Journal: bioRxiv

Article Title: Reprogramming the Immune Suppressive Tumor Microenvironment in Glioma Enhances the Efficacy of Immune-Mediated Gene Therapy

doi: 10.1101/2025.11.11.687828

Figure Lengend Snippet: (A) Expression of NT5E mRNA based on TCGA-GBM and TCGA-LGG combined dataset stratified based on the wildtype ( n = 204) and mutant ( n = 230) IDH1 status of the patients (Mann–Whitney U test). (B) Quantitative PCR analysis of NT5E mRNA expression across wtIDH1 glioma cells (NPA, RPA, SJGBM2-wtIDH1, MGG8-wtIDH1) and mIDH1 glioma cells (NPAI, RPAI, SJGBM2-mIDH1, MGG8-mIDH1) (Student’s t test, n = 2). (C) Representative flow cytometry histograms and (D) quantitation of the percentage of wildtype and mutant IDH1 glioma cells expressing CD73. (Student’s t test, n = 3). (E) Representative flow cytometry histogram and (F) quantitation of the frequency of CD73 expressing glioma cells to total glioma cells. (Student’s t test, n = 5). (G) Western blot image and (H) quantitation of CD73 levels from total glioma tumor in vivo (Student’s t test, n = 3). (I) Representative flow cytometry histogram and (J) quantitation of CD73 expressing human mIDH1 glioma cells (SF10602s) with and without mIDH1 inhibitor (AG881) treatment (Student’s t test, n = 4).

Article Snippet: 100 μL of 150 μg anti-mouse monoclonal antibody CD73 (TY/23; Bio X Cell, BE0209) was administered i.p. on days 7, 10, 13, 16 post glioma cell implantation.

Techniques: Expressing, Mutagenesis, MANN-WHITNEY, Real-time Polymerase Chain Reaction, Flow Cytometry, Quantitation Assay, Western Blot, In Vivo

Journal: bioRxiv

Article Title: Reprogramming the Immune Suppressive Tumor Microenvironment in Glioma Enhances the Efficacy of Immune-Mediated Gene Therapy

doi: 10.1101/2025.11.11.687828

Figure Lengend Snippet: (A) Mechanism of methylation sequencing (methyl-seq) DNA methylation profiling. (B) Methyl-seq readout of the CD73 encoding gene region for NPA (wtIDH1) and NPAI (mIDH1). Red arrow indicates the NT5E promoter region with notable methylation difference between NPA and NPAI glioma cell DNA methylation. (C) Decitabine (DAC) mechanisms of action resulting in DNA methylation by inhibiting DNMT. (D) Timeline of DAC treatment on glioma cells to measure the effect on CD73 expression. (E) Representative flow cytometry histograms and (F) quantitation of CD73 expression from wtIDH1 and mIDH1 glioma cells treated with DAC (one-way ANOVA, n = 3).

Article Snippet: 100 μL of 150 μg anti-mouse monoclonal antibody CD73 (TY/23; Bio X Cell, BE0209) was administered i.p. on days 7, 10, 13, 16 post glioma cell implantation.

Techniques: Methylation, Sequencing, DNA Methylation Assay, Expressing, Flow Cytometry, Quantitation Assay

Journal: bioRxiv

Article Title: Reprogramming the Immune Suppressive Tumor Microenvironment in Glioma Enhances the Efficacy of Immune-Mediated Gene Therapy

doi: 10.1101/2025.11.11.687828

Figure Lengend Snippet: (A) Diagram of Sleeping Beauty (SB) transposon generated CD73 knockout (NPA CD73KO) and CD73 wildtype (NPA CD73wt) glioma models and the generation of implanted glioma mouse models (GMMs). (B) Survival of SB generated CD73-wt glioma models (NPA; n = 8) compared to CD73-KO glioma models (NPA CD73KO; n = 6) (Log-rank test). (C) TCGA (GBMLGG) wtIDH patient survival curve with patient cases stratified on NT5E gene expression (top 25% cases are high NT5E n = 58; bottom 25% of cases are low NT5E n = 59) (Mann-Whitney U test). (D) TCGA (GBMLGG) mIDH patient survival curve with patient cases stratified on NT5E gene expression (top 25% cases are high NT5E n = 107; bottom 25% of cases are low NT5E n = 108) (Mann-Whitney U test). (E) Mechanism of action of Ad-TK/Ad-Flt3L gene therapy (GT). (F) Schematic showing experimental design of NPA CD73wt and NPA CD73KO glioma cell implantation and treatment with or without Ad-TK/Ad-Flt3L GT. (G) Survival curve of NPA CD73wt and NPA CD73KO glioma models ( n = 5) treated on the timeline shown in (F).

Article Snippet: 100 μL of 150 μg anti-mouse monoclonal antibody CD73 (TY/23; Bio X Cell, BE0209) was administered i.p. on days 7, 10, 13, 16 post glioma cell implantation.

Techniques: Generated, Knock-Out, Gene Expression, MANN-WHITNEY

Journal: bioRxiv

Article Title: Reprogramming the Immune Suppressive Tumor Microenvironment in Glioma Enhances the Efficacy of Immune-Mediated Gene Therapy

doi: 10.1101/2025.11.11.687828

Figure Lengend Snippet: (A) Proposed mechanism of action of the adenosinergic pathway mediated immune suppression contributing to glioma progression with the combination of CD73 inhibition with Ad-TK/Ad-Flt3L gene therapy. (B) Experiment timeline schematic of NPA wtIDH1 glioma cell implantation and treatments with CD73 inhibiting monoclonal antibody (aCD73), small molecule inhibitor of CD73 (AB680), and Ad-TK/Ad-Flt3L gene therapy. (C) Survival curve of NPA glioma models ( n = 5) treated on the timeline shown in (B) (Log-rank test). (D) H&E staining of 5 µm paraffin-embedded brain sections from saline, long-term survivors from aCD73 GT and AB680 GT treatment groups (scale bar = 1 mm). Paraffin-embedded 5 µm brain sections for each treatment group were immunohistochemistry stained for (E) glial fibrillary acidic protein (GFAP), (F) myelin basic protein (MBP), (G) CD68, and (H) IBA1. Low magnification (10X) panels (black scale bar = 100 µm), high magnification (40X) panels (black scale bar = 20 µm) indicate positive staining for the areas delineated in the low-magnification panels. Representative images from a single experiment consisting of independent biological replicates are displayed.

Article Snippet: 100 μL of 150 μg anti-mouse monoclonal antibody CD73 (TY/23; Bio X Cell, BE0209) was administered i.p. on days 7, 10, 13, 16 post glioma cell implantation.

Techniques: Inhibition, Staining, Saline, Immunohistochemistry

Journal: bioRxiv

Article Title: Reprogramming the Immune Suppressive Tumor Microenvironment in Glioma Enhances the Efficacy of Immune-Mediated Gene Therapy

doi: 10.1101/2025.11.11.687828

Figure Lengend Snippet: (A) Schematic showing experimental design of NPA glioma cell implantation and treatment with small molecule inhibitor of CD73 (AB680) and Ad-TK/Ad-Flt3L gene therapy. (B-C) Quantitation of the frequency of CD8 that are (B) IFNγ positive, (C) granzyme-B (GzB) positive (one-way ANOVA, n = 5). (D) Schematic showing experimental design of tumor specific CD8 T cell splenocyte activation and proliferation assay. (E) Flow cytometry quantitation data of tumor specific CD8 T cells from the splenocyte T cell proliferation experiment (one-way ANOVA, n = 5). (F) Representative flow cytometry histograms and (G) quantitation of CFSE expression as an indication of CD8 T cell proliferation (one-way ANOVA, n = 5).

Article Snippet: 100 μL of 150 μg anti-mouse monoclonal antibody CD73 (TY/23; Bio X Cell, BE0209) was administered i.p. on days 7, 10, 13, 16 post glioma cell implantation.

Techniques: Quantitation Assay, Activation Assay, Proliferation Assay, Flow Cytometry, Expressing

Journal: Cell reports

Article Title: Neutral ceramidase-dependent regulation of macrophage metabolism directs intestinal immune homeostasis and controls enteric infection

doi: 10.1016/j.celrep.2022.110560

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: CD73 (154 Sm) , Fluidigm , Cat# 3154019B; RRID: AB_2813854.

Techniques: Recombinant, Western Blot, Protease Inhibitor, Enzyme-linked Immunosorbent Assay, SYBR Green Assay, Cell Culture, IP Phosphatase Assay, Software