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Background: Undifferentiated carcinoma (UC) is a rare subtype of pancreatic cancer distinguished from UC with osteoclast-like giant cells (UC-OGC) in 2019, affecting interpretation of literature that does not distinguish these subtypes. We sought to identify translationally relevant differences between these 2 variants and compared with pancreatic ductal adenocarcinoma., Methods: We characterized clinical and multiomic differences between UC (n = 32) and UC-OGC (n = 15) using DNA sequencing, RNA sequencing, and multiplex immunofluorescence and compared these findings with pancreatic ductal adenocarcinoma., Results: Characteristics at diagnosis were similar between UC and UC-OGC, though the latter was more resectable (P = .009). Across all stages, median overall survival was shorter for UC than for UC-OGC (0.4 years vs 10.8 years, respectively; P = .003). This shorter survival was retained after stratification by resection, albeit without statistical significance (1.8 years vs 11.9 years, respectively; P = .08). In a subset of patients with available tissue, the genomic landscape was similar among UC (n = 9), UC-OGC (n = 5), and pancreatic ductal adenocarcinoma (n = 159). Bulk RNA sequencing was deconvoluted and, along with multiplex immunofluorescence in UC (n = 13), UC-OGC (n = 5), and pancreatic ductal adenocarcinoma (n = 16), demonstrated statistically significantly increased antigen-presenting cells, including M2 macrophages and natural killer cells, and decreased cytotoxic and regulatory T cells in UC and UC-OGC vs pancreatic ductal adenocarcinoma. Findings were similar between UC and UC-OGC , except for decreased regulatory T cells in UC-OGC (P = .04)., Conclusions: In this series, UC was more aggressive than UC-OGC, with these variants having more antigen-presenting cells and fewer regulatory T cells than pancreatic ductal adenocarcinoma, suggesting potential for immune-modulating therapies in the treatment of these pancreatic cancer subtypes., (© The Author(s) 2024. Published by Oxford University Press.)

Pancreatic ductal adenocarcinoma has a unique tumor microbiome and the systemic depletion of bacteria or fungi using antibiotic/antifungal cocktails leads to a decrease in pancreatic tumor burden in mice. However, functional studies remain rare due to the limited availability of clinically relevant microbiota. Here, we describe in detail the isolation of bacteria and fungi from the small intestine and tumor of pancreatic cancer patients at the Rogel Cancer Center. We then further characterized the impact of a newly isolated Klebsiella oxytoca strain ( UMKO1 ) on the pancreatic tumor microenvironment using bacterial genome sequencing, untargeted and targeted metabolomics, as well as an ex vivo tumor transplant system. We found that UMKO1 possesses a gene for the long form of cytidine deaminase, which can inactivate the standard PDAC chemotherapeutic agent gemcitabine. In addition, we found that UMKO1 can produce several indoles when grown in tumor-like conditions, metabolites that can lead to an immune suppressive environment and interfere with therapy outcome. To test this in detail, we assessed changes in immune populations in pancreatic tumor explants upon exposure to the supernatant of UMKO1 and other isolated bacteria grown in tumor Interstitial fluid media (TIFM). We found that while none of the bacterial supernatants changed the abundance of CD8 T cells, granzyme B positive CD8 T cells were the lowest in tumor explants exposed to UMKO1 , and not other isolated Klebsiella species or the non-pathogenic laboratory strain E. coli K12 . In summary, the isolated collection of bacteria and fungi from this study are a valuable toolbox to study the impact of microbiota on pancreatic cancer.

Background & Aims: The fundamental biology of pancreatic ductal adenocarcinoma has been greatly impacted by the characterization of genetically engineered mouse models that allow temporal and spatial activation of oncogenic KRAS (KRAS G12D ). One of the most commonly used models involves targeted insertion of a cre-recombinase into the Ptf1a gene. However, this approach disrupts the Ptf1a gene, resulting in haploinsufficiency that likely affects sensitivity to oncogenic KRAS (KRAS G12D ). This study aims to determine if Ptf1a haploinsufficiency affected the acinar cell response to KRAS G12D before and after induction of pancreatic injury., Methods: We performed morphological and molecular analysis of 3 genetically engineered mouse models that express a tamoxifen-inducible cre-recombinase to activate Kras G12D in acinar cells of the pancreas. The cre-recombinase was targeted to the acinar-specific transcription factor genes, Ptf1a or Mist1/Bhlha15, or expressed within a BAC-derived Elastase transgene. Histological and RNA-seq analyses were used to delineate differences between the models., Results: Up to 2 months after tamoxifen induction of KRAS G12D , morphological changes were negligible. However, induction of pancreatic injury by cerulein resulted in widespread PanIN lesions in Ptf1a creERT pancreata within 7 days and maintained for at least 5 weeks post-injury, which was not seen in the models with 2 functional Ptf1a alleles. RNA-sequencing analysis prior to injury induction suggested Ptf1a creERT and Mist1 creERT mice have unique profiles of gene expression that predict a differential response to injury. Multiplex analysis of pancreatic tissue confirmed different inflammatory responses between the models., Conclusions: These findings suggest Ptf1a haploinsufficiency in Ptf1a creERT mouse models promotes KRAS G12D priming of genes for promotion of pancreatic ductal adenocarcinoma., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Trace metal contamination is a widespread issue due to its many natural and anthropogenic sources and known carcinogenic, teratogenic, and reproductive effects. As previous invertebrate trace metal research has primarily focused on model species (Daphnia magna, Chironomidae, etc.), our understanding of effects on non-model invertebrate species remains relatively poor. As such, this study assessed the exposure effects of cadmium, arsenic, and lead on viability, locomotor behavior, and embryonic development of the Seminole ramshorn snail (Planorbella duryi). Exposure treatments of CdCl 2 , Na 2 HAsO 4 • 7H 2 O, or Pb (NO 3 ) 2 were prepared at concentrations of 0, 0.01, 0.1, 1, and 10 mg/L and confirmed using inductively coupled plasma optical emission spectroscopy (ICP-OES). Individual adult P. duryi were exposed for 7 days with viability assessed every 24 h, and locomotor behavior was accessed on Days 1 and 7 using ToxTrac v2.97 automated behavior software. Individual embryos from newly laid (<6 h old) embryonic clutches were exposed for 10 days, during which embryonic development stage was documented every 24 h. Based on our results, an additional follow-up study for cadmium was conducted using a lower range of 0-0.1 mg/L to allow for the observation of sublethal endpoints. Adult lead and cadmium exposure resulted in significant mortality in the highest treatments (1 and 10 mg/L), dose-dependent behavioral effects, and delayed embryonic development. Arsenic exposures resulted in little to no impacts for all assessed endpoints. Our results provide new insight into the sublethal impacts of these contaminants and highlight potential for behavior and embryonic development as useful tools for risk assessment. PRACTITIONER POINTS: The exposure effects of lead, cadmium, and arsenic on the viability, embryonic development, and locomotor behavior of a common freshwater snail species was investigated using environmentally relevant concentrations. The severity of impact differed for each trace metal, with cadmium being the most toxic and arsenic the least toxic at concentrations ranging from 0 to 10 mg/L. Embryonic development appeared to be the most sensitive endpoint of those tested in this study, suggesting that exposure may have prolonged effects that extend to population and community levels. The Seminole ramshorn snail serves as a sensitive alternative model species that can be used to assess the impacts of contaminants on freshwater invertebrates in future studies., (© 2024 The Authors. Water Environment Research published by Wiley Periodicals LLC on behalf of Water Environment Federation.)

Coal ash (CA) is an industrial waste product that has been shown to contain several neurotoxic constituents such as cadmium, selenium, mercury, lead, and arsenic. Contaminant-laced leachates enter the environment via seepage, runoff, permitted discharge, or accidental spills from CA storage ponds or landfills which may pose a risk to wildlife residing in receiving waterways. In this study, we assessed 1) the presence and concentration of thirteen trace elements (Al, Ca, Mg, Cr, Cd, As, Se, Pb, Cu, Zn, Mn, Fe, B) in surface water and sediment grab samples using ICP-OES, 2) the temporal variability of trace elements using Pb-210 dated sediment core samples, 3) differences in species diversity using environmental DNA (eDNA) analyses, and 4) the presence and concentration of trace metals in banded killifish (Fundulus diaphanus) epaxial muscle tissue collected from waterways surrounding the Possum Point Power Station (Stafford, VA). Results showed the highest concentrations of As, Cd, Cr, Cu, Fe, Mg, Se, Zn, and B in Quantico Creek (QC) adjacent to the coal ash ponds and elevated average cadmium and zinc concentrations compared to both upstream and downstream locations along the Potomac River. Sediment core profiles and Pb-210 analyses showed historical enrichment of several trace elements in QC beginning after the commissioning of the power plant in 1948. When compared to upstream and downstream sites, species diversity was drastically reduced in Quantico Creek based on eDNA identification. Muscle tissues of banded killifish collected in Quantico Creek displayed increased Al, Cd, and Zn concentrations compared to upstream and downstream sites. Collectively, our results demonstrate the potential impacts of coal ash landfills on aquatic ecosystems and suggest that further research is needed to fully inform risk assessment and remediation efforts., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Coal fly ash is an industrial waste product generated by coal fired powerplants which has been shown to contain elevated concentrations of several toxic trace metals. When stored in landfills or other repositories, these trace metals can enter nearby surface waters via a number of routes including leaching or runoff. Our study examined 1) the presence and concentration of eleven trace elements in a range of lab-created coal ash leachate solutions at neutral pH using ICP-OES, 2) the physiological effects of these leachate solutions on a freshwater gastropod (Planorbella duryi), and 3) the ability of these trace metals to bioaccumulate in the tissues of exposed individuals. As, Cd, Cu, Mg, Mn, and Pb were detected in solutions at increasing concentrations concurrent with ash concentration. Exposure to leachates caused significant delays in embryonic development, reduced juvenile shell growth, decreases in egg and clutch production, and the display of avoidance behaviors. Tissues of exposed snails contained elevated concentrations of As, Cd, Cu, and Cr, with bioconcentration factors 177,550 times higher in cadmium and 85,468 times higher in arsenic in the highest treatment compared to control organisms. Our results highlight the potential harmful effects of coal ash leachates on a novel freshwater invertebrate species using several unique methodologies, providing key information regarding their potential impacts on surrounding aquatic ecosystems., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Tyler Edward Frankel reports financial support was provided by Morris Animal Foundation., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Early detection of Pancreatic Ductal Adenocarcinoma (PDAC), one of the most aggressive malignancies of the pancreas, is crucial to avoid metastatic spread to other body regions. Detection of pancreatic cancer is typically carried out by assessing the distribution and arrangement of tumor and immune cells in histology images. This is further complicated due to morphological similarities with chronic pancreatitis (CP), and the co-occurrence of precursor lesions in the same tissue. Most of the current automated methods for grading pancreatic cancers rely on extensive feature engineering involving accurate identification of cell features or utilising single number spatially informed indices for grading purposes. Moreover, sophisticated methods involving black-box approaches, such as neural networks, do not offer insights into the model's ability to accurately identify the correct disease grade. In this paper, we develop a novel cell-graph based Cell-Graph Attention (CGAT) network for the precise classification of pancreatic cancer and its precursors from multiplexed immunofluorescence histology images into the six different types of pancreatic diseases. The issue of class imbalance is addressed through bootstrapping multiple CGAT-nets, while the self-attention mechanism facilitates visualization of cell-cell features that are likely responsible for the predictive capabilities of the model. It is also shown that the model significantly outperforms the decision tree classifiers built using spatially informed metric, such as the Morisita-Horn (MH) indices., Competing Interests: AR has a consulting agreement with Voxel analytics LLC and consults for Genophyll, LLC. MB is currently employed with the Division of Data and Decision Sciences, Tata Consultancy Services, India. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The funders were not involved in the study design, collection, analysis, and interpretation of data, the writing of this article or the decision to submit it for publication., (Copyright © 2021 Baranwal, Krishnan, Oneka, Frankel and Rao.)

Purpose : Pancreatic ductal adenocarcinoma (PDAC) has the lowest five-year survival rate of all cancers in the United States. Programmed death 1 receptor (PD-1)-programmed death ligand 1 (PD-L1) immune checkpoint inhibition has been unsuccessful in clinical trials. Myeloid-derived suppressor cells (MDSCs) are known to block anti-tumor CD8+ T cell immune responses in various cancers including pancreas. This has led us to our objective that was to develop a clinically relevant in vitro organoid model to specifically target mechanisms that deplete MDSCs as a therapeutic strategy for PDAC. Method : Murine and human pancreatic ductal adenocarcinoma (PDAC) autologous organoid/immune cell co-cultures were used to test whether PDAC can be effectively treated with combinatorial therapy involving PD-1 inhibition and MDSC depletion. Results : Murine in vivo orthotopic and in vitro organoid/immune cell co-culture models demonstrated that polymorphonuclear (PMN)-MDSCs promoted tumor growth and suppressed cytotoxic T lymphocyte (CTL) proliferation, leading to diminished efficacy of checkpoint inhibition. Mouse- and human-derived organoid/immune cell co-cultures revealed that PD-L1-expressing organoids were unresponsive to nivolumab in vitro in the presence of PMN-MDSCs. Depletion of arginase 1-expressing PMN-MDSCs within these co-cultures rendered the organoids susceptible to anti-PD-1/PD-L1-induced cancer cell death. Conclusions : Here we use mouse- and human-derived autologous pancreatic cancer organoid/immune cell co-cultures to demonstrate that elevated infiltration of polymorphonuclear (PMN)-MDSCs within the PDAC tumor microenvironment inhibit T cell effector function, regardless of PD-1/PD-L1 inhibition. We present a pre-clinical model that may predict the efficacy of targeted therapies to improve the outcome of patients with this aggressive and otherwise unpredictable malignancy.

Pancreatic Cancer (PDA) is an aggressive malignancy characterized by early spread and a high mortality. Current studies suggest that a subpopulation of cells exist within tumors, cancer stem cell (CSC), which are capable of self-renewal and give rise to unique progeny which form the major neoplastic cellular component of tumors. While CSCs constitute a small cellular subpopulation within the tumor, their resistance to chemotherapy and radiation make them an important therapeutic target for eradication. Along with distinctive phenotypic properties, CSCs possess a unique metabolic plasticity allowing them to rapidly respond and adapt to environmental changes. These cells and their progeny also display a significantly altered epigenetic state with distinctive patterns of DNA methylation. Several mechanisms of cross-talk between epigenetic and metabolic pathways in PDA exist which ultimately contribute to the observed cellular plasticity and enhanced tumorigenesis. In this review we discuss various examples of this metabolic-epigenetic interplay and how it may constitute a new avenue for therapy specifically targeting CSCs in PDA., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Microenvironment evaluation of intact tissue for analysis of cell infiltration and spatial organization are essential in understanding the complexity of disease processes. The principle techniques used in the past include immunohistochemistry (IHC) and immunofluorescence (IF) which enable visualization of cells as a snapshot in time using between 1 and 4 markers. Both techniques have shortcomings including difficulty staining poorly antigenic targets and limitations related to cross-species reactivity. IHC is reliable and reproducible, but the nature of the chemistry and reliance on the visible light spectrum allows for only a few markers to be used and makes co-localization challenging. Use of IF broadens potential markers but typically relies on frozen tissue due to the extensive tissue autofluorescence following formalin fixation. Flow cytometry, a technique that enables simultaneous labeling of multiple epitopes, abrogates many of the deficiencies of IF and IHC, however, the need to examine cells as a single cell suspension loses the spatial context of cells discarding important biologic relationships. Multiplex fluorescent immunohistochemistry (mfIHC) bridges these technologies allowing for multi-epitope cellular phenotyping in formalin fixed paraffin embedded (FFPE) tissue while preserving the overall microenvironment architecture and spatial relationship of cells within intact undisrupted tissue. High fluorescent intensity fluorophores that covalently bond to the tissue epitope enables multiple applications of primary antibodies without worry of species specific cross-reactivity by secondary antibodies. Although this technology has been proven to produce reliable and accurate images for the study of disease, the process of creating a useful mfIHC staining strategy can be time consuming and exacting due to extensive optimization and design. In order to make robust images that represent accurate cellular interactions in-situ and to mitigate the optimization period for manual analysis, presented here are methods for slide preparation, optimizing antibodies, multiplex design as well as errors commonly encountered during the staining process.

Progestins are utilized as a component of human contraceptives, and commonly enter the environment via wastewater treatment plant effluent. Certain progestins activate fish androgen receptors and cause decreases in fecundity and masculinization of females. We used a nest acquisition assay and computer-assisted sperm analysis to examine the effects of levonorgestrel on male fathead minnow (Pimephales promelas) reproductive fitness. Males were exposed to 0, 10, or 100 ng/L levonorgestrel for 14 d. Combinations of a control male and a male from one of the treatments were placed into a competitive nesting assay, and the time each male spent holding the nest and time spent exhibiting aggressive behaviors were analyzed at 48 h postexposure. Semen samples were analyzed for total motility, straight-line velocity, curvilinear velocity, average path velocity, linearity, beat cross frequency, and wobble at 0, 30, 60, 90, and 120 s postactivation. Males exposed to either 10 or 100 ng/L of levonorgestrel exhibited increased nest acquisition success and lower levels of aggression compared with control-control pairings, as well as decreases in multiple sperm motion characteristics. Our results suggest that further research is required to ascertain the effects of levonorgestrel on male gamete quality and reproductive behaviors. Environ Toxicol Chem 2018;37:1131-1137. © 2017 SETAC., (© 2017 SETAC.)

The synthetic progestin levonorgestrel is commonly utilized in human oral contraceptives. It enters the environment as a component of wastewater treatment plant effluent, and has been measured at low ng/L concentrations in surface waters. It has been shown to activate fish androgen receptors, causing the physical masculinization of females, changes in reproductive behavior, and decreases in fecundity. In the present study, the effects of levonorgestrel exposure on early-stage oogenesis in the fathead minnow (Pimephales promelas) was examined. Adult females were exposed to 0, 10, or 100 ng/L levonorgestrel for 14 d using a flow-through exposure system. The ovaries from each female were then removed via dissection and weighed for gonadosomatic index (GSI) calculations, and oocytes from one lobe preserved in Serra's fixative. Total numbers of late-stage vitellogenic oocytes exhibiting a germinal vesicle were then quantified. In a second exposure, blood plasma samples were collected from adult females and analyzed for vitellogenin concentrations using enzyme-linked immunosorbent assay. Females exposed to both concentrations of levonorgestrel developed male secondary sexual characteristics in a dose-dependent manner, and ovaries contained significantly fewer late stage oocytes. Exposure to 100 ng/L of levonorgestrel resulted in decreased GSI and blood plasma vitellogenin concentrations. The results suggest that female exposure to levonorgestrel alone may have profound effects on reproduction in progestin-contaminated environments. Environ Toxicol Chem 2017;36:3299-3304. © 2017 SETAC., (© 2017 SETAC.)

The field of tumor immunology and immunotherapy has undergone a renaissance in the past decade do in large part to a better understanding of the tumor immune microenvironment. After suffering countless successes and setbacks in the twentieth century, immunotherapy has now come to the forefront of cancer research and is recognized as an important tool in the anti-tumor armamentarium. The goal of therapy is to aid the immune system in recognition and destruction of tumor cells by enhancing its ability to react to tumor antigens. This traditionally has been accomplished by induction of adaptive immunity through vaccination or through passive delivery of immunologic effectors as in the case of adoptive cell transfer. The recent discovery of immune "checkpoints" whose purpose is to suppress immune activity and prevent auto-immunity has created a new angle by which reactivity to tumors can be enhanced. Blockers of these checkpoints have yielded impressive clinical results and have recently been approved for use in a wide variety of malignancies. With data showing increasing rates of not only treatment response, but complete remissions, immunotherapy is poised to become an increasingly utilized therapy in the treatment of cancer.

Background and Objectives: Hirschsprung's disease (HD) must always be considered in very early-onset constipation. Although HD has a well-described clinical course, little is known about those neonates in whom HD was excluded. We aimed to describe the long-term clinical outcomes of neonates with a clinical suspicion of HD that was excluded by rectal suction biopsy., Methods: This is a single-center double-cohort comparative study. Neonates who underwent rectal mucosa biopsy for suspected HD were age and sex matched with healthy controls. A survey on clinical outcomes, stooling patterns, and other gastrointestinal (GI)-related conditions was sent to parents. Pathology slides were re-reported by an experienced histopathologist blinded to the clinical data., Results: A total of 51 neonates were included [25 cases, 26 controls; 41% males, median time of follow-up 4.25 years (interquartile range 2.7-6.9)]. Nine (36%) of patients in the case group required prolonged laxative use for constipation during the first year of life compared with 0 (0%) controls (P<0.001). This difference was maintained at the end of follow-up, with 5 (20%) versus 0 (0%), respectively (P=0.02). Case neonates were significantly more likely to be hospitalized or to be diagnosed with a chronic GI-related condition than the controls (33 vs. 12%, P=0.01; and 19 vs. 8%, P=0.04, respectively)., Conclusion: Neonatal constipation is associated with long-term GI-related disorders and should be considered clinically significant even when the diagnosis of HD is excluded. Neonates with early-onset abnormal stooling patterns should be monitored with adequate pediatrician or pediatric gastroenterologist follow-up.

Interleukin (IL)-22, a member of the IL-10 family, is a cytokine secreted by several types of immune cells including IL-22(+)CD4(+) T cells (Th22) and IL-22 expressing innate leukocytes (ILC22). Recent studies have demonstrated that IL-22 is a key component in mucosal barrier defense, tissue repair, epithelial cell survival, and proliferation. Furthermore, accumulating evidence has defined both protective and pathogenic properties of IL-22 in a number of conditions including autoimmune disease, infection, and malignancy. In this review, we summarize the expression and signaling pathway and functional characteristics of the IL-22 and IL-22 receptor axis in physiological and pathological scenarios and discuss the potential to target IL-22 signaling to treat human diseases.

Infiltration of tumors with effector T cells is positively associated with therapeutic efficacy and patient survival. However, the mechanisms underlying effector T-cell trafficking to the tumor microenvironment remain poorly understood in patients with colon cancer. The polycomb repressive complex 2 (PRC2) is involved in cancer progression, but the regulation of tumor immunity by epigenetic mechanisms has yet to be investigated. In this study, we examined the relationship between the repressive PRC2 machinery and effector T-cell trafficking. We found that PRC2 components and demethylase JMJD3-mediated histone H3 lysine 27 trimethylation (H3K27me3) repress the expression and subsequent production of Th1-type chemokines CXCL9 and CXCL10, mediators of effector T-cell trafficking. Moreover, the expression levels of PRC2 components, including EZH2, SUZ12, and EED, were inversely associated with those of CD4, CD8, and Th1-type chemokines in human colon cancer tissue, and this expression pattern was significantly associated with patient survival. Collectively, our findings reveal that PRC2-mediated epigenetic silencing is not only a crucial oncogenic mechanism, but also a key circuit controlling tumor immunosuppression. Therefore, targeting epigenetic programs may have significant implications for improving the efficacy of current cancer immunotherapies relying on effective T-cell-mediated immunity at the tumor site., (©2015 American Association for Cancer Research.)