Three-Dimensional Imaging Analysis for Skeletal Muscle.

Skeletal muscle is a highly ordered tissue composed of a complex network of a diverse variety of cells. The dynamic spatial and temporal interaction between these cells during homeostasis and during times of injury gives the skeletal muscle its regenerative capacity. In order to properly understand the process of regeneration, a three-dimensional (3-D) imaging process must be conducted. While there have been several protocols studying 3-D imaging, it has primarily been focused on the nervous system. This protocol aims to outline the workflow for rendering a 3-D image of the skeletal muscle using spatial data from confocal microscope images. This protocol uses the ImageJ, Ilastik, and Imaris software for 3-D rendering and computational image analysis as both are relatively easy to use and have powerful segmentation capabilities.

Phase II study of ceralasertib (AZD6738) in combination with durvalumab in patients with advanced gastric cancer.

BACKGROUND: Targeting the DNA damage repair (DDR) pathways is an attractive strategy for boosting cancer immunotherapy. Ceralasertib (AZD6738) is an oral kinase inhibitor of ataxia telangiectasia and Rad3 related protein, which is a master regulator of DDR. We conducted a phase II trial of ceralasertib plus durvalumab in patients with previously treated advanced gastric cancer (AGC) to demonstrate the safety, tolerability, and clinical activity of the combination. METHODS: This phase II, open-label, single-center, non-randomized study was designed to evaluate the efficacy and safety of ceralasertib in combination with durvalumab in patients with AGC. The study drug regimen was ceralasertib (240 mg two times a day) days 15-28 in a 28-day cycle in combination with durvalumab (1500 mg) at day 1 every 4 weeks. The primary end point was overall response rate (ORR) by Response Evaluation Criteria in Solid Tumors (V.1.1). Exploratory biomarker analysis was performed using fresh tumor biopsies in all enrolled patients. RESULTS: Among 31 patients, the ORR, disease control rate, median progression-free survival (PFS), and overall survival were 22.6% (95% CI 9.6% to 41.1%), 58.1% (95% CI 39.1% to 75.5%), 3.0 (95% CI 2.1 to 3.9) months, and 6.7 (95% CI 3.8 to 9.6) months, respectively. Common adverse events were manageable with dose modification. A subgroup of patients with a loss of ataxia telangiectasia mutated (ATM) expression and/or high proportion of mutational signature attributable to homologous repair deficiency (sig. HRD) demonstrated a significantly longer PFS than those with intact ATM and low sig. HRD (5.60 vs 1.65 months; HR 0.13, 95% CI 0.045 to 0.39; long-rank p<0.001). During the study treatment, upregulation of the innate immune response by cytosolic DNA, activation of intratumoral lymphocytes, and expansion of circulating tumor-reactive CD8 +T cell clones were identified in responders. Enrichment of the tumor vasculature signature was associated with treatment resistance. CONCLUSIONS: Ceralasertib plus durvalumab has promising antitumor activity, with durable responses in patients with refractory AGC. Thus, a biomarker-driven trial is required. TRIAL REGISTRATION: NCT03780608.

Characterizing the Diffusion Property of Hydrogen Sorption and Desorption Processes in Several Spherical-Shaped Polymers.

We developed a method for characterizing permeation parameters in hydrogen sorption and desorption processes in polymers using the volumetric measurement technique. The technique was utilized for three polymers: nitrile butadiene rubber (NBR), ethylene propylene diene monomer (EPDM), and fluoroelastomer (FKM). The total uptake (C∞), total desorbed content (C0), diffusivity in sorption (Ds), and diffusivity in desorption (Dd) of hydrogen in the polymers were determined versus the sample diameter used in both processes. For all the polymers, the diameter dependence was not detected for C∞ and C0. The average C∞ and C0 at 5.75 MPa were 316 wt∙ppm and 291 wt∙ppm for NBR, 270 wt∙ppm and 279 wt∙ppm for EPDM, and 102 wt∙ppm and 93 wt∙ppm for FKM. The coincidence of C∞ and C0 in the sorption and desorption process indicated physisorption upon introducing hydrogen molecules into the polymers. The larger Dd in the desorption process than Ds could be attributed to an increased amorphous phase and volume swelling after decompression. The equilibrium time to reach the saturation of the hydrogen content in both processes was experimentally confirmed as proportional to the squared radius and consistent with the COMSOL simulation. This method could be used to predict the equilibrium time of the sorption time, depending on the radius of the polymers without any measurement.

An indole-based fluorescent chemosensor targeting the autophagosome.

Autophagy is a process for the degradation and recycling of intracellular components and dysfunctional organelles. We developed an indole-embedded fluorescent naphthalimide for the selective imaging of autophagosomes in live cells. It was shown as intense puncta in the fluorescence confocal images and co-localizes with an autophagosome marker, LC3-RFP. In addition, it was applied to cellular autophagic models based on ER stress and starvation to verify its capability.

Determinants of Response and Intrinsic Resistance to PD-1 Blockade in Microsatellite Instability-High Gastric Cancer.

Sequence alterations in microsatellites and an elevated mutational burden are observed in 20% of gastric cancers and associated with clinical response to anti-PD-1 antibodies. However, 50% of microsatellite instability-high (MSI-H) cancers are intrinsically resistant to PD-1 therapies. We conducted a phase II trial of pembrolizumab in patients with advanced MSI-H gastric cancer and included serial and multi-region tissue samples in addition to serial peripheral blood analyses. The number of whole-exome sequencing (WES)-derived nonsynonymous mutations correlated with antitumor activity and prolonged progression-free survival (PFS). Coupling WES to single-cell RNA sequencing, we identified dynamic tumor evolution with greater on-treatment collapse of mutational architecture in responders. Diverse T-cell receptor repertoire was associated with longer PFS to pembrolizumab. In addition, an increase in PD-1+ CD8+ T cells correlated with durable clinical benefit. Our findings highlight the genomic, immunologic, and clinical outcome heterogeneity within MSI-H gastric cancer and may inform development of strategies to enhance responsiveness. SIGNIFICANCE: This study highlights response heterogeneity within MSI-H gastric cancer treated with pembrolizumab monotherapy and underscores the potential for extended baseline and early on-treatment biomarker analyses to identify responders. The observed markers of intrinsic resistance have implications for patient stratification to inform novel combinations among patients with intrinsically resistant features.See related commentary by Fontana and Smyth, p. 2126.This article is highlighted in the In This Issue feature, p. 2113.

Bispecific Antibody Molecule Inhibits Tumor Cell Proliferation More Efficiently Than the Two-Molecule Combination.

BACKGROUND: Monoclonal antibodies (mAbs) have proved to be a valuable tool for the treatment of different cancer types. However, clinical use of an increasing number of mAbs, have also highlighted limitations with monotherapy for cancers, in particular for such with more complex mechanisms, requiring action on additional molecules or pathways, or for cancers quickly acquiring resistance following monotherapy. An example for the latter is the mAb trastuzumab, FDA approved for treatment of metastatic gastric carcinoma. To circumvent this, researchers have reported synergistic, anti-proliferative effects by combination targeting of HER2 and EGFR by trastuzumab and the EGFR-targeting mAb Cetuximab overcoming trastuzumab resistance. METHODS: Maintaining the proven functionality of trastuzumab, we have designed bi-specific antibody molecules, called AffiMabs, by fusing an EGFR-targeting Affibody molecule to trastuzumab's heavy or light chains. Having confirmed binding to EGFR and Her2 and cytotoxicity of our AffiMabs, we analyzed apoptosis rate, receptor surface levels, phosphorylation levels of receptors and associated signaling pathways as well as differentially expressed genes on transcriptome level with the aim to elucidate the mode of action of our AffiMabs. RESULTS: The AffiMabs are able to simultaneously bind HER2 and EGFR and show increased cytotoxic effect compared to the original trastuzumab therapeutic molecule and, more importantly, even to the combination of trastuzumab and EGFR-targeting Affibody molecule. Analyzing the mode of action, we could show that bi-specific AffiMabs lead to reduced surface receptor levels and a downregulation of cell cycle associated genes on transcriptome level. CONCLUSION: Our study shows that transcriptome analysis can be used to validate the choice of receptor targets and guide the design of novel multi-specific molecules. The inherent modularity of the AffiMab format renders it readily applicable to other receptor targets.

Real-Time Monitoring for Hydraulic States Based on Convolutional Bidirectional LSTM with Attention Mechanism.

By monitoring a hydraulic system using artificial intelligence, we can detect anomalous data in a manufacturing workshop. In addition, by analyzing the anomalous data, we can diagnose faults and prevent failures. However, artificial intelligence, especially deep learning, needs to learn much data, and it is often difficult to get enough data at the real manufacturing site. In this paper, we apply augmentation to increase the amount of data. In addition, we propose real-time monitoring based on a deep-learning model that uses convergence of a convolutional neural network (CNN), a bidirectional long short-term memory network (BiLSTM), and an attention mechanism. CNN extracts features from input data, and BiLSTM learns feature information. The learned information is then fed to the sigmoid classifier to find out if it is normal or abnormal. Experimental results show that the proposed model works better than other deep-learning models, such as CNN or long short-term memory (LSTM).

DNA methylation disruption reshapes the hematopoietic differentiation landscape.

Mutations in genes involved in DNA methylation (DNAme; for example, TET2 and DNMT3A) are frequently observed in hematological malignancies1-3 and clonal hematopoiesis4,5. Applying single-cell sequencing to murine hematopoietic stem and progenitor cells, we observed that these mutations disrupt hematopoietic differentiation, causing opposite shifts in the frequencies of erythroid versus myelomonocytic progenitors following Tet2 or Dnmt3a loss. Notably, these shifts trace back to transcriptional priming skews in uncommitted hematopoietic stem cells. To reconcile genome-wide DNAme changes with specific erythroid versus myelomonocytic skews, we provide evidence in support of differential sensitivity of transcription factors due to biases in CpG enrichment in their binding motif. Single-cell transcriptomes with targeted genotyping showed similar skews in transcriptional priming of DNMT3A-mutated human clonal hematopoiesis bone marrow progenitors. These data show that DNAme shapes the topography of hematopoietic differentiation, and support a model in which genome-wide methylation changes are transduced to differentiation skews through biases in CpG enrichment of the transcription factor binding motif.

EpiMethylTag: simultaneous detection of ATAC-seq or ChIP-seq signals with DNA methylation.

Activation of regulatory elements is thought to be inversely correlated with DNA methylation levels. However, it is difficult to determine whether DNA methylation is compatible with chromatin accessibility or transcription factor (TF) binding if assays are performed separately. We developed a fast, low-input, low sequencing depth method, EpiMethylTag, that combines ATAC-seq or ChIP-seq (M-ATAC or M-ChIP) with bisulfite conversion, to simultaneously examine accessibility/TF binding and methylation on the same DNA. Here we demonstrate that EpiMethylTag can be used to study the functional interplay between chromatin accessibility and TF binding (CTCF and KLF4) at methylated sites.

Revealing Protein Aggregates under Thapsigargin-Induced ER Stress Using an ER-Targeted Thioflavin.

Endoplasmic reticulum-thioflavin T (ER-ThT), a thioflavin T-based fluorescent chemosensor, was developed to detect protein aggregates in the endoplasmic reticulum (ER) and was applied to live cells under various forms of ER stress. Upon dithiothreitol (DTT)-induced reductive denaturation of lysozyme and albumin, the intensity was increased in a protein concentration-dependent way, following a nonfluorescent lag phase. ER-ThT detects protein aggregates rather than unfolded proteins in solution, and the protein aggregation can be visualized in the presence of lipid membranes or native proteins. Within live HeLa cells, ER-ThT is localized in the ER and its fluorescence was dramatically increased upon ER stress induction by DTT, Thapsigargin, or Brefeldin A. Moreover, in the presence of ER stress modulators (tauroursodeoxycholic acid, trimethylamine N-oxide, or 4-phenylbutyric acid), also known as chemical chaperones, the fluorescence under Thapsigargin treatment was suppressed to the level of the control group. Thus, ER-ThT is capable of detecting the accumulation of protein aggregates under ER stress in living cells and acts as an in vitro screening tool for ER stress modulators, putative prodrugs against ER-related proteopathy. Overall, the results strongly suggest that protein aggregation is intricately involved in the activation of the unfolded protein response following ER stress.

Impedance spectroscopy for in situ and real-time observations of the effects of hydrogen on nitrile butadiene rubber polymer under high pressure.

Nondestructive impedance spectroscopy (IS) was developed and demonstrated to detect the effects of hydrogen on nitrile butadiene rubber exposed to hydrogen gas (H2) at high pressures up to 10 MPa. IS was applied to obtain an in situ and real-time quantification of H2 penetration into and its desorption out of rubber under high pressure. The diffusion coefficients of H2 were also obtained from the time evolution of the capacitance, which were compared with those obtained by thermal desorption gas analysis. The in situ measurements of the capacitance and the dissipation factor under various pressures during cyclic stepwise pressurization and decompression demonstrated the diffusion behaviour of H2, the phase of the rubber under high pressure, the transport properties of H2 gas, and the physicochemical interaction between H2 and the rubber. These phenomena were supported by a COMSOL simulation based on the electric current conservation equation and scanning electron microscopy (SEM) observations.

Somatic mutations and cell identity linked by Genotyping of Transcriptomes.

Defining the transcriptomic identity of malignant cells is challenging in the absence of surface markers that distinguish cancer clones from one another, or from admixed non-neoplastic cells. To address this challenge, here we developed Genotyping of Transcriptomes (GoT), a method to integrate genotyping with high-throughput droplet-based single-cell RNA sequencing. We apply GoT to profile 38,290 CD34+ cells from patients with CALR-mutated myeloproliferative neoplasms to study how somatic mutations corrupt the complex process of human haematopoiesis. High-resolution mapping of malignant versus normal haematopoietic progenitors revealed an increasing fitness advantage with myeloid differentiation of cells with mutated CALR. We identified the unfolded protein response as a predominant outcome of CALR mutations, with a considerable dependency on cell identity, as well as upregulation of the NF-κB pathway specifically in uncommitted stem cells. We further extended the GoT toolkit to genotype multiple targets and loci that are distant from transcript ends. Together, these findings reveal that the transcriptional output of somatic mutations in myeloproliferative neoplasms is dependent on the native cell identity.

Epigenetic evolution and lineage histories of chronic lymphocytic leukaemia.

Genetic and epigenetic intra-tumoral heterogeneity cooperate to shape the evolutionary course of cancer1. Chronic lymphocytic leukaemia (CLL) is a highly informative model for cancer evolution as it undergoes substantial genetic diversification and evolution after therapy2,3. The CLL epigenome is also an important disease-defining feature4,5, and growing populations of cells in CLL diversify by stochastic changes in DNA methylation known as epimutations6. However, previous studies using bulk sequencing methods to analyse the patterns of DNA methylation were unable to determine whether epimutations affect CLL populations homogeneously. Here, to measure the epimutation rate at single-cell resolution, we applied multiplexed single-cell reduced-representation bisulfite sequencing to B cells from healthy donors and patients with CLL. We observed that the common clonal origin of CLL results in a consistently increased epimutation rate, with low variability in the cell-to-cell epimutation rate. By contrast, variable epimutation rates across healthy B cells reflect diverse evolutionary ages across the trajectory of B cell differentiation, consistent with epimutations serving as a molecular clock. Heritable epimutation information allowed us to reconstruct lineages at high-resolution with single-cell data, and to apply this directly to patient samples. The CLL lineage tree shape revealed earlier branching and longer branch lengths than in normal B cells, reflecting rapid drift after the initial malignant transformation and a greater proliferative history. Integration of single-cell bisulfite sequencing analysis with single-cell transcriptomes and genotyping confirmed that genetic subclones mapped to distinct clades, as inferred solely on the basis of epimutation information. Finally, to examine potential lineage biases during therapy, we profiled serial samples during ibrutinib-associated lymphocytosis, and identified clades of cells that were preferentially expelled from the lymph node after treatment, marked by distinct transcriptional profiles. The single-cell integration of genetic, epigenetic and transcriptional information thus charts the lineage history of CLL and its evolution with therapy.

Corrupted coordination of epigenetic modifications leads to diverging chromatin states and transcriptional heterogeneity in CLL.

Cancer evolution is fueled by epigenetic as well as genetic diversity. In chronic lymphocytic leukemia (CLL), intra-tumoral DNA methylation (DNAme) heterogeneity empowers evolution. Here, to comprehensively study the epigenetic dimension of cancer evolution, we integrate DNAme analysis with histone modification mapping and single cell analyses of RNA expression and DNAme in 22 primary CLL and 13 healthy donor B lymphocyte samples. Our data reveal corrupted coherence across different layers of the CLL epigenome. This manifests in decreased mutual information across epigenetic modifications and gene expression attributed to cell-to-cell heterogeneity. Disrupted epigenetic-transcriptional coordination in CLL is also reflected in the dysregulation of the transcriptional output as a function of the combinatorial chromatin states, including incomplete Polycomb-mediated gene silencing. Notably, we observe unexpected co-mapping of typically mutually exclusive activating and repressing histone modifications, suggestive of intra-tumoral epigenetic diversity. Thus, CLL epigenetic diversification leads to decreased coordination across layers of epigenetic information, likely reflecting an admixture of cells with diverging cellular identities.

Intratumor heterogeneity inferred from targeted deep sequencing as a prognostic indicator.

Tumor genetic heterogeneity may underlie poor clinical outcomes because diverse subclones could be comprised of metastatic and drug resistant cells. Targeted deep sequencing has been used widely as a diagnostic tool to identify actionable mutations in cancer patients. In this study, we evaluated the clinical utility of estimating tumor heterogeneity using targeted panel sequencing data. We investigated the prognostic impact of a tumor heterogeneity (TH) index on clinical outcomes, using mutational profiles from targeted deep sequencing data acquired from 1,352 patients across 8 cancer types. The TH index tended to be increased in high pathological stage disease in several cancer types, indicating clonal expansion of cancer cells as tumor progression proceeds. In colorectal cancer patients, TH index values also correlated significantly with clinical prognosis. Integration of the TH index with genomic and clinical features could improve the power of risk prediction for clinical outcomes. In conclusion, deep sequencing to determine the TH index could serve as a promising prognostic indicator in cancer patients.

High-depth fluorescence imaging using a two-photon FRET system for mitochondrial pH in live cells and tissues.

We developed a fluorescent pH probe (1) capable of two-photon excitation and far-visible-emission based on FRET, composed of naphthalimide-piperazine-rhodamine. It exhibited a pH-dependent reversible and fast ratiometric fluorescence change in the rhodamine emission. Probe 1 was applied to image the pH perturbations of mitochondria in living cells and tissues.

Establishment of a Patient-derived Xenograft for Development of Personalized HER2-targeting Therapy in Gastric Cancer.

BACKGROUND/AIM: To maximize success rate for development of HER2-targeted therapeutics, patient-derived xenograft (PDX) models reflecting HER2-positive gastric cancer (HER2+ GC) patients were established. MATERIALS AND METHODS: GC tissues obtained from surgery of GC patients were implanted into immune-deficient mice, and tumor tissue of HER2+ PDXs were verified of the patient-mimic HER2 expression by immunohistochemistry and explored for the feasibility by testing with Herceptin, the approved therapeutics and novel HER2 antibody therapeutics being developed. RESULTS: We obtained 5 cases of HER2+ GC PDX models reflecting patient's GC tumor, consisting of 2 cases of HER2 3+ and 2 cases of HER2 2+. Novel HER2 antibody displayed significantly improved anti-cancer efficacy in combination with Herceptin. CONCLUSION: The HER2+ GC PDX models were successfully established to be utilized for preclinical evaluation of HER2-targeting drugs and combined therapies for GC treatment, as an ideal platform of personalized tools for precision therapy.

SIDR: simultaneous isolation and parallel sequencing of genomic DNA and total RNA from single cells.

Simultaneous sequencing of the genome and transcriptome at the single-cell level is a powerful tool for characterizing genomic and transcriptomic variation and revealing correlative relationships. However, it remains technically challenging to analyze both the genome and transcriptome in the same cell. Here, we report a novel method for simultaneous isolation of genomic DNA and total RNA (SIDR) from single cells, achieving high recovery rates with minimal cross-contamination, as is crucial for accurate description and integration of the single-cell genome and transcriptome. For reliable and efficient separation of genomic DNA and total RNA from single cells, the method uses hypotonic lysis to preserve nuclear lamina integrity and subsequently captures the cell lysate using antibody-conjugated magnetic microbeads. Evaluating the performance of this method using real-time PCR demonstrated that it efficiently recovered genomic DNA and total RNA. Thorough data quality assessments showed that DNA and RNA simultaneously fractionated by the SIDR method were suitable for genome and transcriptome sequencing analysis at the single-cell level. The integration of single-cell genome and transcriptome sequencing by SIDR (SIDR-seq) showed that genetic alterations, such as copy-number and single-nucleotide variations, were more accurately captured by single-cell SIDR-seq compared with conventional single-cell RNA-seq, although copy-number variations positively correlated with the corresponding gene expression levels. These results suggest that SIDR-seq is potentially a powerful tool to reveal genetic heterogeneity and phenotypic information inferred from gene expression patterns at the single-cell level.

Clinical Application of Targeted Deep Sequencing in Solid-Cancer Patients and Utility for Biomarker-Selected Clinical Trials.

Molecular profiling of actionable mutations in refractory cancer patients has the potential to enable "precision medicine," wherein individualized therapies are guided based on genomic profiling. The molecular-screening program was intended to route participants to different candidate drugs in trials based on clinical-sequencing reports. In this screening program, we used a custom target-enrichment panel consisting of cancer-related genes to interrogate single-nucleotide variants, insertions and deletions, copy number variants, and a subset of gene fusions. From August 2014 through April 2015, 654 patients consented to participate in the program at Samsung Medical Center. Of these patients, 588 passed the quality control process for the 381-gene cancer-panel test, and 418 patients were included in the final analysis as being eligible for any anticancer treatment (127 gastric cancer, 122 colorectal cancer, 62 pancreatic/biliary tract cancer, 67 sarcoma/other cancer, and 40 genitourinary cancer patients). Of the 418 patients, 55 (12%) harbored a biomarker that guided them to a biomarker-selected clinical trial, and 184 (44%) patients harbored at least one genomic alteration that was potentially targetable. This study demonstrated that the panel-based sequencing program resulted in an increased rate of trial enrollment of metastatic cancer patients into biomarker-selected clinical trials. Given the expanding list of biomarker-selected trials, the guidance percentage to matched trials is anticipated to increase. IMPLICATIONS FOR PRACTICE: This study demonstrated that the panel-based sequencing program resulted in an increased rate of trial enrollment of metastatic cancer patients into biomarker-selected clinical trials. Given the expanding list of biomarker-selected trials, the guidance percentage to matched trials is anticipated to increase.

Misfolded polypeptides are selectively recognized and transported toward aggresomes by a CED complex.

Misfolded polypeptides are rapidly cleared from cells via the ubiquitin-proteasome system (UPS). However, when the UPS is impaired, misfolded polypeptides form small cytoplasmic aggregates, which are sequestered into an aggresome and ultimately degraded by aggrephagy. Despite the relevance of the aggresome to neurodegenerative proteinopathies, the molecular mechanisms underlying aggresome formation remain unclear. Here we show that the CTIF-eEF1A1-DCTN1 (CED) complex functions in the surveillance of either pre-existing or newly synthesized polypeptides by linking two molecular events: selective recognition and aggresomal targeting of misfolded polypeptides. These events are accompanied by CTIF sequestration into the aggresome, preventing the additional synthesis of misfolded polypeptides from mRNAs bound by nuclear cap-binding complex. These events render cells more resistant to apoptosis induced by proteotoxic stresses. Collectively, our data provide compelling evidence for a previously unappreciated protein surveillance pathway and a regulatory gene expression network for coping with misfolded polypeptides.