Pancreatic stellate cells (PSCs) differentiate into cancer-associated fibroblasts (CAFs) that produce desmoplastic stroma, thereby modulating disease progression and therapeutic response in ...pancreatic ductal adenocarcinoma (PDA). However, it is unknown whether CAFs uniformly carry out these tasks or if subtypes of CAFs with distinct phenotypes in PDA exist. We identified a CAF subpopulation with elevated expression of α-smooth muscle actin (αSMA) located immediately adjacent to neoplastic cells in mouse and human PDA tissue. We recapitulated this finding in co-cultures of murine PSCs and PDA organoids, and demonstrated that organoid-activated CAFs produced desmoplastic stroma. The co-cultures showed cooperative interactions and revealed another distinct subpopulation of CAFs, located more distantly from neoplastic cells, which lacked elevated αSMA expression and instead secreted IL6 and additional inflammatory mediators. These findings were corroborated in mouse and human PDA tissue, providing direct evidence for CAF heterogeneity in PDA tumor biology with implications for disease etiology and therapeutic development.
Pancreatic cancer is one of the most lethal malignancies due to its late diagnosis and limited response to treatment. Tractable methods to identify and interrogate pathways involved in pancreatic ...tumorigenesis are urgently needed. We established organoid models from normal and neoplastic murine and human pancreas tissues. Pancreatic organoids can be rapidly generated from resected tumors and biopsies, survive cryopreservation, and exhibit ductal- and disease-stage-specific characteristics. Orthotopically transplanted neoplastic organoids recapitulate the full spectrum of tumor development by forming early-grade neoplasms that progress to locally invasive and metastatic carcinomas. Due to their ability to be genetically manipulated, organoids are a platform to probe genetic cooperation. Comprehensive transcriptional and proteomic analyses of murine pancreatic organoids revealed genes and pathways altered during disease progression. The confirmation of many of these protein changes in human tissues demonstrates that organoids are a facile model system to discover characteristics of this deadly malignancy.
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•Pancreas organoids can be derived from mouse and human normal and neoplastic tissue•Orthotopically transplanted organoids recapitulate the stages of PDA tumorigenesis•Organoids can be used to discover new mediators of PDA progression•Human neoplastic organoid cultures can be established from endoscopic biopsies
Organoid cultures are derived from normal and neoplastic murine and human pancreas tissues. Orthotopic transplantation of organoids and analyses of organoid transcriptomes and proteomes provide insights into the molecular bases of pancreatic tumorigenesis.
Despite recent advances in the treatment of cancer, pancreatic ductal adenocarcinoma (PDAC) still retains the worst survival rate of common malignancies. Late diagnosis and lack of curative ...therapeutic options are the most pressing clinical problems for this disease. Therefore, there is a need for patient models and biomarkers that can be applied in the clinic to identify the most effective therapy for a patient. Pancreatic ductal organoids are ex-vivo models of PDAC that can be established from very small biopsies, enabling the study of localized, advanced, and metastatic patients. Organoids models have been applied to pancreatic cancer research and offer a promising platform for precision medicine approaches.
Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis, and new therapies are needed. Altered metabolism is a cancer vulnerability, and several metabolic pathways have been shown to promote ...PDAC. However, the changes in cholesterol metabolism and their role during PDAC progression remain largely unknown. Here we used organoid and mouse models to determine the drivers of altered cholesterol metabolism in PDAC and the consequences of its disruption on tumor progression. We identified sterol O-acyltransferase 1 (SOAT1) as a key player in sustaining the mevalonate pathway by converting cholesterol to inert cholesterol esters, thereby preventing the negative feedback elicited by unesterified cholesterol. Genetic targeting of Soat1 impairs cell proliferation in vitro and tumor progression in vivo and reveals a mevalonate pathway dependency in p53 mutant PDAC cells that have undergone p53 loss of heterozygosity (LOH). In contrast, pancreatic organoids lacking p53 mutation and p53 LOH are insensitive to SOAT1 loss, indicating a potential therapeutic window for inhibiting SOAT1 in PDAC.
The regular arrangements of β-strands around a central axis in β-barrels and of α-helices in coiled coils contrast with the irregular tertiary structures of most globular proteins, and have ...fascinated structural biologists since they were first discovered. Simple parametric models have been used to design a wide range of α-helical coiled-coil structures, but to date there has been no success with β-barrels. Here we show that accurate de novo design of β-barrels requires considerable symmetry-breaking to achieve continuous hydrogen-bond connectivity and eliminate backbone strain. We then build ensembles of β-barrel backbone models with cavity shapes that match the fluorogenic compound DFHBI, and use a hierarchical grid-based search method to simultaneously optimize the rigid-body placement of DFHBI in these cavities and the identities of the surrounding amino acids to achieve high shape and chemical complementarity. The designs have high structural accuracy and bind and fluorescently activate DFHBI in vitro and in Escherichia coli, yeast and mammalian cells. This de novo design of small-molecule binding activity, using backbones custom-built to bind the ligand, should enable the design of increasingly sophisticated ligand-binding proteins, sensors and catalysts that are not limited by the backbone geometries available in known protein structures.
Histone covalent modifications regulate many, if not all, DNA-templated processes, including gene expression and DNA damage response. The biological consequences of histone modifications are mediated ...partially by evolutionarily conserved "reader/effector" modules that bind to histone marks in a modification- and context-specific fashion and subsequently enact chromatin changes or recruit other proteins to do so. Recently, the Plant Homeodomain (PHD) finger has emerged as a class of specialized "reader" modules that, in some instances, recognize the methylation status of histone lysine residues, such as histone H3 lysine 4 (H3K4). While mutations in catalytic enzymes that mediate the addition or removal of histone modifications (i.e., "writers" and "erasers") are already known to be involved in various human diseases, mutations in the modification-specific "reader" proteins are only beginning to be recognized as contributing to human diseases. For instance, point mutations, deletions or chromosomal translocations that target PHD fingers encoded by many genes (such as recombination activating gene 2 (RAG2), Inhibitor of Growth (ING), nuclear receptor-binding SET domain-containing 1 (NSD1) and Alpha Thalassaemia and Mental Retardation Syndrome, X-linked (ATRX)) have been associated with a wide range of human pathologies including immunological disorders, cancers, and neurological diseases. In this review, we will discuss the structural features of PHD fingers as well as the diseases for which direct mutation or dysregulation of the PHD finger has been reported. We propose that misinterpretation of the epigenetic marks may serve as a general mechanism for human diseases of this category. Determining the regulatory roles of histone covalent modifications in the context of human disease will allow for a more thorough understanding of normal and pathological development, and may provide innovative therapeutic strategies wherein "chromatin readers" stand as potential drug targets.
The recent development of human organoids as patient-specific models of pancreatic ductal adenocarcinoma (PDA) has helped set the stage for a new era of personalized medicine. Organoids can be ...generated from a resected PDA tumor in as little as 2-4 weeks, and are amenable to therapeutic screening as well as genetic and biochemical perturbation. Moreover, because these models promote the propagation of the neoplastic PDA cells at the expense of the stromal cells, transcriptome and genome-wide sequencing of organoids offers an unprecedented view of the genetic and expression changes occurring in the neoplastic cells of individual tumors. Here, we describe methods to generate PDA organoid cultures from resected human tumor specimens. We also describe how to propagate, cryopreserve, and thaw human PDA organoid cultures.
Objective
The relationship between adipose tissue fibrosis, adipocyte hypertrophy, and preadipocyte hyperplasia in the context of obesity and the correlation of these tissue‐based phenomena with ...systemic metabolic disease are poorly defined. The goal of this study was to clarify the relationship between adipose tissue fibrosis, adipocyte hypertrophy, and preadipocyte hyperplasia in human obesity and determine the correlation of these adipose‐tissue based phenomena with diabetes.
Methods
Visceral and subcutaneous adipose tissues from humans with obesity collected during bariatric surgery were studied with QRTPCR, immunohistochemistry, and flow cytometry for expression of collagens and fibrosis‐related proteins, adipocyte size, and preadipocyte frequency. Results were correlated with clinical characteristics including diabetes status.
Results
Fibrosis was decreased, hypertrophy was increased, and preadipocyte frequency and fibrotic gene expression were decreased in adipose tissues from diabetic subjects compared to non‐diabetic subjects. These differences were greater in visceral compared to subcutaneous adipose tissue.
Conclusions
These data are consistent with the hypothesis that adipose tissue fibrosis in the context of human obesity limits adipocyte hypertrophy and is associated with a reciprocal increase in adipocyte hyperplasia, with beneficial effects on systemic metabolism. These findings suggest adipose tissue fibrosis as a potential target for manipulation of adipocyte metabolism.
is mutated in the majority of pancreatic ductal adenocarcinoma. MAPK and PI3K-AKT are primary KRAS effector pathways, but combined MAPK and PI3K inhibition has not been demonstrated to be clinically ...effective to date. We explore the resistance mechanisms uniquely employed by malignant cells.
We evaluated the expression and activation of receptor tyrosine kinases in response to combined MEK and AKT inhibition in KPC mice and pancreatic ductal organoids. In addition, we sought to determine the therapeutic efficacy of targeting resistance pathways induced by MEK and AKT inhibition in order to identify malignant-specific vulnerabilities.
Combined MEK and AKT inhibition modestly extended the survival of KPC mice and increased Egfr and ErbB2 phosphorylation levels. Tumor organoids, but not their normal counterparts, exhibited elevated phosphorylation of ERBB2 and ERBB3 after MEK and AKT blockade. A pan-ERBB inhibitor synergized with MEK and AKT blockade in human PDA organoids, whereas this was not observed for the EGFR inhibitor erlotinib. Combined MEK and ERBB inhibitor treatment of human organoid orthotopic xenografts was sufficient to cause tumor regression in short-term intervention studies.
Analyses of normal and tumor pancreatic organoids revealed the importance of ERBB activation during MEK and AKT blockade primarily in the malignant cultures. The lack of ERBB hyperactivation in normal organoids suggests a larger therapeutic index. In our models, pan-ERBB inhibition was synergistic with dual inhibition of MEK and AKT, and the combination of a pan-ERBB inhibitor with MEK antagonists showed the highest activity both
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