The Gustatory Sensory G-Protein GNAT3 Suppresses Pancreatic Cancer Progression in Mice

Abstract

Background & aims: Pancreatic ductal adenocarcinoma (PDA) initiation and progression are accompanied by an immunosuppressive inflammatory response. Here, we evaluated the immunomodulatory role of chemosensory signaling in metaplastic tuft cells (MTCs) by analyzing the role of GNAT3, a gustatory pathway G-protein expressed by MTCs, during PDA progression.

Methods: Gnat3-null (Gnat3^-/-) mice were crossbred with animals harboring a Cre-inducible Kras^LSL-G12D/+ allele with either Ptf1a^Cre/+ (KC) or tamoxifen-inducible Ptf1a^CreERT/+ (KC^ERT) mice to drive oncogenic KRAS expression in the pancreas. Ex vivo organoid conditioned medium generated from KC and Gnat3^-/-;KC acinar cells was analyzed for cytokine secretion. Experimental pancreatitis was induced in KC^ERT and Gnat3^-/-;KC^ERT mice to accelerate tumorigenesis, followed by analysis using mass cytometry and single-cell RNA sequencing. To study PDA progression, KC and Gnat3^-/-;KC mice were aged to morbidity or 52 weeks.

Results: Ablation of Gnat3 in KC organoids increased release of tumor-promoting cytokines in conditioned media, including CXCL1 and CXCL2. Analysis of Gnat3^-/-;KC^ERT pancreata found altered expression of immunomodulatory genes in Cxcr2 expressing myeloid-derived suppressor cells (MDSCs) and an increased number of granulocytic MDSCs, a subset of tumor promoting MDSCs. Importantly, expression levels of CXCL1 and CXCL2, known ligands for CXCR2, were also elevated in Gnat3^-/-;KC^ERT pancreata. Consistent with the tumor-promoting role of MDSCs, aged Gnat3^-/-;KC mice progressed more rapidly to metastatic carcinoma compared with KC controls.

Conclusions: Compromised gustatory sensing, achieved by Gnat3 ablation, enhanced the CXCL1/2-CXCR2 axis to alter the MDSC population and promoted the progression of metastatic PDA.

Keywords: CXCL1; CXCL2; MDSC; Tuft Cell.

Graphical abstract

Figure 1

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.

Figure 2

Metaplastic tuft cells are present 6 weeks after injury, and Gnat3 ablated cells maintain tuft marker expression. (A) Genetic strategy to generate KC^ERT mice with Gnat3 ablation (Gnat3^-/-;KC^ERT). Schematic of tamoxifen (TMX) treatment to induce Kras^G12D/+ 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 KC^ERT 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 KC^ERT and Gnat3^-/-;KC^ERT 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.

Figure 3

Gnat3 ablation has no effect on pancreatic neoplasia formation. Analysis of tamoxifen and cerulein treated KC^ERT and Gnat3^-/-;KC^ERT pancreata harvested 6 weeks after cerulein treatment. (A) H&E staining with pancreas-to-body weight ratios below (n = 27; 35). IHC for amylase (n = 10; 13) and CK19 (n = 10; 12) quantified below by total positive area from total pancreas area. Scale bars: 1000 μm. (B) Staining for picrosirius red (n = 9; 17), Ki67, CC3 (n = 6; 8), and DCLK1 (n = 8) with quantification below for each. Scale bars: 100 μm. Significance was calculated using an unpaired t test; P < .05 statistically significant.

Figure 4

GNAT3 loss increases gMDSC presence during pancreatic transformation. Manually gated mass cytometry data from tamoxifen and cerulein treated KC^ERT and Gnat3^-/-;KC^ERT pancreata harvested 6 weeks after cerulein treatment (n = 10). (A) Percentage of fibroblasts (CD140a⁺) or total immune cells (CD45⁺) from total cell numbers. (B) T cells (CD45⁺CD3⁺) as percentage of total immune cells. Percent of CD4⁺ T cells (CD45⁺CD3⁺CD4⁺) and CD8⁺ T cells (CD45⁺CD3⁺CD8⁺) from T cells. (C) B cells (CD45⁺CD19⁺) as percent of total immune cells. (D) Percent of TAM (CD45⁺CD11b⁺F4/80⁺) from total immune cells. TAM phenotypes were characterized by anti-inflammatory TAM (CD45⁺CD11b⁺F4/80⁺CD206⁺) and proinflammatory TAM (CD45⁺CD11b⁺F4/80⁺CD11c⁺ or Ly6C⁺) as percent of TAM. (E) NK family (CD45⁺NK1.1⁺) split into NK T cells (CD45⁺NK1.1⁺CD3⁺) and NK cells (CD45⁺NK1.1⁺CD3^-) as percent of total immune cells. (F) Non-TAM myeloid cells (CD45⁺CD11b⁺F480^-) were determined as percent of total immune cells. MDSCs were separated into mMDSC (CD45⁺CD11b⁺F4/80^-Ly6G^-Ly6C⁺) or gMDSC (CD45⁺CD11b⁺F4/80^-Ly6G⁺Ly6C⁺) populations as percent of non-TAM myeloid cells. Significance was calculated using the Mann-Whitney nonparametric test with Benjamini-Hochberg correction for multiple comparisons. P adjusted < .05 statistically significant.

Figure 5

Single-cell RNA transcriptomic analysis identifies altered MDSC gene expression in Gnat3 ablated neoplasia. Analysis of tamoxifen and cerulein treated KC^ERT and Gnat3^-/-;KC^ERT single-cell RNA transcriptomes from pancreata collected 6 weeks after cerulein treatment. (A) Unbiased clustering of single cells driven by transcriptome differences and visualized by UMAP (n = 2; 2). (B) Dot plot of gene expression patterns used to identify cell populations from pooled KC^ERT and Gnat3^-/-;KC^ERT cells. Percent of cells expressing each gene per cluster noted by dot size. Average gene expression is represented by color of dot. iCAF, inflammatory cancer associated fibroblasts; myCAF, myofibroblastic cancer associated fibroblasts; T-reg, T regulatory cells. (C and D) Heatmap of single-cell RNA transcriptomes for TAMs (C) and MDSCs (D) displaying the statistically significant differentially expressed genes (rows). Selected genes identified on right of MDSC heatmap. (E) Violin plots displaying frequency and expression levels of selected genes from cells in the MDSC cluster. Genes labeled by group: C-type lectin receptors (Clec12a, Clec4a2), neutrophil granule proteins (Camp, Ngp, Ltf), antimicrobial proteins (Pglyrp1, Lyz2), and secreted proteins (Il1b, Serpinb1a, Anxa1, Anxa3). Significance was calculated using Bonferroni adjusted P values from nonparametric Wilcoxon rank sum test. P adjusted < .05 statistically significant.

Figure 6

Gnat3 ablation increases CXCL1 and CXCL2 expression in the neoplastic epithelial compartment. Analysis of tamoxifen and cerulein treated KC^ERT and Gnat3^-/-;KC^ERT pancreata collected 6 weeks after cerulein treatment. (A) UMAP of Cxcr2 single-cell RNA transcriptome expression from KC^ERT and Gnat3^-/-;KC^ERT 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 KC^ERT and Gnat3^-/-;KC^ERT 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.

Figure 7

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 KC^ERT;ROSA26^LSL-EYFP and Gnat3^-/-;KC^ERT;ROSA26^LSL-EYFP pancreata collected 6 weeks after cerulein treatment. In KC^ERT;ROSA26^LSL-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^-/-;KC^ERT;ROSA26^LSL-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.

Figure 8

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.