4, CD68, IL-6, and IL-10 in tumor tissue of intracranial models. The (scale bar represents 50 µm.) n = 5/group; Mean ± SEM; *P < 0.05, **P < 0.01 by t test.

Fig. 4.

ZIP4 promotes TREM1 expression via ZEB1. (A) Electron microscopy of EVs from ZIP4-expressing GBM cells. (B) Measurement of EV diameters. (C) Western blot for EV markers (CD81, CD9, CD63, and Tsg101) in U251/LN229 ZIP4-overexpressing and knockout cells. (D) Protein array analysis of EV-derived proteins in U251/LN229 VCT/ZIP4 and koVCT/koZIP4 cells. (E) TREM1 expression in EVs by protein array. (F) Western blot of TREM1 in EV lysates. n = 3/group; Mean ± SEM; *P < 0.05 by t test. (G) Expression levels of ZIP4, ZEB1, and TREM1 in GBM stable cell lines. (H) ChIP assay confirms ZEB1 binding to the TREM1 promoter. (I) Luciferase assay showing ZEB1-mediated activation of TREM1 promoter. n = 3/group; Mean ± SEM; *P < 0.05 by t test. (J) Western blot of ZIP4, ZEB1, and TREM1 expression after ZEB1 knockdown.

Fig. 5.

TREM1 induces microglial transformation via the Akt-ERK pathway. (A) Western blot evaluated the phosphorylation of Akt, ERK1/2, and STAT3 in HMC3 cells treated with U251 VCT/ZIP4 and koVCT/koZIP4 EVs. (B) Expression of SYK, PDK, IL-6, and IL-10 in HMC3 cells treated with U251 VCT/ZIP4 and koVCT/koZIP4 EVs. (C) Western blot evaluated the phosphorylation of Akt and ERK1/2 in HMC3 cells after treatment with EVs, EVs with TREM1 depletion, or recombinant TREM1. (D) Antibody neutralization of TREM1 in U251 cell EVs. (E) Expression of SYK, PDK, IL-6, and IL-10 in HMC3 cells after TREM1 treatment. (F) TREM1 activates Akt, ERK1/2, and STAT3 in HMC3 cells. *P < 0.05, **P < 0.01.

Fig. 6.

TREM1 remodels the immune landscape of the GBM TME. (A) Representative images of CD68 and p-ERK1/2 expression in low-grade and high-grade GBM tissues. (B) Percentage of ERK1/2-activated microglia in GBM. (C) Representative images of CD68, p-ERK1/2, IL-6, and IL-10 expression in U251 intracranial models. (D) Percentage of CD68/p-ERK1/2/IL-6/IL-10⁺⁺⁺ microglia in intracranial ZIP4/VCT models. n = 3/group; Mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001 by t test. (E and F) Footprint assay in U251 intracranial models on days 8 and 50. (G) Limb hemiplegia evaluation in intracranial ZIP4/VCT models. n = 10/group; Mean ± SEM. The scale bars represent 200 µm at 10× magnification and 50 µm at 40× magnification.

Fig. 7.

Graphical abstract showing ZIP4-mediated microglia plasticity drives GBM progression. Zinc uptake via ZIP4 activates the transcription factor ZEB1, inducing TREM1 overexpression. TREM1 is subsequently secreted in EVs from glioma cells. These EVs activate microglia via the AKT/ERK/STAT3 pathway, leading to IL-6 and IL-10 secretion, promoting inflammation and tumor growth, which contribute to neuronal necrosis and tumor progression.