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: bioRxiv

Article Title: MEK1/2 inhibition transiently alters the tumor immune microenvironment to enhance immunotherapy efficacy against head and neck cancer

doi: 10.1101/2021.08.22.457244

Figure Lengend Snippet: (A) Key mutations in 4NQO-T and 4NQO-L cell lines found in MSK-IMPACT genome sequencing. ( B) Hot spot mutations of KRAS in HNC, extracted from cBioportal, GENIE cohort. ( C) Tumor volume changes after 20 days of treatment with trametinib compared to vehicle in NSG and WT mice. ( D) IHC staining of pERK (top left) and Ki67 (top right) in NSG and WT mice. Quantification is shown in the bottom panel (scale bars: 20 µm; inset 10 µm). ( E) Intracellular staining of IFNγ in CD8 + T cells isolated from the vehicle-treated or trametinib SE-treated (5 days) mice. Graph showing the percentage of CD8 + IFNγ + with or without activation with phorbol 12-myristate 13-acetate (PMA) and ionomycin (Iono). Brefeldin A was used as protein transport inhibitor. Results of two independent experiments are shown. (F) PD-1 and TIM3 levels in CD8 + T cells in 4NQO-L tumors treated with vehicle or a short exposure with trametinib. (G) Top – Tumor growth curves for four groups of mice: vehicle + IgG, trametinib + IgG, vehicle + anti-CD8 (αCD8), and trametinib + αCD8. Bottom – Volume of 4NQO-L tumors in WT mice treated with vehicle or trametinib with and without depletion of CD8 + T cells. ( H) IHC staining and quantification of CD8 + T cell depletion experiment (scale bar: 20 µm). For statistics, an unpaired 2-sided t-test or one-way ANOVA was performed. * p < 0.05; ** p < 0.01; *** p < 0.001, **** p < 0.0001 were considered statistically significant. Tra - trametinib, Veh - vehicle.

Article Snippet: In vivo PlusTM anti-mouse CD8α (rat anti-mouse CD8α, BP0061-25 Clone 2.43) or IgG In vivo Plus TM rat IgG2b isotype control, both from Bio X Cell, were used for the CD8 depletion experiments.

Techniques: Sequencing, Immunohistochemistry, Staining, Isolation, Activation Assay

Journal: bioRxiv

Article Title: MEK1/2 inhibition transiently alters the tumor immune microenvironment to enhance immunotherapy efficacy against head and neck cancer

doi: 10.1101/2021.08.22.457244

Figure Lengend Snippet: (A) Tumor growth curves and volumes at the experiment endpoint for 4NQO-L and 4NQO-T tumors in WT mice treated with αPD-1 or IgG. B16 tumors in naïve and cured mice and tumor weights in grams (bar diagram). Relative volumes of 4NQO-L tumors in WT mice treated as indicated. ( D) Weights of 4NQO-L tumors and images of the tumors at the end of the experiment (day 31). For statistics, an unpaired 2-sided t -test or one-way ANOVA was performed. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 were considered statistically significant. ns - not significant. Tra - trametinib, Veh - vehicle.

Article Snippet: In vivo PlusTM anti-mouse CD8α (rat anti-mouse CD8α, BP0061-25 Clone 2.43) or IgG In vivo Plus TM rat IgG2b isotype control, both from Bio X Cell, were used for the CD8 depletion experiments.

Techniques:

Journal: bioRxiv

Article Title: MEK1/2 inhibition transiently alters the tumor immune microenvironment to enhance immunotherapy efficacy against head and neck cancer

doi: 10.1101/2021.08.22.457244

Figure Lengend Snippet: (A) Scheme of the experimental setting. ( B) Tumor volumes of 4NQO-L tumors treated as indicated. ( C) viSNE plots of the CyTOF data showing CD8 and PD-1 expression in CD45 + cells from 4NQO-T tumors treated with vehicle, short exposure (SE; 5 days) trametinib, or a prolonged exposure (PE; 33 days) of trametinib. (D) IHC staining (left) of CD8 and PD-1 in 4NQO-L tumors after treatment with vehicle, SE of trametinib, or PE of trametinib (scale bars: 10 µm). Quantification on the right. (E) viSNE plots of the CyTOF data showing CD11c, MHCII and F4/80 expression in CD45 + cells from 4NQO-T tumors treated with vehicle, SE of trametinib, or PE of trametinib. (F) Flow cytometric dot plot analysis of a macrophage-like MDSC population (MDSC-1) and a DC-like MDSC population (MDSC-2) in 4NQO-L tumors treated with vehicle or trametinib for 5 days. ( G) In-vitro proliferation assay of CD8 + T cells in co-culture (ratio 1:10) with two cell populations, MDSC-1 and MDSC-2, derived from 4NQO-L tumor-bearing mice. Geometric mean fluorescent intensities of CFSC (Carboxyfluorescein diacetyl succinimidyl ester) is shown. (H) Tumor growth curves of mice injected with 4NQO-L cells in the lip and treated with veh+IgG, CSF-1R inhibitor (PLX-3397), veh+αPD-1, or combination of CSF-1Ri and αPD-1. One way ANOVA was performed, and * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 were considered statistically significant. Tra - trametinib, Veh - vehicle.

Article Snippet: In vivo PlusTM anti-mouse CD8α (rat anti-mouse CD8α, BP0061-25 Clone 2.43) or IgG In vivo Plus TM rat IgG2b isotype control, both from Bio X Cell, were used for the CD8 depletion experiments.

Techniques: Expressing, Immunohistochemistry, In Vitro, Proliferation Assay, Co-Culture Assay, Derivative Assay, Injection

Journal: bioRxiv

Article Title: MEK1/2 inhibition transiently alters the tumor immune microenvironment to enhance immunotherapy efficacy against head and neck cancer

doi: 10.1101/2021.08.22.457244

Figure Lengend Snippet: (A) Top – H&E stained images of tongues injected with 4NQO-T cells after exposure to vehicle, SE (7 days) trametinib or PE (25 days) trametinib (scale bars: 2000 µm). Bottom – IHC images and quantification of CD8 + T cells in 4NQO-T tumors after treatment with trametinib for 7 and 25 days (scale bars: 20 µm). ( B) IF co-staining (OPAL) of CD8 (green) and PD1 (red) and merge (yellow) of 4NQO-T tumors treated as indicated (scale bars: 50 µm; inset 10 µm). ( C) Pattern of infiltration of CD8 + T cells in the vehicle-treated and SE trametinib-treated lip tissue samples (Scale bars: 500 µm; enlargements 50 µm). ( D) viSNE plots of the CyTOF data showing CD11b, F4/80 and PD-L1 expression in CD45 + cells from 4NQO-T tumors treated with vehicle, SE (5 days) trametinib, or PE (33 days) trametinib. ( E) Flow cytometry analysis of CD45 + cells based on their CD11b, CD11c, MHCII CSF1R and F4/80 expression. (F) viSNE plots representing flow data of the myeloid populations CSF1R, CD11b, CD11c, MHCII, F4/80 and MGL2 (CD301b + ) shown for 4NQO-L tumors treated with vehicle or trametinib for 5 days (SE). ( G) Flow cytometry dot plot analysis of MGL2 (CD301b + ) and F4/80 on CD45 + CD11b + , when 4NQO-L tumors treated with a SE of trametinib or vehicle. ( H) IHC analysis (top) and quantification (bottom) of CD11c and CD8 in tissues of 4NQO-L tumors treated with vehicle + IgG, vehicle + αPD-1, CSF-1Ri (CSF-1R inhibitor) + IgG, or CSF-1R + αPD-1 (scale bars: 20 µm). For statistics, an unpaired 2-sided t -test or one-way ANOVA was performed. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 were considered statistically significant. ns - not significant. Tra - trametinib, Veh - vehicle.

Article Snippet: In vivo PlusTM anti-mouse CD8α (rat anti-mouse CD8α, BP0061-25 Clone 2.43) or IgG In vivo Plus TM rat IgG2b isotype control, both from Bio X Cell, were used for the CD8 depletion experiments.

Techniques: Staining, Injection, Expressing, Flow Cytometry

Journal: Neoplasia (New York, N.Y.)

Article Title: Anti-PD1 prolongs the response of PI3K and farnesyl transferase inhibition in HRAS- and PIK3CA -mutant head and neck cancers

doi: 10.1016/j.neo.2025.101157

Figure Lengend Snippet: Tipifarnib induces AKT activation in tumor cells and promotes immune escape via PD-L1 upregulation in HPV-negative HRAS mutant HNSCC, whereas tipifarnib efficacy depends on CD8 + T cell infiltration in HPV-negative HRAS mutant HNSCC. (A) Representative immunohistochemical staining images showing the expression of cleaved caspase-3, Ki67, pMAPK, pAKT, pS6, CD45, CD8, and PD-L1 in mEERL tumors treated with vehicle or tipifarnib (60 mg/kg) for three days (scale bars: 100 μm; insets 20 μm). Quantification of positive cells (n = 3 tumors and n = 15 analysis fields). Error bars indicate SEM. Statistical significance was calculated using one-way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001); ns denotes not significant). (B) IHC images showing the expression of cleaved caspace-3, Ki67, pMAPK, pAKT, pS6, CD45, CD8, and PD-L1 in FT1 tumors treated with vehicle or tipifarnib (60 mg/kg) for three days (scale bars: 100 μm; insets 20 μm). Quantification of positive cells (n = 3 tumors and n = 15 analysis fields). Error bars indicate SEM. Statistical significance was calculated using a one-way analysis of variance (ANOVA; ** p < 0.01, *** p < 0.001, ****p < 0.0001; ns denotes not significant). (C) Growth of mEERL tumors in WT mice: 3 × 10⁶ cells were injected orthotopically, and mice were randomized into six treatment arms ( n = 6) with tipifarnib, with or without depletion of CD8 + T cells and CD20 + B cells. Right: Fold change in volumes of mEERL tumors treated with tipifarnib with or without depletion of CD8 + T cells and CD20 + B cells.

Article Snippet: In vivo PlusTM anti-mouse CD8α (rat anti-mouse CD8α, BP0061-25 Clone 2.43), mouse anti-mouse CD20 (clone MB20-115, Mouse IgG2c, Bio X Cell) or IgG In vivo Plus TM rat IgG2b isotype control, from Bio X Cell, were used for the CD8 and CD20 depletion experiment.

Techniques: Activation Assay, Mutagenesis, Immunohistochemical staining, Staining, Expressing, Injection