Journal: Journal of Neuroinflammation

Article Title: IL-1β + tumor-associated macrophages accelerate glioblastoma progression by amplifying the PGE2-EP4 signaling

doi: 10.1186/s12974-025-03551-y

Figure Lengend Snippet: GCM induces macrophages to secrete IL-1β. (A) IL1B mRNA expression in THP1 Macs was measured after stimulation with GCM from U251 and T98G cells, as well as LPS (100 ng/ml) at various time points. Statistical significance is indicated by * compared to the control group ( n = 3). (B) ELISA analysis of IL-1β protein levels in the supernatant of THP1 Macs after 48 h of indicated stimulation ( n = 6). (C) Flow cytometry analysis showing representative plots (left) and the quantification of IL-1β + cells as a percentage of total cells (right) in THP1 Macs after 48 h of stimulation ( n = 4). (D) Representative immunofluorescence images of IL-1β and CD68 expression in THP1 Macs after 48 h of stimulation ( n = 5). Blue: DAPI, Green: IL-1β, Red: CD68. The fluorescence intensity of IL-1β in different groups (upper right) and the percentage of IL-1β + cells among CD68 + cells (lower right). (Data are shown as the Mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001)

Article Snippet: The T98G and THP1 cells were gifted by the Department of Neurosurgery, Sun Yat-sen University Cancer Center (Guangzhou, China), while the U251 cell line was purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China) and authenticated.

Techniques: Expressing, Control, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Immunofluorescence, Fluorescence

Journal: Journal of Neuroinflammation

Article Title: IL-1β + tumor-associated macrophages accelerate glioblastoma progression by amplifying the PGE2-EP4 signaling

doi: 10.1186/s12974-025-03551-y

Figure Lengend Snippet: IL-1β promotes the production of inflammatory factors in GBM cells. (A) mRNA expression of IL6, IL1B, and CSF in U251 cells stimulated with various concentrations of IL-1β ( n = 3). Statistical significance compared to the control group is indicated by * p values. (B) Workflow for RNA sequencing of U251 cells treated with 1ng/ml IL-1β. (C) Venn diagram depicting the overlap between 711 upregulated genes in IL-1β-stimulated U251 cells and 1,891 secreted protein-coding genes from The Human Protein Atlas, resulting in 117 intersecting genes. (D) Heatmap illustrating the expression of inflammation-related, cell invasion-related, and chemokine-related genes among the 117 intersecting genes. (E) List of the top 10 upregulated genes from the 117 intersecting genes. (F) Top 20 KEGG pathways enriched in IL-1β-stimulated U251 cells. (G) PTGS2 mRNA expression in U251 and T98G cells following IL-1β stimulation ( n = 3). (H) ELISA analysis of PGE2 secretion in U251 and T98G cells after IL-1β stimulation ( n = 3). (I) mRNA expression of PTGS2 in GBM ( n = 153), LGG ( n = 513), and Normal groups ( n = 2642) from the TCGA database (left) and correlation analysis between PTGS2 and IL1B expression in GBM ( n = 153) patients (right). (Data are presented as mean ± SEM. NS: Not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001)

Article Snippet: The T98G and THP1 cells were gifted by the Department of Neurosurgery, Sun Yat-sen University Cancer Center (Guangzhou, China), while the U251 cell line was purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China) and authenticated.

Techniques: Expressing, Control, RNA Sequencing, Enzyme-linked Immunosorbent Assay

Journal: Journal of Neuroinflammation

Article Title: IL-1β + tumor-associated macrophages accelerate glioblastoma progression by amplifying the PGE2-EP4 signaling

doi: 10.1186/s12974-025-03551-y

Figure Lengend Snippet: PGE2 and TNFα secreted by GBM cells collaboratively induce the IL-1β + TAM phenotype. (A) Experimental setup for collecting conditioned media from U251 cells treated with IL-1β and from control U251 cells. (B) IL1B mRNA expression in macrophages stimulated with U251 GCM or U251 IL−1β GCM for 24 h ( n = 3). (C) IL1B mRNA expression in macrophages treated with various concentrations of PGE2 for 24 h ( n = 3). (D) TNFA mRNA expression in GBM ( n = 153), LGG ( n = 513), and Normal groups ( n = 2642) from the TCGA database (left) and correlation between TNFA and IL1B expression in GBM ( n = 153) patients (right). (E) TNFA mRNA expression in U251 and T98G cells after 24-hour IL-1β stimulation ( n = 3). (F) ELISA analysis of TNFα secretion in U251 and T98G cells treated with IL-1β for 48 h ( n = 3). (G-H) TNFR1 and TNFR2 mRNA expression in macrophages after stimulation with different concentrations of PGE2 for 24 h ( n = 3). (I) IL1B mRNA expression in macrophages stimulated with various concentrations of TNFα and PGE2 for 24 h ( n = 3). (J) ELISA analysis of IL-1β protein levels in macrophage supernatants after stimulation with different concentrations of TNFα and PGE2 for 48 h ( n = 3). (K) Volcano plot showing the upregulated and downregulated genes in THP1 Macs stimulated with U251 GCM ( n = 3). (L) Volcano plot of upregulated and downregulated genes in THP1 Macs stimulated with PGE2/TNFα ( n = 3). (Data are expressed as mean ± SEM. NS: Not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001)

Article Snippet: The T98G and THP1 cells were gifted by the Department of Neurosurgery, Sun Yat-sen University Cancer Center (Guangzhou, China), while the U251 cell line was purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China) and authenticated.

Techniques: Control, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Journal of Neuroinflammation

Article Title: IL-1β + tumor-associated macrophages accelerate glioblastoma progression by amplifying the PGE2-EP4 signaling

doi: 10.1186/s12974-025-03551-y

Figure Lengend Snippet: C/EBPβ is upregulated in macrophages stimulated by PGE2/TNFα or GCM. (A) GO analysis identifies the top 10 biological processes with significant differences comparing PGE2/TNFα vs. control (left) and U251 GCM vs. control (right) ( n = 3). (B) Enrichment of inflammatory response-related genes and their overlap between the two experimental groups compared to the control. (C-D) Expression levels of the top 9 most significantly upregulated genes among the 52 intersecting genes. (E) CEBPB mRNA expression in THP1 Macs after 24-hour stimulation with U251 GCM, T98G GCM, or PGE2/TNF-α ( n = 3). * p indicates statistical significance compared to the control group. (F) mRNA expression of CEBPB in GBM ( n = 153), LGG ( n = 513), and Normal groups ( n = 2642) from the TCGA database (left) and correlation between CEBPB and IL1B gene expression in GBM ( n = 153) patients (right). (G) Expression analysis of CEBPB (left) and IL1B (right) genes in GBM patients ( n = 9) from scRNA-seq data. (Data are presented as mean ± SEM. NS: Not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001)

Article Snippet: The T98G and THP1 cells were gifted by the Department of Neurosurgery, Sun Yat-sen University Cancer Center (Guangzhou, China), while the U251 cell line was purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China) and authenticated.

Techniques: Control, Expressing, Gene Expression

Journal: Journal of Neuroinflammation

Article Title: IL-1β + tumor-associated macrophages accelerate glioblastoma progression by amplifying the PGE2-EP4 signaling

doi: 10.1186/s12974-025-03551-y

Figure Lengend Snippet: GBM-derived PGE2 and TNFα indue IL-1β expression in macrophages via the PKA- C/EBPβ signaling pathway. (A-B) Expression of IL1B (A) and CEBPB (B) mRNA in THP1 Macs after 24 h of stimulation with different sequences of PGE2 (1nM) and TNFα (10ng/ml) ( n = 3). (C) Expression of C/EBPβ protein in THP1 Macs after stimulation under various conditions ( n = 3). (D) Expression of C/EBPβ protein in THP1 Macs after stimulation with PGE2/TNFα or U251 GCM. (E) Immunofluorescence showing co-expression of IL-1β (green) and C/EBPβ (red) in THP1 Macs 6 h post-stimulation under different conditions ( n = 3). Blue, DAPI. (F-G) CEBPB mRNA (F) and IL1B mRNA (G) expression in THP1 Macs 24 h after stimulation with U251 or T98G GCM following a 30-minute pre-treatment with varying concentrations of PKA inhibitor (H89) ( n = 3). (Data are presented as the Mean ± SEM. NS: Not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001)

Article Snippet: The T98G and THP1 cells were gifted by the Department of Neurosurgery, Sun Yat-sen University Cancer Center (Guangzhou, China), while the U251 cell line was purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China) and authenticated.

Techniques: Derivative Assay, Expressing, Immunofluorescence

Journal: Journal of Neuroinflammation

Article Title: IL-1β + tumor-associated macrophages accelerate glioblastoma progression by amplifying the PGE2-EP4 signaling

doi: 10.1186/s12974-025-03551-y

Figure Lengend Snippet: PGE2 is a key factor among tumor-derived soluble molecules that drives the secretion of IL-1β in THP1 Macs. (A) Workflow for collecting NS398 GCM from U251 and T98G cells treated with the PTGS2 inhibitor (NS398). (B) ELISA analysis of PGE2 production by U251 and T98G cells treated with NS398 ( n = 3). (C-D) Effects of NS398 GCM collected from different concentrations of NSG398 treated U251(C) or T98G (D) on the expression of IL1B mRNA in macrophages ( n = 3). (E-F) Effects of NS398 GCM collected from different concentrations of NSG398 treated U251(E) or T98G (F) on the expression of IL-1β protein in macrophages ( n = 3). (G-H) Effects of NS398 GCM collected from different concentrations of NSG398 treated U251(G) or T98G (H) on the expression of CEBPB mRNA in macrophages ( n = 3). (Data are presented as the Mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001)

Article Snippet: The T98G and THP1 cells were gifted by the Department of Neurosurgery, Sun Yat-sen University Cancer Center (Guangzhou, China), while the U251 cell line was purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China) and authenticated.

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Journal of Neuroinflammation

Article Title: IL-1β + tumor-associated macrophages accelerate glioblastoma progression by amplifying the PGE2-EP4 signaling

doi: 10.1186/s12974-025-03551-y

Figure Lengend Snippet: Tumor-derived PGE2 stimulates IL-1β expression via EP4 receptor signaling. (A) t-SNE plot showing the expression levels of PTGER1-4 (EP1-4) mRNA in GBM patients ( n = 9) from scRNA-seq data. (B) Comparison of PTGER1-4 mRNA expression in THP1 Macs ( n = 3). (C) Workflow for using ONO-AE3-208 to block the EP4 receptor in macrophages. (D-G) Effects of ONO-AE3-208 on the expression of IL1B mRNA and protein in macrophages stimulated with GCM from U251 and T98G cells ( n = 3). (H-I) Effects of ONO-AE3-208 on the expression of CEBPB mRNA in macrophages stimulated with U251 and T98G GCM ( n = 3). (Data are presented as the Mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001)

Article Snippet: The T98G and THP1 cells were gifted by the Department of Neurosurgery, Sun Yat-sen University Cancer Center (Guangzhou, China), while the U251 cell line was purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China) and authenticated.

Techniques: Derivative Assay, Expressing, Comparison, Blocking Assay