Search

Numerous studies in hepatocellular carcinoma (HCC) have proposed tissue-based gene signatures for individualized prognostic assessments. Here, we develop a novel circulating tumor cell (CTC)–based transcriptomic profiling assay to translate tissue-based messenger RNA (mRNA) signatures into a liquid biopsy setting for noninvasive HCC prognostication. The HCC-CTC mRNA scoring system combines the NanoVelcro CTC Assay for enriching HCC CTCs and the NanoString nCounter platform for quantifying the HCC-CTC Risk Score (RS) panel in enriched HCC CTCs. The prognostic role of the HCC-CTC RS was assessed in The Cancer Genome Atlas (TCGA) HCC cohort (n = 362) and validated in an independent clinical CTC cohort (n = 40). The HCC-CTC RS panel was developed through our integrated data analysis framework of 8 HCC tissue-based gene signatures and identified the top 10 prognostic genes (discoidin domain receptor tyrosine kinase 1 [DDR1], enoyl-CoA hydratase and 3-hydroxyacyl CoA dehydrogenase [EHHADH], androgen receptor [AR], lumican [LUM], hydroxysteroid 17-beta dehydrogenase 6[HSD17B6], prostate transmembrane protein, androgen induced 1 [PMEPA1], tsukushi, small leucine rich proteoglycan [TSKU], N-terminal EF-hand calcium binding protein 2 [NECAB2], ladinin 1 [LAD1], solute carrier family 27 member 5 [SLC27A5]) highly expressed in HCC with low expressions in white blood cells. The panel accurately discriminated overall survival in TCGA HCC cohort (hazard ratio [HR], 2.0; 95% confidence interval [CI], 1.4-2.9). The combined use of the scoring system and HCC-CTC RS panel successfully distinguished artificial blood samples spiked with an aggressive HCC cell type, SNU-387, from those spiked with PLC/PRF/5 cells (P = 0.02). In the CTC validation cohort (n = 40), HCC-CTC RS remained an independent predictor of survival (HR, 5.7; 95% CI, 1.5-21.3; P = 0.009) after controlling for Model for End-Stage Liver Disease score, Barcelona Clinic Liver Cancer stage, and CTC enumeration count. Our study demonstrates a novel interdisciplinary approach to translate tissue-based gene signatures into a liquid biopsy setting. This noninvasive approach will allow real-time disease profiling and dynamic prognostication of HCC.

In this study, the physical states of cellulose in 1-allyl-3-methylimidazolium chloride (AmimCl) were analyzed via rheological observations under three different conditions: namely, steady state, small amplitude oscillatory shear, and large amplitude oscillatory shear conditions. The physical state of the solution changed from an isotropic phase to a gel structure via a liquid crystalline (LC) phase as the concentration increased. The rheological analysis showed that the 16 wt% solution was physically gelled in the anisotropic phase. This instantaneous gelation at the LC phase markedly enhanced the mechanical performance of the film obtained from the solution. This study provides a fundamental strategy for significant improvement in the mechanical properties of cellulose fibers or films.

Background Two prostate cancer (PC) classification methods based on transcriptome profiles, a de novo method referred to as the “Prostate Cancer Classification System” (PCS) and a variation of the established PAM50 breast cancer algorithm, were recently proposed. Both studies concluded that most human PC can be assigned to one of three tumor subtypes, two categorized as luminal and one as basal, suggesting the two methods reflect consistency in underlying biology. Despite the similarity, differences and commonalities between the two classification methods have not yet been reported. Methods Here, we describe a comparison of the PCS and PAM50 classification systems. PCS and PAM50 signatures consisting of 37 (PCS37) and 50 genes, respectively, were used to categorize 9,947 PC patients into PCS and PAM50 classes. Enrichment of hallmark gene sets and luminal and basal marker gene expression were assessed in the same datasets. Finally, survival analysis was performed to compare PCS and PAM50 subtypes in terms of clinical outcomes. Results PCS and PAM50 subtypes show clear differential expression of PCS37 and PAM50 genes. While only three genes are shared in common between the two systems, there is some consensus between three subtype pairs (PCS1 versus Luminal B, PCS2 versus Luminal A, and PCS3 versus Basal) with respect to gene expression, cellular processes, and clinical outcomes. PCS categories displayed better separation of cellular processes and luminal and basal marker gene expression compared to PAM50. Although both PCS1 and Luminal B tumors exhibited the worst clinical outcomes, outcomes between aggressive and less aggressive subtypes were better defined in the PCS system, based on larger hazard ratios observed. Conclusion The PCS and PAM50 classification systems are similar in terms of molecular profiles and clinical outcomes. However, the PCS system exhibits greater separation in multiple clinical outcomes and provides better separation of prostate luminal and basal characteristics.

We report a covalent chemistry-based hepatocellular carcinoma (HCC)-specific extracellular vesicle (EV) purification system for early detection of HCC by performing digital scoring on the purified EVs. Earlier detection of HCC creates more opportunities for curative therapeutic interventions. EVs are present in circulation at relatively early stages of disease, providing potential opportunities for HCC early detection. We develop an HCC EV purification system (i.e., EV Click Chips) by synergistically integrating covalent chemistry-mediated EV capture/release, multimarker antibody cocktails, nanostructured substrates, and microfluidic chaotic mixers. We then explore the translational potential of EV Click Chips using 158 plasma samples of HCC patients and control cohorts. The purified HCC EVs are subjected to reverse-transcription droplet digital PCR for quantification of 10 HCC-specific mRNA markers and computation of digital scoring. The HCC EV-derived molecular signatures exhibit great potential for noninvasive early detection of HCC from at-risk cirrhotic patients with an area under receiver operator characteristic curve of 0.93 (95% CI, 0.86 to 1.00; sensitivity = 94.4%, specificity = 88.5%)., Extracellular vesicles (EVs) are present in circulation at relatively early stages of disease, providing potential opportunities for early cancer diagnosis. Here, the authors report a covalent chemistry-based hepatocellular carcinoma (HCC)-specific EV purification system for early detection of HCC by performing digital scoring on the purified EVs.

Despite advances in therapy for melanoma, heterogeneous responses with limited durability represent a major gap in treatment outcomes. The purpose of this study was to determine whether alteration in tumor blood flow could augment drug delivery and improve antitumor responses in a regional model of melanoma. This approach to altering tumor blood flow was termed “dynamic control.” Dynamic control of tumor vessels in C57BL/6 mice bearing B16 melanoma was performed using volume expansion (saline bolus) followed by phenylephrine. Intravital microscopy (IVM) was used to observe changes directly in real time. Our approach restored blood flow in non-functional tumor vessels. It also resulted in increased chemotherapy (melphalan) activity, as measured by formation of DNA adducts. The combination of dynamic control and melphalan resulted in superior outcomes compared to melphalan alone (median time to event 40.0 vs 25.0 days, respectively, p = 0.041). Moreover, 25% (3/12) of the mice treated with the combination approach showed complete tumor response. Importantly, dynamic control plus melphalan did not result in increased adverse events. In summary, we showed that dynamic control was feasible, directly observable, and augmented antitumor responses in a regional model of melanoma. Early clinical trials to determine the translational feasibility of dynamic control are ongoing.

Background Woodchucks (Marmota monax) are a well-accepted animal model for the investigation of spontaneous hepatocellular carcinoma (HCC). As HCC tumors obtain nutrient blood supply exclusively from the hepatic artery, hepatic artery infusion (HAI) has been applied to HCC. However, there is a scarcity of experimental animal models to standardize drug regimens and examine novel agents. The purpose of this study was to establish an HAI model in woodchucks. Materials and methods HAI ports were placed in the gastroduodenal artery (GDA) of 11 woodchucks. The ports were infused with either a vehicle (dextrose 5% in water) or an experimental drug, CBL0137, once a week for 3 wk. Technical success rates, anatomical variation, morbidity and mortality, and tumor responses between groups were analyzed. Results The GDA access was feasible and reproducible in all woodchucks (11/11). The average operation time was 95 ± 20 min with no increase in the levels of liver enzymes detected from either infusate. The most common morbidity of CBL0137 therapy was anorexia after surgery. One woodchuck died due to hemorrhage at the gallbladder removal site from hepatic coagulopathy. Significantly higher CBL0137 concentrations were measured in the liver compared with blood after each HAI. Tumor growth was suppressed after multiple CBL0137 HAI treatments which corresponded to greater T cell infiltration and increased tumor cell apoptosis. Conclusions HAI via GDA was a feasible and reproducible approach with low morbidity and mortality in woodchucks. The described techniques serve as a reliable platform for the identification and characterization of therapeutics for HCC.

Simple Summary Extracellular vesicles (EVs) are heterogenous in size, cargo, and mechanism of biogenesis. While the mechanism of formation of small EVs, such as exosomes, has been widely investigated, little is known about the pathobiology of large EVs. We identify here a microRNA that alters cellular vesicologenesis increasing shedding of large EVs and slightly reducing shedding of small EVs. We also demonstrate that this microRNA, miR1227, targets SEC23A to promote this phenotype. Importantly, large EVs are released by cells undergoing a mesenchymal-amoeboid transition that functionally translates into a more metastatic phenotype. Abstract Cancer cells shed a heterogenous mixture of extracellular vesicles (EVs), differing in both size and composition, which likely influence physiological processes in different manners. However, how cells differentially control the shedding of these EV populations is poorly understood. Here, we show that miR-1227, which is enriched in prostate cancer EVs, compared to the cell of origin, but not in EVs derived from prostate benign epithelial cells, induces the shedding of large EVs (such as large oncosomes), while inhibiting the shedding of small EVs (such as exosomes). RNA sequencing from cells stably expressing miR-1227, a modified RISCTRAP assay that stabilizes and purifies mRNA-miR-1227 complexes for RNA sequencing, and in silico target prediction tools were used to identify miR-1227 targets that may mediate this alteration in EV shedding. The COPII vesicle protein SEC23A emerged and was validated by qPCR, WBlot, and luciferase assays as a direct target of miR-1227. The inhibition of SEC23A was sufficient to induce the shedding of large EVs. These results identify a novel mechanism of EV shedding, by which the inhibition of SEC23A by miR-1227 induces a shift in EV shedding, favoring the shedding of large EV over small EV.

Purpose: Pioglitazone, an antihyperglycemic drug, is widely used in diabetes mellitus patients with insulin resistance. Although pioglitazone is known to have a potential link to bladder cancer (BC), there have been contradictory results. This present study is designed to understand the regulatory mechanisms that drive the effects of pioglitazone on the bladder epithelial cells.Methods: Labeled liquid chromatography-tandem mass spectrometry-based proteomics profiling characterized the global proteomes of normal human bladder epithelial cells treated with or without pioglitazone.Results: This approach detected approximately 5,769 proteins in total. Of those 5,769 proteins, 124 were identified as being differentially expressed due to pioglitazone treatment. Further analysis identified 95 upregulated and 29 downregulated proteins (absolute log2 fold change >0.58 and P-value

Protein S-palmitoylation is a powerful post-translational modification that regulates protein trafficking, localization, turnover, and signal transduction. Palmitoylation controls several important cellular processes, and, if dysregulated, can lead to cancer, cardiovascular disease, and neurological disorders. The role of protein palmitoylation in mediating resistance to systemic cisplatin-based chemotherapies in cancer is currently unknown. This is of particular interest because cisplatin is currently the gold standard of treatment for bladder cancer (BC), and there are no feasible options after resistance is acquired. Using unbiased global proteomic profiling of purified S-palmitoylated peptides combined with intensive bioinformatics analyses, we identified 506 candidate palmitoylated proteins significantly enriched in cisplatin-resistant BC cells. One of these proteins included PD-L1, which is highly palmitoylated in resistant cells. Pharmacological inhibition of fatty acid synthase (FASN) suppressed PD-L1 palmitoylation and expression, which suggests the potential use of FASN-PD-L1-targeted therapeutic strategies in BC patients. Taken together, these results highlight the role of protein palmitoylation in mediating BC chemoresistance.

Stromal androgen-receptor (AR) action is essential for prostate development, morphogenesis and regeneration. However, mechanisms underlying how stromal AR maintains the cell niche in support of pubertal prostatic epithelial growth are unknown. Here, using advanced mouse genetic tools, we demonstrate that selective deletion of stromal AR expression in prepubescent Shh-responsive Gli1-expressing cells significantly impedes pubertal prostate epithelial growth and development. Single-cell transcriptomic analyses showed that AR loss in these prepubescent Gli1-expressing cells dysregulates androgen signaling-initiated stromal-epithelial paracrine interactions, leading to growth retardation of pubertal prostate epithelia and significant development defects. Specifically, AR loss elevates Shh-signaling activation in both prostatic stromal and adjacent epithelial cells, directly inhibiting prostatic epithelial growth. Single-cell trajectory analyses further identified aberrant differentiation fates of prostatic epithelial cells directly altered by stromal AR deletion. In vivo recombination of AR-deficient stromal Gli1-lineage cells with wild-type prostatic epithelial cells failed to develop normal prostatic epithelia. These data demonstrate previously unidentified mechanisms underlying how stromal AR-signaling facilitates Shh-mediated cell niches in pubertal prostatic epithelial growth and development.

Vascularized composite tissue allotransplantation (VCA) can replace severely damaged body parts but the unavoidable toxicity of high doses of immunosuppressive drugs, such as tacrolimus, required results in significant morbidity. Here we tested whether we could suppress immune activity in a mouse model of VCA by mimicking the natural immune suppression generated by nervous system-induced signaling of adrenergic receptors (AR) by using a safe and well-studied β-AR agonist (terbutaline). Using wild-type and β2-AR-knockout (KO) mice, we found that increased β2-AR signaling results in delayed rejection in VCA recipients, even with subtherapeutic doses of tacrolimus, and this was associated with changes in immune contexture, expression of pro-inflammatory cytokines and chemokines, and function of endothelial adhesion molecules. We propose that β-AR agonists can be used safely to mimic the natural suppression of immune responses generated by adrenergic stress signaling and thereby reduce the dose needed of other more toxic immunosuppressive drugs.

Background: Myocardial Gal3 (galectin-3) expression is associated with cardiac inflammation and fibrosis. Increased Gal3 portends susceptibility to heart failure and death. There are no data reporting the causative role of Gal3 to mediate cardiac fibro-inflammatory response and heart failure. Methods: We developed a cardioselective Gal3 gain-of-function mouse ( Gal3+/+ ) using α-myosin heavy chain promotor. We confirmed Gal3-transgene expression with real-time polymerase chain reaction and quantified cardiac/circulating Gal3 with Western blot and immunoassays. We used echocardiogram and cardiac magnetic resonance imaging to measure cardiac volumes, function, and myocardial velocities. Ex vivo, we studied myocardial inflammation/fibrosis and downstream TGF (transforming growth factor) β1-mRNA expression. We examined the effects of acute myocardial ischemia in presence of excess Gal3 by inducing acute myocardial infarction in mice. Two subsets of mice including mice treated with N-acetyl-seryl-aspartyl-lysyl-proline (a Gal3-inhibitor) and mice with genetic Gal3 loss-of-function ( Gal3 −/−) were studied for comparative analysis of Gal3 function. Results: Gal3+/+ mice had increased cardiac/circulating Gal3. Gal3+/+ mice showed excess pericardial fat pad, dilated ventricles and cardiac dysfunction, which was partly normalized by N-acetyl-seryl-aspartyl-lysyl-proline. Cardiac magnetic resonance imaging showed reduced myocardial contractile velocities in Gal3+/+ . The majority of Gal3+/+ mice did not survive acute myocardial infarction, and the survivors had profound cardiac dysfunction. Myocardial histology of Gal3+/+ mice showed macrophage/mast-cell infiltration, fibrosis and higher TGFβ1-mRNA expression, which were mitigated by both Gal3 gene deletion and N-acetyl-seryl-aspartyl-lysyl-proline administration. Conclusions: Our study shows that cardioselective Gal3 overexpression leads to multiple cardiac phenotypic defects including ventricular dilation and cardiac dysfunction. Pharmacological Gal3 inhibition conferred protective effects with reduction of inflammation and fibrosis. Our study highlights the importance of translational studies to counteract Gal3 function and prevent cardiac dysfunction.

Synucleinopathies are neurodegenerative disorders characterized by abnormal α-synuclein deposition that include Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. The pathology of these conditions also includes neuronal loss and neuroinflammation. Neuron-released α-synuclein has been shown to induce neurotoxic, proinflammatory microglial responses through Toll-like receptor 2, but the molecular mechanisms involved are poorly understood. Here, we show that leucine-rich repeat kinase 2 (LRRK2) plays a critical role in the activation of microglia by extracellular α-synuclein. Exposure to α-synuclein was found to enhance LRRK2 phosphorylation and activity in mouse primary microglia. Furthermore, genetic and pharmacological inhibition of LRRK2 markedly diminished α-synuclein-mediated microglial neurotoxicity via lowering of tumor necrosis factor-α and interleukin-6 expression in mouse cultures. We determined that LRRK2 promoted a neuroinflammatory cascade by selectively phosphorylating and inducing nuclear translocation of the immune transcription factor nuclear factor of activated T cells, cytoplasmic 2 (NFATc2). NFATc2 activation was seen in patients with synucleinopathies and in a mouse model of synucleinopathy, where administration of an LRRK2 pharmacological inhibitor restored motor behavioral deficits. Our results suggest that modulation of LRRK2 and its downstream signaling mediator NFATc2 might be therapeutic targets for treating synucleinopathies.

Rationale: Our objective was to develop a circulating tumor cell (CTC)-RNA assay for characterizing clinically relevant RNA signatures for the assessment of androgen receptor signaling inhibitor (ARSI) sensitivity in metastatic castration-resistant prostate cancer (mCRPC) patients. Methods: We developed the NanoVelcro CTC-RNA assay by combining the Thermoresponsive (TR)-NanoVelcro CTC purification system with the NanoString nCounter platform for cellular purification and RNA analysis. Based on the well-validated, tissue-based Prostate Cancer Classification System (PCS), we focus on the most aggressive and ARSI-resistant PCS subtype, i.e., PCS1, for CTC analysis. We applied a rigorous bioinformatic process to develop the CTC-PCS1 panel that consists of prostate cancer (PCa) CTC-specific RNA signature with minimal expression in background white blood cells (WBCs). We validated the NanoVelcro CTC-RNA assay and the CTC-PCS1 panel with well-characterized PCa cell lines to demonstrate the sensitivity and dynamic range of the assay, as well as the specificity of the PCS1 Z score (the likelihood estimate of the PCS1 subtype) for identifying PCS1 subtype and ARSI resistance. We then selected 31 blood samples from 23 PCa patients receiving ARSIs to test in our assay. The PCS1 Z scores of each sample were computed and compared with ARSI treatment sensitivity. Results: The validation studies using PCa cell line samples showed that the NanoVelcro CTC-RNA assay can detect the RNA transcripts in the CTC-PCS1 panel with high sensitivity and linearity in the dynamic range of 5-100 cells. We also showed that the genes in CTC-PCS1 panel are highly expressed in PCa cell lines and lowly expressed in background WBCs. Using the artificial CTC samples simulating the blood sample conditions, we further demonstrated that the CTC-PCS1 panel is highly specific in identifying PCS1-like samples, and the high PCS1 Z score is associated with ARSI resistance samples. In patient bloods, ARSI-resistant samples (ARSI-R, n=14) had significantly higher PCS1 Z scores as compared with ARSI-sensitive samples (ARSI-S, n=17) (Rank-sum test, P=0.003). In the analysis of 8 patients who were initially sensitive to ARSI (ARSI-S) and later developed resistance (ARSI-R), we found that the PCS1 Z score increased from the time of ARSI-S to the time of ARSI-R (Pairwise T-test, P=0.016). Conclusions: Using our new methodology, we developed a first-in-class CTC-RNA assay and demonstrated the feasibility of transforming clinically-relevant tissue-based RNA profiling such as PCS into CTC tests. This approach allows for detecting RNA expression relevant to clinical drug resistance in a non-invasive fashion, which can facilitate patient-specific treatment selection and early detection of drug resistance, a goal in precision oncology.

Supplemental Digital Content is available in the text., Adoptive cell transfer therapy for cancer has existed for decades and is experiencing a resurgence in popularity that has been facilitated by improved methods of production, techniques for genetic modification, and host preconditioning. The trafficking of adoptively transferred lymphocytes and infiltration into the tumor microenvironment is sine qua non for successful tumor eradication; however, the paradox of extremely poor trafficking of lymphocytes into the tumor microenvironment raises the issue of how best to deliver these cells to optimize entry into tumor tissue. We examined the route of administration as a potential modifier of both trafficking and antitumor efficacy. Femoral artery cannulation and tail vein injection for the intra-arterial (IA) and IV delivery, respectively, were utilized in the B16-OVA/OT-I mouse model system. Both IV and IA infusions showed decreased tumor growth and prolonged survival. However, although significantly increased T-cell tumor infiltration was observed in IA mice, tumor growth and survival were not improved as compared with IV mice. These studies suggest that IA administration produces increased early lymphocyte trafficking, but a discernable survival benefit was not seen in the murine model examined.

Alterations in DNA methylation are important epigenetic markers in bladder cancer (BC). These epigenome modifications may drive the mechanisms of aggressive chemo-resistant BC. Clinicopathological biomarkers that indicate chemotherapeutic resistance are critical for better assessing treatment strategies for individual patients. Thus, in this study, we aimed to determine whether DNA methylation of certain metabolic enzymes is significantly altered in cisplatin-resistant BC cells. Methods: To characterize CpG methylation and nucleosome accessibility in cisplatin-resistant BC cells, the Illumina Infinium HM450 DNA methylation assay was performed. Perturbed gene expression was found to be associated with cisplatin resistance, and the biological roles of spermidine/spermine N1-acetyltransferase (SAT1) and argininosuccinate synthase 1 (ASS1) were further studied using qRT-PCR analysis and various cell biology assays, including western blot. Results: ASS1 and SAT1, genes for amino acid and polyamine metabolism catalysts, respectively, were found to be vastly hypermethylated, resulting in greatly downregulated expression. ASS1 expression is of particular interest because prior studies have demonstrated its potential association with BC stage and recurrence. In regard to chemoresistance, we found that aberrant expression or induced stimulation of SAT1 restored cisplatin sensitivity in the cell culture system. We also found that the addition of exogenous arginine deiminase through administration of ADI-PEG 20 (pegylated arginine deiminase) increased ASS1 expression and enhanced cisplatin's apoptotic effects. Conclusions: Our study demonstrates a novel mechanistic link between the epigenetic perturbation of SAT1 and ASS1 and cancer metabolism in cisplatin-resistant bladder cancer cells. These findings suggest potential utility of SAT1 and ASS1 as predictive biomarkers in re-sensitizing bladder cancer to chemotherapy and personalizing therapy.

BACKGROUND: The liver has unique anatomy in that most blood flow to normal hepatocytes is derived from the portal venous system, while liver tumors obtain their nutrient blood supply exclusively from the hepatic artery. The focused arterial delivery of anticancer agents to liver tumors has been performed for decades; however, preclinical models to standardize drug regimens and examine novel agents have been lacking. The purpose of this study was to establish preclinical hepatic artery infusion (HAI) models in a mouse, and to evaluate the safety and delivery capability of the models. MATERIAL AND METHODS: Female C57BL/6 and BALB/c mice aged 8–12 wk were used to develop models of HAI via the hepatic artery (HA), superior pancreaticoduodenal artery (SPDA), or lienogastric artery (LGA). Success rates, distribution of perfusion, and associated morbidity and mortality were analyzed between groups. RESULTS: All three models were feasible and reproducible in mice, and there was no statistical difference on body weight change between models. The HA model had a 13.3% mortality from acute liver failure, and the SPDA model demonstrated significant duodenal and pancreatic toxicity. SPDA and LGA routes had the highest success rates (96.7% and 91.4%, respectively) with low mortality, better drug delivery, and preserved physiologic liver function compared to the HA model. CONCLUSIONS: The optimal route of HAI was mouse breed-specific; SPDA access in BALB/c mice, and the LGA access in C57BL/6 mice. The described techniques serve as a reproducible platform for the identification and characterization of therapeutics for diverse metastatic liver tumors.

Background Water lavage (WL) during gastrointestinal cancer surgery has osmotically mediated lytic effects on tumor cells. We investigated the safety and efficacy of WL with CRS-HIPEC. Methods This is a retrospective review, 1/2003-7/2014, of a single institution experience with CRS-HIPEC comparing patients who had WL (WL+) to those who did not (WL−). Results Of 157 CRS-HIPECs, 16 (10.2%) were WL+. WL+ had more PCI scores >20 compared to WL− (56.3% vs 19.4%, respectively, p = 0.003); however, the completeness of cytoreduction (CC) was similar. There were no differences in hospital length of stay or post-operative complications. The average POD 1 sodium (Na) level was statistically lower in the WL+ group (133.6 ± 2.5 vs 135.5 ± 3.2 mEq/L, p = 0.023); however, the average Na at discharge for each group was 140 mEq/L. There were no differences in 3-year OS (3WL+:0.63 vs WL−:0.68, p = 0.97) or RFS (WL+:0.32 vs WL−:0.39, p = 0.47). A subset analysis for patients with PCI >20 showed no difference between groups. Conclusions WL offers a low cost, safe and theoretically efficacious method of tumor cell lysis for peritoneal malignancy.

During roasting, major changes occur in the composition and physiological effects of coffee beans. In this study, in vitro antioxidant effects and anti-inflammatory effects of Coffea arabica green coffee extracts were investigated at different roasting levels corresponding to Light, Medium, City, and French roast. Total caffeine did not show huge difference according to roasting level, but total chlorogenic acid contents were higher in light roasted coffee extract than other roasted groups. In addition, light roasted coffee extract had the highest antioxidant activity in the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. To determine the in vitro antioxidant property, coffee extracts were used to treat AML-12 cells. Intracellular glutathione (GSH) concentration and mRNA expression levels of genes related to GSH synthesis were negatively related to roasting levels. The anti-inflammatory effects of coffee extracts were investigated in lipopolysaccharide-treated RAW 264.7 macrophage cells. The cellular antioxidant activity of coffee extracts exhibited similar patterns as the AML-12 cells. The expression of mRNA for tumor necrosis factor-alpha and interleukin-6 was decreased in cells treated with the coffee extracts and the expression decreased with increasing roasting levels. These data suggest that coffee has physiological antioxidant and anti-inflammatory activities and these effects are negatively correlated with roasting levels in the cell models.

Background Metastatic prostate cancer (PC) is highly lethal. The ability to identify primary tumors capable of dissemination is an unmet need in the quest to understand lethal biology and improve patient outcomes. Previous studies have linked chromosomal instability (CIN), which generates aneuploidy following chromosomal missegregation during mitosis, to PC progression. Evidence of CIN includes broad copy number alterations (CNAs) spanning > 300 base pairs of DNA, which may also be measured via RNA expression signatures associated with CNA frequency. Signatures of CIN in metastatic PC, however, have not been interrogated or well defined. We examined a published 70-gene CIN signature (CIN70) in untreated and castration-resistant prostate cancer (CRPC) cohorts from The Cancer Genome Atlas (TCGA) and previously published reports. We also performed transcriptome and CNA analysis in a unique cohort of untreated primary tumors collected from diagnostic prostate needle biopsies (PNBX) of localized (M0) and metastatic (M1) cases to determine if CIN was linked to clinical stage and outcome. Methods PNBX were collected from 99 patients treated in the VA Greater Los Angeles (GLA-VA) Healthcare System between 2000 and 2016. Total RNA was extracted from high-grade cancer areas in PNBX cores, followed by RNA sequencing and/or copy number analysis using OncoScan. Multivariate logistic regression analyses permitted calculation of odds ratios for CIN status (high versus low) in an expanded GLA-VA PNBX cohort (n = 121). Results The CIN70 signature was significantly enriched in primary tumors and CRPC metastases from M1 PC cases. An intersection of gene signatures comprised of differentially expressed genes (DEGs) generated through comparison of M1 versus M0 PNBX and primary CRPC tumors versus metastases revealed a 157-gene “metastasis” signature that was further distilled to 7-genes (PC-CIN) regulating centrosomes, chromosomal segregation, and mitotic spindle assembly. High PC-CIN scores correlated with CRPC, PC-death and all-cause mortality in the expanded GLA-VA PNBX cohort. Interestingly, approximately 1/3 of M1 PNBX cases exhibited low CIN, illuminating differential pathways of lethal PC progression. Conclusions Measuring CIN in PNBX by transcriptome profiling is feasible, and the PC-CIN signature may identify patients with a high risk of lethal progression at the time of diagnosis.

Gene expression profiling across various brain areas at the single-cell resolution enables the identification of molecular markers of neuronal subpopulations and comprehensive characterization of their functional roles. Despite the scientific importance and experimental versatility, systematic methods to analyze such data have not been established yet. To this end, we developed a statistical approach based on in situ hybridization data in the Allen Brain Atlas and thereby identified specific genes for each type of neuron in the ventral tegmental area (VTA). This approach also allowed us to demarcate subregions within the VTA comprising specific neuronal subpopulations. We further identified WW domain-containing oxidoreductase as a molecular marker of a population of VTA neurons that co-express tyrosine hydroxylase and vesicular glutamate transporter 2, and confirmed their region-specific distribution by immunohistochemistry. The results demonstrate the utility of our analytical approach for uncovering expression signatures representing specific cell types and neuronal subpopulations enriched in a given brain area.

Microglia (MG), the principal neuroimmune sentinels in the brain, continuously sense changes in their environment and respond to invading pathogens, toxins, and cellular debris, thereby affecting neuroinflammation. Microbial pathogens produce small metabolites that influence neuroinflammation, but the molecular mechanisms that determine whether pathogen-derived small metabolites affect microglial activation of neuroinflammation remain to be elucidated. We hypothesized that odorant receptors (ORs), the largest subfamily of G protein-coupled receptors, are involved in microglial activation by pathogen-derived small metabolites. We found that MG express high levels of two mouse ORs, Olfr110 and Olfr111, which recognize a pathogenic metabolite, 2-pentylfuran, secreted by Streptococcus pneumoniae. These interactions activate MG to engage in chemotaxis, cytokine production, phagocytosis, and reactive oxygen species generation. These effects were mediated through the Gαs -cyclic adenosine monophosphate-protein kinase A-extracellular signal-regulated kinase and Gβγ -phospholipase C-Ca2+ pathways. Taken together, our results reveal a novel interplay between the pathogen-derived metabolite and ORs, which has major implications for our understanding of microglial activation by pathogen recognition. DATABASE: Model data are available in the PMDB database under the accession number PM0082389.

The multikinase inhibitor sorafenib is the only standard first-line therapy for hepatocellular carcinoma (HCC). Here, we report the dose-dependent effects of sorafenib on the immune response, which is related to nuclear factor of activated T cells 1 (NFAT1) activity. In vitro and in vivo experiments were performed with low and high doses of sorafenib using human T cells and spontaneous developed woodchuck HCC models. In vitro studies demonstrated that following exposure to a high dose of sorafenib the baseline activity of NFAT1 in T cells was significantly increased. In a parallel event, high dose sorafenib resulted in a significant decrease in T cell proliferation and increased the proportion of PD-1 expressing CD8+ T cells with NFAT1 activation. In the in vivo model, smaller tumors were detected in the low-dose sorafenib treated group compared to the placebo and high-dose treated groups. The low-dose sorafenib group showed a significant tumor growth delay with significantly more CD3+ cells in tumor. This study demonstrates that sorafenib has immunomodulatory effects in a dose- and time-dependent manner. Higher dose of sorafenib treatment was associated with immunosuppressive action. This observed effect of sorafenib should be taken into consideration in the selection of optimum starting dose for future trials.

Transcriptomic profiling of tumor tissues introduces a large database, which has led to improvements in the ability of cancer diagnosis, treatment, and prevention. However, performing tumor transcriptomic profiling in the clinical setting is very challenging since the procurement of tumor tissues is inherently limited by invasive sampling procedures. Here, we demonstrated the feasibility of purifying hepatocellular carcinoma (HCC) circulating tumor cells (CTCs) from clinical patient samples with improved molecular integrity using Click Chips in conjunction with a multimarker antibody cocktail. The purified CTCs were then subjected to mRNA profiling by NanoString nCounter platform, targeting 64 HCC-specific genes, which were generated from an integrated data analysis framework with 8 tissue-based prognostic gene signatures from 7 publicly available HCC transcriptomic studies. After bioinformatics analysis and comparison, the HCC CTC-derived gene signatures showed high concordance with HCC tissue-derived gene signatures from TCGA database, suggesting that HCC CTCs purified by Click Chips could enable the translation of HCC tissue molecular profiling into a noninvasive setting.

Background: Genome and transcriptome-based analysis has begun to reshape the approach to prostate cancer (PC). Two different gene expression signatures have shown that PC can be divided into 3 subclasses reflecting luminal-basal biology. These subtypes point toward biological drivers that may strongly influence how care should be personalized including optimization of androgen receptor targeted therapy. The majority of work done in this area has been based on tissue-based gene expression. With the advent of newer nanotechnology platforms for isolation of circulating tumor cells (CTCs), profiling of PC gene expression from blood is now possible. Methods: We recruited 34 patients with metastatic castration resistant PC who had available blood specimens prior to initiation of androgen receptor signaling inhibitor (ARSI, e.g. abiraterone, enzalutamide and apalutamide) therapy. We combined variations of the NanoVelcro Assays (thermoresponsive, click-chemistry) allowing for capture and release of CTCs with intact mRNA. Gene sets from the PCS and PAM50 signatures were re-reviewed to optimize signal detection in the blood and enriched for genes upregulated in PC. The NanoString nCounter platform was applied to profile the resulting genes. A pilot study was conducted using banked samples available through the Urologic Oncology Program Blood and Biospecimen Bank at Cedars-Sinai Medical Center. Results: The final assay was tested in banked blood samples and provided classifications of patients that associated with clinical responsiveness to therapy. Validation was conducted to examine the performance of the CTC-specific PCS/PAM50 panel in public databases (including Prostate Cancer Transcriptome Atlas and GenomeDx). Our pilot study showed that the median overall survival was significantly worse in PCS1 patients. Conclusions: This study shows initial proof of principle that genomic classification in blood is possible using contemporary tool for blood component isolation and RNA profiling. Additional technical and clinical validations are needed prior to widespread implementation, but these methods may make it possible to increase the utilization of genomic classifiers in clinical studies and in practice. Citation Format: PAI-CHI TENG, Minhyung Kim, Yu Jen Jan, Jie-Fu Chen, Nu Yao, Gina C. Chu, Pin-Jung Chen, Jasmine J. Wang, Yi-Te Lee, Yazhen Zhu, Leland W. Chung, Felix Y. Feng, Michael R. Freeman, Sungyong You, Hsian-Rong Tseng, Edwin M. Posadas. Gene expression of circulating tumor cells is predictive of treatment response in patients with advanced prostate cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5447.

Hepatocellular carcinoma (HCC) is the 4th most common cause of cancer-related deaths worldwide. The poor prognosis of HCC is due to the fact that diagnosis is often made at an advanced stage in disease development. It is crucial to develop a non-invasive liquid biopsy-based diagnostic solution for early detection of HCC. In this study, we developed a covalent chemistry-based nanostructured silicon substrate (“EV Click Chip”) for the isolation of HCC extracellular vesicles (EVs). The EV Click Chip leverages specific click chemistry reactions and sensitive multi-marker cocktail antibody identification of the HCC EVs. The EV Click Chip also allows for the subsequent release of the captured HCC EVs and is optimal for the downstream molecular analysis. A well-validated 10 liver-specific genes were quantified using reverse transcription droplet digital PCR (RT-ddPCR) in HCC EVs purified by the optimized EV Click Chips for the detection of HCC. Our EV Click Chip-based HCC-EV Assay is able to differentiate HCC from non-HCC controls (chronic liver diseases, healthy donors and other cancers) and outperformed clinical AFP test for distinguishing early-stage HCC from at-risk cirrhotic patients. Citation Format: Yi-Te Lee, Na Sun, Ryan Y. Zhang, Rueihung Kao, Pin-Jung Chen, Pai-Chi Teng, Jasmine J. Wang, Yingying Yang, Minhyung Kim, Edwin M. Posadas, Sungyong You, Ju Dong Yang, Vatche G. Agopian, Hsian-Rong Tseng, Yazhen Zhu. Purification and mRNA profiling of extracellular vesicles for early detection of hepatocellular carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5436.

Introduction and Objective: Alterations in DNA methylation are important epigenetic markers in bladder cancer (BC). These epigenome modifications drive the mechanisms of aggressive chemoresistant BC. Clinicopathologic biomarkers that indicate chemotherapeutic resistance are critical for better assessing treatment strategies for individual patients. Thus, in this study, we aimed to determine whether DNA methylation of certain metabolic enzymes is significantly altered in cisplatin-resistant BC cells. Methods: To characterize CpG methylation and nucleosome accessibility in cisplatin-resistant BC cells, the Illumina Infinium HM450 DNA methylation assay was performed. Perturbed gene expression was found to be associated with cisplatin resistance, and the biologic roles of spermidine/spermine N1-acetyltransferase (SAT1) and argininosuccinate synthase 1 (ASS1) were further studied using qRT-PCR analysis and various cell biology assays, including Western blot. Results: ASS1 and SAT1, genes for amino acid and polyamine metabolism catalysts, respectively, were found to be vastly hypermethylated, resulting in greatly downregulated expression. ASS1 expression is of particular interest because prior studies have demonstrated its potential association with BC stage and recurrence. In regard to chemoresistance, we found that aberrant expression or induced stimulation of SAT1 restored cisplatin sensitivity in the cell culture system. We also found that the addition of exogenous arginine deiminase through administration of ADI-PEG 20 (pegylated arginine deiminase) increased ASS1 expression and enhanced cisplatin's apoptotic effects. Conclusions: Our study demonstrates a novel mechanistic link between the epigenetic perturbation of SAT1 and ASS1 and cancer metabolism in cisplatin-resistant bladder cancer cells. These findings suggest potential utility of SAT1 and ASS1 as predictive biomarkers in resensitizing bladder cancer to chemotherapy and personalizing therapy. Citation Format: Austin Yeon, Sungyong You, Minhyung Kim, Amit Gupta, Myung Hee Park, Daniel Weisenberger, Gangning Liang, Jayoung Kim. Epigenetic remodeling of cancer metabolisms in cisplatin-resistant bladder cancer [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2019 May 18-21; Denver, CO. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(15_Suppl):Abstract nr B14.

Extracellular vesicles (EVs) are membrane-enclosed particles that play an important role in cancer progression and have emerged as a promising source of circulating biomarkers. ProteinS-acylation, also known as palmitoylation, has been proposed as a post-translational mechanism that modulates the dynamics of EV biogenesis and protein cargo sorting. However, technical challenges have limited large-scale profiling of the whole palmitoyl-proteins of EVs. We successfully employed a novel approach that combines low-background acyl-biotinyl exchange (LB-ABE) with label-free proteomics to analyze the palmitoyl proteome of large EVs (L-EVs) and small EVs (S-EVs) from prostate cancer cells. Here we report the first palmitoyl-protein signature of EVs, and demonstrate that L- and S-EVs harbor proteins associated with distinct biological processes and subcellular origin. We identified STEAP1, STEAP2, and ABBC4 as prostate cancer-specific palmitoyl proteins enriched in both EV populations in comparison with the originating cell lines. Importantly, the presence of the above proteins in EVs was significantly reduced upon inhibition of palmitoylation in the producing cells. These results suggest that palmitoylation may be involved in the differential sorting of proteins to distinct EV populations and allow for better detection of disease biomarkers.

e17509 Background: Genomic profiling has strongly impacted the contemporary understanding of prostate cancer (PCa). Clinical trials are now testing the utility of genomic classifiers such as the PCS (You, Ca Res 2016) and PAM50 (Zhao, JAMA Onc 2017) systems to optimize therapy selection. As contemporary tissue is not always readily available, especially in metastatic, castration-resistant PCa (mCRPC), a blood-based test would be better suited for assessing patients and predicting treatment response. Methods: The CTC-RNA assay combines the Thermoresponsive (TR)-NanoVelcro system with the NanoString nCounter platform. This allows for CTC purification and RNA analysis. Using a novel bioinformatics approach that accounts for differences in background signals between tissue and blood, we reconstructed the PCS and PAM50 panels to recapitulate both classifiers in this blood-based assay. A weighted Z-score and nearest centroid classifier were used to calculate gene expression and to assign PCS and PAM50 subtypes. Performance of the revised signatures and CTC-RNA assay was benchmarked on simulated spiked-blood specimens. An initial clinical test was performed using clinically annotated, banked blood specimens within the Translational Oncology Program Blood and Biospecimen Bank. Results: CTC-RNA profiles of C4-2B AR signaling inhibitor (ARSI)-resistant sublines were compared to parental C4-2B. C4-2B ARSI-resistant cells had significantly higher PCS1 Z scores, PCS1 probability, and basal probability compared to the parental C4-2B cells. Blood samples from 34 mCRPC patients prior to initiation of therapy with ARSIs (abiraterone, enzalutamide, or apalutamide) were then analyzed. Samples were classified as PCS1 (n = 3), PCS2 (n = 20), and PCS3 (n = 11); luminal A (n = 12), luminal B (n = 11), and basal (n = 11). The biochemical progression-free survival (bPFS) on ARSI and overall survival (OS) for PCS1/Basal vs. other are shown in the table. Conclusions: The CTC-RNA assay is capable of generating luminal-basal classifications such as those in the PCS and PAM50 systems. Given early data of these classifiers and their potential to guide therapeutic decisions, this approach may be useful as an alternative to biopsy to facilitate such decisions. Larger prospective studies will be needed to confirm and validate its clinical utility. [Table: see text]

Metabolic alterations in prostate cancer (PC) are associated with progression and aggressiveness. However, the underlying mechanisms behind PC metabolic functions are unknown. Our group recently reported on the important role of centromere protein F (CENPF), a protein associated with the centromere-kinetochore complex and chromosomal segregation during mitosis, in PC MRI visibility. In this study, we focused on discerning the role of CENPF in metabolic perturbation in human PC3 cells. A series of bioinformatics analyses showed that CENPF is one gene that is strongly associated with aggressive PC, and that its expression is positively correlated with metastasis. By identifying and reconstructing the CENPF network, we found additional associations with lipid regulation. Further untargeted metabolomics analysis using gas chromatography-time-of-flight-mass spectrometry (GC-TOF-MS) revealed that silencing of CENPF alters the global metabolic profiles of PC cells and inhibits cell proliferation, which suggests that CENPF may be a critical regulator of PC metabolism. These findings provide useful scientific insights that can be applied in future studies investigating potential targets for PC treatment.

Tumour vessels have been studied extensively as they are critical sites for drug delivery, anti-angiogenic therapies and immunotherapy. As a preclinical tool, intravital microscopy (IVM) allows for in vivo real-time direct observation of vessels at the cellular level. However, to date there are no reports of intravital high-resolution imaging of human tumours in the clinical setting. Here we report the feasibility of IVM examinations of human malignant disease with an emphasis on tumour vasculature as the major site of tumour-host interactions. Consistent with preclinical observations, we show that patient tumour vessels are disorganized, tortuous and ∼50% do not support blood flow. Human tumour vessel diameters are larger than predicted from immunohistochemistry or preclinical IVM, and thereby have lower wall shear stress, which influences delivery of drugs and cellular immunotherapies. Thus, real-time clinical imaging of living human tumours is feasible and allows for detection of characteristics within the tumour microenvironment., Intravital microscopy has been used in laboratory animals to visualise the blood vessels in tumours. Here, the authors use this technique in melanoma patients undergoing surgery and show that vessels in situ have a larger diameter than excised tissue

Historically, intra-arterial (IA) drug administration for malignant brain tumors including glioblastoma multiforme (GBM) was performed as an attempt to improve drug delivery. With the advent of percutaneous neuorovascular techniques and modern microcatheters, intracranial drug delivery is readily feasible; however, the question remains whether IA administration is safe and more effective compared to other delivery modalities such as intravenous (IV) or oral administrations. Preclinical large animal models allow for comparisons between treatment routes and to test novel agents, but can be expensive and difficult to generate large numbers and rapid results. Accordingly, we developed a murine model of IA drug delivery for GBM that is reproducible with clear readouts of tumor response and neurotoxicities. Herein, we describe a novel mouse model of IA drug delivery accessing the internal carotid artery (ICA) to treat ipsilateral implanted GBM tumors that is consistent and reproducible with minimal experience. The intent of establishing this unique platform is to efficiently interrogate targeted anti-tumor agents that may be designed to take advantage of a directed, regional therapy approach for brain tumors.

Background Vascularized composite tissue allotransplantation (VCA) from a cadaveric donor has now become a clinical reality and the treatment modality of choice for patients with devastating injuries, deformities, and complex tissue defects. However, many VCA patients experience severe toxicities due to the strong immunosuppression required secondary to high antigenicity of the grafts. To improve immunosuppressive protocols for VCA, feasible and reliable preclinical models are necessary. The purpose of this study was to introduce new techniques to an established preclinical VCA model to accelerate future investigations. Methods C57BL/6 (H-2b) and BALB/c (H-2d) mice were used to perform VCA as recipients and donors, respectively. Surgery time, success rate, associated complications, and mortality were analyzed. Blood flow in grafts was interrogated with laser speckle image (LSI). Results A nonsuture cuff technique was used with the abdominal aorta for end-to-end anastomosis. The cuff technique demonstrated efficiency for donor surgery (52 ± 10 minutes for donor vs. 45 ± 8 minutes for recipient surgery). Successful revascularization was achieved in 27 (90%) of 30 transplants. The majority of surgical complications occurred within 48 hours including artery occlusion, venous occlusion, cerebral stroke, and minor bleeding without mortality. LSI was useful in detecting intraoperative vascular complications with display patterns predictive of complication type. Conclusions The described techniques may facilitate a more efficient heterotopic hind limb transplantation mouse model of VCA.

Hyperactivated mTOR signaling in the developing brain has been implicated in multiple forms of pathology including tuberous sclerosis complex (TSC). To date, various phenotypic defects such as cortical lamination irregularity, subependymal nodule formation, dysmorphic astrocyte differentiation and dendritic malformation have been described for patients and animal models. However, downstream networks affected in the developing brain by hyperactivated mTOR signaling have yet to be characterized. Here, we present an integrated analysis of transcriptomes and proteomes generated from wild-type and Tsc1/Emx1-Cre forebrains. This led to comprehensive lists of genes and proteins whose expression levels were altered by hyperactivated mTOR signaling. Further incorporation of TSC patient data followed by functional enrichment and network analyses pointed to changes in molecular components and cellular processes associated with neuronal differentiation and morphogenesis as the key downstream events underlying developmental and morphological defects in TSC. Our results provide novel and fundamental molecular bases for understanding hyperactivated mTOR signaling-induced brain defects which can in turn facilitate identification of potential diagnostic markers and therapeutic targets for mTOR signaling-related neurological disorders.

170 Background: Transcriptome-based analysis has begun to reshape the approach to prostate cancer (PC). Two different gene expression signatures have shown that PC can be divided into 3 subclasses reflecting luminal-basal biology. These subtypes point toward biological drivers that may strongly influence how care should be personalized including optimization of androgen receptor targeted therapy. The majority of work done in this area has been based on tissue-based gene expression. With the advent of newer nanotechnology platforms for isolation of circulating tumor cells (CTCs), profiling of PC gene expression from blood is now possible. Methods: We recruited 34 patients with metastatic castration resistant PC at Cedars-Sinai Medical Center who had available blood specimens prior to initiation of androgen receptor signaling inhibitor (ARSI, e.g. abiraterone, enzalutamide and apalutamide) therapy.We utilized the NanoVelcro Assays which allow for capture and release of CTCs with intact mRNA. Gene sets from the PCS and PAM50 signatures were re-reviewed to optimize signal detection in the blood and enriched for genes upregulated in PC. The NanoString nCounter platform was used for RNA profiling. Results: The final assay was tested in banked blood samples and provided classifications of patients that associated with clinical responsiveness to therapy. Validation was conducted to examine the performance of the CTC-specific PCS/PAM50 panel in public databases (including Prostate Cancer Transcriptome Atlas and GenomeDx). Our pilot study showed that the median overall survival was significantly worse in PCS1 patients. Conclusions: This study shows initial proof of principle that genomic classification in blood is possible using contemporary tool for blood component isolation and RNA profiling. Additional technical and clinical validations are needed prior to widespread implementation, but these methods may make it possible to increase the utilization of genomic classifiers in clinical studies and in practice.

Solid-pseudopapillary neoplasm is an uncommon pancreatic tumor with distinct clinicopathologic features. Solid-pseudopapillary neoplasms are characterized by mutations in exon 3 of CTNNB1. However, little is known about the gene and microRNA expression profiles of solid-pseudopapillary neoplasms. Thus, we sought to characterize solid-pseudopapillary neoplasm-specific gene expression and identify the signaling pathways activated in these tumors. Comparisons of gene expression in solid-pseudopapillary neoplasm to pancreatic ductal carcinomas, neuroendocrine tumors, and non-neoplastic pancreatic tissues identified solid-pseudopapillary neoplasm-specific mRNA and microRNA profiles. By analyzing 1686 (1119 upregulated and 567 downregulated) genes differentially expressed in solid-pseudopapillary neoplasm, we found that the Wnt/β-catenin, Hedgehog, and androgen receptor signaling pathways, as well as genes involved in epithelial mesenchymal transition, are activated in solid-pseudopapillary neoplasms. We validated these results experimentally by assessing the expression of β-catenin, WIF-1, GLI2, androgen receptor, and epithelial-mesenchymal transition-related markers with western blotting and immunohistochemistry. Our analysis also revealed 17 microRNAs, especially the miR-200 family and miR-192/215, closely associated with the upregulated genes associated with the three pathways activated in solid-pseudopapillary neoplasm and epithelial mesenchymal transition. Our results provide insight into the molecular mechanisms underlying solid-pseudopapillary neoplasm tumorigenesis and its characteristic less epithelial cell differentiation than the other common pancreatic tumors.

The clinical practice of hyperthermic intraperitoneal chemoperfusion (HIPEC) for carcinomatosis has lacked preclinical justification. A standardized mouse model was created to evaluate the independent effects of intraperitoneal chemotherapy. Diffuse colorectal carcinomatosis was generated in mice prior to intraperitoneal lavage with mitomycin C (MMC) at clinically comparable dosing for variable lengths of time. Tumor volumes, MMC tissue concentrations and survival were measured in comparison to saline lavage and intravenous MMC. Magnetic resonance imaging revealed a direct correlation between tumor volume, MMC dose and exposure time and survival. Intravenous MMC demonstrated a rapid clearance from the blood, lower peritoneal tissue concentrations, less tumor growth inhibition and decreased survival compared to intraperitoneal administration. Intraperitoneal chemotherapy inhibited tumor growth independent of cytoreduction or hyperthermia, demonstrated improved peritoneal tissue concentration and was associated with increased survival. These data support the clinical utility of the intraperitoneal chemotherapy component of HIPEC.

Myeloid-derived suppressor cells (MDSC) contribute to an immunosuppressive network that drives cancer escape by disabling T cell adaptive immunity. The prevailing view is that MDSC-mediated immunosuppression is restricted to tissues where MDSC co-mingle with T cells. Here we show that splenic or, unexpectedly, blood-borne MDSC execute far-reaching immune suppression by reducing expression of the L-selectin lymph node (LN) homing receptor on naïve T and B cells. MDSC-induced L-selectin loss occurs through a contact-dependent, post-transcriptional mechanism that is independent of the major L-selectin sheddase, ADAM17, but results in significant elevation of circulating L-selectin in tumor-bearing mice. Even moderate deficits in L-selectin expression disrupt T cell trafficking to distant LN. Furthermore, T cells preconditioned by MDSC have diminished responses to subsequent antigen exposure, which in conjunction with reduced trafficking, severely restricts antigen-driven expansion in widely-dispersed LN. These results establish novel mechanisms for MDSC-mediated immunosuppression that have unanticipated implications for systemic cancer immunity. DOI: http://dx.doi.org/10.7554/eLife.17375.001

Background Isolated limb perfusion (ILP) for extremity melanoma has been used clinically for over half a century. Mouse modeling of ILP may offer significant experimental advantages compared with existing models. We propose a novel mouse model and report our initial experience. Methods We injected female C57BL/6 mice (22–25 g) with 1 × 106 B16 melanoma cells subcutaneously in the distal right thigh. After 7 d of tumor establishment, we cannulated the superficial femoral artery (inflow) and vein (outflow) of anesthetized mice and placed a proximal tourniquet. Non-oxygenated perfusate included low-dose or high-dose melphalan and saline (control). We analyzed endpoints of cannulation time, procedural complications, morbidity, toxicity, and tumor response. Results We performed 11 superficial femoral vessel cannulations. Median cannulation time was 19 min (range, 15–32 min). Intact perfusion models were obtained in 10 of 11 cases (91%); one case failed owing to superficial femoral vein dissection. Morbidity rate was 20% (one wound dehiscence and one hematoma). Both high- and low-dose melphalan perfusion groups (4 mice/group) trended to growth delay and regression compared with saline-perfused groups. Toxicity was greater in the high-dose melphalan-treated mice. Conclusions We have established the first reproducible mouse model of ILP for melanoma. Future experiments will take advantage of the large number of established mouse knockout models and reagents to dissect the precise mechanisms of tumor control after ILP, and examine to novel agents.

Lower urinary tract symptoms (LUTSs) are highly prevalent among the elderly and negatively impact quality of life. Since caffeinated beverages are enjoyed worldwide and the relationship between LUTS and caffeine is still not fully understood, it would be of particular interest to examine the underlying mechanisms that drive caffeine's influence on LUTS development and progression. The aim of this study is to characterize the effects of caffeine on hTert-immortalized normal bladder epithelial cells by investigating whether exposure to caffeine can cause potential changes in the bladder proteome and/or biological pathways. In labeled LC-MS/MS proteomic analysis, 57 proteins are found as being differentially expressed in caffeine-treated bladder epithelial cells, compared to controls; this included 32 upregulated and 25 downregulated proteins. Further functional gene enrichment analysis reveals that caffeine affects major biological pathways, including those for "muscle contraction" and "chromatin assembly." These findings provide new scientific insights that may be useful in future studies investigating the role of caffeine in bladder dysfunctions.

Introduction: Increased galectin-3 is associated with ischemic cardiomyopathy, although its role in early remodeling post-myocardial infarction (MI) has not been fully elucidated. There are no data demonstrating that blocking galectin-3 expression would have an impact on the heart and that its relationship to remodeling is not simply an epiphenomenon. The direct association between galectin-3 and myocardial inflammation, dysfunction, and adverse cardiovascular outcomes post-MI was examined using clinical and translational studies. Methods: We performed expression analysis of 9753 genes in murine model of acute MI. For galectin-3 loss of function studies, homozygous galectin-3 knock-out (KO) mice were subjected to coronary artery ligation procedure to induce acute MI (MI, N = 6; Sham, N = 6). For clinical validation, serum galectin-3 levels were measured in 96 patients with ST-elevation MI. Echocardiographic and angiographic parameters of myocardial dysfunction and 3-month composite outcome including mortality, recurrent MI, stroke, and heart failure hospitalization were measured. Results: In the infarct regions of murine models, galectin-3 was a robustly expressed gene. Elevated galectin-3 expression strongly correlated with macrophage-mediated genes. Galectin-3 KO mice showed reduced myocardial macrophage infiltration after acute MI. Galectin-3 levels were higher in patients with early systolic dysfunction, and predicted 3-month major adverse cardiovascular events (area under the curve [AUC]: 0.917 ± 0.063; P = .001). Conclusions: Galectin-3 is directly associated with early myocardial inflammation post-MI and may represent a potential target for therapeutic inhibition.