PURPOSE: Docetaxel resistance is a significant obstacle in the treatment of prostate cancer (PCa), resulting in unfavorable patient prognoses. Intratumoral heterogeneity, often associated with epithelial-to-mesenchymal transition (EMT), has previously emerged as a phenomenon that facilitates adaptation to various stimuli, thus promoting cancer cell diversity and eventually resistance to chemotherapy, including docetaxel. Hence, understanding intratumoral heterogeneity is essential for better patient prognosis and the development of personalized treatment strategies. METHODS: To address this, we employed a high-throughput single-cell flow cytometry approach to identify a specific surface fingerprint associated with docetaxel-resistance in PCa cells and complemented it with proteomic analysis of extracellular vesicles. We further validated selected antigens using docetaxel-resistant patient-derived xenografts in vivo and probed primary PCa specimens to interrogate of their surface fingerprint. RESULTS: Our approaches revealed a 6-molecule surface fingerprint linked to docetaxel resistance in primary PCa specimens. We observed consistent overexpression of CD95 (FAS/APO-1), and SSEA-4 surface antigens in both in vitro and in vivo docetaxel-resistant models, which was also observed in a cell subpopulation of primary PCa tumors exhibiting EMT features. Furthermore, CD95, along with the essential enzymes involved in SSEA-4 synthesis, ST3GAL1, and ST3GAL2, displayed a significant increase in patients with PCa undergoing docetaxel-based therapy, correlating with poor survival outcomes. CONCLUSION: In summary, we demonstrate that the identified 6-molecule surface fingerprint associated with docetaxel resistance pre-exists in a subpopulation of primary PCa tumors before docetaxel treatment. Thus, this fingerprint warrants further validation as a promising predictive tool for docetaxel resistance in PCa patients prior to therapy initiation.
- MeSH
- Drug Resistance, Neoplasm * MeSH
- Docetaxel * pharmacology therapeutic use MeSH
- Epithelial-Mesenchymal Transition drug effects MeSH
- Humans MeSH
- Mice MeSH
- Cell Line, Tumor MeSH
- Prostatic Neoplasms * pathology drug therapy metabolism MeSH
- Antineoplastic Agents pharmacology therapeutic use MeSH
- Xenograft Model Antitumor Assays MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Male MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
Cancer cells display complex genomic aberrations that include large-scale genetic rearrangements and epigenetic modulation that are not easily captured by short-read sequencing. This study presents a novel approach for simultaneous profiling of long-range genetic and epigenetic changes in matched cancer samples, focusing on clear cell renal cell carcinoma (ccRCC). ccRCC is a common kidney cancer subtype frequently characterized by a 3p deletion and the inactivation of the von Hippel-Lindau (VHL) gene. We performed integrated genetic, cytogenetic, and epigenetic analyses on paired tumor and adjacent nontumorous tissue samples. Optical genome mapping identified genomic aberrations as structural and copy number variations, complementing exome-sequencing findings. Single-molecule methylome and hydroxymethylome mapping revealed a significant global reduction in 5hmC level in both sample pairs, and a correlation between both epigenetic signals and gene expression was observed. The single-molecule epigenetic analysis identified numerous differentially modified regions, some implicated in ccRCC pathogenesis, including the genes VHL, PRCC, and PBRM1. Notably, pathways related to metabolism and cancer development were significantly enriched among these differential regions. This study demonstrates the feasibility of integrating optical genome and epigenome mapping for comprehensive characterization of matched tumor and adjacent tissue, uncovering both established and novel somatic aberrations.
- MeSH
- DNA-Binding Proteins MeSH
- Epigenesis, Genetic * genetics MeSH
- Epigenome * genetics MeSH
- Carcinoma, Renal Cell * genetics pathology MeSH
- Middle Aged MeSH
- Humans MeSH
- Chromosome Mapping methods MeSH
- DNA Methylation * genetics MeSH
- Von Hippel-Lindau Tumor Suppressor Protein genetics MeSH
- Kidney Neoplasms * genetics pathology MeSH
- Gene Expression Regulation, Neoplastic MeSH
- Transcription Factors MeSH
- DNA Copy Number Variations * genetics MeSH
- Check Tag
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Research Support, N.I.H., Extramural MeSH
Quantitative genomic mapping of DNA damage may provide insights into the underlying mechanisms of damage and repair. Sequencing based approaches are bound to the limitations of PCR amplification bias and read length which hamper both the accurate quantitation of damage events and the ability to map them to structurally complex genomic regions. Optical Genome mapping in arrays of parallel nanochannels allows physical extension and genetic profiling of millions of long genomic DNA fragments, and has matured to clinical utility for characterization of complex structural aberrations in cancer genomes. Here we present a new mapping modality, Repair-Assisted Damage Detection - Optical Genome Mapping (RADD-OGM), a method for single-molecule level mapping of DNA damage on a genome-wide scale. Leveraging ultra-long reads to assemble the complex structure of a sarcoma cell-line genome, we mapped the genomic distribution of oxidative DNA damage, identifying regions more susceptible to DNA oxidation. We also investigated DNA repair by allowing cells to repair chemically induced DNA damage, pinpointing locations of concentrated repair activity, and highlighting variations in repair efficiency. Our results showcase the potential of the method for toxicogenomic studies, mapping the effect of DNA damaging agents such as drugs and radiation, as well as following specific DNA repair pathways by selective induction of DNA damage. The facile integration with optical genome mapping enables performing such analyses even in highly rearranged genomes such as those common in many cancers, a challenging task for sequencing-based approaches.
- MeSH
- Bromates toxicity MeSH
- Humans MeSH
- Chromosome Mapping * instrumentation methods MeSH
- Microfluidic Analytical Techniques * instrumentation methods MeSH
- Cell Line, Tumor MeSH
- Nanotechnology * instrumentation methods MeSH
- DNA Repair genetics MeSH
- Oxidative Stress drug effects genetics MeSH
- DNA Damage * genetics MeSH
- Gene Expression Regulation MeSH
- Gene Expression Profiling MeSH
- Toxicogenetics * instrumentation methods MeSH
- DNA Copy Number Variations MeSH
- Single Molecule Imaging * instrumentation methods MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
The GPCR signalling cascade is a key pathway responsible for the signal transduction of a multitude of physical and chemical stimuli, including light, odorants, neurotransmitters and hormones. Understanding the structural and functional properties of the GPCR cascade requires direct observation of signalling processes in high spatial and temporal resolution, with minimal perturbation to endogenous systems. Optical microscopy and spectroscopy techniques are uniquely suited to this purpose because they excel at multiple spatial and temporal scales and can be used in living objects. Here, we review recent developments in microscopy and spectroscopy technologies which enable new insights into GPCR signalling. We focus on advanced techniques with high spatial and temporal resolution, single-molecule methods, labelling strategies and approaches suitable for endogenous systems and large living objects. This review aims to assist researchers in choosing appropriate microscopy and spectroscopy approaches for a variety of applications in the study of cellular signalling. LINKED ARTICLES: This article is part of a themed issue Complexity of GPCR Modulation and Signaling (ERNST). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v182.14/issuetoc.
- MeSH
- Humans MeSH
- Microscopy * methods MeSH
- Receptors, G-Protein-Coupled * chemistry metabolism MeSH
- Signal Transduction MeSH
- Spectrum Analysis * methods MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Review MeSH
The viscoelastic properties of biological membranes are crucial in controlling cellular functions and are determined primarily by the lipids' composition and structure. This work studies these properties by varying the structure of the constituting lipids in order to influence their interaction with high-density lipoprotein (HDL) particles. Various fluorescence-based techniques were applied to study lipid domains, membrane order, and the overall lateral as well as the molecule-internal glycerol region mobility in HDL-membrane interactions (i.e., binding and/or cargo transfer). The analysis of interactions with HDL particles and various lipid phases revealed that both fully fluid and some gel-phase lipids preferentially interact with HDL particles, although differences were observed in protein binding and cargo exchange. Both interactions were reduced with ordered lipid mixtures containing cholesterol. To investigate the mechanism, membranes were prepared from single-lipid components, enabling step-by-step modification of the lipid building blocks. On a biophysical level, the different mixtures displayed varying stiffness, fluidity, and hydrogen bond network changes. Increased glycerol mobility and a strengthened hydrogen bond network enhanced anchoring interactions, while fluid membranes with a reduced water network facilitated cargo transfer. In summary, the data indicate that different lipid classes are involved depending on the type of interaction, whether anchoring or cargo transfer.
- Publication type
- Journal Article MeSH
Závěrečná zpráva o řešení grantu Agentury pro zdravotnický výzkum MZ ČR
nestr.
V současnosti nejsme schopni určit příčiny vzácných geneticky podmíněných nemocí u ~50% případů a počet nově objevovaných kauzálních genů v posledních letech klesá. Naším záměrem je provést integrovanou analýzu genomu, transkriptomu, proteomu a metabolomu k určení genetické diagnózy ve skupině ~40 případů a rodin se vzácným geneticky podmíněným onemocněním, u kterých předchozí cílená klinická, biochemická a genetická vyšetření včetně exomového sekvenování nevedly k diagnóze. Využijeme našich zkušeností s novými technologiemi celogenomového sekvenování (NovaSeq), sekvenování dlouhých fragmentů jednotlivých molekul DNA (Oxford Nanopore, PacBio) a možností korelací genomové informace s výsledky analýz tělních tekutin, tkání, tkáňových kultur a vhodných buněčných modelů. Uplatníme nové bioinformatické nástroje analýzy multi-OMIC dat a nástroje umožňující globální sdílení fenotypových a genomických dat. Cílem je zrychlit poznání doposud nediagnostikovaných vzácných nemocí , zlepšení diagnostické výtěžňosti a zajištění inovativní péče o pacienty s vzácnými nemocemi v České Republice.; The failure to diagnose the cause of a rare genetic disease occurs in ~50% of the cases and the rate of discovery of novel genes and disease-gene relations appears to be declining. We intend to apply multi-OMIC approaches to identify causal genetic defects in ~40 selected cases from our previous studies, in which we have negative results from standard genetic and genomic analyses. We will benefit from our access and experience with new platforms for whole-genome analysis (NovaSeq), single molecule long read length sequencing (Oxford Nanopore, PacBio) and our ability to correlate genomic information with transcriptome, proteome and metabolome analyses of affected tissues, body fluids and patient cell-derived models. We will also apply new bioinformatics tools allowing effective integration of OMIC data and use tools enabling exchange of phenotypic and genomic information via shared platforms and tools worldwide. The ultimate goal is to accelerate understanding of these unsolved diseases, improve diagnostic yield, and deliver innovative care for rare genetic diseases in Czech Republic.
INTRODUCTION: The gold standard for serum neurofilament light chain (sNfL) determination is the single molecule array (SIMOA), the use of which is limited by availability and cost. The VEUS method is a fully automated, user-friendly diagnostic system requiring no sample preparation, with high reported sensitivity, multiplexing capability, and rapid diagnostics. The aim of this study was to compare the SIMOA and VEUS methods for determining sNfL levels in patients with multiple sclerosis (MS). METHODOLOGY: A single-centre cross-sectional study was conducted at the MS Centre of University Hospital Ostrava. Patients were enrolled in the study from January 18 to January 31, 2024. Inclusion criteria were: 1) diagnosis of MS according to the revised 2017 McDonald criteria, 2) age ≥18 years, and 3) signed informed consent. The NF-light V2 diagnostic kit (SIMOA, Quanterix) and the Singleplex Neurology assay kit (VEUDx, EZDiatech) were used to determine sNfL concentrations. The two methods were compared by use of Spearman correlation, Passing-Bablok regression, and Bland-Altman analysis. RESULTS: A total of 49 patients were included in the study, of whom 39 (79.6 %) were female. The median sNfL concentration was 7.73 (IQR 5.80-9.93) ng/L determined by SIMOA and 1.31 (IQR 1.18-1.65) ng/L by VEUS. We did not find a correlation between SIMOA and VEUS (rs = 0.025, p = 0.866). Passing-Bablok regression demonstrated a systematic and proportional difference between the two methods. A significant disagreement between them was also confirmed by the Bland-Altman plots. On average, sNfL values measured by SIMOA were 3.56 ng/L (95 % CI 0.78 to 6.34) higher than those measured by VEUS. CONCLUSION: Our investigation uncovered noteworthy disparities between the SIMOA and VEUS techniques in determining sNfL levels. Specifically, the VEUS technique systematically produces lower estimates of sNFL levels. This substantial variance emphasizes the importance of carefully evaluating assay methods when quantifying sNfL.
- MeSH
- Biomarkers blood MeSH
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Neurofilament Proteins * blood MeSH
- Cross-Sectional Studies MeSH
- Multiple Sclerosis * blood diagnosis MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Comparative Study MeSH
Early-stage diagnosis of prostatic carcinoma is essential for successful treatment and, thus, significant prognosis improvement. In laboratory practice, the standard non-invasive diagnostic approach is the immunochemical detection of the associated biomarker, prostate-specific antigen (PSA). Ultrasensitive detection of PSA is essential for both diagnostic and recurrence monitoring purposes. To achieve exceptional sensitivity, we have developed a microfluidic device with a flow-through cell for single-molecule analysis using photon-upconversion nanoparticles (UCNPs) as a detection label. For this purpose, magnetic microparticles (MBs) were first optimized for the capture and preconcentration of PSA and then used to implement a bead-based upconversion-linked immunoassay (ULISA) in the microfluidic device. The digital readout based on counting single nanoparticle-labeled PSA molecules on MBs enabled a detection limit of 1.04 pg mL-1 (36 fM) in 50% fetal bovine serum, which is an 11-fold improvement over the respective analog MB-based ULISA. The microfluidic technique conferred several other advantages, such as easy implementation and the potential for achieving high-throughput analysis. Finally, it was proven that the microfluidic setup is suitable for clinical sample analysis, showing a good correlation with a reference electrochemiluminescence assay (recovery rates between 97% and 105%).
- MeSH
- Immunoassay instrumentation methods MeSH
- Humans MeSH
- Limit of Detection MeSH
- Microfluidic Analytical Techniques instrumentation MeSH
- Prostatic Neoplasms diagnosis blood MeSH
- Nanoparticles chemistry MeSH
- Prostate-Specific Antigen * analysis blood MeSH
- Check Tag
- Humans MeSH
- Male MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Advancements in genomics are transforming the clinical management of chronic myeloid leukemia (CML) toward precision medicine. The impact of somatic mutations on treatment outcomes is still under debate. We studied the association of somatic mutations in epigenetic modifier genes and activated signaling/myeloid transcription factors (AS/MTFs) with disease progression and treatment failure in patients with CML after tyrosine kinase inhibitor (TKI) therapy. A total of 394 CML samples were sequenced, including 254 samples collected at initial diagnosis and 140 samples taken during follow-up. Single-molecule molecular inversion probe (smMIP)-based next-generation sequencing (NGS) was conducted targeting recurrently mutated loci in 40 genes, with a limit of detection of 0.2%. Seventy mutations were detected in 57 diagnostic samples (22.4%), whereas 64 mutations were detected in 39 of the follow-up samples (27.9%). Carrying any mutation at initial diagnosis was associated with worse outcomes after TKI therapy, particularly in AS/MTF genes. Patients having these mutations at initial diagnosis and treated with imatinib showed higher risks of treatment failure (hazard ratio, 2.53; 95% confidence interval, 1.13-5.66; P = .0239). The adverse prognostic impact of the mutations was not clear for patients treated with second-generation TKIs. The multivariate analysis affirmed that mutations in AS/MTF genes independently serve as adverse prognostic factors for molecular response, failure-free survival, and progression risk. Additionally, there was an observable nonsignificant trend indicating a heightened risk of progression to advanced disease and worse overall survival. In conclusion, mutations in the AS/MTF genes using smMIP-based NGS can help identify patients with a potential risk of both treatment failure and progression and may help upfront TKI selection.
- MeSH
- Leukemia, Myelogenous, Chronic, BCR-ABL Positive * genetics drug therapy mortality diagnosis MeSH
- Adult MeSH
- Protein Kinase Inhibitors therapeutic use MeSH
- Middle Aged MeSH
- Humans MeSH
- Young Adult MeSH
- Mutation * MeSH
- Prognosis MeSH
- Disease Progression MeSH
- Aged, 80 and over MeSH
- Aged MeSH
- Signal Transduction MeSH
- Transcription Factors genetics MeSH
- Treatment Outcome MeSH
- High-Throughput Nucleotide Sequencing MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Young Adult MeSH
- Male MeSH
- Aged, 80 and over MeSH
- Aged MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
Early identification of resistant cancer cells is currently a major challenge, as their expansion leads to refractoriness. To capture the dynamics of these cells, we made a comprehensive analysis of disease progression and treatment response in a chronic lymphocytic leukemia (CLL) patient using a combination of single-cell and bulk genomic methods. At diagnosis, the patient presented with unfavorable genetic markers, including notch receptor 1 (NOTCH1) mutation and loss(11q). The initial and subsequent treatment lines did not lead to a durable response and the patient developed refractory disease. Refractory CLL cells featured substantial dysregulation in B-cell phenotypic markers such as human leukocyte antigen (HLA) genes, immunoglobulin (IG) genes, CD19 molecule (CD19), membrane spanning 4-domains A1 (MS4A1; previously known as CD20), CD79a molecule (CD79A) and paired box 5 (PAX5), indicating B-cell de-differentiation and disease transformation. We described the clonal evolution and characterized in detail two cell populations that emerged during the refractory disease phase, differing in the presence of high genomic complexity. In addition, we successfully tracked the cells with high genomic complexity back to the time before treatment, where they formed a rare subpopulation. We have confirmed that single-cell RNA sequencing enables the characterization of refractory cells and the monitoring of their development over time.
