A fluorescent splice-switching mouse model enables high-throughput, sensitive quantification of antisense oligonucleotide delivery and activity.

While antisense oligonucleotides (ASOs) are used in the clinic, therapeutic development is hindered by the inability to assay ASO delivery and activity in vivo. Accordingly, we developed a dual-fluorescence, knockin mouse model that constitutively expresses mKate2 and an engineered EGFP that is alternatively spliced in the presence of ASO to induce expression. We first examined free ASO activity in the brain following intracerebroventricular injection revealing EGFP splice-switching is both ASO concentration and time dependent in major central nervous system cell types. We then assayed the impact of lipid nanoparticle delivery on ASO activity after intravenous administration. Robust EGFP fluorescence was observed in the liver and EGFP+ cells were successfully isolated using fluorescence-activated cell sorting. Together, these results show the utility of this animal model in quantifying both cell-type- and organ-specific ASO delivery, which can be used to advance ASO therapeutics for many disease indications.

Preventing VEGF-Mediated Vascular Permeability by Experimentally Potentiating BBB Characteristics in Endothelial Cells.

Difficulties with poor reproducibility and translatability of animal model-based research, along with increased efforts to abide by the 3Rs tenet of animal welfare, are driving demand for more relevant human cellular systems. This is especially true for central nervous system (CNS) vasculatures with specialized properties and barriers, namely the blood-brain and blood-retinal barriers (BBB and BRB, respectively) which are difficult to model in vitro. The BBB and BRB protect neurovascular units by regulating nutrient homeostasis, maintaining local ion levels, protecting against exposure from circulating toxins and pathogens, and restricting passage of peripheral immune factors. In this manuscript, we will describe transgenic and pharmacological-based protocols to generate relevant BBB and BRB models both from human pluripotent stem cell-derived endothelial cells (hPSC-ECs) and from primary human umbilical vein endothelial cells (HUVECs). When followed, researchers can expect to generate well-characterized, anatomical and functional BBB and BRB EC monolayers in 36-48 h that are stable up to 90 h. The ability to generate more relevant BBB and BRB EC cultures will improve drug discovery efforts and inform future therapies for neurovascular disorders.

Decoding Neuronal Diversification by Multiplexed Single-cell RNA-Seq.

Cellular reprogramming is driven by a defined set of transcription factors; however, the regulatory logic that underlies cell-type specification and diversification remains elusive. Single-cell RNA-seq provides unprecedented coverage to measure dynamic molecular changes at the single-cell resolution. Here, we multiplex and ectopically express 20 pro-neuronal transcription factors in human dermal fibroblasts and demonstrate a widespread diversification of neurons based on cell morphology and canonical neuronal marker expressions. Single-cell RNA-seq analysis reveals diverse and distinct neuronal subtypes, including reprogramming processes that strongly correlate with the developing brain. Gene mapping of 20 exogenous pro-neuronal transcription factors further unveiled key determinants responsible for neuronal lineage specification and a regulatory logic dictating neuronal diversification, including glutamatergic and cholinergic neurons. The multiplex scRNA-seq approach is a robust and scalable approach to elucidate lineage and cellular specification across various biological systems.

Inducers of the endothelial cell barrier identified through chemogenomic screening in genome-edited hPSC-endothelial cells.

The blood-retina barrier and blood-brain barrier (BRB/BBB) are selective and semipermeable and are critical for supporting and protecting central nervous system (CNS)-resident cells. Endothelial cells (ECs) within the BRB/BBB are tightly coupled, express high levels of Claudin-5 (CLDN5), a junctional protein that stabilizes ECs, and are important for proper neuronal function. To identify novel CLDN5 regulators (and ultimately EC stabilizers), we generated a CLDN5-P2A-GFP stable cell line from human pluripotent stem cells (hPSCs), directed their differentiation to ECs (CLDN5-GFP hPSC-ECs), and performed flow cytometry-based chemogenomic library screening to measure GFP expression as a surrogate reporter of barrier integrity. Using this approach, we identified 62 unique compounds that activated CLDN5-GFP. Among them were TGF-ß pathway inhibitors, including RepSox. When applied to hPSC-ECs, primary brain ECs, and retinal ECs, RepSox strongly elevated barrier resistance (transendothelial electrical resistance), reduced paracellular permeability (fluorescein isothiocyanate-dextran), and prevented vascular endothelial growth factor A (VEGFA)-induced barrier breakdown in vitro. RepSox also altered vascular patterning in the mouse retina during development when delivered exogenously. To determine the mechanism of action of RepSox, we performed kinome-, transcriptome-, and proteome-profiling and discovered that RepSox inhibited TGF-ß, VEGFA, and inflammatory gene networks. In addition, RepSox not only activated vascular-stabilizing and barrier-establishing Notch and Wnt pathways, but also induced expression of important tight junctions and transporters. Taken together, our data suggest that inhibiting multiple pathways by selected individual small molecules, such as RepSox, may be an effective strategy for the development of better BRB/BBB models and novel EC barrier-inducing therapeutics.

Characterization of Tumor Blood Vasculature Expression of Human Invasive Bladder Cancer by Laser Capture Microdissection and Transcriptional Profiling.

Tumor-associated blood vessels differ from normal vessels and play key roles in tumor progression. We aimed to identify biomolecules that are expressed differentially in human bladder cancer-associated blood vessels to find novel biomarkers and mechanisms involved in tumor-associated angiogenesis. The transcriptome of tumor blood vasculature from human invasive bladder carcinoma (I-BLCA) and normal bladder tissue vasculature was compared using differential expression and unsupervised hierarchical clustering analyses. Pathway analysis identified up-regulation of genes involved in the proliferation, cell cycle, angiogenesis, inflammation, and transforming growth factor-ß signaling in tumor blood vasculature. A common consensus gene expression signature was identified between bladder cancer tumor blood vasculature with tumor blood vasculature of other solid cancers, which correlated with the overall survival of patients with several of the solid cancers investigated in The Cancer Genome Atlas data set. In bladder tumor blood vasculature, the secreted factor angiopoietin-like protein 2 (ANGPTL2), was confirmed to be up-regulated by quantitative RT-PCR and immunohistochemical staining. The up-regulation of ANGPTL2 in plasma was also observed in non-invasive bladder carcinoma and I-BLCA. We semiquantitatively analyzed expression of ANGPTL2 in tissue microarrays from I-BLCA and surprisingly found an opposite correlation between staining intensity and progression-free survival. Our results indicate that ANGPTL2 might serve as a potential biomarker to predict progression-free survival in I-BLCA.

Functional Genomics for Cancer Drug Target Discovery.

Functional genomics describes a field of biology that uses a range of approaches for assessing gene function with high-throughput molecular, genetic, and cellular technologies. The near limitless potential for applying these concepts to study the activities of all genetic loci has completely upended how today's cancer biologists tackle drug target discovery. We provide an overview of contemporary functional genomics platforms, highlighting areas of distinction and complementarity across technologies, so as to aid in the development or interpretation of cancer-focused screening efforts.

Patient hiPSCs Identify Vascular Smooth Muscle Arylacetamide Deacetylase as Protective against Atherosclerosis.

Although susceptibility to cardiovascular disease (CVD) is different for every patient, why some patients with type 2 diabetes mellitus (T2DM) develop CVD while others are protected has not yet been clarified. Using T2DM-patient-derived human induced pluripotent stem cells (hiPSCs), we found that in patients protected from CVD, there was significantly elevated expression of an esterase, arylacetamide deacetylase (AADAC), in vascular smooth muscle cells (VSMCs). We overexpressed this esterase in human primary VSMCs and VSMCs differentiated from hiPSCs and observed that the number of lipid droplets was significantly diminished. Further metabolomic analyses revealed a marked reduction in storage lipids and an increase in membrane phospholipids, suggesting changes in the Kennedy pathway of lipid bioassembly. Cell migration and proliferation were also significantly decreased in AADAC-overexpressing VSMCs. Moreover, apolipoprotein E (Apoe)-knockout mice overexpressing VSMC-specific Aadac showed amelioration of atherosclerotic lesions. Our findings suggest that higher AADAC expression in VSMCs protects T2DM patients from CVD.

Identification of a combination of transcription factors that synergistically increases endothelial cell barrier resistance.

Endothelial cells (ECs) display remarkable plasticity during development before becoming quiescent and functionally mature. EC maturation is directed by several known transcription factors (TFs), but the specific set of TFs responsible for promoting high-resistance barriers, such as the blood-brain barrier (BBB), have not yet been fully defined. Using expression mRNA data from published studies on ex vivo ECs from the central nervous system (CNS), we predicted TFs that induce high-resistance barrier properties of ECs as in the BBB. We used our previously established method to  generate ECs from human pluripotent stem cells (hPSCs), and then we overexpressed the candidate TFs in hPSC-ECs and measured barrier resistance and integrity using electric cell-substrate impedance sensing, trans-endothelial electrical resistance and FITC-dextran permeability assays. SOX18 and TAL1 were the strongest EC barrier-inducing TFs, upregulating Wnt-related signaling and EC junctional gene expression, respectively, and downregulating EC proliferation-related genes. These TFs were combined with SOX7 and ETS1 that together effectively induced EC barrier resistance, decreased paracellular transport and increased protein expression of tight junctions and induce mRNA expression of several genes involved in the formation of EC barrier and transport. Our data shows identification of a transcriptional network that controls barrier resistance in ECs. Collectively this data may lead to novel approaches for generation of in vitro models of the BBB.

Modeling the Effects of Severe Metabolic Disease by Genome Editing of hPSC-Derived Endothelial Cells Reveals an Inflammatory Phenotype.

The kinase AKT2 (PKB) is an important mediator of insulin signaling, for which loss-of-function knockout (KO) mutants lead to early onset diabetes mellitus, and dominant active mutations lead to early development of obesity and endothelial cell (EC) dysfunction. To model EC dysfunction, we used edited human pluripotent stem cells (hPSCs) that carried either a homozygous deletion of AKT2 (AKT2 KO) or a dominant active mutation (AKT2 E17K), which, along with the parental wild type (WT), were differentiated into ECs. Profiling of EC lines indicated an increase in proinflammatory and a reduction in anti-inflammatory fatty acids, an increase in inflammatory chemokines in cell supernatants, increased expression of proinflammatory genes, and increased binding to the EC monolayer in a functional leukocyte adhesion assay for both AKT2 KO and AKT2 E17K. Collectively, these findings suggest that vascular endothelial inflammation that results from dysregulated insulin signaling (homeostasis) may contribute to coronary artery disease, and that either downregulation or upregulation of the insulin pathway may lead to inflammation of endothelial cells. This suggests that the standard of care for patients must be expanded from control of metabolic parameters to include control of inflammation, such that endothelial dysfunction and cardiovascular disorders can ultimately be prevented.

Cellular Resistance Mechanisms to Targeted Protein Degradation Converge Toward Impairment of the Engaged Ubiquitin Transfer Pathway.

Proteolysis targeting chimeras are bifunctional small molecules capable of recruiting a target protein of interest to an E3 ubiquitin ligase that facilitates target ubiquitination followed by proteasome-mediated degradation. The first molecules acting on this novel therapeutic paradigm have just entered clinical testing. Here, by using Bromodomain Containing 4 (BRD4) degraders engaging cereblon and Von Hippel-Lindau E3 ligases, we investigated key determinants of resistance to this new mode of action. A loss-of-function screen for genes required for BRD4 degradation revealed strong dependence on the E2 and E3 ubiquitin ligases as well as for members of the COP9 signalosome complex for both cereblon- and Von Hippel-Lindau-engaging BRD4 degraders. Cancer cell lines raised to resist BRD4 degraders manifested a degrader-specific mechanism of resistance, resulting from the loss of components of the ubiquitin proteasome system. In addition, degrader profiling in a cancer cell line panel revealed a differential pattern of activity of Von Hippel-Lindau- and cereblon-based degraders, highlighting the need for the identification of degradation-predictive biomarkers enabling effective patient stratification.

Monolayer Generation of Vascular Endothelial Cells from Human Pluripotent Stem Cells.

The use of human pluripotent stem cells (hPSCs) for modeling human diseases and therapeutic applications requires differentiation methods that generate physiologically relevant cell types in a robust and standardized way. Herein, we describe an efficient and scalable monolayer protocol to convert pluripotent stem cells into vascular endothelial cells using defined culture conditions.The combinatorial use of small molecule compounds, growth factors as well as morphogens directs human pluripotent stem cells toward endothelial cells within 6 days. The protocol has the capacity to generate endothelial cells with high efficiencies of up to 80%. An additional immunomagnetic cell purification step that is based on the surface marker VE-cadherin results in a virtually pure population of endothelial cells. In a subsequent expansion step human PSC-derived endothelial cells can be further propagated, while maintaining their endothelial identity. Thus, our differentiation protocol enables the generation of hPSC-derived endothelial cells at a scale that is relevant for drug discovery campaigns or clinical applications.

Alternative transcription of a shorter, non-anti-angiogenic thrombospondin-2 variant in cancer-associated blood vessels.

Thrombospondin-2 (TSP2) is an anti-angiogenic matricellular protein that inhibits tumor growth and angiogenesis. Tumor-associated blood vascular endothelial cells (BECs) were isolated from human invasive bladder cancers and from matched normal bladder tissue by immuno-laser capture microdissection. Exon expression profiling analyses revealed a particularly high expression of a short TSP2 transcript containing only the last 9 (3') exons of the full-length TSP2 transcript. Using 5' and 3' RACE (rapid amplification of cDNA ends) and Sanger sequencing, we confirmed the existence of the shorter transcript of TSP2 (sTSP2) and determined its sequence which completely lacked the anti-angiogenic thrombospondin type 1 repeats domain. The largest open reading frame predicted within the transcript comprises 209 amino acids and matches almost completely the C-terminal lectin domain of full-length TSP2. We produced recombinant sTSP2 and found that unlike the full-length TSP2, sTSP2 did not inhibit vascular endothelial growth factor-A-induced proliferation of cultured human BECs, but in contrast when combined with TSP2 blocked the inhibitory effects of TSP2 on BEC proliferation. In vivo studies with stably transfected A431 squamous cell carcinoma cells revealed that full-length TSP2, but not sTSP2, inhibited tumor growth and angiogenesis. This study reveals that the transcriptional program of tumor stromal cells can change to transcribe a new version of an endogenous angiogenesis inhibitor that has lost its anti-angiogenic activity.

Requirements for Using iPSC-Based Cell Models for Assay Development in Drug Discovery.

A prevalent challenge in drug discovery is the translation of findings from preclinical research into clinical success. Currently, more physiological in vitro systems are being developed to overcome some of these challenges. In particular, induced pluripotent stem cells (iPSCs) have provided the opportunity to generate human cell types that can be utilized for developing more disease-relevant cellular assay models. As the use of these complex models is lengthy and fairly complicated, we lay out our experiences of the cultivation, differentiation, and quality control requirements to successfully utilize pluripotent stem cells in drug discovery.

An integrated expression atlas of miRNAs and their promoters in human and mouse.

MicroRNAs (miRNAs) are short non-coding RNAs with key roles in cellular regulation. As part of the fifth edition of the Functional Annotation of Mammalian Genome (FANTOM5) project, we created an integrated expression atlas of miRNAs and their promoters by deep-sequencing 492 short RNA (sRNA) libraries, with matching Cap Analysis Gene Expression (CAGE) data, from 396 human and 47 mouse RNA samples. Promoters were identified for 1,357 human and 804 mouse miRNAs and showed strong sequence conservation between species. We also found that primary and mature miRNA expression levels were correlated, allowing us to use the primary miRNA measurements as a proxy for mature miRNA levels in a total of 1,829 human and 1,029 mouse CAGE libraries. We thus provide a broad atlas of miRNA expression and promoters in primary mammalian cells, establishing a foundation for detailed analysis of miRNA expression patterns and transcriptional control regions.

High expression of insulin receptor on tumour-associated blood vessels in invasive bladder cancer predicts poor overall and progression-free survival.

Bladder cancer is a frequently recurring disease with a very poor prognosis once progressed to invasive stages, and tumour-associated blood vessels play a crucial role in this process. In order to identify novel biomarkers associated with progression, we isolated blood vascular endothelial cells (BECs) from human invasive bladder cancers and matched normal bladder tissue, and found that tumour-associated BECs greatly up-regulated the expression of insulin receptor (INSR). High expression of INSR on BECs of invasive bladder cancers was significantly associated with shorter progression-free and overall survival. Furthermore, increased expression of the INSR ligand IGF-2 in invasive bladder cancers was associated with reduced overall survival. INSR may therefore represent a novel biomarker to predict cancer progression. Mechanistically, we observed pronounced hypoxia in human bladder cancer tissue, and found a positive correlation between the expression of the hypoxia marker gene GLUT1 and vascular INSR expression, indicating that hypoxia drives INSR expression in tumour-associated blood vessels. In line with this, exposure of cultured BECs and human bladder cancer cell lines to hypoxia led to increased expression of INSR and IGF-2, respectively, and IGF-2 increased BEC migration through the activation of INSR in vitro. Taken together, we identified vascular INSR expression as a potential biomarker for progression in bladder cancer. Furthermore, our data suggest that IGF-2/INSR mediated paracrine crosstalk between bladder cancer cells and endothelial cells is functionally involved in tumour angiogenesis and may thus represent a new therapeutic target. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Transcriptional profiling of macrophage and tumor cell interactions in vitro.

Macrophages are important mediators of tumor progression and their function is broadly influenced by different microenvironmental stimuli. To understand the molecular basis of the tumor-supporting role of macrophages in aggressive breast cancer we co-cultured human peripheral monocytes with two breast cancer cell lines representing distinct aggressive cellular phenotype and transcriptionally profiled the changes occurring in both cells during in vitro activated crosstalk. Here we provide a detailed description of the experimental design, sample identity and analysis of the Illumina RNA-Seq data, which have been deposited into Gene Expression Omnibus (GEO): GSE75130.

CD73 Predicts Favorable Prognosis in Patients with Nonmuscle-Invasive Urothelial Bladder Cancer.

AIMS: CD73 is a membrane associated 5'-ectonucleotidase that has been proposed as prognostic biomarker in various solid tumors. The aim of this study is to evaluate CD73 expression in a cohort of patients with primary bladder cancer in regard to its association with clinicopathological features and disease course. METHODS: Tissue samples from 174 patients with a primary urothelial carcinoma were immunohistochemically assessed on a tissue microarray. Associations between CD73 expression and retrospectively obtained clinicopathological data were evaluated by contingency analysis. Survival analysis was performed to investigate the predictive value of CD73 within the subgroup of pTa and pT1 tumors in regard to progression-free survival (PFS). RESULTS: High CD73 expression was found in 46 (26.4%) patients and was significantly associated with lower stage, lower grade, less adjacent carcinoma in situ and with lower Ki-67 proliferation index. High CD73 immunoreactivity in the subgroup of pTa and pT1 tumors (n = 158) was significantly associated with longer PFS (HR: 0.228; p = 0.047) in univariable Cox regression analysis. CONCLUSION: High CD73 immunoreactivity was associated with favorable clinicopathological features. Furthermore, it predicts better outcome in the subgroup of pTa and pT1 tumors and may thus serve as additional tool for the selection of patients with favorable prognosis.

Connexin 43 expression predicts poor progression-free survival in patients with non-muscle invasive urothelial bladder cancer.

OBJECTIVES: To evaluate the protein expression of connexin 43 (Cx43) in primary urothelial bladder cancer and test its association with the histopathological characteristics and clinical outcome. METHODS: A tissue microarray containing 348 tissue samples from 174 patients with primary urothelial carcinomas of the bladder was immunohistochemically stained for Cx43. The intensity of staining was semiquantitatively evaluated (score 0, 1+, 2+), and the association with clinicopathological features was assessed. Univariable and multivariable analyses were performed to identify predictors for progression-free survival (PFS). RESULTS: Membranous Cx43 immunoreactivity was detected in 118 (67.8%) of 174 analysable urothelial carcinomas, of which 31 (17.8%) showed even a strong (score 2+) and mainly homogeneous staining. Strong expression levels of Cx43 (score 2+) were associated with higher tumour grade, multiplicity and increased proliferation (all p<0.05). In the subgroup of patients with stage pTa and pT1 bladder tumours (n=158), strong Cx43 expression (p<0.001), solid growth pattern (p<0.001) and increased Ki-67 proliferation fraction (p<0.05) were significantly associated with shorter PFS in an univariable Cox regression analysis. In multivariable Cox regression models, Cx43 immunoreactivity and histological growth pattern remained highly significant and adverse risk factors for PFS. CONCLUSIONS: The expression levels of Cx43 are frequent in non-muscle invasive bladder cancer (NMIBC), with high expression levels being associated with poor prognosis. Routine assessment of Cx43 expression may improve the identification of high-risk NMIBC.

Characterization of macrophage--cancer cell crosstalk in estrogen receptor positive and triple-negative breast cancer.

Tumor heterogeneity may broadly influence the activation of tumor-associated macrophages. We aimed to dissect how breast cancer cells of different molecular characteristics contribute to macrophage phenotype and function. Therefore, we performed whole transcriptome sequencing of human monocytes that were co-cultured with estrogen receptor positive (ER(+)) or triple-negative (TNBC) breast cancer cell lines and studied the biological responses related to the differential gene activation in both monocytes and cancer cells by pathway analysis. ER(+) and TNBC cancer cell lines induced distinctly different macrophage phenotypes with different biological functions, cytokine and chemokine secretion, and morphology. Conversely, ER(+) and TNBC breast cancer cell lines were distinctly influenced by the presence of macrophages. ER(+) cells demonstrated up-regulation of an acute phase inflammatory response, IL-17 signaling and antigen presentation pathway, whereas thioredoxin and vitamin D3 receptor pathways were down-regulated in the respective macrophages. The TNBC educated macrophages down-regulated citrulline metabolism and differentiated into M2-like macrophages with increased MMR protein expression and CCL2 secretion. These data demonstrate how different cancer cells educate the host cells to support tumor growth and might explain why high infiltration of macrophages in TNBC tumors associates with poor prognosis.