Circulating tumor cells (CTCs) are rare cells that can be found in the peripheral blood of cancer patients. They have been demonstrated to be useful prognostic markers in many cancer types. Within the last decade various methods have been developed to detect rare cells within a liquid biopsy from a cancer patient. These methods have revealed the phenotypic diversity of CTCs and how they can represent the complement of cells that are found in a tumor. Single-cell proteogenomics has emerged as an all-encompassing next-generation technological approach for CTC research. This allows for the deconstruction of cellular heterogeneity, dynamics of metastatic initiation and progression, and response or resistance to therapeutics in the clinical settings. We take advantage of this opportunity to investigate CTC heterogeneity and understand their full potential in precision medicine.The high-definition single-cell analysis (HD-SCA) workflow combines detection of the entire population of CTCs and rare cancer related cells with single-cell genomic analysis and may therefore provide insight into their subpopulations based on molecular as well as morphological data. In this chapter we describe in detail the protocols from isolation of a candidate cell from a microscopy slide, through whole-genome amplification and library preparation, to CNV analysis of identified cells from the HD-SCA workflow. This process may also be applicable to any platform starting with a standard microscopy slide or isolated cell of interest.

• Klíčová slova
• CNV, CTC, Circulating tumor cells, Copy number variation, Liquid biopsy, Precision medicine, Single-cell analysis,
• MeSH
• analýza jednotlivých buněk metody   MeSH
• individualizovaná medicína MeSH
• lidé MeSH
• mutace MeSH
• nádorové cirkulující buňky MeSH
• nádory diagnóza   genetika   MeSH
• proteogenomika metody   MeSH
• variabilita počtu kopií segmentů DNA * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Analysis of cellular composition and metabolism at a single-cell resolution allows gaining more information about complex relationships of cells within tissues or whole living organisms by resolving the variance stemming from the cellular heterogeneity. Mass spectrometry (MS) is a perfect analytical tool satisfying the demanding requirements of detecting and identifying compounds present in such ultralow-volume samples of high chemical complexity. However, the method of sampling and sample ionization is crucial in obtaining relevant information. In this work, we present a microfluidic sampling platform that integrates single-cell extraction from MS-incompatible media with electrical cell lysis and nanoESI-MS analysis of human erythrocytes. Hemoglobin alpha and beta chains (300 amol/cell) were successfully identified in mass spectra of single-erythrocyte lysates.

• Klíčová slova
• bioanalysis, hemoglobin, mass spectrometry, microfluidics, single-cell analysis,
• MeSH
• erytrocyty MeSH
• hmotnostní spektrometrie s elektrosprejovou ionizací * metody   MeSH
• laboratoř na čipu MeSH
• lidé MeSH
• mikrofluidika * metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Correct determination of the instantaneous level and changes of relevant proteins inside individual cells is essential for correct interpretation and understanding of physiological, diagnostic, and therapeutic events. Thus, single-cell analyses are important for quantification of natural cellular heterogeneity, which cannot be evaluated from averaged data of a cell population measurements. Here, we developed an original highly sensitive and selective instrumentation and methodology based on homogeneous single-step bioluminescence assay to quantify caspases and evaluate their heterogeneity in individual cells. Individual suspended cells are selected under microscope and reliably transferred into the 7 µl detection vials by a micromanipulator. The sensitivity of the method is given by implementation of photomultiplying tube with a cooled photocathode working in the photon counting mode. By optimization of our device and methodology, the limits of detection and quantitation were decreased down to 2.1 and 7.0 fg of recombinant caspase-3, respectively. These masses are lower than average amounts of caspase-3/7 in individual apoptotic and even non-apoptotic cells. As a proof of concept, the content of caspase-3/7 in single treated and untreated HeLa cells was determined to be 154 and 25 fg, respectively. Based on these results, we aim to use the technology for investigations of non-apoptotic functions of caspases.

• Klíčová slova
• Bioluminescence, Caspase detection and quantification, Photon counting, Single-cell analysis,
• MeSH
• apoptóza * MeSH
• HeLa buňky MeSH
• kaspasa 3 MeSH
• kaspasy * MeSH
• lidé MeSH
• technologie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• kaspasa 3 MeSH
• kaspasy * MeSH

Development of techniques for the analysis of the content of individual cells represents an important direction in modern bioanalytical chemistry. While the analysis of chromosomes, organelles, or location of selected proteins has been traditionally the domain of microscopic techniques, the advances in miniaturized analytical systems bring new possibilities for separations and detections of molecules inside the individual cells including smaller molecules such as hormones or metabolites. It should be stressed that the field of single cell analysis is very broad, covering advanced optical, electrochemical and mass spectrometry instrumentation, sensor technology and separation techniques. The number of papers published on single cell analysis has reached several hundred in recent years. Thus a complete literature coverage is beyond the limits of a journal article. The following text provides a critical overview of some of the latest developments with the main focus on mass spectrometry, microseparation methods, electrophoresis in capillaries and microfluidic devices and respective detection techniques for performing single cell analyses.

• Klíčová slova
• Capillary electrophoresis, Electrochemistry, Fluorescence, Mass spectrometry, Microfluidic devices, Single cell analysis,
• MeSH
• analýza jednotlivých buněk trendy   MeSH
• Bacteria metabolismus   MeSH
• chromatografie kapalinová MeSH
• fluorescenční barviva chemie   MeSH
• hmotnostní spektrometrie MeSH
• houby metabolismus   MeSH
• lidé MeSH
• proteiny analýza   izolace a purifikace   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• fluorescenční barviva MeSH
• proteiny MeSH

Electrical characteristics of living cells have been proven to reveal important details about their internal structure, charge distribution and composition changes in the cell membrane, as well as the extracellular context. An impedance flow cytometry is a common approach to determine the electrical properties of a cell, having the advantage of label-free and high throughput. However, the current techniques are complex and costly for the fabrication process. For that reason, we introduce an integrated dual microneedle-microchannel for single-cell detection and electrical properties extraction. The dual microneedles utilized a commercially available tungsten needle coated with parylene. When a single cell flows through the parallel-facing electrode configuration of the dual microneedle, the electrical impedance at multiple frequencies is measured. The impedance measurement demonstrated the differential of normal red blood cells (RBCs) with three different sizes of microbeads at low and high frequencies, 100 kHz and 2 MHz, respectively. An electrical equivalent circuit model (ECM) was used to determine the unique membrane capacitance of individual cells. The proposed technique demonstrated that the specific membrane capacitance of an RBC is 9.42 mF/m-2, with the regression coefficients, ρ at 0.9895. As a result, this device may potentially be used in developing countries for low-cost single-cell screening and detection.

• Klíčová slova
• Single-cell analysis, impedance analysis, impedance flow cytometry, microfluidic,
• MeSH
• analýza jednotlivých buněk MeSH
• buněčná membrána fyziologie   MeSH
• elektrická impedance * MeSH
• elektrická kapacitance MeSH
• erytrocyty * fyziologie   MeSH
• lidé MeSH
• průtoková cytometrie * přístrojové vybavení   metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Analysing the chemical content of individual cells has already been proven to reveal unique information on various biological processes. Single-cell analysis provides more accurate and reliable results for biology and medicine than analyses of extracts from cell populations, where a natural heterogeneity is averaged. To meet the requirements in the research of important biologically active molecules, such as caspases, we have developed a miniaturized device for simultaneous analyses of individual cells. A stainless steel body with a carousel holder enables high-sensitivity parallel detections in eight microvials. The holder is mounted in front of a photomultiplier tube with cooled photocathode working in photon counting mode. The detection of active caspase-3/7, central effector caspases in apoptosis, in single cells is based on the bioluminescence chemistry commercially available as Caspase-Glo® 3/7 reagent developed by Promega. Individual cells were captured from a culture medium under microscope and transferred by micromanipulator into detection microvial filled with the reagent. As a result of testing, the limits of detection and quantification were determined to be 0.27/0.86 of active caspase-3/7 content in an average apoptotic cell and 0.46/2.92 for non-apoptotic cells. Application potential of this technology in laboratory diagnostics and related medical research is discussed. Graphical abstract Miniaturized device for simultaneous analyses of individual cells.

• Klíčová slova
• Apoptosis, Bioluminescence, Caspase-3/7, Single-cell analysis,
• MeSH
• analýza jednotlivých buněk přístrojové vybavení   metody   MeSH
• apoptóza * MeSH
• design vybavení MeSH
• enzymatické testy přístrojové vybavení   metody   MeSH
• kaspasa 3 analýza   metabolismus   MeSH
• kaspasa 7 analýza   metabolismus   MeSH
• kultivované buňky MeSH
• luminiscenční měření přístrojové vybavení   metody   MeSH
• myši MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• kaspasa 3 MeSH
• kaspasa 7 MeSH

DNA double stranded breaks (DSBs) are the most serious type of lesions introduced into chromatin by ionizing radiation. During DSB repair, cells recruit different proteins to the damaged sites in a manner dependent on local chromatin structure, DSB location in the nucleus, and the repair pathway entered. 53BP1 is one of the important players participating in repair pathway decision of the cell. Although many molecular biology details have been investigated, the architecture of 53BP1 repair foci and its development during the post-irradiation time, especially the period of protein recruitment, remains to be elucidated. Super-resolution light microscopy is a powerful new tool to approach such studies in 3D-conserved cell nuclei. Recently, we demonstrated the applicability of single molecule localization microscopy (SMLM) as one of these highly resolving methods for analyses of dynamic repair protein distribution and repair focus internal nano-architecture in intact cell nuclei. In the present study, we focused our investigation on 53BP1 foci in differently radio-resistant cell types, moderately radio-resistant neonatal human dermal fibroblasts (NHDF) and highly radio-resistant U87 glioblastoma cells, exposed to high-LET 15N-ion radiation. At given time points up to 24 h post irradiation with doses of 1.3 Gy and 4.0 Gy, the coordinates and spatial distribution of fluorescently tagged 53BP1 molecules was quantitatively evaluated at the resolution of 10⁻20 nm. Clusters of these tags were determined as sub-units of repair foci according to SMLM parameters. The formation and relaxation of such clusters was studied. The higher dose generated sufficient numbers of DNA breaks to compare the post-irradiation dynamics of 53BP1 during DSB processing for the cell types studied. A perpendicular (90°) irradiation scheme was used with the 4.0 Gy dose to achieve better separation of a relatively high number of particle tracks typically crossing each nucleus. For analyses along ion-tracks, the dose was reduced to 1.3 Gy and applied in combination with a sharp angle irradiation (10° relative to the cell plane). The results reveal a higher ratio of 53BP1 proteins recruited into SMLM defined clusters in fibroblasts as compared to U87 cells. Moreover, the speed of foci and thus cluster formation and relaxation also differed for the cell types. In both NHDF and U87 cells, a certain number of the detected and functionally relevant clusters remained persistent even 24 h post irradiation; however, the number of these clusters again varied for the cell types. Altogether, our findings indicate that repair cluster formation as determined by SMLM and the relaxation (i.e., the remaining 53BP1 tags no longer fulfill the cluster definition) is cell type dependent and may be functionally explained and correlated to cell specific radio-sensitivity. The present study demonstrates that SMLM is a highly appropriate method for investigations of spatiotemporal protein organization in cell nuclei and how it influences the cell decision for a particular repair pathway at a given DSB site.

• Klíčová slova
• 15N ion irradiation, repair cluster formation, repair cluster persistence, repair foci nano-architecture, single molecule localization microscopy (SMLM),
• MeSH
• 53BP1 metabolismus   MeSH
• konfokální mikroskopie metody   MeSH
• kultivované buňky MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• rekombinační oprava DNA * MeSH
• transport proteinů MeSH
• zobrazení jednotlivé molekuly metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• 53BP1 MeSH

The protein heterogeneity at the single-cell level has been recognized to be vital for an understanding of various life processes during animal development. In addition, the knowledge of accurate quantity of relevant proteins at cellular level is essential for appropriate interpretation of diagnostic and therapeutic results. Some low-copy-number proteins are known to play a crucial role during cell proliferation, differentiation, and also in apoptosis. The fate decision is often based on the concentration of these proteins in the individual cells. This is likely to apply also for caspases, cysteine proteases traditionally associated with cell death via apoptosis but recently being discovered also as important factors in cell proliferation and differentiation. The hypothesis was tested in bone-related cells, where modulation of fate from apoptosis to proliferation/differentiation and vice versa is particularly challenging, e.g., towards anti-osteoporotic treatments and anti-cancer strategies. An ultrasensitive and highly selective method based on bioluminescence photon counting was used to quantify activated caspase-3/7 in order to demonstrate protein-level heterogeneity in individual cells within one population and to associate quantitative measurements with different cell fates (proliferation, differentiation, apoptosis). The results indicate a gradual increase of caspase-3/7 activation from the proliferative status to differentiation (more than three times) and towards apoptosis (more than six times). The findings clearly support one of the putative key mechanisms of non-apoptotic functions of pro-apoptotic caspases based on fine-tuning of their activation levels.

• Klíčová slova
• Apoptosis, Bioluminescence detection, Caspase-3/7, Cell death and differentiation, Single-cell detection and quantification,
• MeSH
• aktivace enzymů MeSH
• apoptóza MeSH
• buněčná diferenciace MeSH
• buněčné linie MeSH
• kaspasa 3 chemie   genetika   metabolismus   MeSH
• kaspasa 7 chemie   genetika   metabolismus   MeSH
• myši MeSH
• osteoblasty cytologie   fyziologie   MeSH
• proliferace buněk MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• kaspasa 3 MeSH
• kaspasa 7 MeSH

Single cell gel electrophoresis, also known as the comet assay, is widely used for the detection and measurement of DNA strand breaks. With the addition of a step in which DNA is incubated with specific endonucleases recognising damaged bases, these lesions can be measured, too. In the standard protocol, electrophoresis is carried out at high pH. If, instead, electrophoresis is in neutral buffer, the effect of DNA damage seems to be much reduced--either because alkaline conditions are needed to reveal certain lesions, or because the effect of the same number of breaks on DNA migration is greater at high pH. A lower sensitivity can be useful in some circumstances, as it extends the range of DNA damage levels over which the assay can be used. Here we compare the performance of standard and modified techniques with a variety of DNA-damaging agents and offer possible explanations for the differences in behaviour of DNA under alternative electrophoretic conditions.

• MeSH
• chinoliziny farmakologie   MeSH
• DNA-formamidopyrimidinglykosylasa MeSH
• elektroforéza v agarovém gelu metody   MeSH
• exodeoxyribonukleasy metabolismus   MeSH
• Fabaceae genetika   MeSH
• HeLa buňky účinky léků   MeSH
• koncentrace vodíkových iontů MeSH
• léčivé rostliny MeSH
• lidé MeSH
• methylmethansulfonát farmakologie   MeSH
• N-glykosylhydrolasy metabolismus   MeSH
• poškození DNA * účinky léků   MeSH
• pufry MeSH
• pyrimidinové dimery analýza   MeSH
• pyrrolidiny farmakologie   MeSH
• senzitivita a specificita MeSH
• vitamin K farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chinoliziny MeSH
• DNA-formamidopyrimidinglykosylasa MeSH
• exodeoxyribonuclease III MeSH Prohlížeč
• exodeoxyribonukleasy MeSH
• methylmethansulfonát MeSH
• N-glykosylhydrolasy MeSH
• pufry MeSH
• pyrimidinové dimery MeSH
• pyrrolidiny MeSH
• Ro 19-8022 MeSH Prohlížeč
• vitamin K MeSH

Triple-negative breast cancer (TNBC) is an aggressive and complex subtype of breast cancer that lacks targeted therapy. TNBC manifests characteristic, extensive intratumoral heterogeneity that promotes disease progression and influences drug response. Single-cell techniques in combination with next-generation computation provide an unprecedented opportunity to identify molecular events with therapeutic potential. Here, we describe the generation of a comprehensive mass cytometry panel for multiparametric detection of 23 phenotypic markers and 13 signaling molecules. This single-cell proteomic approach allowed us to explore the landscape of TNBC heterogeneity, with particular emphasis on the tumor microenvironment. We prospectively profiled freshly resected tumors from 26 TNBC patients. These tumors contained phenotypically distinct subpopulations of cancer and stromal cells that were associated with the patient's clinical status at the time of surgery. We further classified the epithelial-mesenchymal plasticity of tumor cells, and molecularly defined phenotypically diverse populations of tumor-associated stroma. Furthermore, in a retrospective tissue-microarray TNBC cohort, we showed that the level of CD97 at the time of surgery has prognostic potential.

• Klíčová slova
• mass cytometry, phenotypic plasticity, single-cell profiles, triple-negative breast cancer, tumor heterogeneity, unsupervised machine learning algorithm,
• MeSH
• buňky stromatu metabolismus   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nádorové mikroprostředí MeSH
• proteomika MeSH
• retrospektivní studie MeSH
• signální transdukce MeSH
• triple-negativní karcinom prsu * metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH