Journal: Advanced Science

Article Title: BCAT2 Shapes a Noninflamed Tumor Microenvironment and Induces Resistance to Anti‐PD‐1/PD‐L1 Immunotherapy by Negatively Regulating Proinflammatory Chemokines and Anticancer Immunity

doi: 10.1002/advs.202207155

Figure Lengend Snippet: BCAT2 negatively correlates with anticancer immunity in TME of BLCA. A) Expression pattern of chemokines, chemokine receptors, MHC molecules, and immunostimulators in high and low BCAT2 groups. B) Activity of cancer immunity cycle in high and low BCAT2 groups. Seven colors represent seven steps in the cycle. * p < 0.05; ** p < 0.01; *** p < 0.001. C) Correlations between BCAT2 and multiple types of immune cell (CD8 + T cell, CD4 + T cell, DC cell, and NK cell) in six independent algorithms. D) Expression patterns of multiple subtypes of immune cell (CD8 + T cell, NK cell, Macrophage, Th1 cell, and DC cell) related effector genes in high and low BCAT2 groups. E) Correlations between BCAT2 and ICB related effector genes. Number in the circle means correlation coefficient.

Article Snippet: Accompanying combination therapy,100 μ g InVivoPlus anti‐mouse CD8 α (Cat: BP0117, Bioxcell, USA) and IgG2b isotype control (Cat: BP0090, Bioxcell, USA) were employed to deplete CD8 + T cells in mice.

Techniques: Expressing, Activity Assay

Journal: Advanced Science

Article Title: BCAT2 Shapes a Noninflamed Tumor Microenvironment and Induces Resistance to Anti‐PD‐1/PD‐L1 Immunotherapy by Negatively Regulating Proinflammatory Chemokines and Anticancer Immunity

doi: 10.1002/advs.202207155

Figure Lengend Snippet: BCAT2 varies expression patterns of CD8 + T‐cell‐related chemokines and inhibits cytotoxic capacity of CTLs. A,B) Heatmaps of ProcartaPlex multiple immunoassays display secretion variation of common cytokines and chemokines in BCAT2 OE, BCAT2 KD and negative control groups ( n = 3 per group). C,D) Histograms show normalized mRNA expression levels and protein secretion concentrations of CCL3, CCL4, CCL5, CXCL9, and CXCL10 in BCAT2 OE (left), BCAT2 KD (right) and negative control groups ( n = 3 per group). * p < 0.05; ** p < 0.01; *** p < 0.001. E) Chemotaxis assay indicates different chemotaxis ability of CTLs in BCAT2 OE, BCAT2 KD, and negative control groups ( n = 3 per group). * p < 0.05; ** p < 0.01. F) T‐cell‐mediated cancer cell killing assay indicates different killing abilities of T cells cocultured with BCAT2 OE, BCAT2 KD, and negative control cell lines ( n = 3 per group). G) Flow cytometry analysis indicates different activities of CD8 + T cells in different coculture groups ( n = 3 per group).

Article Snippet: Accompanying combination therapy,100 μ g InVivoPlus anti‐mouse CD8 α (Cat: BP0117, Bioxcell, USA) and IgG2b isotype control (Cat: BP0090, Bioxcell, USA) were employed to deplete CD8 + T cells in mice.

Techniques: Expressing, Negative Control, Chemotaxis Assay, Flow Cytometry

Journal: Advanced Science

Article Title: BCAT2 Shapes a Noninflamed Tumor Microenvironment and Induces Resistance to Anti‐PD‐1/PD‐L1 Immunotherapy by Negatively Regulating Proinflammatory Chemokines and Anticancer Immunity

doi: 10.1002/advs.202207155

Figure Lengend Snippet: Validation of exclusive spatial relationships of BCAT2 + tumor cells and CD8 + T cells by TMA of Xiangya BLCA cohort. A) IHC image of BCAT2 and CD8 in inflamed and noninflamed types of TME. Scale bar: 50 µm. B) Correlation between BCAT2 and CD8 on the basis of IHC scores of them in overall TMA. C) Multicolor IF image of BCAT2, CK19, CD8, and combination index in inflamed and noninflamed types of TME. BCAT2 + cell (pink), CK19 + cell (cyan), CD8+T cell (orange), and cell nucleus (blue). Scale bar: 50 µm. D,E) Detailed coexpression rates of BCAT2 + CK19 + and BCAT2 + CD8 + cells in the typical sample. F) Distance gradient analysis (0–25, 25–50, 50–100, and 100–150 µm) of CD8 + T cells around BCAT2 + CK19 + cells in the typical sample.

Article Snippet: Accompanying combination therapy,100 μ g InVivoPlus anti‐mouse CD8 α (Cat: BP0117, Bioxcell, USA) and IgG2b isotype control (Cat: BP0090, Bioxcell, USA) were employed to deplete CD8 + T cells in mice.

Techniques: Biomarker Discovery

Journal: Advanced Science

Article Title: BCAT2 Shapes a Noninflamed Tumor Microenvironment and Induces Resistance to Anti‐PD‐1/PD‐L1 Immunotherapy by Negatively Regulating Proinflammatory Chemokines and Anticancer Immunity

doi: 10.1002/advs.202207155

Figure Lengend Snippet: Loss of BCAT2 enhances efficacy of anti‐PD‐1 therapy. A) Flow diagram of treatment plan. B) Harvested tumors of different therapy regimens ( n = 5 per group). C–E) Quantification of tumor volume, body weight and survival time in different therapy regimens ( n = 5 per group). ns: no significance; * p < 0.05; ** p < 0.01; *** p < 0.001. F) Contour plots indicate proportion of CD8 + T cells; G) proportions of GZMB + CD8 + T cells, IFN‐ γ + CD8 + T cells, TNF‐ α + CD8 + T cells, and Perforin + CD8 + T cells in different therapy regimens. H) Quantified scatter plots exhibit proportion of CD8 + T cells; I–L) proportions of GZMB + CD8 + T cells, IFN‐ γ + CD8 + T cells, TNF‐ α + CD8 + T cells, and Perforin + CD8 + T cells in different therapy regimens ( n = 5 per group). ns: no significance; * p <0.05; ** p < 0.01; *** p < 0.001. M,N) IF image and quantified histogram show densities of CD8 + T cells in different therapy regimens ( n = 3 per group). Scale bar: 20 µm. CD8 + T cell (green) and cell nucleus (blue). ns: no significance, * p <0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Accompanying combination therapy,100 μ g InVivoPlus anti‐mouse CD8 α (Cat: BP0117, Bioxcell, USA) and IgG2b isotype control (Cat: BP0090, Bioxcell, USA) were employed to deplete CD8 + T cells in mice.

Techniques:

Journal: bioRxiv

Article Title: Resolution of SARS-CoV-2 infection in human lung tissues is driven by extravascular CD163+ monocytes

doi: 10.1101/2024.03.08.583965

Figure Lengend Snippet: (A) BLT-L mice were administered 200 μg of anti-CD3e (OKT3), anti-CD4 (OKT4), anti-CD8 (OKT8) or IgG2a isotype. (B) Viral titer (log(PFU/mg)) in fLX extracted from BLT-L mice treated with isotype, OKT3, OKT4, or OKT8 antibody at 12 dpi. (C) MHC-I staining of fLX tissue sections (anti-MHC class I (EMR8-5) CST 88274) extracted from naïve mice, OKT4-treated mice (12 dpi), or from infected fLX at 2 and 12 dpi. Scale bar = 100 μM (D a-g ) Multiplex immunohistochemistry of fLX tissue sections extracted from Isotype (a), OKT3 (b,c), OKT4 (d,e), or OKT8 (f,g)-treated BLT-L mice at 12 dpi. CD163: teal, CD4: yellow, CD3: orange, PPARγ: green, Dapi: gray. Viral titer (PFU/mg) of the respective fLX is inlaid in white. Scale bar = 100 μM (E-H) Quantification of selected cytokines (CCL2, CCL3, CXCL10 and CCL19) in the serum of naïve and infected BLT-L mice (2 dpi or 12 dpi), and of OKT4-treated BLT-L mice (12 dpi). (I) Histopathological scoring of fLX extracted from isotype, OKT3, OKT4, or OKT8-treated BLT-L mice. One -way ANOVA. p-values are indicated on graphs. Error bars indicate mean ± Standard error of the mean .

Article Snippet: BLT-L mice were administered 200 mg of anti-CD3e (OKT3) (BioxCell; cat. # BE0001-2), anti-CD4 (OKT4) (BioxCell; cat. # BE0003-2), anti-CD8 (OKT8) (BioxCell; cat. # BE0004-2), or isotype IgG2a (Thermofisher; cat # 02-6200) antibody 3-, 2-, and 1-day prior to inoculation and 4- and 8-days post inoculation with 1×10 6 PFU SARS-CoV-2 WA-1.

Techniques: Staining, Infection, Multiplex Assay, Immunohistochemistry

Journal: bioRxiv

Article Title: Metabolomic Profiling Reveals Potential of Fatty Acids as Regulators of Stem-like Exhausted CD8 T Cells During Chronic Viral Infection

doi: 10.1101/2024.10.07.617124

Figure Lengend Snippet: C57BL/6 mice were infected with LCMV ARM, Cl13 or left uninfected (Un) and analyses were performed on days 8 and 20 p.i.. (A) Plasma free fatty acids (FFA) at indicated timepoints from days 1 through 30 p.i. normalized to Un levels. Horizontal lines indicate average Un (dotted line) ± 2 standard deviations (grey box). (B) Adipocyte area in visceral white adipose tissue (WAT) at day 8 p.i. and representative images (right). (C) Immunodetection of HSL and P-HSL (Ser660) in WAT at day 8 p.i.. Band intensity was normalized to GAPDH and Un mice. (D-H&M) Cl13-infected mice treated with isotype control (IgG2a) or anti-CD8-depleting antibodies (7CD8) and analyzed at day 8 p.i.. (D) Plasma FFA levels. (E) Adipocyte area in WAT and representative images (right). (F) Immunodetection of HSL and P-HSL (Ser660) in WAT. Band intensity was normalized to ACTB and isotype control mice. (G-H) Overlap of plasma metabolites (G) and predicted pathways (H) from isotype vs. anti-CD8 treated LCMV Cl13-infected mice and from 48hr fasted vs. ad libitum uninfected mice. (I-J) Overlap of plasma metabolites (I) and predicted pathways (J) from isotype vs. anti-CD8 treated LCMV Cl13-infected mice and human fasting metabolic signatures. (K – M) Number of differentially expressed metabolites with short-, medium-, or long-acyl chains that were elevated at day 8 p.i. in LCMV Cl13 vs ARM infected mice (K), LCMV Cl13-infected mice vs. human fasting signature (L), and isotype vs. anti-CD8 treated LCMV Cl13-infected mice (M). (A-F) Representative of 2 (B-F) or 3 (A) independent experiments with n=3-5 mice/group. (G-M) 3 pooled experiments with n=4-10 mice per group (CD8 depletion experiments) or 2 pooled experiments with n=9-10 mice/group (fasting experiments). (A-F) Averages ± SEM. (A) 2-way ANOVA. (B-C) One-way ANOVA with Tukey’s correction. (D-F) Two tailed Student’s t-test. *p<0.05, **<0.01, ***<0.001, ****<0.0001.

Article Snippet: For CD8 T cell depletion studies, mice were intraperitoneally injected with anti-CD8α (53-6.72, BioXcell) or IgG2a (2A3, BioXcell) antibodies on day -2, -1, and 5 p.i. (250 µg/mouse) as well as on the day of infection (200 µg/mouse).

Techniques: Infection, Immunodetection, Control, Two Tailed Test

Journal: bioRxiv

Article Title: Metabolomic Profiling Reveals Potential of Fatty Acids as Regulators of Stem-like Exhausted CD8 T Cells During Chronic Viral Infection

doi: 10.1101/2024.10.07.617124

Figure Lengend Snippet: C57BL/6 mice were infected with LCMV ARM or Cl13 and splenocytes were isolated and stained with BODIPY 493/503, BODIPY C1,C12, or BODIPY C16 at day 8 p.i.. Neutral lipid content (A,D), medium-chain FA uptake (B,E) and long-chain FA uptake (C,F) in CD8 + D b /GP 33-41 tetramer + cells from LCMV ARM vs. Cl13 infected mice (A-C) and across CD8 + D b /GP 33-41 tetramer + Tex STEM (LY108 + CX3CR1 - ) and Tex EFF (LY108 - CX3CR1 + or LY108 - CX3CR1 - ) from LCMV Cl13-infected mice. (A-C) Averages ± SEM. (D-F) Values from different Tex subsets corresponding to the same mouse are connected with a line. (A-F) Data are representative of two (A-C) or three (D-F) experiments, with n=4-5 mice/group. (A-C) Two-tailed Student’s t-test. (D-F) One-way ANOVA with Tukey’s correction. *p<0.05, **<0.01, ***<0.001.

Article Snippet: For CD8 T cell depletion studies, mice were intraperitoneally injected with anti-CD8α (53-6.72, BioXcell) or IgG2a (2A3, BioXcell) antibodies on day -2, -1, and 5 p.i. (250 µg/mouse) as well as on the day of infection (200 µg/mouse).

Techniques: Infection, Isolation, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: Metabolomic Profiling Reveals Potential of Fatty Acids as Regulators of Stem-like Exhausted CD8 T Cells During Chronic Viral Infection

doi: 10.1101/2024.10.07.617124

Figure Lengend Snippet: C57BL/6 mice were infected with LCMV Cl13, and CD8 + PD-1 + Tex STEM (LY108 + CX3CR1 - ) or Tex EFF-LIKE (LY108 - CX3CR1 + or LY108 - CX3CR1 -/low ) splenocytes were FACS-purified at day 8 p.i.. (A) Flow cytometry showing CX3CR1 and LY108 expression in FACS-purified Tex subsets. (B-C) Tex subsets were incubated with palmitate-BSA (FA) or BSA alone (BSA) for 8 hours and processed for RNAseq. GO biological processes (B & C, right) and KEGG pathways (C, left) enriched by upregulated (left) or downregulated (right) genes in Tex EFF-LIKE CX3CR1 -/low (B) and Tex EFF-LIKE CX3CR1 + (C) after BSA vs. FA incubation. (A-C) Data pooled across 3 independent repeats with n=4-5 mice/experiment.

Article Snippet: For CD8 T cell depletion studies, mice were intraperitoneally injected with anti-CD8α (53-6.72, BioXcell) or IgG2a (2A3, BioXcell) antibodies on day -2, -1, and 5 p.i. (250 µg/mouse) as well as on the day of infection (200 µg/mouse).

Techniques: Infection, Purification, Flow Cytometry, Expressing, Incubation

Journal: bioRxiv

Article Title: Metabolomic Profiling Reveals Potential of Fatty Acids as Regulators of Stem-like Exhausted CD8 T Cells During Chronic Viral Infection

doi: 10.1101/2024.10.07.617124

Figure Lengend Snippet: C57BL/6 mice were infected with LCMV Cl13, and CD8 + PD1 + Tex STEM (LY108 + CX3CR1 - ) or Tex EFF-LIKE (LY108 - CX3CR1 + or LY108 - CX3CR1 -/low ) splenocytes from day 8 p.i. were FACS-purified and incubated with palmitate-BSA (FA) or BSA alone (BSA). (A-E) Eight hours post-culture, Tex subsets were processed for RNAseq. (A) PCA plot showing individual Tex subsets after BSA vs. FA incubation. (B) Number of differentially expressed genes (p-value<0.05, log2FC>0.5) in BSA vs. FA incubated Tex subsets. (C-D) GO biological processes enriched by upregulated (C) or downregulated (D) genes after FA vs. BSA incubation. (E) Venn diagram showing overlap of differentially expressed genes in BSA vs. FA incubated in different Tex subsets. (F) Seahorse assay traces of oxygen consumption rates (OCR) over time before and after the addition of Oligomycin (Oligo), FCCP and Rotenone/Antimycin A (Rot/AA). (G-J) Average basal oxygen consumption rate (G), ATP-production linked oxygen consumption (H), spare respiratory capacity (I), and mitochondrial-independent oxygen consumption (J) in each Tex subset incubated with BSA vs. FA. (A-E) Data pooled across 3 independent repeats with n=4-5 mice/experiment. (F) Data representative of 3 individual repeats with n=8-10 mice/experiment. (G-J) Each data point represents a single experimental repeat, paired data from the same repeat are connected with a line. (G-J) Paired student’s t-test. *p< 0.05, **<0.01, ***<0.001.

Article Snippet: For CD8 T cell depletion studies, mice were intraperitoneally injected with anti-CD8α (53-6.72, BioXcell) or IgG2a (2A3, BioXcell) antibodies on day -2, -1, and 5 p.i. (250 µg/mouse) as well as on the day of infection (200 µg/mouse).

Techniques: Infection, Purification, Incubation

Journal: bioRxiv

Article Title: Metabolomic Profiling Reveals Potential of Fatty Acids as Regulators of Stem-like Exhausted CD8 T Cells During Chronic Viral Infection

doi: 10.1101/2024.10.07.617124

Figure Lengend Snippet: C57BL/6 mice were infected with LCMV Cl13 and treated i.p. with 3.0 μmol or 0.3 mmol of FA (1:1 lauric and palmitic acid) or PBS every 12 hours from days 16 to 20 p.i.. CD8 + PD1 + Tex STEM (LY108 + CX3CR1 - ), Tex INT (LY108 - CX3CR1 + ) or Tex TERM (LY108 - CX3CR1 - ) were FACS purified for RNAseq (B-F) or stained for FACS analysis (G) at day 21 p.i.. (A) Experimental design. (B) PCA showing individual Tex subsets from FA-treated vs. untreated mice. (C) Number of differentially expressed genes (p-value<0.05, log2FC>0.5). (D,E) GO biological processes enriched by upregulated (D) or downregulated (E) genes in Tex STEM from FA treated vs. untreated mice. (F) Venn diagram showing overlap of differentially expressed genes in Tex subsets from FA treated vs. untreated mice. (G) Averages ± SEM of mitochondrial polarization (charge/mass) in CD8 + D b /GP 33-41 tetramer + Tex subsets. (A-F) Data are combined across three experiments with n=4-5 mice/group. (G) Data are representative of three experiments with n=4-5 mice/group. Unpaired two-tailed Student’s t-test. *p<0.05.

Article Snippet: For CD8 T cell depletion studies, mice were intraperitoneally injected with anti-CD8α (53-6.72, BioXcell) or IgG2a (2A3, BioXcell) antibodies on day -2, -1, and 5 p.i. (250 µg/mouse) as well as on the day of infection (200 µg/mouse).

Techniques: Infection, Purification, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: Metabolomic Profiling Reveals Potential of Fatty Acids as Regulators of Stem-like Exhausted CD8 T Cells During Chronic Viral Infection

doi: 10.1101/2024.10.07.617124

Figure Lengend Snippet: C57BL/6 mice were infected with LCMV Cl13 and treated i.p. with 0.3 mmol of FA (1:1 lauric and palmitic acid) or PBS every 12 hours from days 16 to 20 p.i.. CD8 + PD-1 + Tex STEM (LY108 + CX3CR1 - ), Tex INT (LY108 - CX3CR1 + ) or Tex TERM (LY108 - CX3CR1 - ) splenocytes were FACS purified for RNAseq at day 21 p.i.. (A) Flow cytometry showing CX3CR1 and LY108 expression in FACS-purified Tex subsets. (B) GO analysis showing biological processes enriched by genes that were overlapping in all three Tex subsets and were downregulated in FA treated vs. untreated mice. Data combined across three experiments with n=4-5 mice/group.

Article Snippet: For CD8 T cell depletion studies, mice were intraperitoneally injected with anti-CD8α (53-6.72, BioXcell) or IgG2a (2A3, BioXcell) antibodies on day -2, -1, and 5 p.i. (250 µg/mouse) as well as on the day of infection (200 µg/mouse).

Techniques: Infection, Purification, Flow Cytometry, Expressing

Journal: bioRxiv

Article Title: Metabolomic Profiling Reveals Potential of Fatty Acids as Regulators of Stem-like Exhausted CD8 T Cells During Chronic Viral Infection

doi: 10.1101/2024.10.07.617124

Figure Lengend Snippet: C57BL/6 mice were infected with LCMV Cl13 and treated i.p. with either 0.3 mmol (A-F) or 3 mmol (A-E) of FA (1:1 lauric and palmitic acid mix) or PBS every 12 hours from days 16 to 20 p.i.. (A-F) Splenocytes (A-E) or blood (F) were obtained at day 21 p.i.. (A) Flow cytometry showing CX3CR1 and TCF1 expression in CD8 + D b /GP 33-41 tetramer + cells. (B-D) Fold change of numbers (left) or percentages (right) of CD8 + D b /GP 33-41 tetramer + Tex STEM (LY108 + CX3CR1 - ) (B), Tex INT (LY108 - CX3CR1 + ) (C) or Tex TERM (LY108 - CX3CR1 - ) (D) normalized to the average from untreated mice at day 21 p.i.. (E) Mean fluorescence intensity (MFI) of PD-1 in CD8 + D b /GP 33-41 tetramer + Tex STEM (left), Tex INT (middle) or Tex TERM (right) normalized to the average from untreated mice at day 21 p.i.. (F) Plasma viral titers from FA treated mice at day 21 p.i.. (B-F) Averages ± SEM. (A-E) Data are combined across three independent repeats with n=4-5 mice/group. (F) Data are combined across four independent repeats with n=4-8 mice/group. (B-E) One-way ANOVA with Tukey’s correction. (F) Individual two-tailed Student’s t-test. *p<0.05, **<0.01, ***<0.001, ****<0.0001.

Article Snippet: For CD8 T cell depletion studies, mice were intraperitoneally injected with anti-CD8α (53-6.72, BioXcell) or IgG2a (2A3, BioXcell) antibodies on day -2, -1, and 5 p.i. (250 µg/mouse) as well as on the day of infection (200 µg/mouse).

Techniques: Infection, Flow Cytometry, Expressing, Fluorescence, Two Tailed Test

Journal: bioRxiv

Article Title: Metabolomic Profiling Reveals Potential of Fatty Acids as Regulators of Stem-like Exhausted CD8 T Cells During Chronic Viral Infection

doi: 10.1101/2024.10.07.617124

Figure Lengend Snippet: C57BL/6 mice were infected with LCMV Cl13 and treated i.p. with either 0.3 mmol or 3 mmol of FA (1:1 lauric and palmitic acid mix) or PBS every 12 hours from days 16 to 20 p.i.. Splenocytes were harvested and analyzed at day 21 p.i. (A) Numbers (left) and percentages (right) of interferon-ψ producing CD8 T cells after 5 hr ex vivo stimulation with LCMV GP 33-41 peptide. (B) Expression of TIM3 expression in CD8 + D b /GP 33-41 tetramer + Tex STEM (LY108 + CX3CR1 - ) (left), Tex INT (LY108 - CX3CR1 + ) (middle) or Tex TERM (LY108 - CX3CR1 - ) (right) normalized to average from untreated mice. (A-B) Averages ± SEM. Data are combined across three experiments with n = 4-5 mice/group. (A) Unpaired two-tailed Student’s t-test. (B) One-way ANOVA with Tukey’s correction.

Article Snippet: For CD8 T cell depletion studies, mice were intraperitoneally injected with anti-CD8α (53-6.72, BioXcell) or IgG2a (2A3, BioXcell) antibodies on day -2, -1, and 5 p.i. (250 µg/mouse) as well as on the day of infection (200 µg/mouse).

Techniques: Infection, Ex Vivo, Expressing, Two Tailed Test

Journal: Journal for Immunotherapy of Cancer

Article Title: METTL3 promotes an immunosuppressive microenvironment in bladder cancer via m6A-dependent CXCL5/CCL5 regulation

doi: 10.1136/jitc-2024-011108

Figure Lengend Snippet: METTL3 is highly expressed in tumors and is associated with an immunosuppressive microenvironment. (A) Flowchart for screening key N6-methyladenosine (m6A) modification genes related to immunotherapy response in bladder cancer (BLCA). (B) Pearson correlation analysis bar chart of the 10 target genes with the percentage of complete response (CR) patients to immunotherapy in the IMvigor210 cohort, and a scatter plot of METTL3 expression level versus CR patient percentage. (C) Proportion of immunotherapy responses among different Lund subtypes in the IMvigor210 cohort. (D) Violin plot of METTL3 expression levels in bladder tissues of patients with different Lund subtypes. (E–F) Expression and statistical analysis of METTL3 in normal and tumor cells from single-cell sequencing of clinical bladder cancer samples. Histogram of METTL3 expression levels in cancer tissues versus adjacent normal tissues in (G) non-paired samples and (H) paired samples from the The Cancer Genome Atlas (TCGA) bladder cancer cohort. (I) Representative immunohistochemistry staining of METTL3 in clinical BLCA samples. (J–K) Scatter plots of METTL3 expression levels with CD8+T cell, cytotoxic cell, and myeloid-derived suppressor cell (MDSC) infiltration levels based on ssGSEA algorithm and TIMER V.2.0 database. (L) Statistical plot of METTL3 expression levels and immune scores in BLCA from the CAMOIP database. *p<0.05; **p<0.01; ***p<0.001.

Article Snippet: Anti-mouse Programmed Cell Death Protein 1 (PD-1) antibody (Bioxcell, #BE0146), anti-mouse CD8α antibody (Bioxcell, #BE0061), and anti-mouse Gr-1 antibody (Bioxcell, #BE0075) were also dissolved in PBS and administered intraperitoneally.

Techniques: Modification, Expressing, Sequencing, Immunohistochemistry, Staining, Derivative Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: METTL3 promotes an immunosuppressive microenvironment in bladder cancer via m6A-dependent CXCL5/CCL5 regulation

doi: 10.1136/jitc-2024-011108

Figure Lengend Snippet: METTL3 regulates bladder cancer progression by chemotactic CD8+T cell infiltration through the IGF2BP1-AHR-CCL5 axis. (A) Venn diagram illustrating the screening process for key transcription factors regulated by METTL3-mediated m6A modification and involved in CCL5 transcription. (B) Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) analysis of AHR and CCL5 mRNA expression levels after AHR knockdown in MB49 cells. (C) Assessment of CCL5 mRNA expression levels after overexpression of METTL3 and/or knockdown of AHR in MB49 cells. (D) Schematic representation of AHR binding sites within the CCL5 promoter region as predicted by JASPAR. (E) CHIP-qPCR analysis of AHR enrichment at the CCL5 promoter region. (F) mRNA and (G) protein expression levels of AHR after METTL3 knockdown in MB49 cells. (H) Peak plot of m6A modification sites in AHR in MB49 cells. (I) MeRIP-qPCR analysis showing changes in AHR m6A modification levels following METTL3 knockdown in MB49 cells. (J) RIP-qPCR analysis of METTL3 enrichment in AHR mRNA in MB49 cells. (K) MeRIP-qPCR showing changes in AHR m6A modification levels after treatment with the METTL3 inhibitor STM2457 in MB49 cells. (L) RT-qPCR analysis of AHR mRNA levels after STM2457 treatment to inhibit METTL3 in MB49 cells. (M) RNA decay assay showing AHR mRNA stability after silencing METTL3. (N) RNA decay assay showing AHR mRNA stability after treatment with METTL3 inhibitor STM2457 (2 µg/mL, 72 hours) in MB49 cells. (O) RT-qPCR analysis of IGF2BP1 and AHR mRNA expression levels in MB49 cells after silencing IGF2BP1. (P) RT-qPCR analysis of IGF2BP2 and AHR mRNA expression levels in MB49 cells after silencing IGF2BP2. (Q) RT-qPCR analysis of METTL3, IGF2BP1, and AHR mRNA expression levels in MB49 cells after overexpression of METTL3 and/or silencing of IGF2BP1. (R) Images of tumors formed by MB49 stable cell lines (control, AHR overexpression, METTL3 knockdown, METTL3 knockdown with AHR overexpression) subcutaneously implanted into the backs of C57BL/6J mice. (S) Growth curves of mouse bladder cancer tumors. (T) Volume of mouse bladder cancer tumors. (U) Schematic of the animal experiment. (V) Images of bladder cancer tumors in mice. (W) Growth curves of bladder cancer tumors in mice. (X) Tumor weights of bladder cancer tumors in mice; ns, no significance. *p<0.05; **p<0.01; ***p<0.001.

Article Snippet: Anti-mouse Programmed Cell Death Protein 1 (PD-1) antibody (Bioxcell, #BE0146), anti-mouse CD8α antibody (Bioxcell, #BE0061), and anti-mouse Gr-1 antibody (Bioxcell, #BE0075) were also dissolved in PBS and administered intraperitoneally.

Techniques: Modification, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Knockdown, Over Expression, Binding Assay, ChIP-qPCR, Stable Transfection, Control

Journal: Journal for Immunotherapy of Cancer

Article Title: METTL3 promotes an immunosuppressive microenvironment in bladder cancer via m6A-dependent CXCL5/CCL5 regulation

doi: 10.1136/jitc-2024-011108

Figure Lengend Snippet: Targeting METTL3 enhances the efficacy of anti-Programmed Cell Death Protein 1 (PD-1) immunotherapy in bladder cancer. (A) Control and METTL3-knockdown MB49 stable cell lines were subcutaneously injected into mice. Anti-PD-1 antibody (200 µg/mouse, every 3 days) was administered intraperitoneally starting on day 6. Tumors were harvested on day 12 for flow cytometric analysis of the immune microenvironment (n=5). (B–D) Images, growth curves, and tumor weights of subcutaneous bladder cancer tumors in mice. (E–F) Flow cytometric analysis of MDSCs and CD8+T cell infiltration levels in the tumor tissues of mouse bladder cancer. (G) Wild-type MB49 cells were subcutaneously injected into mice, and on day 6, the mice were randomly divided into groups. Treatment included anti-PD-1 antibody (200 µg/mouse, every 3 days, intraperitoneally), IgG antibody (200 µg/mouse, every 3 days, intraperitoneally), the METTL3 inhibitor STM2457 (250 µg/tumor, once daily, intratumorally), and a combination of STM2457 and anti-PD-1 antibody. (H, J) Images, growth curves, and tumor weights of bladder cancer tumors in mice. (K) Control or METTL3 knockdown MB49 stable cell lines were orthotopically injected into the mouse bladder wall to establish an orthotopic bladder cancer model. Anti-PD-1 antibody (200 µg/mouse, every 3 days, intraperitoneally) or IgG antibody (200 µg/mouse, every 3 days, intraperitoneally) was administered starting on day 6 (n=5). (L) In vivo imaging system (IVIS) Living imaging of tumor growth in the orthotopic bladder cancer model. (M) Images of orthotopic bladder cancer tumors in mice. (N) Statistical analysis of fluorescence signal values from IVIS Living imaging on day 16. (O) Tumor volume in the orthotopic bladder cancer model. (P) Tumor weight in the orthotopic bladder cancer model. (Q) Schematic diagram of the study content. ns, no significance. *p<0.05; **p<0.01; ***p<0.001.

Article Snippet: Anti-mouse Programmed Cell Death Protein 1 (PD-1) antibody (Bioxcell, #BE0146), anti-mouse CD8α antibody (Bioxcell, #BE0061), and anti-mouse Gr-1 antibody (Bioxcell, #BE0075) were also dissolved in PBS and administered intraperitoneally.

Techniques: Control, Knockdown, Stable Transfection, Injection, In Vivo Imaging, Imaging, Fluorescence