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G protein α s (GNAS) mediates receptor-stimulated cAMP signalling, which integrates diverse environmental cues with intracellular responses. GNAS is mutationally activated in multiple tumour types, although its oncogenic mechanisms remain elusive. We explored this question in pancreatic tumourigenesis where concurrent GNAS and KRAS mutations characterize pancreatic ductal adenocarcinomas (PDAs) arising from intraductal papillary mucinous neoplasms (IPMNs). By developing genetically engineered mouse models, we show that Gnas R201C cooperates with Kras G12D to promote initiation of IPMN, which progress to invasive PDA following Tp53 loss. Mutant Gnas remains critical for tumour maintenance in vivo. This is driven by protein-kinase-A-mediated suppression of salt-inducible kinases (Sik1–3), associated with induction of lipid remodelling and fatty acid oxidation. Comparison of Kras -mutant pancreatic cancer cells with and without Gnas mutations reveals striking differences in the functions of this network. Thus, we uncover Gnas-driven oncogenic mechanisms, identify Siks as potent tumour suppressors, and demonstrate unanticipated metabolic heterogeneity among Kras -mutant pancreatic neoplasms. Bardeesy and colleagues show that mutant GNAS suppresses salt-inducible kinases by activating PKA, leading to lipid remodelling and pancreatic tumourigenesis

Pancreatic ductal adenocarcinoma (PDAC) lethality is due to metastatic dissemination. Characterization of rare, heterogeneous circulating tumor cells (CTCs) can provide insight into metastasis and guide development of novel therapies. Using the CTC-iChip to purify CTCs from PDAC patients for RNA-seq characterization, we identify three major correlated gene sets, with stemness genes LIN28B/KLF4 , WNT5A , and LGALS3 enriched in each correlated gene set; only LIN28B CTC expression was prognostic. CRISPR knockout of LIN28B —an oncofetal RNA-binding protein exerting diverse effects via negative regulation of let-7 miRNAs and other RNA targets—in cell and animal models confers a less aggressive/metastatic phenotype. This correlates with de-repression of let-7 miRNAs and is mimicked by silencing of downstream let-7 target HMGA2 or chemical inhibition of LIN28B/let-7 binding. Molecular characterization of CTCs provides a unique opportunity to correlated gene set metastatic profiles, identify drivers of dissemination, and develop therapies targeting the “seeds” of metastasis. Metastatic dissemination contributes to the lethality in pancreatic ductal adenocarcinoma (PDAC). Here, the authors perform RNA-sequencing on patient derived circulating tumor cells (CTCs) and identify three major CTC subgroups, and show the therapeutic potential of targeting LIN28B/let-7 pathway to halt cancer metastasis.

Increased use of cross-sectional imaging has resulted in frequent detection of incidental cystic pancreatic lesions. Serous cystadenomas (SCAs) are benign cysts that do not require surgical intervention unless symptomatic. Unfortunately, up to half of SCAs do not have typical imaging findings (“atypical SCAs”), overlap with potentially malignant precursor lesions, and thus pose a diagnostic challenge. We tested whether the analysis of circulating extracellular vesicle (EV) biomarkers using a digital EV screening technology (DEST) could enhance the discrimination of cystic pancreatic lesions and avoid unnecessary surgical intervention in these atypical SCAs. Analysis of 25 different protein biomarkers in plasma EV from 68 patients identified a putative biomarker signature of Das-1, Vimentin, Chromogranin A, and CAIX with high discriminatory power (AUC of 0.99). Analysis of plasma EV for multiplexed markers may thus be helpful in clinical decision-making.

Tolerance of mouse kidney allografts arises in grafts that develop regulatory tertiary lymphoid organs (rTLOs). Single-cell RNA-seq (scRNA-seq) data and adoptive transfer of alloreactive T cells after transplantation showed that cytotoxic CD8+ T cells are reprogrammed within the accepted graft to an exhausted/regulatory-like phenotype mediated by IFN-γ. Establishment of rTLOs was required because adoptive transfer of alloreactive T cells prior to transplantation results in kidney allograft rejection. Despite the presence of intragraft CD8+ cells with a regulatory phenotype, they were not essential for the induction and maintenance of kidney allograft tolerance since renal allotransplantation into CD8-KO recipients resulted in acceptance and not rejection. Analysis of scRNA-seq data from allograft kidneys and malignant tumors identified similar regulatory-like cell types within the T cell clusters and trajectory analysis showed that cytotoxic CD8+ T cells are reprogrammed into an exhausted/regulatory-like phenotype intratumorally. Induction of cytotoxic CD8+ T cell dysfunction of infiltrating cells appears to be a beneficial mechanistic pathway that protects the kidney allotransplant from rejection through a process we call \"defensive tolerance.\" This pathway has implications for our understanding of allotransplant tolerance and tumor resistance to host immunity.

The question of whether dedicated progenitor cells exist in adult vertebrate pancreas remains controversial. Centroacinar cells and terminal duct (CA/TD) cells lie at the junction between peripheral acinar cells and the adjacent ductal epithelium, and are frequently included among cell types proposed as candidate pancreatic progenitors. However these cells have not previously been isolated in a manner that allows formal assessment of their progenitor capacities. We have found that a subset of adult CA/TD cells are characterized by high levels of ALDH1 enzymatic activity, related to high-level expression of both Aldh1a1 and Aldh1a7. This allows their isolation by FACS using a fluorogenic ALDH1 substrate. FACS-isolated CA/TD cells are relatively depleted of transcripts associated with differentiated pancreatic cell types. In contrast, they are markedly enriched for transcripts encoding Sca1, Sdf1, c-Met, Nestin, and Sox9, markers previously associated with progenitor populations in embryonic pancreas and other tissues. FACS-sorted CA/TD cells are uniquely able to form self-renewing \"pancreatospheres\" in suspension culture, even when plated at clonal density. These spheres display a capacity for spontaneous endocrine and exocrine differentiation, as well as glucose-responsive insulin secretion. In addition, when injected into cultured embryonic dorsal pancreatic buds, these adult cells display a unique capacity to contribute to both the embryonic endocrine and exocrine lineages. Finally, these cells demonstrate dramatic expansion in the setting of chronic epithelial injury. These findings suggest that CA/TD cells are indeed capable of progenitor function and may contribute to the maintenance of tissue homeostasis in adult mouse pancreas.

Patient-derived xenograft (PDX) tumors are powerful tools to study cancer biology. However, the ability of PDX tumors to model the biological and histological diversity of pancreatic ductal adenocarcinoma (PDAC) is not well known. In this study, we subcutaneously implanted 133 primary and metastatic PDAC tumors into immunodeficient mice. Fifty-seven tumors were successfully engrafted and even after extensive passaging, the histology of poorly-, moderately-, and well-differentiated tumors was maintained in the PDX models. Moreover, the fibroblast and collagen contents in the stroma of patient tumors were recapitulated in the corresponding PDX models. Analysis of the clinicopathological features of patients revealed xenograft tumor engraftment was associated with lymphovascular invasion (P = 0.001) and worse recurrence-free (median, 7 vs. 16 months, log-rank P = 0.047) and overall survival (median, 13 vs. 21 months, log-rank P = 0.038). Among successful engraftments, median time of growth required for reimplantation into new mice was 151 days. Reflective of the inherent biological diversity between PDX tumors with rapid (<151 days) and slow growth, differences in their growth were maintained during extensive passaging. Rapid growth was additionally associated with lymph node metastasis (P = 0.022). The association of lymphovascular invasion and lymph node metastasis with PDX formation and rapid growth may reflect an underlying biological mechanism that allows these tumors to adapt and grow in a new environment. While the ability of PDX tumors to mimic the cellular and non-cellular features of the parental tumor stroma provides a valuable model to study the interaction of PDAC cells with the tumor microenvironment, the association of successful engraftment with adverse clinicopathological features suggests PDX models over represent more aggressive forms of this disease.

Pancreatic intraepithelial neoplasia (PanIN) and intraductal papillary mucinous neoplasms (IPMNs) are pancreatic ductal adenocarcinoma (PDAC) precursor lesions. Detecting these precursors and monitoring their progression are crucial for early PDAC diagnosis. Digital PCR (dPCR) is a highly sensitive nucleic acid quantification technique and offers a cost‐effective option for patient follow‐up. However, the clinical utility of conventional dPCR is restricted by multiplexing constraints, particularly due to the challenge of simultaneously quantifying multiple mutations and amplifications. In this study, we applied highly multiplexed dPCR and melting curve analysis to simultaneously measure single nucleotide mutations and amplifications of KRAS and GNAS. The developed 14‐plex assay included both wild‐type and mutant KRAS, a common driver gene in both PanIN and IPMN, and GNAS, which is specifically mutated in IPMN, along with RPP30, a reference gene for copy number alterations (CNAs). This multiplex dPCR method detected all target mutations with a limit of detection below 0.2% while quantifying CNAs. Additionally, the assay accurately quantified variant allele frequencies in liquid biopsy and tissue samples from both pancreatic neoplasm precursor and PDAC patients, indicating its potential for use in comprehensive patient follow‐up. Combining melting curve analysis enhances the multiplexing capability of digital PCR. Here, we developed a 14‐plex assay to simultaneously measure single nucleotide mutations and amplifications of KRAS and GNAS, which are common driver genes in pancreatic cancer precursors. This assay accurately quantified variant allele frequencies in clinical samples from both pancreatic neoplasm precursor and PDAC patients.

Background Patient-derived xenograft (PDX) mouse models of cancer have been recognized as better mouse models that recapitulate the characteristics of original malignancies including preserved tumor heterogeneity, lineage hierarchy, and tumor microenvironment. However, common challenges of PDX models are the significant time required for tumor expansion, reduced tumor take rates, and higher costs. Here, we describe a fast, simple, and cost-effective method of expanding PDX of pancreatic ductal adenocarcinoma (PDAC) in mice. Methods We used two established frozen PDAC PDX tissues (derived from two different patients) and implanted them subcutaneously into SCID mice. After tissues reached 10–20 mm in diameter, we performed survival surgery on each mouse to harvest 90–95% of subcutaneous PDX (incomplete resection), allowing the remaining 5–10% of PDX to continue growing in the same mouse. Results We expanded three consecutive passages (P1, P2, and P3) of PDX in the same mouse. Comparing the times required for in vivo expansion, P2 and P3 (expanded through incomplete resection) grew 26-60% faster than P1. Moreover, such expanded PDX tissues were successfully implanted orthotopically into mouse pancreases. Within 20 weeks using only 14 mice, we generated sufficient PDX tissue for future implantation of 200 mice. Our histology study confirmed that the morphologies of cancer cells and stromal structures were similar across all three passages of subcutaneous PDX and the orthotopic PDX and were reflective of the original patient tumors. Conclusions Taking advantage of incomplete resection of tumors associated with high local recurrence, we established a fast method of PDAC PDX expansion in mice.

Pancreatic ductal adenocarcinoma (PDAC) has an extraordinarily dense fibrotic stroma that impedes tumor perfusion and delivery of anticancer drugs. Since the extracellular matrix (ECM) comprises the bulk of the stroma, it is primarily responsible for the increased interstitial tissue pressure and stiff mechanical properties of the stroma. Besides its mechanical influence, the ECM provides important biochemical and physical cues that promote survival, proliferation, and metastasis. By serving as a nutritional source, the ECM also enables PDAC cells to survive under the nutrient-poor conditions. While therapeutic strategies using stroma-depleting drugs have yielded disappointing results, an increasing body of research indicates the ECM may offer a variety of potential therapeutic targets. As preclinical studies of ECM-targeted drugs have shown promising effects, a number of clinical trials are currently investigating agents with the potential to advance the future treatment of PDAC. Thus, the present review seeks to give an overview of the complex relationship between the ECM and PDAC.

Absorptive and secretory cells of the small intestine are derived from a single population of Lgr5-expressing stem cells. While key genetic pathways required for differentiation into specific lineages have been defined, epigenetic programs contributing to this process remain poorly characterized. Members of the BET family of chromatin adaptors contain tandem bromodomains that mediate binding to acetylated lysines on target proteins to regulate gene expression. In this study, we demonstrate that mice treated with a small molecule inhibitor of BET bromodomains, CPI203, exhibit greater than 90% decrease in tuft and enteroendocrine cells in both crypts and villi of the small intestine, with no changes observed in goblet or Paneth cells. BET bromodomain inhibition did not alter the abundance of Lgr5-expressing stem cells in crypts, but rather exerted its effects on intermediate progenitors, in part through regulation of Ngn3 expression. When BET bromodomain inhibition was combined with the chemotherapeutic gemcitabine, pervasive apoptosis was observed in intestinal crypts, revealing an important role for BET bromodomain activity in intestinal homeostasis. Pharmacological targeting of BET bromodomains defines a novel pathway required for tuft and enteroendocrine differentiation and provides an important tool to further dissect the progression from stem cell to terminally differentiated secretory cell.