pe cy7 anti mouse trem1  (Bioss)

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: scRNA-seq reveals a new MES-MDM in patients with GBM. (A) T-SNE plots showing unsupervised clusters of nine cell types superclusters. The nine superclusters are: cancer cell, monocyte-derived macrophages (MDM), monocyte, microglial Cells (MGcells), endothelial cells (ECs), lymphocytes, oligodendrocyte, stromal cells (SCs), and neutrophils. Dots represent individual cells, and colors represent different cell populations. (B) The SCENIC analysis identified five transcription factor (TF) groups associated with five MDM clusters. (C) Heatmap showing the scores of the MES-signatures among MDM clusters and other myeloid clusters. (D) Distribution of monocytes and MDM clusters along the pseudotime trajectory using Monocle2 (up); Ratio of MDMC2 cells in stage 1, stage 2 and stage 3-4-5 of pseudotime trajectory (down). (E) Density plots of switching genes for significantly over-represented functional ontologies. (F) T-SNE plots showing different identified types of MDM. (G) Intersection of switchgenes in our data and up-regulated genes in published data. (H) Feature plots and violin plots showing the expression of TREM1 and SLC2A3 in MDM clusters and determining that MDMC2 specifically overexpressed TREM1. (I) Scatterplots showing significant correlations between TREM1 expression and the scoring of MES-myeloid signature in the MDMs of each sample. (J) Representative images of Multiplexed immunostaining of LGG, nGBM, and rGBM patients (n = 3). (K) Intersection signature genes of MES-MDM, MP-MES and MES-myeloid. (L) Representative FCM plots and summary data showing the percentage of TREM1 hi MDM in GBM tissues (n = 9). (M) Expression of the MES signature genes in TREM1 hi MDM and TREM1 lo MDM in primary 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: Derivative Assay, Functional Assay, Expressing, Immunostaining

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: Pro-tumor function of MES-MDM. (A) Heatmap showing the proportions of MDM clusters in GBM using TCGA-GBM/LGG cohort (n = 493). LGG: low grade gliomas. MGMT: O6-methylguanine-DNA methyltransferase. EGFR: epidermal growth factor receptor. TERT: telomerase reverse transcriptase. Chr: chromosome. Co: co-deletion. CL: classical. PN: proneural. NE: neural. MES: mesenchymal. BRAF V600E: BRAF proto-oncogene, serine/threonine kinase V600E mutation. NA: not applicable. G2: Grade 2. G3: Grade 3. G4: Grade 4. (B) and (C) Boxplots display the estimated proportions of MDM clusters in different histology (B) or subtypes (C) using TCGA-GBM/LGG cohort (n = 493) CL: classical. PN: proneural. NE: neural. MES: mesenchymal. G2: Grade 2. G3: Grade 3. G4: Grade 4. Center line shows median, box limits indicate the upper and lower quartiles, and whiskers extend 1.5 times the interquartile range. *, p < 0.05. ns, not significant. A two-sided unpaired Wilcoxon test was conducted. (D) The overall survival of patients in the CGGA-GBM/LGG cohort (n = 229) was analyzed using multivariate Cox regression. Forest plots with error bars display the confidence interval, indicating the lower bound at 2.5 % and the higher bound at 97.5 %. (E) Feature plots and violin plots showing the scoring of the MES-MDM signature in LGG, nGBM, and rGBM using GSE182109 data. (F) Schematic illustration of mPBMC-derived MDM treatment schedule in an orthotopic GBM model. (G) Bioluminescence analysis of orthotopic tumor growth over time, n = 5. (H) Survival curve of GBM-bearing mice with mPBMC-derived TREM1 hi MDM or TREM1 lo MDM treatment (n = 5 mice). (I) Bioluminescence images in tumor-bearing mice treated with mPBMC-derived MDM were quantified at day 20, 30, and 40.

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: Reverse Transcription, Mutagenesis, Derivative Assay

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: Hypoxia induces the MES-MDM signature. (A) Boxplots displaying estimated proportions of MDM clusters in different spatial position of hGBMs using bulk RNA-seq data from the Ivy-hGBM cohort (n = 270). ∗, p < 0.05. (B) Surface plots showing the transcriptional programs of MDM clusters at the spatial level using published hGBM spatial transcriptomics data. (C) Immunostaining of CD68, TMEM119, TREM1, and GLUT1 in the peri-necrotic region of hGBM. Scale bar = 100 μm. (D) Visualization of distinct switching genes from the two paths filtered by the McFadden’s Pseudo R2. (E) Intersection of TFs of switch genes in branch1 and branch2, and the correlations between intersection TFs and TREM1 expression in CGGA-GBM cohort (n = 386). (F) Representative images of western blotting of p65 in hPBMC-derived MDM with the indicated treatment. (G) Relevant images depicting the western blot analysis of HIF-1a, p65, ATF3, FOSL2, TREM1 in hPBMC-derived MDM treated as indicated are presented. (H) STRING analysis revealed the interaction between p65, ATF3, and FOSL2. (I) Relative expression of MES-MDM signature genes after knockdown of ATF3 and FOSL2 in hPBMC-derived MDM.

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: RNA Sequencing, Immunostaining, Expressing, Western Blot, Derivative Assay, Knockdown

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

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: Targeting TREM1 enhances the efficacy of anti-PD-1 immunotherapy. (A) Relative expression of MES-cancer cells signature genes after TREM1 inhibitor treatment. (B) Relative expression of MES-cancer cells signature genes in the co-culture system. (C) and (D) Representative immunostaining images and quantitative analysis depicting CD44 in GBO which were co-cultured with hPBMC-derived MES-MDM. (E) Representative immunostaining images and quantitative analysis depicting KI67 in GBO which were co-cultured with hPBMC-derived MES-MDM with the treatment indicated. (F) Expression score of the IL7R + CD4 + T-cells signature genes of non-responder and responder after anti-PD-1 therapy. (G) Schematic illustration of anti-PD-1 and/or LP17 treatment in GBM-bearing mice. (H) Survival curve of GBM-bearing mice (n = 5 mice/group). (I) Percentage of Il7r + CD4 + T-cells in CD4 + T-cells of GBM-bearing mice with anti-PD-1 and/or LP17 treatment. (J) Percentage of exhaustion Il7r + CD4 + T-cells in Il7r + CD4 + T-cells of GBM-bearing mice with anti-PD-1 and/or LP17 treatment. (K) Representative images of multiplexed immunostaining from brains of GBM-bearing mice with anti-PD-1 and/or LP17 treatment. ∗∗, p < 0.01. ∗∗∗, p < 0.001.

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, Co-Culture Assay, Immunostaining, Cell Culture, Derivative Assay