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Extracellular vesicles (EVs) are lipid bound vesicles secreted by cells into the extracellular environment. Studies have implicated EVs in cell proliferation, epithelial-mesenchymal transition, metastasis, angiogenesis, and mediating the interaction of tumor cells and microenvironment. A systematic characterization of EVs from pancreatic cancer cells and cancer-associated fibroblasts (CAFs) would be valuable for studying the roles of EV proteins in pancreatic tumorigenesis.Proteomic and functional analyses were applied to characterize the proteomes of EVs released from 5 pancreatic cancer lines, 2 CAF cell lines, and a normal pancreatic epithelial cell line (HPDE).More than 1400 nonredundant proteins were identified in each EV derived from the cell lines. The majority of the proteins identified in the EVs from the cancer cells, CAFs, and HPDE were detected in all 3 groups, highly enriched in the biological processes of vesicle-mediated transport and exocytosis. Protein networks relevant to pancreatic tumorigenesis, including epithelial-mesenchymal transition, complement, and coagulation components, were significantly enriched in the EVs from cancer cells or CAFs.These findings support the roles of EVs as a potential mediator in transmitting epithelial-mesenchymal transition signals and complement response in the tumor microenvironment and possibly contributing to coagulation defects related to cancer development.

Cognitive psychology considers the environment as providing information, not affecting fundamental information processes. Thus, cognitive psychology’s traditional paradigms study responses to precisely timed stimuli in controlled environments. However, new research demonstrates the environment does influence cognitive processes and offers cognitive psychology new methods. The authors examine one such proposal: cognitive ethology. Cognitive ethology improves cognitive psychology’s ecological validity through first drawing inspiration from robust phenomena in the real world, then moving into the lab to test those phenomena. To support such methods, cognitive ethologists appeal to embodied cognition, or 4E cognition, for its rich relationships between agents and environments. However, the authors note while cognitive ethology focuses on new methods (epistemology) inspired by embodied cognition, it preserves most traditional assumptions about cognitive processes (ontology). But embodied cognition—particularly its radical variants—also provides strong ontological challenges to cognitive psychology, which work against cognitive ethology. The authors argue cognitive ethology should align with the ontology of less radical embodied cognition, which produces epistemological implications, offering alternative methodologies. For example, cognitive ethology can explore differences between real-world and lab studies to fully understand how cognition depends on environments.

Global efforts to upgrade water, drainage, and sanitation services are hampered by hydrometeorological data-scarcity plus uncertainty about climate change. Intensity–duration–frequency (IDF) tables are used routinely to design water infrastructure so offer an entry point for adapting engineering standards. This paper begins with a novel procedure for guiding downscaling predictor variable selection for heavy rainfall simulation using media reports of pluvial flooding. We then present a three-step workflow to: (1) spatially downscale daily rainfall from grid-to-point resolutions; (2) temporally scale from daily series to sub-daily extreme rainfalls and; (3) test methods of temporal scaling of extreme rainfalls within Regional Climate Model (RCM) simulations under changed climate conditions. Critically, we compare the methods of moments and of parameters for temporal scaling annual maximum series of daily rainfall into sub-daily extreme rainfalls, whilst accounting for rainfall intermittency. The methods are applied to Kampala, Uganda and Kisumu, Kenya using the Statistical Downscaling Model (SDSM), two RCM simulations covering East Africa (CP4 and P25), and in hybrid form (RCM-SDSM). We demonstrate that Gumbel parameters (and IDF tables) can be reliably scaled to durations of 3 h within observations and RCMs. Our hybrid RCM-SDSM scaling reduces errors in IDF estimates for the present climate when compared with direct RCM output. Credible parameter scaling relationships are also found within RCM simulations under changed climate conditions. We then discuss the practical aspects of applying such workflows to other city-regions.

Background: Transcriptional remodeling is known to contribute to heart failure (HF). Targeting stress-dependent gene expression mechanisms may represent a clinically relevant gene therapy option. We recently uncovered a salutary mechanism in the heart whereby JP2 (junctophilin-2), an essential component of the excitation-contraction coupling apparatus, is site-specifically cleaved and releases an N-terminal fragment (JP2NT [N-terminal fragment of JP2]) that translocates into the nucleus and functions as a transcriptional repressor of HF-related genes. This study aims to determine whether JP2NT can be leveraged by gene therapy techniques for attenuating HF progression in a preclinical pressure overload model. Methods: We intraventricularly injected adeno-associated virus (AAV) (2/9) vectors expressing eGFP (enhanced green fluorescent protein), JP2NT, or DNA-binding deficient JP2NT (JP2NT ΔbNLS/ARR ) into neonatal mice and induced cardiac stress by transaortic constriction (TAC) 9 weeks later. We also treated mice with established moderate HF from TAC stress with either AAV-JP2NT or AAV-eGFP. RNA-sequencing analysis was used to reveal changes in hypertrophic and HF-related gene transcription by JP2NT gene therapy after TAC. Echocardiography, confocal imaging, and histology were performed to evaluate heart function and pathological myocardial remodeling following stress. Results: Mice preinjected with AAV-JP2NT exhibited ameliorated cardiac remodeling following TAC. The JP2NT DNA-binding domain is required for cardioprotection as its deletion within the AAV-JP2NT vector prevented improvement in TAC-induced cardiac dysfunction. Functional and histological data suggest that JP2NT gene therapy after the onset of cardiac dysfunction is effective at slowing the progression of HF. RNA-sequencing analysis further revealed a broad reversal of hypertrophic and HF-related gene transcription by JP2NT overexpression after TAC. Conclusions: Our prevention- and intervention-based approaches here demonstrated that AAV-mediated delivery of JP2NT into the myocardium can attenuate stress-induced transcriptional remodeling and the development of HF when administered either before or after cardiac stress initiation. Our data indicate that JP2NT gene therapy holds great potential as a novel therapeutic for treating hypertrophy and HF.

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Supplementary Methods

Supplementary Figure S3 shows analysis for microvascularization (A), alcian blue staining of tissue sections (B), and ICAM-1 expression in human samples (C).

Figures S1-4 and Table S1 Figure S1. N-CAFs and R-CAFs were exposed to the indicated concentrations of gemcitabine for 72h, and viability was measured via MTT assay. 3 biologic replicates were included in the experiment. **p{less than or equal to}0.01, **** p{less than or equal to}0.0001 Figure S2. MiaPaCa-2 cells (1 X 105) were subcutaneously injected with N-CAF cells expressing GFP (3 X 105) in NSG mice. At 6 weeks, tumors were harvested and fluorescence microscopy was performed. Representative images of H&E stained sections as well as fluorescence microscopy are displayed. Only occasional GFP+ CAFs were present after 6 weeks. Figure S3. Primary CAFs were treated with 1µM gemcitabine plus DMSO or the above listed small molecule inhibitors for 96h and SASP mediator expression was compared to untreated cells via qRT-PCR. Because of limited primary cell numbers, the experiment was performed once in multiple cell lines rather than being replicated in any single line. Drug concentrations and targets: SB203580 10µM- p38 MAPK, SP600125 20µM- JNK, BEZ235 1µM- ATM/ATR/DNA-PK/PI3K/mTOR, AZD77622 200nM- Chk1/Chk2, VE821 2µM- ATR, KU55933 20µM- ATM. Figure S4. Combined data from Figure S3. The mean normalized expression of each marker from CAF1-3 is displayed. In this combined analysis, none of the changes reached statistical significance.

Pancreatic ductal adenocarcinoma (PDAC) has a characteristically dense stroma comprised predominantly of cancer-associated fibroblasts (CAF). CAFs promote tumor growth, metastasis, and treatment resistance. This study aimed to investigate the molecular changes and functional consequences associated with chemotherapy treatment of PDAC CAFs. Chemoresistant immortalized CAFs (R-CAF) were generated by continuous incubation in gemcitabine. Gene expression differences between treatment-naïve CAFs (N-CAF) and R-CAFs were compared by array analysis. Functionally, tumor cells (TC) were exposed to N-CAF– or R-CAF–conditioned media and assayed for migration, invasion, and viability in vitro. Furthermore, a coinjection (TC and CAF) model was used to compare tumor growth in vivo. R-CAFs increased TC viability, migration, and invasion compared with N-CAFs. In vivo, TCs coinjected with R-CAFs grew larger than those accompanied by N-CAFs. Genomic analysis demonstrated that R-CAFs had increased expression of various inflammatory mediators, similar to the previously described senescence-associated secretory phenotype (SASP). In addition, SASP mediators were found to be upregulated in response to short duration treatment with gemcitabine in both immortalized and primary CAFs. Inhibition of stress-associated MAPK signaling (P38 MAPK or JNK) attenuated SASP induction as well as the tumor-supportive functions of chemotherapy-treated CAFs in vitro and in vivo. These results identify a negative consequence of chemotherapy on the PDAC microenvironment that could be targeted to improve the efficacy of current therapeutic regimens.Implications: Chemotherapy treatment of pancreatic cancer–associated fibroblasts results in a proinflammatory response driven by stress-associated MAPK signaling that enhances tumor cell growth and invasiveness. Mol Cancer Res; 14(5); 437–47. ©2016 AACR.

Supplemental Figure 1. Calcipotriol does not inhibit Wnt signaling through VDR sequestration of β-catenin or induction of DKK1. (A) AsPC-1 cells were treated with 100 nM calcipotriol for 4 hours. Whole cell lysates were collected and immunoprecipitated with anti- β-catenin or isotype control antibodies followed by western blots for β-catenin, E-cadherin or VDR. (B) Western blot for DKK-1 and tubulin (loading control) and (C) BAR reporter activity for indicated conditions. AsPC-1 cells were transfected with control or DKK1 siRNA for 48 hours and treated with vehicle or 100 nM calcipotriol for an additional 24 hours. BAR was normalized to vehicle and siRNA control and is reported as mean {plus minus} SD, with one representative experiment of 3 biological repeats shown.

Supplemental Figure 3. Calcipotriol reduces VDR message while paradoxically increasing VDR protein levels. (A) VDR expression in AsPC-1 by qPCR after treatments described in Figure 3E. (B) Western blots for VDR or tubulin (loading control) for AsPC-1 lysates at indicated timepoints after calcipotriol treatment. Arrowhead indicates specific band representing VDR protein. qPCR reported as mean {plus minus} SEM. Representative experiments from 2-3 biologic replicates are shown. **P

Supplemental Table 1. QPCR PRIMERS

Supplementary Methods

Supplemental Figure 5. Gene ontology terms enriched in gene expression microarray analysis following β-catenin knockdown.

Supplemental Figure 2. Calcipotriol does not alter LRP6 expression in Low-BAR/Low-VDR cell lines. (A) Western blot for LRP6 or tubulin (loading control) following treatment with vehicle or 10 μM calcipotriol. Representative experiments from 2-3 biologic replicates are shown.

Supplemental Table 2. RNA-Seq analysis showing significant gene expression changes following 6 hour calcipotriol treatment of AsPC-1 cell line

Desmoplasia and an inflammatory environment are defining features of pancreatic cancer. Unclear is how pancreatic cells that undergo oncogenic transformation can cross-talk with immune cells and how this contributes to the development of pancreatic lesions. Here, we demonstrate that pancreatic acinar cells expressing mutant KRAS can expedite their transformation to a duct-like phenotype by inducing local inflammation. Specifically, we show that KRASG12D induces the expression of intercellular adhesion molecule-1 (ICAM-1), which serves as chemoattractant for macrophages. Infiltrating macrophages amplify the formation of KRASG12D-caused abnormal pancreatic structures by remodeling the extracellular matrix and providing cytokines such as TNF. Depletion of macrophages or treatment with a neutralizing antibody for ICAM-1 in mice expressing oncogenic Kras under an acinar cell–specific promoter resulted in both a decreased formation of abnormal structures and decreased progression of acinar-to-ductal metaplasia to pancreatic intraepithelial neoplastic lesions.Significance: We here show that oncogenic KRAS in pancreatic acinar cells upregulates the expression of ICAM-1 to attract macrophages. Hence, our results reveal a direct cooperative mechanism between oncogenic Kras mutations and the inflammatory environment to drive the initiation of pancreatic cancer. Cancer Discov; 5(1); 52–63. ©2014 AACR.This article is highlighted in the In This Issue feature, p. 1

Supplementary Figure from Porcupine Inhibition Disrupts Mitochondrial Function and Homeostasis in WNT Ligand–Addicted Pancreatic Cancer

High pressure (20 MPa), cyclic, supercritical carbon dioxide (scCO2) treatments can reduce the moisture content of green Pinus radiata sapwood from 150–200% to 35–40%. Such treatments can be used as a dewatering pre-treatment before the kiln-drying of timber. Kiln-drying can utilise various temperature and humidity schedules, targeting around 10% moisture content, with a final stress-relieving steam-conditioning step. After scCO2 treatment and kiln-drying of samples, kiln brown stain was evaluated using the CIE L*a*b* colour space while drying stress was assessed by stress-cup measurements. The most significant results of scCO2 pre-treatment of Pinus radiata sapwood followed by kiln-drying plus steam-conditioning were as follows: Drying from green (36 h from a moisture content (MC) of 164%) using a conventional temperature schedule (90 °C/60 °C) took 2–5 times longer than kiln-drying scCO2 pre-treated boards (37.5% MC) to a target of 10% MC. Colour measurements proved that kiln brown stain does not occur. The use of a steam-conditioning step in reducing internal drying stresses was important irrespective of whether or not there was a scCO2 pre-treatment step. Over all drying schedule combinations, internal drying stress of both green and scCO2 pre-treated timber was similar after kiln-drying plus steam-conditioning. However, using only 90 °C/60 °C schedule data, with steam-conditioning, drying stresses were lower using kiln-drying without the scCO2 pre-treatment. This was surprising since the scCO2 step reduced the moisture content to around 37.5% without significant moisture gradients and so a secondary kiln-drying to 10% moisture content could have been expected to yield lower internal stress levels by preventing large moisture gradients to develop during drying. This result confirms the efficacy of the steam-conditioning step following standard kiln-drying. The colour data demonstrating the prevention of kiln brown stain using kiln-drying schedules offers a path to increasing timber quality for interior applications.

Figure S6. Global TGFBR-Deficiency does not Alter Non-Peripheral Tregs

Purpose:Prostate-specific membrane antigen (PSMA) targeting radioligands deliver radiation to PSMA-expressing cells. However, the relationship between PSMA levels and intralesion heterogeneity of PSMA expression, and cytotoxic radiation by radioligand therapy (RLT) is unknown. Here we investigate RLT efficacy as function of PSMA levels/cell, and the fraction of PSMA+ cells in a tumor.Experimental Design:RM1 cells expressing different levels of PSMA (PSMA−, PSMA+, PSMA++, PSMA+++; study 1) or a mix of PSMA+ and PSMA− RM1 (study 2, 4) or PC-3/PC-3-PIP (study 3) cells at various ratios were injected into mice. Mice received 177Lu- (studies 1–3) or 225Ac- (study 4) PSMA617. Tumor growth was monitored. Two days post-RLT, tumors were resected in a subset of mice. Radioligand uptake and DNA damage were quantified.Results:177Lu-PSMA617 efficacy increased with increasing PSMA levels (study 1) and fractions of PSMA positive cells (studies 2, 3) in both, the RM1 and PC-3-PIP models. In tumors resected 2 days post-RLT, PSMA expression correlated with 177Lu-PSMA617 uptake and the degree of DNA damage. Compared with 177Lu-PSMA617, 225Ac-PSMA617 improved overall antitumor effectiveness and tended to enhance the differences in therapeutic efficacy between experimental groups.Conclusions:In the current models, both the degree of PSMA expression and the fraction of PSMA+ cells correlate with 177Lu-/225Ac-PSMA617 tumor uptake and DNA damage, and thus, RLT efficacy. Low or heterogeneous PSMA expression represents a resistance mechanism to RLT.See related commentary by Ravi Kumar and Hofman, p. 2774

Supplemental Methods and Legend

Purpose: To carry out an integrative profile of human pancreatic ductal adenocarcinoma (PDAC) to identify prognosis-significant genes and their related pathways.Experimental Design: A concordant survival-based whole genome in silico array analysis of DNA copy number, and mRNA and miRNA expression in 25 early-stage PDAC was carried out. A novel composite score simultaneously integrated gene expression with regulatory mechanisms to identify the signature genes with the most levels of prognosis-significant evidence. The predominant signaling pathways were determined via a pathway-based approach. Independent patient cohorts (n = 148 and 42) were then used as in vitro validation of the array findings.Results: The composite score identified 171 genes in which expressions were able to define two prognosis subgroups (P = 3.8e-5). Eighty-eight percent (151 of 171) of the genes were regulated by prognosis-significant miRNAs. The phosphoinositide 3-kinase/AKT pathway and SRC signaling were densely populated by prognosis-significant genes and driven by genomic amplification of SRC and miRNA regulation of p85α and CBL. On tissue microarray validation (n = 148), p85α protein expression was associated with improved survival for all patients (P = 0.02), and activated P-SRC (Y418) was associated shorter survival for patients with low-grade histology tumors (P = 0.04). Interacting P-SRC and p85α revealed that they define two distinct PDAC patient subgroups (P = 0.0066). Furthering the importance of these pathways, CBL protein expression was associated with improved survival (P = 0.03) on a separate cohort (n = 42).Conclusions: These pathways and related genes may represent putative clinical biomarkers and possible targets of individualized therapy in the distinct patient subgroups they define. Clin Cancer Res; 18(5); 1352–63. ©2012 AACR.

In early pancreatic carcinogenesis, TGFβ acts as a tumor suppressor due to its growth-inhibitory effects in epithelial cells. However, in advanced disease, TGFβ appears to promote tumor progression. Therefore, to better understand the contributions of TGFβ signaling to pancreatic carcinogenesis, we generated mouse models of pancreatic cancer with either epithelial or systemic TGFBR deficiency. We found that epithelial suppression of TGFβ signals facilitated pancreatic tumorigenesis, whereas global loss of TGFβ signaling protected against tumor development via inhibition of tumor-associated fibrosis, stromal TGFβ1 production, and the resultant restoration of antitumor immune function. Similarly, TGFBR-deficient T cells resisted TGFβ-induced inactivation ex vivo, and adoptive transfer of TGFBR-deficient CD8+ T cells led to enhanced infiltration and granzyme B–mediated destruction of developing tumors. These findings paralleled our observations in human patients, where TGFβ expression correlated with increased fibrosis and associated negatively with expression of granzyme B. Collectively, our findings suggest that, despite opposing the proliferation of some epithelial cells, TGFβ may promote pancreatic cancer development by affecting stromal and hematopoietic cell function. Therefore, the use of TGFBR inhibition to target components of the tumor microenvironment warrants consideration as a potential therapy for pancreatic cancer, particularly in patients who have already lost tumor-suppressive TGFβ signals in the epithelium. Cancer Res; 76(9); 2525–39. ©2016 AACR.