Dual targeting macrophages and microglia is a therapeutic vulnerability in models of PTEN-deficient glioblastoma
Yang Liu, … , Amy B. Heimberger, Peiwen Chen
Yang Liu, … , Amy B. Heimberger, Peiwen Chen
Published October 1, 2024
Citation Information: J Clin Invest. 2024;134(22):e178628. https://doi.org/10.1172/JCI178628.
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Dual targeting macrophages and microglia is a therapeutic vulnerability in models of PTEN-deficient glioblastoma

Abstract

Tumor-associated macrophages and microglia (TAMs) are critical for tumor progression and therapy resistance in glioblastoma (GBM), a type of incurable brain cancer. We previously identified lysyl oxidase (LOX) and olfactomedin like-3 (OLFML3) as essential macrophage and microglia chemokines, respectively, in GBM. Here, single-cell transcriptomics and multiplex sequential immunofluorescence followed by functional studies demonstrate that macrophages negatively correlate with microglia in the GBM tumor microenvironment. LOX inhibition in PTEN-deficient GBM cells upregulates OLFML3 expression via the NF-κB-PATZ1 signaling pathway, inducing a compensatory increase of microglia infiltration. Dual targeting macrophages and microglia via inhibition of LOX and the CLOCK-OLFML3 axis generates potent antitumor effects and offers a complete tumor regression in more than 60% of animals when combined with anti-PD1 therapy in PTEN-deficient GBM mouse models. Thus, our findings provide a translational triple therapeutic strategy for this lethal disease.

Authors

Yang Liu, Junyan Wu, Hinda Najem, Yiyun Lin, Lizhi Pang, Fatima Khan, Fei Zhou, Heba Ali, Amy B. Heimberger, Peiwen Chen

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Figure 3

LOX negatively regulates OLFML3 expression and microglia infiltration in GBM.

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LOX negatively regulates OLFML3 expression and microglia infiltration in...
(A and B) Identification (A) and expression heatmap (B) of 4 overlapping PTEN-LOX axis-regulated genes encoding secreted factors in PTEN-KO versus WT SF763 cells and in LOX shRNA (shLOX) versus shRNA control (shC) U87 cells. Red signal indicates higher expression and blue signal denotes lower expression. (C) Immunoblots for OLFML3 and LOX in lysates of U87 and PTEN-KO SF763 cells expressing shC and shLOX. (D and E) Immunoblots for OLFML3 in lysates of U87 and PTEN-KO SF763 cells (D) and CT2A cells and 005 GSCs (E) treated with BAPN at indicated concentrations. (F and G) Representative images (F) and quantification (G) of relative migration of HMC3 microglia following stimulation with the conditioned media (CM) from U87 cells pretreated with or without BAPN (200 μM). Scale bar: 400 μm. n = 3 independent samples. Student’s t test. (H) Immunoblots for OLFML3 and LOX in lysates of GL261 cells in the presence or absence of LOX overexpression (OE). (I and J) IF (I) and quantification (J) of relative CX3CR1+ microglia (green) in tumors from CT2A-bearing mice treated with or without BAPN (2 g/L in drinking water) on day 4. DAPI (blue). Scale bar: 50 μm. n = 3 independent samples. Student’s t test. (K and L) Representative images (K) and quantification (L) of flow cytometry for the percentage of intratumoral CD45loCD11b+CX3CR1+ microglia in size-matched tumors from CT2A tumor-bearing mice treated with or without BAPN. n = 3 independent samples. Student’s t test. (M–P) IF (M) and quantification of relative F4/80+ macrophages (N, green), CX3CR1+ microglia (O, green), and OLFML3+ cells (P, red) in tumors from mice implanted with control and LOX-overexpressed GL261 cells. DAPI (blue). Scale bar: 50 μm. n = 3 independent samples. Student’s t test. *P < 0.05; **P < 0.01; and ***P < 0.001.