Journal: Journal of Advanced Research

Article Title: Targeting mesenchymal monocyte-derived macrophages to enhance the sensitivity of glioblastoma to temozolomide by inhibiting TNF/CELSR2/p65/Kla-HDAC1/EPAS1 axis

doi: 10.1016/j.jare.2025.05.032

Figure Lengend Snippet: MES-MDM promote MES subtype transition of cancer cells. (A) T-SNE plots and percentage of cell types showing the single-cell landscape of samples with and without MES-MDM enrichment. The pie chart presents the proportion of cancer cells of the four subtypes among all cancer cells in the two groups: Enrichment group (G3, G4, G14, G18, G19, G22 sample), non-enrichment group (G15, G17, G21 sample). (B) Relative expression of MES-cancer cells signature genes after co-culturing with primary-TREM1 hi MDM or primary-TREM1 lo MDM. (C) A schematic diagram of the sorting and identification of NPC/OPC/MES/AC cancer cells from GBO. (D) Immunostaining for subtype-specific markers was performed on NPC/OPC/MES/AC cancer cells cultured by special culture medium. (E) Relative expression of signature genes in the cultured NPC/OPC/MES/AC-cancer cells. (F) Proportion of MES-cancer cells after co-culture of NPC-cancer cells and primary-MDM by FCM detection. (G) Inferred interaction ligand receptor pair between MES-MDM and cancer cells was analyzed using our hGBM scRNA-seq data by CellphoneDB analysis. (H) Representative images of western blotting of CD44 in GBO with the indicated treatment. (I) Representative images depicting the western blot analysis of CD44, p65, EPAS1, CEBPD, HDAC1 in prim-NPC-cancer cells treated as indicated. (J) Representative images depicting the western blot analysis of Pan-Kla in the proteins after IP by anti-HDAC1 antibody with the indicated treatment. (K) Representative images depicting the western blot analysis of Pan-Kla in the proteins after IP by anti-HDAC1 antibody with the indicated treatment, mut1 (K200: AAG-CGA), mut2 (K200: AAG-GCG). (L) Relative expression of MES-cancer cells signature genes with the indicated treatment in NPC-cancer cells.

Article Snippet: The antibodies used included anti-human CD68 (abcam, ab955), anti-human TMEM119 (abcam, ab306583), anti-human TREM1 (abcam, ab225861), anti-human GLUT1 (abcam, ab115730), anti-human CD44 (abcam, ab254530), anti-human CD24, anti-human EGFR (abcam, ab52894), anti-human HIF1α (abcam, ab51608), anti-human p65 (abcam, ab32536), anti-human ATF3 (abcam, ab254268), anti-human FOSL2 (proteintech, 15832-1-AP), anti-human GAPDH (proteintech, 10494-1-AP), anti-human EPAS1 (proteintech, 83790-1-RR), anti-human CEBPD (huabio, ER62841), anti-human TNF (abcam, ab183218), anti-human CELSR2 (Biomatik, CAU22255), anti-human Pan-Lactyl-lysine (huabio, PSH03-74), anti-human HDAC1 (huabio, SY12-04), anti-mouse CD4 (huabio, ET1609-52), anti-mouse Il7r (huabio, HA721214), anti-mouse PD-1 (abcam, ab214421), anti-mouse CD16/32 (BioLegend, #101302), APC/cy7 anti-mouse CD45 (BioLegend, #103115), PE/Dazzle 594 anti-mouse CD3 (BioLegend, #100245), PerCP/cy5.5 anti-mouse CD4 (BioLegend, #100434), AF647 anti-mouse FOXP3 (BioLegend, #126407), PE anti-mouse PD-1 (BioLegend, #135205), Brilliant Violet 711 anti- mouse CD11b (BioLegend, #101242), AF488 anti-mouse IL7R (BioLegend, #135017), FITC anti-mouse Ly-6G (BioLegend, #127605), PB anti-mouse Ly-6C (BioLegend, #128013), BV421 anti-mouse PD-1 (BD Biosciences, #562584), FITC anti-mouse IL7R (BioLegend, #135007), PE anti-mouse F4/80 (BioLegend, #123109), PE-cy7 anti-mouse TREM1 (Bioss, bs-4886R), AF488 anti-mouse P2Y12 (Bioss, 12072R), anti-human FcX (BioLegend, #163403), PB anti-human CD44 (BioLegend, #338823), PE-cy7 anti-human CD24 (BD Biosciences, #561646), PE anti-human PDGFRA (ebioscience, #12140181), AF488 anti-human EGFR (BioLegend, #352907), APC/cy7 anti-human CD45 (BioLegend, #368516), FITC anti-human CD3 (BioLegend, #981002), PerCP/cy5.5 anti-human CD4 (BioLegend, #300529), APC anti-human Foxp3 (ebioscience, #17477642), APC anti-human IL7R (ebioscience, #17127842), PE anti-human PD-1 (Bioss, bc12075463), AF647 anti-human CD68 (BioLegend, #333820), AF488 anti-human P2Y12 (Bioss, bc08034611), PE anti-human TREM1 (BioLegend, #314906).

Techniques: Expressing, Immunostaining, Cell Culture, Co-Culture Assay, Western Blot