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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.

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.

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.

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.

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.

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.

Here, we investigated the relationship of the age-associated expansion of IL-7 receptor alpha low (IL-7Rαlow ) effector memory (EM) CD8+ T cells with the global transcriptomic profile of peripheral blood cells in humans. We found 231 aging signature genes of IL-7Rαlow EM CD8+ T cells that corresponded to 15% of the age-associated genes (231/1,497) reported by a meta-analysis study on human peripheral whole blood from approximately 15,000 individuals, having high correlation with chronological age. These aging signature genes were the target genes of several transcription factors including MYC, SATB1, and BATF, which also belonged to the 231 genes, supporting the upstream regulatory role of these transcription factors in altering the gene expression profile of peripheral blood cells with aging. We validated the differential expression of these transcription factors between IL-7Rαlow and high EM CD8+ T cells as well as in peripheral blood mononuclear cells (PBMCs) of young and older adults. Finally, we found a significant association with influenza vaccine responses in older adults, suggesting the possible biological significance of the aging signature genes of IL-7Rαlow EM CD8+ T cells. The results of our study support the relationship of the expansion of IL-7Rαlow EM CD8+ T cells with the age-associated changes in the gene expression profile of peripheral blood cells and its possible biological implications.

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.

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.

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.

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

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.

Life-long regeneration of healthy muscle by cell transplantation is an ideal therapy for patients with degenerative muscle diseases. Yet, obtaining muscle stem cells from patients is very limited due to their exhaustion in disease condition. Thus, development of a method to obtain healthy myogenic stem cells is required. Here, we showed that the four transcription factors, Six1, Eya1, Esrrb, and Pax3, converts fibroblasts into induced myogenic stem cells (iMSCs). The iMSCs showed effective differentiation into multinucleated myotubes and also higher proliferation capacity than muscle derived stem cells both in vitro and in vivo. The iMSCs do not lose their proliferation capacity though the passaging number is increased. We further isolated CD106-negative and α7-integrin-positive iMSCs (sort-iMSCs) showing higher myogenic differentiation capacity than iMSCs. Moreover, genome-wide transcriptomic analysis of iMSCs and sort-iMSCs, followed by network analysis, revealed the genes and signaling pathways associated with enhanced proliferation and differentiation capacity of iMSCs and sort-iMSCs, respectively. The stably expandable iMSCs provide a new source for drug screening and muscle regenerative therapy for muscle wasting disease.

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.

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.

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.

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

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.

The advances in mass spectrometry-based proteomics technologies have enabled the generation of global proteome data from tissue or body fluid samples collected from a broad spectrum of human diseases. Comparative proteomic analysis of global proteome data identifies and prioritizes the proteins showing altered abundances, called differentially expressed proteins (DEPs), in disease samples, compared to control samples. Protein biomarker candidates that can serve as indicators of disease states are then selected as key molecules among these proteins. Recently, it has been addressed that cellular pathways can provide better indications of disease states than individual molecules and also network analysis of the DEPs enables effective identification of cellular pathways altered in disease conditions and key molecules representing the altered cellular pathways. Accordingly, a number of network-based approaches to identify disease-related pathways and representative molecules of such pathways have been developed. In this review, we summarize analytical platforms for network-based protein biomarker discovery and key components in the platforms.

Isolated limb perfusion (ILP) with the chemotherapeutic agent melphalan is an effective treatment option for extremity in-transit melanoma but is toxic and technically challenging to deliver locoregionally. CBL0137 is an experimental clinical drug with broad anticancer activity in animal models, owing to its ability to bind DNA in a nongenotoxic manner and inactivate the FACT chromatin modulator essential for tumor cell viability. Here, we report that CBL0137 delivered by ILP in a murine melanoma model is as efficacious as melphalan, displaying antitumor activity at doses corresponding to only a fraction of the systemic MTD of CBL0137. The ability to bind DNA quickly combined with a favorable safety profile made it possible to substitute CBL0137 in the ILP protocol, using an intra-arterial infusion method, to safely achieve effective tumor suppression. Our findings of a preclinical proof of concept for CBL0137 and its administration via intra-arterial infusion as a superior treatment compared with melphalan ILP allows for locoregional treatment anywhere a catheter can be placed. Cancer Res; 76(22); 6620–30. ©2016 AACR.

Purpose While surgical resection is a cornerstone of cancer treatment, local and distant recurrences continue to adversely affect outcome in a significant proportion of patients. Evidence that an alternative debulking strategy involving radiofrequency ablation (RFA) induces antitumor immunity prompted the current investigation of the efficacy of performing RFA prior to surgical resection (pre-resectional RFA) in a preclinical mouse model. Experimental Design Therapeutic efficacy and systemic immune responses were assessed following pre-resectional RFA treatment of murine CT26 colon adenocarcinoma. Results Treatment with pre-resectional RFA significantly delayed tumor growth and improved overall survival compared to sham surgery, RFA, or resection alone. Mice in the pre-resectional RFA group that achieved a complete response demonstrated durable antitumor immunity upon tumor re-challenge. Failure to achieve a therapeutic benefit in immunodeficient mice confirmed that tumor control by pre-resectional RFA depends on an intact adaptive immune response rather than changes in physical parameters that make ablated tumors more amenable to a complete surgical excision. RFA causes a marked increase in intratumoral CD8+ T lymphocyte infiltration, thus substantially enhancing the ratio of CD8+ effector T cells: FoxP3+ regulatory T cells. Importantly, pre-resectional RFA significantly increases the number of antigen-specific CD8+ T cells within the tumor microenvironment and tumor-draining lymph node but had no impact on infiltration by myeloid-derived suppressor cells, M1 macrophages or M2 macrophages at tumor sites or in peripheral lymphoid organs (i.e., spleen). Finally, pre-resectional RFA of primary tumors delayed growth of distant tumors through a mechanism that depends on systemic CD8+ T cell-mediated antitumor immunity. Conclusion Improved survival and antitumor systemic immunity elicited by pre-resectional RFA support the translational potential of this neoadjuvant treatment for cancer patients with high-risk of local and systemic recurrence.