Journal: Cancer Immunology Research

Article Title: β-Catenin Drives Butyrophilin-like Molecule Loss and γδ T-cell Exclusion in Colon Cancer

doi: 10.1158/2326-6066.CIR-22-0644

Figure Lengend Snippet: HNF4A and HNF4G regulate butyrophilin-like molecule expression in normal gut tissue. A, Schematic of mouse and human promoter regions of indicated genes. Putative HNF4A/G-binding sites are shown in green; CDX1 and CDX2 binding sites are shown in orange. B, Images of CDX1, CDX2, HNF4A and HNF4G protein expressions in SI of WT mice ( n = 4); scale bar, 500 μm. C, Integrative Genomics Viewer analysis of HNF4A/HNF4G ChIP-seq data at mouse Btnl gene loci. D, Fold change in expression levels of indicated genes in WT organoids transduced with shRNA constructs targeting Hnf4 g transcripts. Each dot represents one organoid from one mouse ( n = 4). Data are presented as mean ± SD. E, Butyrophilin-like molecule expression determined by RNA-seq analysis of SI in indicated mouse models. Each dot represents one mouse ( n = 3). Data are presented as mean ± SD. F, Images of organoids from WT mice treated with DMSO control or HNF4A/G inhibitor (HNF4i) representative of 3/group. Fold change in expression levels of indicated genes. Each dot represents one organoid from one mouse ( n = 3). Data are presented as mean ± SD. G, Correlation between HNF4G expression as determined by TempO-seq and γδ T-cell density determined by IHC in the Scotland cohort. Units on axes are normalized counts x 10 3 . Each dot represents one tumor ( n = 77). P and r values determined by Pearson correlation. H, Correlation between BTNL3 or BTNL8 expression and HNF4G expression units on axes are normalized counts x 10 3 . Each dot represents one tumor ( n = 82 Scotland cohort, 258 Marisa cohort). P and r values determined by Pearson correlation. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (unpaired t test or one-way ANOVA followed by Tukey post hoc test).

Article Snippet: Primary antibodies used for IHC were as follows: CDX1 (1:250; Invitrogen #PA5–23056), CDX2 (1:200; Abcam #ab76541), HNF4A (1:10,000; Perseus Proteomics #pph1414–00), HNF4G (1:1,000; Novus Biologicals #NBP1–82531), and SOX9 (1:500; Millipore #AB5535).

Techniques: Expressing, Binding Assay, ChIP-sequencing, Transduction, shRNA, Construct, RNA Sequencing, Control

Journal: Cancer Immunology Research

Article Title: β-Catenin Drives Butyrophilin-like Molecule Loss and γδ T-cell Exclusion in Colon Cancer

doi: 10.1158/2326-6066.CIR-22-0644

Figure Lengend Snippet: Activation of β-catenin decreases Hnf4a and Hnf4g expressions. A, Expressions of HNF4A and HNF4G in normal human colonic tissue and tumor tissue from TCGA ( n = 19 normal, 101 tumor) and Skrypczak ( n = 24 normal, 45 tumor) datasets. Data are presented as median ± minimum/maximum. *, P < 0.05 (Mann–Whitney U test). B, Hnf4a and Hnf4g expressions are determined by RNA-seq analysis of SI in WT ( n = 9), VA F/F ( n = 36), and VA F/F K ( n = 17) mice. Each dot represents one mouse. C, Images of HNF4A and HNF4G protein expressions in SI of indicated models ( n = 4); scale bar, 500 μm. D, Images of intestinal tissue from tumor-bearing VA mice stained for indicated proteins; scale bar, 500 μm. E, Fold change in expression levels of Hnf4a and Hnf4g in organoids from various genotypes measured at indicated days after tamoxifen treatment. Each dot represents one organoid from one mouse ( n = 3). F, Fold change in expression levels of Hnf4a and Hnf4g in WT organoids treated with 3 or 10 μmol/L CHIR-99021 for indicated days. Each dot represents one organoid from one mouse ( n = 3). Data are presented as mean ± SD. **, P < 0.01; ***, P < 0.001 (one-way ANOVA followed by Dunnett post hoc test).

Article Snippet: Primary antibodies used for IHC were as follows: CDX1 (1:250; Invitrogen #PA5–23056), CDX2 (1:200; Abcam #ab76541), HNF4A (1:10,000; Perseus Proteomics #pph1414–00), HNF4G (1:1,000; Novus Biologicals #NBP1–82531), and SOX9 (1:500; Millipore #AB5535).

Techniques: Activation Assay, MANN-WHITNEY, RNA Sequencing, Staining, Expressing

Journal: Cancer Immunology Research

Article Title: β-Catenin Drives Butyrophilin-like Molecule Loss and γδ T-cell Exclusion in Colon Cancer

doi: 10.1158/2326-6066.CIR-22-0644

Figure Lengend Snippet: Inhibition of β-catenin transcriptional activity increases expression of HNF4A, HNF4G, and butyrophilin-like molecules. A, Vγ7 + cell viability in cocultures with CT26 or CT26-B1/6 cells. Each dot represents one paired biological replicate ( n = 4). B, CD25 expression by Vγ7 + cells in cocultures with CT26 or CT26-B1/6 cells. Each dot represents one paired biological replicate ( n = 4). C, CT26 and CT26-B1/6 cancer cell death using flow cytometry after coculture with Vγ7 + cell as indicated. Each dot represents one biological replicate ( n = 4). D, Kaplan–Meier survival analysis of doxycycline-treated CT26 and CT26-B1/6 tumor-bearing mice ( n = 4 CT26, 5 CT26-B1/6) using the log-rank test. E, γδ T-cell numbers in tumors from doxycycline-treated CT26 and CT26-B1/6 tumor-bearing mice. Each dot represents one mouse ( n = 4 CT26, 5 CT26-B1/6). F and H, Images taken from serially stained sections of indicated protein in tumors from indicated mouse models ( n = 3–4; scale bar, 500 μm. G and I, Images of Trdc expression in tumors from indicated mouse models; scale bar, 500 μm. γδ T-cell numbers in tumors. Each dot represents one mouse. Data are presented as mean ± SD. *, P < 0.05; **, P < 0.01 (paired t test or unpaired t test or one-way ANOVA followed by Tukey post hoc test).

Article Snippet: Primary antibodies used for IHC were as follows: CDX1 (1:250; Invitrogen #PA5–23056), CDX2 (1:200; Abcam #ab76541), HNF4A (1:10,000; Perseus Proteomics #pph1414–00), HNF4G (1:1,000; Novus Biologicals #NBP1–82531), and SOX9 (1:500; Millipore #AB5535).

Techniques: Inhibition, Activity Assay, Expressing, Flow Cytometry, Staining