Fig 1: Ablation ofGnat3increases PDA genesis, grade and metastasis. Analysis of KC and Gnat3-/-;KC mice aged to moribund or 52 weeks. (A) Kaplan-Meier survival curve, with endpoint of study indicated by black line at 52 weeks (n = 12; 17). (B and C) H&E analysis of tissues to determine number of mice with carcinoma (n = 12; 16) (B) and grading of carcinoma samples (poor, moderate, or well) (n = 4; 11) (C). (D) H&E and IHC for CK19. Inset boxes are magnified area. Scale bar: full pancreas = 2000 µm, inset = 100 µm. (E) IHC for CK19 on KC or Gnat3-/-;KC lung and liver (black dashed line) and quantification of mice with macrometastasis (n = 12; 16). Scale bar: 2000 µm. (F) IHC for CXCL1 on KC or Gnat3-/-;KC pancreas quantified by positive stain from total pancreas area (n = 6; 7). Scale bar: 100 µm. (G) In situ hybridization for Cxcl2 transcript quantified by positive puncta area (red) of total pancreas area from KC or Gnat3-/-;KC pancreata (n = 7, 7). Scale bar: 50 µm. Significance was calculated using the log-rank (Mantel-Cox) test and unpaired t tests; P < .05 statistically significant.
Fig 2: Single-cell sequencing of pancreata identifying a tuft cell specific gene cluster. Analysis of KC single-cell RNA transcriptomes. (A) Unbiased clustering of single cells driven by transcriptome differences and visualized by UMAP. MTC cluster is circled. EC, endothelial cells. (B) UMAP of tuft cell markers Dclk1, Vav1, and Pou2f3 single-cell RNA transcriptome expression from KC cells with MTC cluster circled. Color scale denotes expression level. (C) Dot plot of gene expression patterns for MTC markers Dclk1, Vav1, and Pou2f3 as well as Cxcl1 and Cxcl2 expression per cell populations from KC cells. MTC cluster is outlined with other cell populations indicated beneath. Percent of cells expressing each gene per cluster is noted by dot size. Average gene expression is represented by color of dot. (D) Co-immunofluorescence for CXCL1 (red) and MTC marker COX1 (white) in tamoxifen and cerulein treated KCERT;ROSA26LSL-EYFP and Gnat3-/-;KCERT;ROSA26LSL-EYFP pancreata collected 6 weeks after cerulein treatment. In KCERT;ROSA26LSL-EYFP pancreata a high level CXCL1 expression (red) is found primarily in non-epithelial cells (YFP-, white arrows) and low expression in MTCs (YFP+, COX1+, yellow arrows). In Gnat3-/-;KCERT;ROSA26LSL-EYFP, non-MTC epithelia (YFP+, COX1-, purple arrows) express high levels of CXCL1, whereas relatively low expression is found in MTCs (yellow arrows) and in stromal cells. DAPI (blue) counterstains nuclei. Scale bar: 20 µm.
Fig 3: Gnat3 ablation increases CXCL1 and CXCL2 expression in the neoplastic epithelial compartment. Analysis of tamoxifen and cerulein treated KCERT and Gnat3-/-;KCERT pancreata collected 6 weeks after cerulein treatment. (A) UMAP of Cxcr2 single-cell RNA transcriptome expression from KCERT and Gnat3-/-;KCERT cells. MDSC cluster circled. Color scale denotes expression level. (B) IHC for CXCL1 quantified from positive area of stroma or neoplasia area (n = 4). Scale bar: 50 µm. (C) CXCL2 protein levels measured by ELISA from pancreas lysate (n = 3; 4). (D) In situ hybridization for Cxcl2 transcript quantified by positive puncta area (red) of stroma or neoplasia area (n = 3). Inset boxes are magnified area. Scale bar: 50 µm, inset = 5 µm. (E and F) UMAP of single-cell RNA transcriptome expression from pooled KCERT and Gnat3-/-;KCERT data where gene expression is denoted by red per each cell for Cxcl1 (E), primarily in fibroblasts, and Cxcl2 (F), primarily in TAM and MDSC compartments as circled. Significance was calculated using an unpaired t test; P < .05 statistically significant.
Fig 4: Metaplastic tuft cells are present 6 weeks after injury, and Gnat3 ablated cells maintain tuft marker expression. (A) Genetic strategy to generate KCERT mice with Gnat3 ablation (Gnat3-/-;KCERT). Schematic of tamoxifen (TMX) treatment to induce KrasG12D/+ expression in the acinar compartment, followed by cerulein (CER) to promote inflammation and harvest of pancreas at 1 or 6 weeks. (B) H&E and IHC for DCLK1 to mark MTCs from pancreata harvested from KCERT mice 1 or 6 weeks after cerulein. Quantitation of DCLK1-positive MTCs from neoplastic area in 1- or 6-week tissue (n = 3; 8). Scale bar: 100 µm. (C) Staining from 6-week post injury KCERT and Gnat3-/-;KCERT pancreatic tissues for known MTC markers: GNAT3, DCLK1, COX1, COX2, and VAV1 (all green), counterstained by phalloidin (red) to mark the luminal tufts and DAPI (blue) to label nuclei. Scale bars: 5 µm. Significance was calculated using an unpaired t test; P < .05 statistically significant.
Fig 5: Gnat3 ablation increases epithelial cytokine release in ex vivo organoid culture model. (A) Analysis of WT or Gnat3-/- adult pancreata by H&E, including magnified inset from box, and pancreas to body weight ratios (n = 11; 18). Scale bar: 2000 μm, inset = 50 μm. (B) IHC for DCLK1 to mark MTCs on WT or Gnat3-/- common biliary ducts. Magnified inset indicated in black box in bottom right. Scale bar: 50 μm, inset = 10 μm. (C) Genetic strategy to generate KC mice with Gnat3 ablation (Gnat3-/-;KC). (D) 3D organoid culture analysis of KC and Gnat3-/-;KC transdifferentiated acinar cells. Phalloidin (red) highlights the structure of individual 3D organoids (white dotted outline) stained for VAV1 (green) to mark MTCs and DAPI (blue) to mark nuclei. White box denotes inset image in bottom right of VAV1-positive MTCs with tufts marked by phalloidin. MTC numbers were determined from average of 130 organoids per sample (n = 4). Scale bar: 20 μm, inset = 5 μm. (E) Cytokine proteomic analysis of conditioned media derived from KC and Gnat3-/-;KC organoids. Representative cytokine array blot (left) and quantitation (right) show 7 differentially expressed proteins (numbered). Differential protein levels from Gnat3-/-;KC (blue bars) were normalized by KC (green line) (n = 3). R, reference points; 1, amphiregulin; 2, chitinase 2-like 1; 3, CXCL1; 4, CXCL2; 5, CXCL16; 6, osteoprotegerin; 7, WISP1. Significance was calculated using an unpaired t test; P < .05 statistically significant.
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