Downregulation of 5-hydroxymethylcytosine is an early event in pancreatic tumorigenesis.

Epigenetic alterations are increasingly recognized as important contributors to the development and progression of pancreatic ductal adenocarcinoma. 5-hydroxymethylcytosine (5hmC) is an epigenetic DNA mark generated through the ten-eleven translocation (TET) enzyme-mediated pathway and is closely linked to gene activation. However, the timing of alterations in epigenetic regulation in the progression of pancreatic neoplasia is not well understood. In this study, we hypothesized that aberrant expression of ten-eleven translocation methylcytosine dioxygenase 1 (TET1) and subsequent global 5hmC alteration are linked to early tumorigenesis in the pancreas. Therefore, we evaluated alterations of 5hmC and TET1 levels using immunohistochemistry in pancreatic neoplasms (n = 380) and normal ducts (n = 118). The study cohort included representation of the full spectrum of precancerous lesions from low- and high-grade pancreatic intraepithelial neoplasia (n = 95), intraductal papillary mucinous neoplasms (all subtypes, n = 129), intraductal oncocytic papillary neoplasms (n = 12), and mucinous cystic neoplasms (n = 144). 5hmC and TET1 were significantly downregulated in all types of precancerous lesion and associated invasive pancreatic ductal adenocarcinomas compared with normal ductal epithelium (all p < 0.001), and expression of 5hmC positively correlated with expression of TET1. Importantly, downregulation of both 5hmC and TET1 was observed in most low-grade precancerous lesions. There were no clear associations between 5hmC levels and clinicopathological factors, thereby suggesting a common epigenetic abnormality across precancerous lesions. We conclude that downregulation of 5hmC and TET1 is an early event in pancreatic tumorigenesis. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Molecular characterization of organoids derived from pancreatic intraductal papillary mucinous neoplasms.

Intraductal papillary mucinous neoplasms (IPMNs) are commonly identified non-invasive cyst-forming pancreatic neoplasms with the potential to progress into invasive pancreatic adenocarcinoma. There are few in vitro models with which to study the biology of IPMNs and their progression to invasive carcinoma. Therefore, we generated a living biobank of organoids from seven normal pancreatic ducts and ten IPMNs. We characterized eight IPMN organoid samples using whole genome sequencing and characterized five IPMN organoids and seven normal pancreatic duct organoids using transcriptome sequencing. We identified an average of 11,344 somatic mutations in the genomes of organoids derived from IPMNs, with one sample harboring 61,537 somatic mutations enriched for T→C transitions and T→A transversions. Recurrent coding somatic mutations were identified in 15 genes, including KRAS, GNAS, RNF43, PHF3, and RBM10. The most frequently mutated genes were KRAS, GNAS, and RNF43, with somatic mutations identified in six (75%), four (50%), and three (37.5%) IPMN organoid samples, respectively. On average, we identified 36 structural variants in IPMN derived organoids, and none had an unstable phenotype (> 200 structural variants). Transcriptome sequencing identified 28 genes differentially expressed between normal pancreatic duct organoid and IPMN organoid samples. The most significantly upregulated and downregulated genes were CLDN18 and FOXA1. Immunohistochemical analysis of FOXA1 expression in 112 IPMNs, 113 mucinous cystic neoplasms, and 145 pancreatic ductal adenocarcinomas demonstrated statistically significant loss of expression in low-grade IPMNs (p < 0.0016), mucinous cystic neoplasms (p < 0.0001), and pancreatic ductal adenocarcinoma of any histologic grade (p < 0.0001) compared to normal pancreatic ducts. These data indicate that FOXA1 loss of expression occurs early in pancreatic tumorigenesis. Our study highlights the utility of organoid culture to study the genetics and biology of normal pancreatic duct and IPMNs. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Generation and characterization of a cell line from an intraductal tubulopapillary neoplasm of the pancreas.

Intraductal tubulopapillary neoplasm (ITPN) is a distinct precancerous lesion in the pancreas with unique clinical and molecular features. Although in vitro studies in two-dimensional culture have led to numerous important insights in pancreatic cancer, such models are currently lacking for precancerous lesions. In this study, we report the generation and characterization of a cell line from a human pancreatic ITPN. Neoplastic cells were initially cultured in a three-dimensional organoid system, followed by transfer to two-dimensional culture. RNA sequencing revealed a gene expression profile consistent with pancreatic ductal origin, and whole genome sequencing identified many somatic mutations (including in genes involved in DNA repair and Wnt signaling) and structural rearrangements. In vitro characterization of the tumorigenic potential demonstrated a phenotype between that of normal pancreatic ductal cells and cancer cell lines. This cell line represents a valuable resource for interrogation of unique ITPN biology, as well as precancerous pancreatic lesions more generally.

Pattern of Invasion in Human Pancreatic Cancer Organoids Is Associated with Loss of SMAD4 and Clinical Outcome.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy characterized by extensive local invasion and systemic spread. In this study, we employed a three-dimensional organoid model of human pancreatic cancer to characterize the molecular alterations critical for invasion. Time-lapse microscopy was used to observe invasion in organoids from 25 surgically resected human PDAC samples in collagen I. Subsequent lentiviral modification and small-molecule inhibitors were used to investigate the molecular programs underlying invasion in PDAC organoids. When cultured in collagen I, PDAC organoids exhibited two distinct, morphologically defined invasive phenotypes, mesenchymal and collective. Each individual PDAC gave rise to organoids with a predominant phenotype, and PDAC that generated organoids with predominantly mesenchymal invasion showed a worse prognosis. Collective invasion predominated in organoids from cancers with somatic mutations in the driver gene SMAD4 (or its signaling partner TGFBR2). Reexpression of SMAD4 abrogated the collective invasion phenotype in SMAD4-mutant PDAC organoids, indicating that SMAD4 loss is required for collective invasion in PDAC organoids. Surprisingly, invasion in passaged SMAD4-mutant PDAC organoids required exogenous TGFß, suggesting that invasion in SMAD4-mutant organoids is mediated through noncanonical TGFß signaling. The Rho-like GTPases RAC1 and CDC42 acted as potential mediators of TGFß-stimulated invasion in SMAD4-mutant PDAC organoids, as inhibition of these GTPases suppressed collective invasion in our model. These data suggest that PDAC utilizes different invasion programs depending on SMAD4 status, with collective invasion uniquely present in PDAC with SMAD4 loss. SIGNIFICANCE: Organoid models of PDAC highlight the importance of SMAD4 loss in invasion, demonstrating that invasion programs in SMAD4-mutant and SMAD4 wild-type tumors are different in both morphology and molecular mechanism.

Kinetic assay of starvation sensitivity in yeast autophagy mutants allows for the identification of intermediary phenotypes.

OBJECTIVE: A classical method to quantitatively determine the starvation sensitivity phenotype of autophagy mutant budding yeast strains is to starve them for a period of time and then to assess the proportion of cells that retain the ability to form colonies when the availability of nutrients is restored. The readout of this colony-formation assay is generally evaluated after a fixed period of time following the restoration of nutrients, so that it can be considered an endpoint assay. One drawback we have identified is the inability to characterize subtle intermediary phenotypes that are detectable at the molecular level but fail to reach statistical significance in the colony formation experiment. We set out to determine whether a more dynamic measurement of growth during recovery after starvation would increase the sensitivity with which we are able to detect partial loss-of-function phenotypes. RESULTS: We describe a 96-well plate-based assay to kinetically assess starvation sensitivity in budding yeast that allows for the quantitative detection of very modest starvation sensitivity phenotypes with statistical significance in autophagy mutant yeast strains lacking the ATG27 gene.

Biphenotypic Differentiation of Pancreatic Cancer in 3-Dimensional Culture.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) is the third most common cause of cancer death in the United States. Improved characterized models of PDAC are needed for drug screening. METHODS: We grew 4 established pancreatic cancer cell lines in hanging drop cultures to produce spheroids. We also grew organoids from explanted xenografted PDAC and surgically resected primary PDAC. We performed transmission and scanning electron microscopy and compared findings with those of the normal pancreatic duct. We also performed single-cell cloning to determine the potential options for differentiation. RESULTS: Spheroids contained tight junctions and desmosomes but lacked zymogen granules, as expected. The former features were present in normal pancreatic duct but absent from PDAC cell lines grown in standard 2-dimensional culture. Spheroids functionally excluded macromolecules in whole mounts. Cells on the surface of PDAC spheroids were carpeted by microvilli except for rare cells with prominent stereocilia. Carpets of microvilli were also seen in low passage organoids produced from xenografts and surgically resected human PDAC, in addition to normal human pancreatic duct. We performed single-cell cloning and resulting spheroids produced both cell phenotypes at the same approximate ratios as those from bulk cultures. CONCLUSIONS: Pancreatic cancer spheroids/organoids are capable of biphenotypic differentiation.

Identification of novel cyclooxygenase-2 selective inhibitors using pharmacophore models.

In the present study we have investigated whether pharmacophore models may account for the activity and selectivity of the known cyclooxygenase-2 (COX-2) selective inhibitors of the phenylsulfonyl tricyclic series, i.e., Celecoxib (1) and Rofecoxib (3), and whether transferring this structural information onto the frame of a nonsteroidal antiinflammatory drug (NSAID), known to tightly bind the enzyme active site, may be useful for designing novel COX-2 selective inhibitors. With this aim we have developed a pharmacophore based on the geometric disposition of chemical features in the most favorable conformation of the COX-2 selective inhibitors SC-558 (2; analogue of Celecoxib (1)) and Rofecoxib (3) and the more restrained compounds 4 (DFU) and 5. The pharmacophore model contains a sulfonyl S atom, an aromatic ring (ring plane A) with a fixed position of the normal to the plane, and an additional aromatic ring (ring plane B), both rings forming a dihedral angle of 290 degrees +/- 10 degrees. The final disposition of the pharmacophoric groups parallels the geometry of the ligand SC-558 (2) in the known crystal structure of the COX-2 complex. Moreover, the nonconserved residue 523 is known to be important for COX-2 selective inhibition; thus, the crystallographic information was used to position an excluded volume in the pharmacophore, accounting for the space limits imposed by this nonconserved residue. The geometry of the final five-feature pharmacophore was found to be consistent with the crystal structure of the nonselective NSAID indomethacin (6) in the COX-2 complex. This result was used to design indomethacin analogues 8 and 9 that exhibited consistent structure-activity relationships leading to the potent and selective COX-2 inhibitor 8a. Compound 8a (LM-1685) was selected as a promising candidate for further pharmacological evaluation.

Enhanced mineralization of diuron using a cyclodextrin-based bioremediation technology.

The phenylurea herbicide diuron [N-(3,4-dichlorophenyl)-N,N-dimethylurea] is widely used in a broad range of herbicide formulations and, consequently, it is frequently detected as a major soil and water contaminant in areas where there is extensive use. Diuron has the unfortunate combination of being strongly adsorbed by soil organic matter particles and, hence, slowly degraded in the environment due to its reduced bioavailability. N-Phenylurea herbicides seem to be biodegraded in soil, but it must be kept in mind that this biotic or abiotic degradation could lead to accumulation of very toxic derived compounds, such as 3,4-dichloroaniline. Research was conducted to find procedures that might result in an increase in the bioavailability of diuron in contaminated soils, through solubility enhancement. For this purpose a double system composed of hydroxypropyl-ß-cyclodextrin (HPBCD), which is capable of forming inclusion complexes in solution, and a two-member bacterial consortium formed by the diuron-degrading Arthrobacter sulfonivorans (Arthrobacter sp. N2) and the linuron-degrading Variovorax soli (Variovorax sp. SRS16) was used. This consortium can achieve a complete biodegradation of diuron to CO2 with regard to that observed in the absence of the CD solution, where only a 45% biodegradation was observed. The cyclodextrin-based bioremediation technology here described shows for the first time an almost complete mineralization of diuron in a soil system, in contrast to previous incomplete mineralization based on single or consortium bacterial degradation.

Detección de la Enterotoxina del Clostridium Perfringens tipo A, en muestras de heces de lechones lactantes con diarrea provenientes de granjas porcinas del estado Aragua

En lechones lactantes, las diarreas de origen bacteriano son las mas comunes. Una de ellas es la causada por C. perfringens tipo A, que a pesar de ser parte de la flora intestinal normal, se ha reportado como agente causante de enteritis a partir de la primera semana de vida. El objetivo de este estudio fue detectar la presencia de la enterotoxina de C. perfringens tipo A en lechones lactantes con diarrea, en edades comprendidas entre 1 y 15 días. La investigación se llevó a cabo en 20 granjas porcinas ubicadas en los municipios José Félix Ribas y Revenga del estado Aragua, que poseen una alta densidad de granjas porcinas. Los lechones fueron seleccionados mediante un muestreo al azar simple. Se obtuvo un total de 20 muestras, correspondientes a 20 granjas, las cuales fueron procesadas para la detección de la enterotoxina de C. perfringens tipo A, aplicando la prueba serológica de Aglutinación Pasiva Reversa en Látex (RPLA), resultando 6 granjas positivas (30%), lo que confirma la presencia de la enterotoxina de C. perfringens tipo A, asociado a diarreas en lechones lactantes entre 1 y 15 días de edad en granjas porcinas del estado Aragua.

Detection of Clostridium perfringens Enterotoxine Type A in Samples of Nursing Suckling Pigs with diarrhea from Porcine Farms of Aragua State Abstract Diarrheas of bacterial origin are the most common diarrheas in suckling pigs, especially those caused by C. perfringens Type A, which is a natural inhabitant of their normal intestinal flora. The C. perfringens Type A has been reported as a causative agent of enteritis from the first week of life. The purpose of this study was to detect the presence of C. perfringens enterotoxin Type A in suckling pigs with diarrhea from 1 to 15 days of age. The study was carried out in 20 porcine farms, located at José Félix Ribas and Revenga municipalities of Aragua State, Venezuela, which have a high density of porcine farms. Suckling pigs were selected by means of a random sampling. A total of 20 stool samples from 20 farms was obtained. The detection of C. perfringens enterotoxin Type A was made using the reverse passive latex agglutination test (RPLA). Results of the present study show that 6 farms (30 %) were detected positive, which confirms the presence of C. perfringens enterotoxin Type A, in association with diarrheas in suckling pigs between 1 to 15 days of age in porcine farms of Aragua State.