Tuft cells transdifferentiate to neural-like progenitor cells in the progression of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDA) is partly initiated through the transdifferentiation of acinar cells to metaplastic ducts that act as precursors of neoplasia and cancer. Tuft cells are solitary chemosensory cells not found in the normal pancreas but arise in metaplasia and neoplasia, diminishing as neoplastic lesions progress to carcinoma. Metaplastic tuft cells (mTCs) function to suppress tumor progression through communication with the tumor microenvironment, but their fate during progression is unknown. To determine the fate of mTCs during PDA progression, we have created a lineage tracing model that uses a tamoxifen-inducible tuft-cell specific Pou2f3CreERT/+ driver to induce transgene expression, including the lineage tracer tdTomato or the oncogene Myc. mTC lineage trace models of pancreatic neoplasia and carcinoma were used to follow mTC fate. We found that mTCs, in the carcinoma model, transdifferentiate into neural-like progenitor cells (NRPs), a cell type associated with poor survival in PDA patients. Using conditional knock-out and overexpression systems, we found that Myc activity in mTCs is necessary and sufficient to induce this Tuft-to-Neuroendocrine-Transition (TNT).

ROR2 regulates cellular plasticity in pancreatic neoplasia and adenocarcinoma.

Cellular plasticity is a hallmark of pancreatic ductal adenocarcinoma (PDAC) starting from the conversion of normal cells into precancerous lesions to the progression of carcinoma subtypes associated with aggressiveness and therapeutic response. We discovered that normal acinar cell differentiation, maintained by the transcription factor Pdx1, suppresses a broad gastric cell identity that is maintained in metaplasia, neoplasia, and the classical subtype of PDAC in mouse and human. We have identified the receptor tyrosine kinase Ror2 as marker of a gastric metaplasia (SPEM)-like identity in the pancreas. Ablation of Ror2 in a mouse model of pancreatic tumorigenesis promoted a switch to a gastric pit cell identity that largely persisted through progression to the classical subtype of PDAC. In both human and mouse pancreatic cancer, ROR2 activity continued to antagonize the gastric pit cell identity, strongly promoting an epithelial to mesenchymal transition, conferring resistance to KRAS inhibition, and vulnerability to AKT inhibition.

Arginase 1 is a key driver of immune suppression in pancreatic cancer.

An extensive fibroinflammatory stroma rich in macrophages is a hallmark of pancreatic cancer. In this disease, it is well appreciated that macrophages are immunosuppressive and contribute to the poor response to immunotherapy; however, the mechanisms of immune suppression are complex and not fully understood. Immunosuppressive macrophages are classically defined by the expression of the enzyme Arginase 1 (ARG1), which we demonstrated is potently expressed in pancreatic tumor-associated macrophages from both human patients and mouse models. While routinely used as a polarization marker, ARG1 also catabolizes arginine, an amino acid required for T cell activation and proliferation. To investigate this metabolic function, we used a genetic and a pharmacologic approach to target Arg1 in pancreatic cancer. Genetic inactivation of Arg1 in macrophages, using a dual recombinase genetically engineered mouse model of pancreatic cancer, delayed formation of invasive disease, while increasing CD8+ T cell infiltration. Additionally, Arg1 deletion induced compensatory mechanisms, including Arg1 overexpression in epithelial cells, namely Tuft cells, and Arg2 overexpression in a subset of macrophages. To overcome these compensatory mechanisms, we used a pharmacological approach to inhibit arginase. Treatment of established tumors with the arginase inhibitor CB-1158 exhibited further increased CD8+ T cell infiltration, beyond that seen with the macrophage-specific knockout, and sensitized the tumors to anti-PD1 immune checkpoint blockade. Our data demonstrate that Arg1 drives immune suppression in pancreatic cancer by depleting arginine and inhibiting T cell activation.

Combinatorial Gli activity directs immune infiltration and tumor growth in pancreatic cancer.

Proper Hedgehog (HH) signaling is essential for embryonic development, while aberrant HH signaling drives pediatric and adult cancers. HH signaling is frequently dysregulated in pancreatic cancer, yet its role remains controversial, with both tumor-promoting and tumor-restraining functions reported. Notably, the GLI family of HH transcription factors (GLI1, GLI2, GLI3), remain largely unexplored in pancreatic cancer. We therefore investigated the individual and combined contributions of GLI1-3 to pancreatic cancer progression. At pre-cancerous stages, fibroblast-specific Gli2/Gli3 deletion decreases immunosuppressive macrophage infiltration and promotes T cell infiltration. Strikingly, combined loss of Gli1/Gli2/Gli3 promotes macrophage infiltration, indicating that subtle changes in Gli expression differentially regulate immune infiltration. In invasive tumors, Gli2/Gli3 KO fibroblasts exclude immunosuppressive myeloid cells and suppress tumor growth by recruiting natural killer cells. Finally, we demonstrate that fibroblasts directly regulate macrophage and T cell migration through the expression of Gli-dependent cytokines. Thus, the coordinated activity of GLI1-3 directs the fibroinflammatory response throughout pancreatic cancer progression.

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

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 KrasLSL-G12D/+ allele with either Ptf1aCre/+ (KC) or tamoxifen-inducible Ptf1aCreERT/+ (KCERT) 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 KCERT and Gnat3-/-;KCERT 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-/-;KCERT 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-/-;KCERT 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.