Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Dual inhibition of HERs and PD-1 counteract resistance in KRAS G12C -mutant head and neck cancer

doi: 10.1186/s13046-024-03227-0

Figure Lengend Snippet: Additive effect of the MRTX849/lapatinib combination on 4NQO-L sensitive tumors is modulated by the presence of CD8 + T-cells. A Growth curve (left) and tumor weight (right) of 4NQO-L tumors in WT mice treated with vehicle ( n = 5 mice, 10 tumors), MRTX849 ( n = 5 mice, 10 tumors), lapatinib ( n = 5 mice, 10 tumors), or the MRTX849/lapatinib combination ( n = 6 mice, 12 tumors). Error bars indicate SEM. Statistical significance was calculated using one-way ANOVA (**** p < 0.0001), ns denotes not significant. B IHC images showing the expression of Ki67 in tissue sections of 4NQO-L tumors treated with vehicle MRTX849, lapatinib, or the MRTX849/lapatinib combination (scale bars: 100 µm; inset 10 µm). n = 4 tumors and n = 27 analysis fields. Error bars indicate SD. Statistical significance was calculated using one-way ANOVA (** p < 0.01, **** p < 0.0001). C IHC images showing the infiltration of CD8 + T-cells in the tissue sections of 4NQO-L tumors treated with vehicle, MRTX849, lapatinib or the MRTX849/lapatinib combination (scale bar: 100 µm; inset: 10 µm). n = 4 tumors and n = 27 analysis fields. Error bars indicate SD. Statistical significance was calculated using one-way ANOVA (**** p < 0.0001). D Top: Tumor volume of the orthotopic 4NQO-L tumors in WT mice (left; n = 8 mice, 8 tumors) or NSG mice (right; n = 6 mice, 6 tumors) treated with vehicle or the MRTX849/lapatinib combination. Statistical significance was calculated using one-way ANOVA (**** p < 0.0001) Error bars indicate SEM. Bottom: Change in tumor volume from first to last day of treatment. E Top: Scheme of the experiment investigating the effect of CD8 + T-cell depletion on MRTX849/lapatinib efficacy. Bottom: Growth of 4NQO-L tumors in WT mice treated with IgG ( n = 6 mice, 11 tumors), αCD8 T cells depletion ( n = 6 mice, 12 tumors), IgG/MRTX849/lapatinib ( n = 5 mice, 10 tumors) or αCD8/MRTX849/lapatinib ( n = 6 mice, 12 tumors). Statistical significance was calculated using one-way ANOVA (**** p < 0.0001), ns denotes not significant

Article Snippet: In vivo PlusTM anti-mouse CD8α or IgG (In vivo PlusTM rat IgG2b) isotype controls, both from Bio X Cell, were used for CD8 depletion experiments.

Techniques: Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Dual inhibition of HERs and PD-1 counteract resistance in KRAS G12C -mutant head and neck cancer

doi: 10.1186/s13046-024-03227-0

Figure Lengend Snippet: MRTX849/lapatinib/αPD-1 treatment is ineffective in MRTX849-resistant tumors. A Growth curve of 4NQO-L KRAS G12C i acquired resistant tumors in WT mice treated with vehicle, MRTX849, lapatinib or MRTX849/lapatinib combination ( n = 6 mice, 12 tumors). B IHC images showing the infiltration of CD8 + T cells in the tissue sections of 4NQO-L KRAS G12C i acquired-resistance tumors treated with vehicle MRTX849, lapatinib, or MRTX849/lapatinib (scale bars: 100 µm; inset 10 µm). n = 4 tumors and n = 40 analysis fields. Error bars indicate SD. Statistical significance was calculated using one-way ANOVA (** p < 0.01, *** p < 0.001, **** p < 0.0001). C IHC images showing expression of PD-L1 in the tissue sections of 4NQO-L KRAS G12C i acquired resistant tumors treated with vehicle, MRTX849, lapatinib or MRTX849/lapatinib (scale bars: 100 µm; inset 10 µm). n = 4 tumors and n = 38 analysis fields. Error bars indicate SD. Statistical significance was calculated using one-way ANOVA (* p < 0.05, *** p < 0.001, **** p < 0.0001), ns denotes not significant. D IHC images showing the expression of αSMA in the tissue sections of 4NQO-L KRAS G12C i acquired resistant tumors treated with vehicle, MRTX849, lapatinib or MRTX849/lapatinib (scale bars: 100 µm; inset 10 µm). n = 4 tumors and n = 40 analysis fields. Error bars indicate SD. Statistical significance was calculated using one-way ANOVA (**** p < 0.0001), ns denotes not significant. E IHC images showing the infiltration of CD8 + T-cells in the tissue sections of 4NQO-L KRAS G12C i-acquired-resistance tumors (scale bars: 100 µm; inset 10 µm). n = 4 tumors and n = 41 analysis fields. Error bars indicate SD. Statistical significance was calculated using one-way ANOVA (**** p < 0.0001). F Growth curves of 4NQO-L KRAS G12C i-acquired-resistance tumors treated with IgG, IgG/MRTX849/lapatinib, MRTX849/αPD-1, or the combination of MRTX849/ lapatinib and αPD-1 ( n = 6 mice, 12 tumors). Statistical significance was calculated using one-way ANOVA, ns denotes not significant

Article Snippet: In vivo PlusTM anti-mouse CD8α or IgG (In vivo PlusTM rat IgG2b) isotype controls, both from Bio X Cell, were used for CD8 depletion experiments.

Techniques: Expressing

Journal: Signal transduction and targeted therapy

Article Title: Targeting BCL9/BCL9L enhances antigen presentation by promoting conventional type 1 dendritic cell (cDC1) activation and tumor infiltration.

doi: 10.1038/s41392-024-01838-9

Figure Lengend Snippet: Fig. 1 Pharmacological inhibition of BCL9 induces tumor regression and increases antigen presentation. a The BCL9 expression between tumors and normal tissues in TCGA COAD datasets (Normal, n = 41; Tumor, n = 462). b The antigen processing and presentation signature (left) and HLA-I signature (right) between low and high BCL9 expression (median value) in TCGA COAD datasets (BCL9Low , n = 209; BCL9High, n = 236). c Tumor growth of 30 mg/kg hsBCL9z96-treated CT26 tumor-bearing mice (n = 6). d Tumor growth of MC38 tumor-bearing Bcl9f/fBcl9lf/f mice and Bcl9f/fBcl9lf/f Cre-ERT2 mice treated i.p. with tamoxifen (1 mg/100 μL) in olive oil on days −7, −6, −5, +1, +6, and +11 post inoculation (n = 6). e Heatmap of the genes included in the GO:0019882 from 30 mg/kg hsBCL9z96-treated CT26 tumors (Vehicle, n = 4; hsBCL9z96, n = 5). f, g The relative expression of Tap1, Tap2, B2m and Psmb9 of tumors from hsBCL9z96-treated CT26 tumor-bearing mice (f) and MC38 tumor- bearing Bcl9f/fBcl9lf/f Cre-ERT2 mice (g) analyzed by qPCR (n = 4–7). h–k Representative plot (h, j) and quantitative analysis (i, k) of OVA257-264-specific CD8+ T cells in TILs of tumors from MC38-OVA tumor-bearing Bcl9f/fBcl9lf/f Cre-ERT2 mice (h, i) and hsBCL9z96-treated MC38- OVA tumor-bearing mice (j, k) treated i.p. with tamoxifen (1 mg/100 μL) in olive oil on days −7, −6, −5, +1, +6, and +11 post inoculation and analyzed by flow cytometry (n = 3). l Tumor growth of C57BL/6 WT (n = 6) and Batf3−/−mice (n = 5) that had been injected subcutaneously with MC38 tumor cells and were treated i.p. with vehicle or 40 mg/kg hsBCL9z96 every day for 2 weeks. These data are representative values expressed as the mean ± SD of each group; n indicates biological replicate; **p < 0.01; ***p < 0.001; ****p < 0.0001; Unpaired Student’s t test (a, b, i, k); Two-way ANOVA followed by Bonferroni test (c, d, f, g)

Article Snippet: For CD4 and CD8 T cells depletion, individual mice were injected i.p. with anti-mouse CD4 monoclonal antibodies (Abs) (100 μg/100 μL; BE0003-1, BioXcell) or anti-mouse CD8 Abs (100 μg/100 μL; BE0004-1, BioXcell) on days +2, +4, +6, +8 and +10 after inoculation.

Techniques: Inhibition, Immunopeptidomics, Expressing, Cytometry, Injection

Journal: Signal transduction and targeted therapy

Article Title: Targeting BCL9/BCL9L enhances antigen presentation by promoting conventional type 1 dendritic cell (cDC1) activation and tumor infiltration.

doi: 10.1038/s41392-024-01838-9

Figure Lengend Snippet: Fig. 2 Inhibition of BCL9/BCL9L enhances cDC1 activation and facilitates cross-priming of CD8+ T cells. a, b CD40 (left) and CD86 (right) expression by CD103+ cDC1 of TdLNs (a) and tumors (b) from 30 mg/kg hsBCL9z96-treated CT26 tumor-bearing mice analyzed by flow cytometry (n = 3-4). c, d CD40 (left) and CD86 (right) expression by CD103+ cDC1 of TdLNs (c) and tumors (d) from MC38 tumor-bearing Bcl9f/fBcl9lf/f Cre-ERT2 mice treated i.p. with tamoxifen (1 mg/100 μL) in olive oil on days −7, −6, −5, +1, +6, and +11 post inoculation analyzed by flow cytometry (n = 3–4). e The representative plot of OT-I CD8+ T cells in TdLNs from hsBCL9z96-treated MC38-OVA tumor- bearing mice analyzed by flow cytometry. f and g Quantitative analysis of the percentage of OT-I CD8+ T cells (f) and CFSE dilution of OT-I CD8+ T cells (mean fluorescent intensity, MFI) (g) based on the result of (e) (n = 3). h The representative plot of OT-I CD8+ T cells in TdLNs from MC38-OVA tumor-bearing Bcl9f/fBcl9lf/f Cre-ERT2 mice treated i.p. with tamoxifen (1 mg/100 μL) in olive oil on days −7, −6, −5, +1, and +6 post inoculation analyzed by flow cytometry. i, j Quantitative analysis of the percentage of OT-I CD8+ T cells (i) and CFSE dilution of OT-I CD8+ T cells (j) based on the result of (h) (n = 3). These data are representative values expressed as the mean ± standard deviation (SD) for each group, derived from three independent experiments; “n” denotes the number of biological replicates. An unpaired Student’s t test was used for statistical analysis of the data in groups a–d, f, g, i, and j

Article Snippet: For CD4 and CD8 T cells depletion, individual mice were injected i.p. with anti-mouse CD4 monoclonal antibodies (Abs) (100 μg/100 μL; BE0003-1, BioXcell) or anti-mouse CD8 Abs (100 μg/100 μL; BE0004-1, BioXcell) on days +2, +4, +6, +8 and +10 after inoculation.

Techniques: Inhibition, Activation Assay, Expressing, Cytometry, Standard Deviation, Derivative Assay

Journal: Signal transduction and targeted therapy

Article Title: Targeting BCL9/BCL9L enhances antigen presentation by promoting conventional type 1 dendritic cell (cDC1) activation and tumor infiltration.

doi: 10.1038/s41392-024-01838-9

Figure Lengend Snippet: Fig. 3 Single-cell transcriptional profiling of CD8+ T cells and cDC1 in tumors and TdLNs from B16-OVA tumor-bearing Bcl9/Bcl9l deficiency mice. a Illustration of experiment and analysis process of single-cell transcriptional analysis. b TSNE plots of clustering process and marker genes (Zbtb46 for DCs, Cd68 for myeloid cells, Mlana for B16-OVA tumor cells, Cd3e for T cells, Cd4 for CD4+ T cells and Cd8a for CD8+ T cells) in tumors from B16-OVA tumor-bearing Bcl9f/fBcl9lf/f mice and Bcl9f/fBcl9lf/f Cre-ERT2 mice treated i.p. with tamoxifen (1 mg/100 μL) in olive oil on days −7, −6, −5, +1, +6 and +11 post inoculation. c–e TSNE plots of DC reclustering (c, e) and marker genes (Xcr1 for cDC1 and Clec10a for cDC2) (d) in tumors from B16-OVA tumor-bearing Bcl9f/fBcl9lf/f mice and Bcl9f/fBcl9lf/f Cre-ERT2 mice treated i.p. with tamoxifen (1 mg/100 μL) in olive oil on days −7, −6, −5, +1, +6, and +11 post inoculation

Article Snippet: For CD4 and CD8 T cells depletion, individual mice were injected i.p. with anti-mouse CD4 monoclonal antibodies (Abs) (100 μg/100 μL; BE0003-1, BioXcell) or anti-mouse CD8 Abs (100 μg/100 μL; BE0004-1, BioXcell) on days +2, +4, +6, +8 and +10 after inoculation.

Techniques: Marker

Journal: Signal transduction and targeted therapy

Article Title: Targeting BCL9/BCL9L enhances antigen presentation by promoting conventional type 1 dendritic cell (cDC1) activation and tumor infiltration.

doi: 10.1038/s41392-024-01838-9

Figure Lengend Snippet: Fig. 4 Bcl9/Bcl9l deficient cDC1 are superior to WT cDC1 in activation, antigen presentation and cross-priming of CD8+ T cells. a Expression of genes related to cDC1 maturation and antigen presentation in tumors and TdLNs from B16-OVA tumor-bearing Bcl9f/fBcl9lf/f mice and Bcl9f/f

Article Snippet: For CD4 and CD8 T cells depletion, individual mice were injected i.p. with anti-mouse CD4 monoclonal antibodies (Abs) (100 μg/100 μL; BE0003-1, BioXcell) or anti-mouse CD8 Abs (100 μg/100 μL; BE0004-1, BioXcell) on days +2, +4, +6, +8 and +10 after inoculation.

Techniques: Activation Assay, Immunopeptidomics, Expressing

Journal: Signal transduction and targeted therapy

Article Title: Targeting BCL9/BCL9L enhances antigen presentation by promoting conventional type 1 dendritic cell (cDC1) activation and tumor infiltration.

doi: 10.1038/s41392-024-01838-9

Figure Lengend Snippet: Fig. 6 Targeting of BCL9/BCL9L increases cDC1 accumulation in tumors through XCL1-XCR1 axis. a Gating strategy of XCR+ cDC1 (CD45+ CD11b−CD11c+ MHC-II+ CD103+ XCR1+) in TILs. b The XCR+ cDC1 in TILs of 30 mg/kg hsBCL9z96-treated CT26 tumor-bearing mice (left) and MC38 tumor-bearing Bcl9f/fBcl9lf/fCre-ERT2 mice (right) treated i.p. with tamoxifen (1 mg/100 μL) in olive oil on days −7, −6, −5, +1, +6, and +11 post inoculation were analyzed by flow cytometry (n = 4). c iCD103+ DC migration toward XCL1 for 3 h by trans well assay (n = 3). d Heatmap of the genes included in GO:0070098 of 30 mg/kg hsBCL9z96-treated CT26 tumors (vehicle, n = 4; hsBCL9z96, n = 5). e, f Xcl1 mRNA (left) and XCL1 protein (right) levels in tumors from 30 mg/kg hsBCL9z96-treated CT26 tumor-bearing mice (e) and MC38 tumor-bearing Bcl9f/fBcl9lf/fCre-ERT2 mice (f) treated i.p. with tamoxifen (1 mg/100 μL) in olive oil on days −7, −6, −5, +1, +6, and +11 post inoculation were analyzed by qPCR and ELISA, respectively (n = 4-5). g, h Representative plot (left) and quantitative analysis (right) of XCL1 expression of CD8+ T cells and NK cells in TILs from 30 mg/kg hsBCL9z96-treated CT26 tumor-bearing mice analyzed by flow cytometry (n = 4). g Representative plot (left) and quantitative analysis (right) of XCL1 expression among CD8+ T cells and NK cells in TILs from MC38 tumor-bearing Bcl9f/fBcl9lf/f Cre-ERT2 mice treated i.p. with tamoxifen (1 mg/100 μL) in olive oil on days −7, −6, −5, +1, +6, and +11 post inoculation were analyzed by flow cytometry (n = 4). h Results are presented as the mean ± standard deviation (SD) for each group, derived from three independent experiments; “n” denotes the number of biological replicates; Unpaired Student’s t test (b, c, e, f); Two-way ANOVA followed by Bonferroni test (g, h)

Article Snippet: For CD4 and CD8 T cells depletion, individual mice were injected i.p. with anti-mouse CD4 monoclonal antibodies (Abs) (100 μg/100 μL; BE0003-1, BioXcell) or anti-mouse CD8 Abs (100 μg/100 μL; BE0004-1, BioXcell) on days +2, +4, +6, +8 and +10 after inoculation.

Techniques: Cytometry, Migration, Enzyme-linked Immunosorbent Assay, Expressing, Standard Deviation, Derivative Assay

Journal: Signal transduction and targeted therapy

Article Title: Targeting BCL9/BCL9L enhances antigen presentation by promoting conventional type 1 dendritic cell (cDC1) activation and tumor infiltration.

doi: 10.1038/s41392-024-01838-9

Figure Lengend Snippet: Fig. 7 Targeting BCL9/BCL9L results in CD8+ T cells accumulation in tumors through CXCL9-CXCR3 axis. a Significantly upregulated GO terms related to IFN-γ response of 30 mg/kg hsBCL9z96-treated CT26 tumors are depicted (vehicle, n = 4; hsBCL9z96, n = 5). b and c Relative Ifng mRNA (left) and IFN-γ protein (right) levels in tumors from 30 mg/kg hsBCL9z96-treated CT26 tumor-bearing mice (b) and MC38 tumor-bearing Bcl9f/fBcl9lf/f Cre-ERT2 mice (c) treated i.p. with tamoxifen (1 mg/100 μL) in olive oil on days −7, −6, −5, +1, +6, and +11 post inoculation analyzed by qPCR and ELISA, respectively (n = 4–7). d, e Relative Cxcl9 mRNA (left) and CXCL9 protein (right) expression of tumors from 30 mg/ kg hsBCL9z96-treated CT26 tumor-bearing mice (d) and MC38 tumor-bearing Bcl9f/fBcl9lf/f Cre-ERT2 mice (e) treated i.p. with tamoxifen (1 mg/ 100 μL) in olive oil on days −7, −6, −5, +1, +6, and +11 post inoculation analyzed by qPCR and ELISA, respectively (n = 4-7). f Assessment of CD8+ T cell migration toward CXCL9 or with the indicated doses of antibodies or chemokine for 4 h by trans well assay (n = 3). g Representative plot (left) and quantitative analysis (right) of CXCL9 expression in cDC1 of tumors from 30 mg/kg hsBCL9z96-treated CT26 tumor-bearing mice analyzed by flow cytometry (n = 3–4). h Representative plot (left) and quantitative analysis (right) of CXCL9 expression in cDC1 of tumors from MC38 tumor-bearing Bcl9/Bcl9l deficiency mice analyzed by flow cytometry (n = 4). i The expression of CXCR3 in CD8+ T cells of tumors from 30 mg/kg hsBCL9z96-treated CT26 tumor-bearing mice (left) and MC38 tumor-bearing Bcl9f/fBcl9lf/f Cre-ERT2 mice (right) treated i.p. with tamoxifen (1 mg/100 μL) in olive oil on days −7, −6, −5, +1, +6, and +11 post inoculation analyzed by flow cytometry (n = 4). Results are presented as the mean ± standard deviation (SD) for each group, derived from three independent experiments; “n” denotes the number of biological replicates; Unpaired Student’s t test (b–e, g–i); One-way ANOVA followed by Bonferroni test (f)

Article Snippet: For CD4 and CD8 T cells depletion, individual mice were injected i.p. with anti-mouse CD4 monoclonal antibodies (Abs) (100 μg/100 μL; BE0003-1, BioXcell) or anti-mouse CD8 Abs (100 μg/100 μL; BE0004-1, BioXcell) on days +2, +4, +6, +8 and +10 after inoculation.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Migration, Cytometry, Standard Deviation, 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 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

Journal: Immunity & Ageing : I & A

Article Title: Ascorbic acid attenuates immunosenescence and cognitive decline via MYH9-Mediated CD8⁺ T cell differentiation

doi: 10.1186/s12979-025-00538-4

Figure Lengend Snippet: Ascorbic acid improves the cognitive level of aged mice and increases the number of CD8 + cells in aged mice. A Trajectory tracking map of mouse movement in the open field test, illustrating time spent in the central zone (seconds), average speed during movement episodes (cm/s), and total distance traveled within the central area. B Statistical chart of New Object Cognition Index. C Flow cytometry was utilized to analyze immune cell populations, including CD3 + T, CD4 + T, CD8 + T, CD11b + , and B cells, in the whole blood of middle-aged mice assigned to either the control group or the AA group ( D ). E Statistical analysis of the flow cytometry data was presented in a histogram format, with significance levels denoted as follows: ns ( P > 0.05), * ( P < 0.05), ** ( P < 0.01), and *** ( P < 0.001)

Article Snippet: The Rat IgG2b isotype and Anti-mouse CD8α-InVivo antibodies (Selleckchem) were each diluted to a final volume of 1 ml using sterile PBS and injected intraperitoneally at a dosage of 200 μg per mouse.

Techniques: Flow Cytometry, Control

Journal: Immunity & Ageing : I & A

Article Title: Ascorbic acid attenuates immunosenescence and cognitive decline via MYH9-Mediated CD8⁺ T cell differentiation

doi: 10.1186/s12979-025-00538-4

Figure Lengend Snippet: AA facilitates the early differentiation of T cells while concurrently suppressing myeloid differentiation. A A schematic depicting the early and long-term differentiation of T cells in an OP9-DL1 and lin-CD117 + HSC co-culture system is presented. B The diagram illustrates the progression of T cell maturation from CD44 and CD25 negative to positive selection. C Flow cytometry analysis and statistical bar graphs are utilized to demonstrate the AA-promoted changes in T cell DN stages, including the DN1 phase (CD44 + CD25 − ), DN2 phase (CD44 + CD25 + ), DN3 phase (CD44 − CD25 + ), and DN4 phase (CD44 − CD25 − ). D Flow cytometry analysis is utilized to examine the inhibition of myeloid differentiation, as well as to assess the long-term T cell differentiation promoted by AA in vitro ( E ). F and G Statistical bar graphs are generated to represent the flow cytometry results of CD8 + and CD4 + T cells, with significance levels denoted as ns: not significant ( P > 0.05), * ( P < 0.05), and ** ( P < 0.01), ( n = 3 for each group)

Article Snippet: The Rat IgG2b isotype and Anti-mouse CD8α-InVivo antibodies (Selleckchem) were each diluted to a final volume of 1 ml using sterile PBS and injected intraperitoneally at a dosage of 200 μg per mouse.

Techniques: Co-Culture Assay, Selection, Flow Cytometry, Inhibition, Cell Differentiation, In Vitro, Generated

Journal: Immunity & Ageing : I & A

Article Title: Ascorbic acid attenuates immunosenescence and cognitive decline via MYH9-Mediated CD8⁺ T cell differentiation

doi: 10.1186/s12979-025-00538-4

Figure Lengend Snippet: The Myh9 protein exhibits binding affinity towards AA and affects the proliferation of CD8 + T cells. A MetPro protein-metabolite interaction experimental workflow diagram. B Predicted binding differential protein KEGG pathway enrichment diagram. C Mass spectrometry predicted binding differential protein heatmap. D Protein interaction average degree analysis diagram. E Protein interaction network analysis diagram. F Concentration gradient binding curves of AA with Rhoa and Myh9 highlight the binding dynamics between these proteins. G Flow cytometry diagrams of CD8 + T cells for (Ga) control group, (Gb) AA group, (Gc) blebbistatin group, and (Gd) blebbistatin + AA group with statistical results ( H ) ( n = 3 for each group. ** p < 0.01, * p < 0.05). I Flow cytometry diagrams of CD11b + cells for (Ia) control group, (Ib) AA group, (Ic) blebbistatin group, and (Id) blebbistatin + AA group with statistical results ( J ) ( n = 3 for each group. **** p < 0.0001, *** p < 0.001, ** p < 0.01)

Article Snippet: The Rat IgG2b isotype and Anti-mouse CD8α-InVivo antibodies (Selleckchem) were each diluted to a final volume of 1 ml using sterile PBS and injected intraperitoneally at a dosage of 200 μg per mouse.

Techniques: Binding Assay, Mass Spectrometry, Concentration Assay, Flow Cytometry, Control

Journal: Immunity & Ageing : I & A

Article Title: Ascorbic acid attenuates immunosenescence and cognitive decline via MYH9-Mediated CD8⁺ T cell differentiation

doi: 10.1186/s12979-025-00538-4

Figure Lengend Snippet: Anti-CD8α antibody injection reduced the cognitive level of young mice. A Flow cytometry analysis of CD8 + T cells versus CD4 + T cells in the peripheral blood of mice after injection of IgG2b antibody and Anti-CD8α antibody ( B ). C Statistical histogram of CD8 + T flow cytometry results. D CD4 + T flow cytometry results statistical histogram. E Flow cytometry analysis of NK cells in mouse peripheral blood after IgG2b and Anti-CD8α antibody injection. F Statistical histogram of flow cytometry results of FNK cells. G Tracking diagram of mouse movement trajectory in open field test after antibody injection. H Time (s), speed, and distance traveled by the mouse in the central region during the open field test. I Statistical chart of new object cognition index ( n = 5 for each group, ns P > 0.05, * p < 0.05, *** P < 0.001)

Article Snippet: The Rat IgG2b isotype and Anti-mouse CD8α-InVivo antibodies (Selleckchem) were each diluted to a final volume of 1 ml using sterile PBS and injected intraperitoneally at a dosage of 200 μg per mouse.

Techniques: Injection, Flow Cytometry