scAmpi-A versatile pipeline for single-cell RNA-seq analysis from basics to clinics.

Single-cell RNA sequencing (scRNA-seq) has emerged as a powerful technique to decipher tissue composition at the single-cell level and to inform on disease mechanisms, tumor heterogeneity, and the state of the immune microenvironment. Although multiple methods for the computational analysis of scRNA-seq data exist, their application in a clinical setting demands standardized and reproducible workflows, targeted to extract, condense, and display the clinically relevant information. To this end, we designed scAmpi (Single Cell Analysis mRNA pipeline), a workflow that facilitates scRNA-seq analysis from raw read processing to informing on sample composition, clinically relevant gene and pathway alterations, and in silico identification of personalized candidate drug treatments. We demonstrate the value of this workflow for clinical decision making in a molecular tumor board as part of a clinical study.

Correction: Immunosuppressive FK506 treatment leads to more frequent EBV-associated lymphoproliferative disease in humanized mice.

[This corrects the article DOI: 10.1371/journal.ppat.1008477.].

Immunosuppressive FK506 treatment leads to more frequent EBV-associated lymphoproliferative disease in humanized mice.

Post-transplant lymphoproliferative disorder (PTLD) is a potentially fatal complication after organ transplantation frequently associated with the Epstein-Barr virus (EBV). Immunosuppressive treatment is thought to allow the expansion of EBV-infected B cells, which often express all eight oncogenic EBV latent proteins. Here, we assessed whether HLA-A2 transgenic humanized NSG mice treated with the immunosuppressant FK506 could be used to model EBV-PTLD. We found that FK506 treatment of EBV-infected mice led to an elevated viral burden, more frequent tumor formation and diminished EBV-induced T cell responses, indicative of reduced EBV-specific immune control. EBV latency III and lymphoproliferation-associated cellular transcripts were up-regulated in B cells from immunosuppressed animals, akin to the viral and host gene expression pattern found in EBV-PTLD. Utilizing an unbiased gene expression profiling approach, we identified genes differentially expressed in B cells of EBV-infected animals with and without FK506 treatment. Upon investigating the most promising candidates, we validated sCD30 as a marker of uncontrolled EBV proliferation in both humanized mice and in pediatric patients with EBV-PTLD. High levels of sCD30 have been previously associated with EBV-PTLD in patients. As such, we believe that humanized mice can indeed model aspects of EBV-PTLD development and may prove useful for the safety assessment of immunomodulatory therapies.

A random motility assay based on image correlation spectroscopy.

We demonstrate the random motility (RAMOT) assay based on image correlation spectroscopy for the automated, label-free, high-throughput characterization of random cell migration. The approach is complementary to traditional migration assays, which determine only the collective net motility in a particular direction. The RAMOT assay is less demanding on image quality compared to single-cell tracking, does not require cell identification or trajectory reconstruction, and performs well on live-cell, time-lapse, phase contrast video microscopy of hundreds of cells in parallel. Effective diffusion coefficients derived from the RAMOT analysis are in quantitative agreement with Monte Carlo simulations and allowed for the detection of pharmacological effects on macrophage-like cells migrating on a planar collagen matrix. These results expand the application range of image correlation spectroscopy to multicellular systems and demonstrate a novel, to our knowledge, migration assay with little preparative effort.

scROSHI: robust supervised hierarchical identification of single cells.

Identifying cell types based on expression profiles is a pillar of single cell analysis. Existing machine-learning methods identify predictive features from annotated training data, which are often not available in early-stage studies. This can lead to overfitting and inferior performance when applied to new data. To address these challenges we present scROSHI, which utilizes previously obtained cell type-specific gene lists and does not require training or the existence of annotated data. By respecting the hierarchical nature of cell type relationships and assigning cells consecutively to more specialized identities, excellent prediction performance is achieved. In a benchmark based on publicly available PBMC data sets, scROSHI outperforms competing methods when training data are limited or the diversity between experiments is large.

Advanced Diffusion-Weighted Imaging Sequences for Breast MRI: Comprehensive Comparison of Improved Sequences and Ultra-High B-Values to Identify the Optimal Combination.

This study investigated the image quality and choice of ultra-high b-value of two DWI breast-MRI research applications. The study cohort comprised 40 patients (20 malignant lesions). In addition to s-DWI with two m-b-values (b50 and b800) and three e-b-values (e-b1500, e-b2000, and e-b2500), z-DWI and IR m-b1500 DWI were applied. z-DWI was acquired with the same measured b-values and e-b-values as the standard sequence. For IR m-b1500 DWI, b50 and b1500 were measured, and e-b2000 and e-b2500 were mathematically extrapolated. Three readers used Likert scales to independently analyze all ultra-high b-values (b1500-b2500) for each DWI with regards to scan preference and image quality. ADC values were measured in all 20 lesions. z-DWI was the most preferred (54%), followed by IR m-b1500 DWI (46%). b1500 was significantly preferred over b2000 for z-DWI and IR m-b1500 DWI (p = 0.001 and p = 0.002, respectively). Lesion detection was not significantly different among sequences or b-values (p = 0.174). There were no significant differences in measured ADC values within lesions between s-DWI (ADC: 0.97 [±0.09] × 10-3 mm2/s) and z-DWI (ADC: 0.99 [±0.11] × 10-3 mm2/s; p = 1.000). However, there was a trend toward lower values in IR m-b1500 DWI (ADC: 0.80 [±0.06] × 10-3 mm2/s) than in s-DWI (p = 0.090) and z-DWI (p = 0.110). Overall, image quality was superior and there were fewer image artifacts when using the advanced sequences (z-DWI + IR m-b1500 DWI) compared with s-DWI. Considering scan preferences, we found that the optimal combination was z-DWI with a calculated b1500, especially regarding examination time.

Quantitative Assessment of Iteratively Denoised 3D SPACE with Inner-Volume Excitation and Simultaneous Multi-Slice BLADE for Optimizing Female Pelvis Magnetic Resonance Imaging at 1.5 T.

RATIONALE AND OBJECTIVES: High-resolution T2-weighted magnetic resonance imaging (MRI) of the pelvis is the main technique used for diagnosing benign and malignant uterine diseases. However, the procedure may be time-consuming and requires training and experience. Therefore, this study was performed to compare the image quality of standard clinical BLADE (stBLADE) with a prototypical accelerated simultaneous multi-slice (SMS) BLADE procedure with either improved temporal resolution (tr) at the same slice thickness (SL) or improved spatial resolution (sr) with the same examination time and a prototypical isotropic 3D SPACE procedure with inner-volume excitation and iterative denoising. MATERIALS AND METHODS: Patients who underwent clinically indicated MRI of the uterus were included in this prospective study and underwent stBLADE (acquisition time, 2 min 59 s; SL, 4 mm) and SMS BLADE (tr) with the same SL (4 mm) but reduced examination time (1 min 20 s) as well as SMS BLADE (sr) with thinner slices (3 mm) and comparable examination time (3 min 16 s). In addition, 3D SPACE was acquired in a sagittal orientation (5 min 36 s). The short axis of the cervix and the long axis of the corpus uteri were reconstructed in 1-mm and 3-mm SLs, retrospectively. Subjective overall image impression, delineation of anatomy/organs, lesion demarcation, and motion artifacts were assessed using a 5-point Likert scale and compared among the different techniques. The preferred sequence was then selected by three independent assessors. RESULTS: The analysis was based on 38 women (mean age, 44 ± 15 years). The overall image impression was similar for stBLADE, SMS BLADE (sr), and SMS BLADE (tr) but was significantly lower for 3D SPACE than stBLADE (p = 0.01). SMS BLADE (sr) was considered the preferred sequence because of slightly better performance in terms of overall image impression, organ delineation, and lesion demarcation, but without statistical significance. Both SMS BLADE (tr) and (sr) produced significantly fewer motion artifacts than stBLADE (p < 0.01 and p = 0.01), with no significant difference between SMS BLADE (tr) and (sr), while 3D SPACE had a significantly lower rating than stBLADE (p < 0.01). Image quality was rated as the least diagnostic criterion in all sequences and all cases. CONCLUSION: SMS BLADE (sr) was the preferred sequence for MRI of the female pelvis, with higher sr than stBLADE. SMS BLADE (tr) may also be used to reduce the acquisition time without compromising image quality. Despite its lower image quality, 3D SPACE can also reduce the examination time and improve the workflow because of the possibility of retrospective multiplanar reconstructions.

Real-time cellular impedance measurements detect Ca(2+) channel-dependent oscillations of morphology in human H295R adrenoma cells.

Endocrine cells, such as H295R have been widely used to study secretion of steroid and other hormones. Exocytosis-dependent hormone release is accompanied by an increase in plasma membrane surface area and a decrease in vesicle content. Recovery of vesicles and decrease in plasma membrane area is achieved by endocytotic processes. These changes in the extent of the surface area lead to morphological changes which can be determined by label-free real-time impedance measurements. Exo- and endocytosis have been described to be triggered by activation of L-type Ca(2+) channels. The present study demonstrates that activation of L-type calcium channels induces prolonged oscillating changes in cellular impedance. The data support the hypothesis that a tight regulation of the intracellular Ca(2+) concentration is a prerequisite for the observed cellular impedance oscillations. Furthermore evidence is presented for a mechanism in which the oscillations depend on a Ca(2+)-triggered calmodulin-dependent cascade involving myosin light chain kinase, nonmuscle myosin II and ultimately actin polymerization, a known determinant for cell shape changes and exocytosis in secretory cells. The described assay provides a method to determine continuously prolonged changes in cellular morphology such as exo/endocytosis cycles. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

Can Dynamic Contrast-enhanced MRI Contribute to Improved Assessment of Rectosigmoid Involvement in Deep Infiltrating Endometriosis?

BACKGROUND/AIM: To determine whether a prototypical compressed-sensing volume-interpolated breath-hold (csVIBE) provides diagnostic value in detecting rectosigmoid infiltration in deep infiltrating endometriosis (DIE). PATIENTS AND METHODS: csVIBE was employed in 151 women undergoing pelvic magnetic resonance imaging, of whom 43 had undergone surgery for suspected endometriosis. The accuracy of T2-weighted BLADE and BLADE/csVIBE, additional diagnostic value of csVIBE, and diagnostic confidence were rated by two readers. Additionally, the presence of the "mushroom cap sign" was assessed on BLADE and csVIBE. RESULTS: The diagnostic accuracy, sensitivity, and specificity of BLADE and BLADE/csVIBE were not significantly different between Readers A and B. For both readers, the confidence in the diagnosis increased with csVIBE, but this increase in the odds ratio was not significant for both readers. Both readers preferred csVIBE over BLADE with regard to detection of the "mushroom cap sign." CONCLUSION: csVIBE may provide a diagnostic benefit for surgical strategy selection through better delineation of the "mushroom cap sign."

The Tumor Profiler Study: integrated, multi-omic, functional tumor profiling for clinical decision support.

The application and integration of molecular profiling technologies create novel opportunities for personalized medicine. Here, we introduce the Tumor Profiler Study, an observational trial combining a prospective diagnostic approach to assess the relevance of in-depth tumor profiling to support clinical decision-making with an exploratory approach to improve the biological understanding of the disease.

Enhancing gene set enrichment using networks.

Differential gene expression (DGE) studies often suffer from poor interpretability of their primary results, i.e., thousands of differentially expressed genes. This has led to the introduction of gene set analysis (GSA) methods that aim at identifying interpretable global effects by grouping genes into sets of common context, such as, molecular pathways, biological function or tissue localization. In practice, GSA often results in hundreds of differentially regulated gene sets. Similar to the genes they contain, gene sets are often regulated in a correlative fashion because they share many of their genes or they describe related processes. Using these kind of neighborhood information to construct networks of gene sets allows to identify highly connected sub-networks as well as poorly connected islands or singletons. We show here how topological information and other network features can be used to filter and prioritize gene sets in routine DGE studies. Community detection in combination with automatic labeling and the network representation of gene set clusters further constitute an appealing and intuitive visualization of GSA results. The RICHNET workflow described here does not require human intervention and can thus be conveniently incorporated in automated analysis pipelines.

A novel anti-HER2 anthracycline-based antibody-drug conjugate induces adaptive anti-tumor immunity and potentiates PD-1 blockade in breast cancer.

Increasing evidence suggests that antibody-drug conjugates (ADCs) can enhance anti-tumor immunity and improve clinical outcome. Here, we elucidate the therapeutic efficacy and immune-mediated mechanisms of a novel HER2-targeting ADC bearing a potent anthracycline derivate as payload (T-PNU) in a human HER2-expressing syngeneic breast cancer model resistant to trastuzumab and ado-trastuzumab emtansine. Mechanistically, the anthracycline component of the novel ADC induced immunogenic cell death leading to exposure and secretion of danger-associated molecular signals. RNA sequencing derived immunogenomic signatures and TCRß clonotype analysis of tumor-infiltrating lymphocytes revealed a prominent role of the adaptive immune system in the regulation of T-PNU mediated anti-cancer activity. Depletion of CD8 T cells severely reduced T-PNU efficacy, thus confirming the role of cytotoxic T cells as drivers of the T-PNU mediated anti-tumor immune response. Furthermore, T-PNU therapy promoted immunological memory formation in tumor-bearing animals protecting those from tumor rechallenge. Finally, the combination of T-PNU and checkpoint inhibition, such as α-PD1, significantly enhanced tumor eradication following the treatment. In summary, a novel PNU-armed, HER2-targeting ADC elicited long-lasting immune protection in a murine orthotopic breast cancer model resistant to other HER2-directed therapies. Our findings delineate the therapeutic potential of this novel ADC payload and support its clinical development for breast cancer patients and potentially other HER2 expressing malignancies.

Machine learning-powered antibiotics phenotypic drug discovery.

Identification of novel antibiotics remains a major challenge for drug discovery. The present study explores use of phenotypic readouts beyond classical antibacterial growth inhibition adopting a combined multiparametric high content screening and genomic approach. Deployment of the semi-automated bacterial phenotypic fingerprint (BPF) profiling platform in conjunction with a machine learning-powered dataset analysis, effectively allowed us to narrow down, compare and predict compound mode of action (MoA). The method identifies weak antibacterial hits allowing full exploitation of low potency hits frequently discovered by routine antibacterial screening. We demonstrate that BPF classification tool can be successfully used to guide chemical structure activity relationship optimization, enabling antibiotic development and that this approach can be fruitfully applied across species. The BPF classification tool could be potentially applied in primary screening, effectively enabling identification of novel antibacterial compound hits and differentiating their MoA, hence widening the known antibacterial chemical space of existing pharmaceutical compound libraries. More generally, beyond the specific objective of the present work, the proposed approach could be profitably applied to a broader range of diseases amenable to phenotypic drug discovery.

Combined mutation in Vhl, Trp53 and Rb1 causes clear cell renal cell carcinoma in mice.

Clear cell renal cell carcinomas (ccRCCs) frequently exhibit inactivation of the von Hippel-Lindau tumor-suppressor gene, VHL, and often harbor multiple copy-number alterations in genes that regulate cell cycle progression. We show here that modeling these genetic alterations by combined deletion of Vhl, Trp53 and Rb1 specifically in renal epithelial cells in mice caused ccRCC. These tumors arose from proximal tubule epithelial cells and shared molecular markers and mRNA expression profiles with human ccRCC. Exome sequencing revealed that mouse and human ccRCCs exhibit recurrent mutations in genes associated with the primary cilium, uncovering a mutational convergence on this organelle and implicating a subset of ccRCCs as genetic ciliopathies. Different mouse tumors responded differently to standard therapies for advanced human ccRCC, mimicking the range of clinical behaviors in the human disease. Inhibition of hypoxia-inducible factor (HIF)-α transcription factors with acriflavine as third-line therapy had therapeutic effects in some tumors, providing preclinical evidence for further investigation of HIF-α inhibition as a ccRCC treatment. This autochthonous mouse ccRCC model represents a tool to investigate the biology of ccRCC and to identify new treatment strategies.

Molecular Phenotyping Combines Molecular Information, Biological Relevance, and Patient Data to Improve Productivity of Early Drug Discovery.

Today, novel therapeutics are identified in an environment which is intrinsically different from the clinical context in which they are ultimately evaluated. Using molecular phenotyping and an in vitro model of diabetic cardiomyopathy, we show that by quantifying pathway reporter gene expression, molecular phenotyping can cluster compounds based on pathway profiles and dissect associations between pathway activities and disease phenotypes simultaneously. Molecular phenotyping was applicable to compounds with a range of binding specificities and triaged false positives derived from high-content screening assays. The technique identified a class of calcium-signaling modulators that can reverse disease-regulated pathways and phenotypes, which was validated by structurally distinct compounds of relevant classes. Our results advocate for application of molecular phenotyping in early drug discovery, promoting biological relevance as a key selection criterion early in the drug development cascade.

MiR-99b-5p expression and response to tyrosine kinase inhibitor treatment in clear cell renal cell carcinoma patients.

A number of treatments targeting VEGF or mTOR pathways have been approved for metastatic clear cell Renal Cell Carcinoma (ccRCC), but the majority of patients show disease progression after first line therapy with a very low rate of complete or long-term responders. It has been shown that miRs may play a role in prediction of treatment response in various cancer types. The aim of our study was to identify a miR signature predictive for RCC patients' response to antiangiogenic tyrosine kinase inhibitor (TKI) treatment in the first line therapy. Sequencing of 40 paired normal/tumor formalin fixed and paraffin embedded ccRCC tissues revealed separate clustering via unsupervised dendrograms. With supervised analysis, the strongest differential expression was obtained with miR-99b-5p, which was significantly lower in patients with short progression free survival (<8 months) and TKI non-responders (progressive disease patients according to RECIST) (p<0.0001, each). Validation using RTqPCR and a second patient cohort compiled from three different hospitals (n=65) showed higher expression of miR-99b-5p in complete responders, but this trend did not reach statistical significance. It is concluded that low miR-99b-5p expression analyzed with sequencing methodology may correlate with tumor progression in TKI-treated ccRCC patients.

White-to-brown metabolic conversion of human adipocytes by JAK inhibition.

The rising incidence of obesity and related disorders such as diabetes and heart disease has focused considerable attention on the discovery of new therapeutics. One promising approach has been to increase the number or activity of brown-like adipocytes in white adipose depots, as this has been shown to prevent diet-induced obesity and reduce the incidence and severity of type 2 diabetes. Thus, the conversion of fat-storing cells into metabolically active thermogenic cells has become an appealing therapeutic strategy to combat obesity. Here, we report a screening platform for the identification of small molecules capable of promoting a white-to-brown metabolic conversion in human adipocytes. We identified two inhibitors of Janus kinase (JAK) activity with no precedent in adipose tissue biology that stably confer brown-like metabolic activity to white adipocytes. Importantly, these metabolically converted adipocytes exhibit elevated UCP1 expression and increased mitochondrial activity. We further found that repression of interferon signalling and activation of hedgehog signalling in JAK-inactivated adipocytes contributes to the metabolic conversion observed in these cells. Our findings highlight a previously unknown role for the JAK-STAT pathway in the control of adipocyte function and establish a platform to identify compounds for the treatment of obesity.

Generation of vascular endothelial and smooth muscle cells from human pluripotent stem cells.

The use of human pluripotent stem cells for in vitro disease modelling and clinical applications requires protocols that convert these cells into relevant adult cell types. Here, we report the rapid and efficient differentiation of human pluripotent stem cells into vascular endothelial and smooth muscle cells. We found that GSK3 inhibition and BMP4 treatment rapidly committed pluripotent cells to a mesodermal fate and subsequent exposure to VEGF-A or PDGF-BB resulted in the differentiation of either endothelial or vascular smooth muscle cells, respectively. Both protocols produced mature cells with efficiencies exceeding 80% within six days. On purification to 99% via surface markers, endothelial cells maintained their identity, as assessed by marker gene expression, and showed relevant in vitro and in vivo functionality. Global transcriptional and metabolomic analyses confirmed that the cells closely resembled their in vivo counterparts. Our results suggest that these cells could be used to faithfully model human disease.

Coupled rotation within single F0F1 enzyme complexes during ATP synthesis or hydrolysis.

F0F1 ATP synthases are the smallest rotary motors in nature and work as ATP factories in bacteria, plants and animals. Here we report on the first observation of intersubunit rotation in fully coupled single F0F1 molecules during ATP synthesis or hydrolysis. We investigate the Na+-translocating ATP synthase of Propionigenium modestum specifically labeled by a single fluorophore at one c subunit using polarization-resolved confocal microscopy. Rotation during ATP synthesis was observed with the immobilized enzyme reconstituted into proteoliposomes after applying a diffusion potential, but not with a Na+ concentration gradient alone. During ATP hydrolysis, stepwise rotation of the labeled c subunit was found in the presence of 2 mM NaCl, but not without the addition of Na+ ions. Moreover, upon the incubation with the F0-specific inhibitor dicyclohexylcarbodiimide the rotation was severely inhibited.