Precision and Accuracy of Receptor Quantification on Synthetic and Biological Surfaces Using DNA-PAINTClick to copy article linkArticle link copied!
- Roger RieraRoger RieraDepartment of Biomedical Engineering, Institute for Complex Molecular Systems (ICMS), Eindhoven University of Technology, P.O. Box 513, Eindhoven5600 MB, NetherlandsMore by Roger Riera
- Emmanouil ArchontakisEmmanouil ArchontakisDepartment of Biomedical Engineering, Institute for Complex Molecular Systems (ICMS), Eindhoven University of Technology, P.O. Box 513, Eindhoven5600 MB, NetherlandsMore by Emmanouil Archontakis
- Glenn CremersGlenn CremersLaboratory of Chemical Biology and Institute for Complex Molecular Systems, Eindhoven University of Technology, P.O. Box 513, Eindhoven5600 MB, The NetherlandsComputational Biology Group, Department of Biomedical Engineering, Eindhoven University of Technology, P.O. Box 513, Eindhoven5600 MB, The NetherlandsMore by Glenn Cremers
- Tom de GreefTom de GreefLaboratory of Chemical Biology and Institute for Complex Molecular Systems, Eindhoven University of Technology, P.O. Box 513, Eindhoven5600 MB, The NetherlandsComputational Biology Group, Department of Biomedical Engineering, Eindhoven University of Technology, P.O. Box 513, Eindhoven5600 MB, The NetherlandsInstitute for Molecules and Materials, Radboud University, Heyendaalseweg 135, AJ Nijmegen6525, The NetherlandsMore by Tom de Greef
- Peter Zijlstra*Peter Zijlstra*Email: [email protected]Department of Applied Physics and Institute for Complex Molecular Systems, Eindhoven University of Technology, P.O. Box 513, Eindhoven5600 MB, The NetherlandsMore by Peter Zijlstra
- Lorenzo Albertazzi*Lorenzo Albertazzi*Email: [email protected]Department of Biomedical Engineering, Institute for Complex Molecular Systems (ICMS), Eindhoven University of Technology, P.O. Box 513, Eindhoven5600 MB, NetherlandsNanoscopy for Nanomedicine, Institute for Bioengineering of Catalonia, Barcelona08028, SpainMore by Lorenzo Albertazzi
Abstract
Characterization of the number and distribution of biological molecules on 2D surfaces is of foremost importance in biology and biomedicine. Synthetic surfaces bearing recognition motifs are a cornerstone of biosensors, while receptors on the cell surface are critical/vital targets for the treatment of diseases. However, the techniques used to quantify their abundance are qualitative or semi-quantitative and usually lack sensitivity, accuracy, or precision. Detailed herein a simple and versatile workflow based on super-resolution microscopy (DNA-PAINT) was standardized to improve the quantification of the density and distribution of molecules on synthetic substrates and cell membranes. A detailed analysis of accuracy and precision of receptor quantification is presented, based on simulated and experimental data. We demonstrate enhanced accuracy and sensitivity by filtering out non-specific interactions and artifacts. While optimizing the workflow to provide faithful counting over a broad range of receptor densities. We validated the workflow by specifically quantifying the density of docking strands on a synthetic sensor surface and the densities of PD1 and EGF receptors (EGFR) on two cellular models.
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License Summary*
You are free to share(copy and redistribute) this article in any medium or format and to adapt(remix, transform, and build upon) the material for any purpose, even commercially within the parameters below:
Creative Commons (CC): This is a Creative Commons license.
Attribution (BY): Credit must be given to the creator.
*Disclaimer
This summary highlights only some of the key features and terms of the actual license. It is not a license and has no legal value. Carefully review the actual license before using these materials.
License Summary*
You are free to share(copy and redistribute) this article in any medium or format and to adapt(remix, transform, and build upon) the material for any purpose, even commercially within the parameters below:
Creative Commons (CC): This is a Creative Commons license.
Attribution (BY): Credit must be given to the creator.
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Results and Discussion
name | docking sequence | supplier | imager sequence | supplier | experiment |
---|---|---|---|---|---|
Sequence 1 (9mer) ATTO655 | Biotin- TTA TAC ATC TA | IDT | CTA GAT GTA T─ATTO655 | IDT | Figures 1,2 and 3 and S1,S2 and S4 |
Sequence 1 (10mer) ATTO655 | NH2- TTA TAC ATC TAG | IDT | CTA GAT GTA T─ATTO655 | Eurofins | Figure S6 |
Sequence 1 (9mer) ATTO647N | NH2- TTA TAC ATC TA | IDT | CTA GAT GTA T─ATTO647N | IDT | Figure S5 |
Sequence 1 (10mer) ATTO647N | NH2- TTA TAC ATC TAG | IDT | CTA GAT GTA T─ATTO647N | IDT | Figures 5 and S5 |
Sequence 2 ATTO647N | - | - | TAT GTA GAT C─ATTO647N | IDT | Figure S5 |
Sequence 2 ATTO655 | - | - | TAT GTA GAT C─ATTO655 | IDT | Figure S4 |
Direct Counting on Synthetic Surfaces with Mean-Shift Clustering
Kinetic Counting on Synthetic Surfaces with qPAINT
Direct Counting of PD1 Receptors on CHO Cells
Conclusions
Materials and Methods
Materials
DNA-PAINT Sequences
Synthetic Surface Preparation for DNA-PAINT Imaging
Cell Culture and Immunostaining
Optical Setup and Image Acquisition
Direct Counting Analysis
Kinetic Counting Analysis
Logistic Fit
DNA-PAINT Simulations
Supporting Information
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acssensors.2c01736.
Typical field of view of single-molecule fluorescence events, histograms of dwell times (dark-and-bright) of binding events, simulation of the direct and kinetic counting performance, control experiments, cumulative distribution functions and calibration, time traces, and number of events on cell membranes (PDF)
Terms & Conditions
Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.
Acknowledgments
The authors acknowledge L. Fitzpatrick from the Technical University of Eindhoven for proofreading the manuscript and supportive information. R.R. and L.A. thank the European Research Council/Horizon 2020 for financial support (no. ERC-StG-757397). E.A. and L.A. thank the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie Grant Agreement No. 765497 (THERACAT). L.A. thanks NWO for support (VIDI grant no. 192.028). This project has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement no. 864772).
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- 15Lubken, R. M.; de Jong, A. M.; Prins, M. W. J. How Reactivity Variability of Biofunctionalized Particles Is Determined by Superpositional Heterogeneities. ACS Nano 2021, 15, 1331– 1341, DOI: 10.1021/acsnano.0c08578Google Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhsFKgsg%253D%253D&md5=fde4a71d9b491536c9c245de7bc3ab09How Reactivity Variability of Biofunctionalized Particles Is Determined by Superpositional HeterogeneitiesLubken, Rafiq M.; de Jong, Arthur M.; Prins, Menno W. J.ACS Nano (2021), 15 (1), 1331-1341CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)The biofunctionalization of particles with specific targeting moieties forms the foundation for mol. recognition in biomedical applications such as targeted nanomedicine and particle-based biosensing. To achieve a high precision of targeting for nanomedicine and high precision of sensing for biosensing, it is important to understand the consequences of heterogeneities of particle properties. Here, we present a comprehensive methodol. to study with expts. and simulations the collective consequences of particle heterogeneities on multiple length scales, called superpositional heterogeneities, in generating reactivity variability per particle. Single-mol. techniques are used to quantify stochastic, interparticle, and intraparticle variabilities, in order to show how these variabilities collectively contribute to reactivity variability per particle, and how the influence of each contributor changes as a function of the system parameters such as particle interaction area, the particle size, the targeting moiety d., and the no. of particles. The results give insights into the consequences of superpositional heterogeneities for the reactivity variability in biomedical applications and give guidelines on how the precision can be optimized in the presence of multiple independent sources of variability.
- 16Horáček, M.; Engels, D. J.; Zijlstra, P. Dynamic Single-Molecule Counting for the Quantification and Optimization of Nanoparticle Functionalization Protocols. Nanoscale 2020, 12, 4128– 4136, DOI: 10.1039/C9NR10218CGoogle Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhslWhurk%253D&md5=3eaa15c54c9acf68dc470e3ab8eb6115Dynamic single-molecule counting for the quantification and optimization of nanoparticle functionalization protocolsHoracek, Matej; Engels, Dion J.; Zijlstra, PeterNanoscale (2020), 12 (6), 4128-4136CODEN: NANOHL; ISSN:2040-3372. (Royal Society of Chemistry)Applications of colloidal particles in the fields of i.e. biosensors, mol. targeting, or drug-delivery require their functionalization with biol. active and specific mol. ligands. Functionalization protocols often result in a heterogeneous population of particles with a varying d., spatial distribution and orientation of the functional groups on the particle surface. A lack of methods to directly resolve these mol. properties of the particle's surface hampers optimization of functionalization protocols and applications. Here quant. single-mol. interaction kinetics is used to count the no. of ligands on the surface of hundreds of individual nanoparticles simultaneously. By analyzing the waiting-time between single-mol. binding events we quantify the particle functionalization both accurately and precisely for a large range of ligand densities. We observe significant particle-to-particle differences in functionalization which are dominated by the particle-size distribution for high mol. densities, but are substantially broadened for sparsely functionalized particles. From time-dependent studies we find that ligand reorganization on long timescales drastically reduces this heterogeneity, a process that has remained hidden up to now in ensemble-averaged studies. The quant. single-mol. counting therefore provides a direct route to quantification and optimization of coupling protocols towards molecularly controlled colloidal interfaces.
- 17Delcanale, P.; Miret-Ontiveros, B.; Arista-Romero, M.; Pujals, S.; Albertazzi, L. Nanoscale Mapping Functional Sites on Nanoparticles by Points Accumulation for Imaging in Nanoscale Topography (PAINT). ACS Nano 2018, 12, 7629– 7637, DOI: 10.1021/acsnano.7b09063Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtl2js7vJ&md5=c04b4297511e06ae763f8aa53441f3ffNanoscale Mapping Functional Sites on Nanoparticles by Points Accumulation for Imaging in Nanoscale Topography (PAINT)Delcanale, Pietro; Miret-Ontiveros, Bernat; Arista-Romero, Maria; Pujals, Silvia; Albertazzi, LorenzoACS Nano (2018), 12 (8), 7629-7637CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)The ability of nanoparticles to selectively recognize a mol. target constitutes the key toward nanomedicine applications such as drug delivery and diagnostics. The activity of such devices is mediated by the presence of multiple copies of functional mols. on the nanostructure surface. Therefore, understanding the no. and the distribution of nanoparticle functional groups is of utmost importance for the rational design of effective materials. Anal. methods are available, but to obtain quant. information at the level of single particles and single functional sites, i.e., going beyond the ensemble, remains highly challenging. Here the authors introduce the use of an optical nanoscopy technique, DNA points accumulation for imaging in nanoscale topog. (DNA-PAINT), to address this issue. Combining subdiffraction spatial resoln. with mol. selectivity and sensitivity, DNA-PAINT provides both geometrical and functional information at the level of a single nanostructure. DNA-PAINT can be used to image and quantify relevant functional proteins such as antibodies and streptavidin on nanoparticles and microparticles with nanometric accuracy in 3D and multiple colors. The generality and the applicability of the authors' method without the need for fluorescent labeling hold great promise for the robust quant. nanocharacterization of functional nanomaterials.
- 18Strauss, S.; Nickels, P. C.; Strauss, M. T.; Jimenez Sabinina, V. J.; Ellenberg, J.; Carter, J. D.; Gupta, S.; Janjic, N.; Jungmann, R. Modified Aptamers Enable Quantitative Sub-10-Nm Cellular DNA-PAINT Imaging. Nat. Methods 2018, 15, 685, DOI: 10.1038/s41592-018-0105-0Google Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhsFGjur3K&md5=11808272f7077ba2e2e6e157a01bea3aModified aptamers enable quantitative sub-10-nm cellular DNA-PAINT imagingStrauss, Sebastian; Nickels, Philipp C.; Strauss, Maximilian T.; Jimenez Sabinina, Vilma; Ellenberg, Jan; Carter, Jeffrey D.; Gupta, Shashi; Janjic, Nebojsa; Jungmann, RalfNature Methods (2018), 15 (9), 685-688CODEN: NMAEA3; ISSN:1548-7091. (Nature Research)Although current implementations of super-resoln. microscopy are tech. approaching true mol.-scale resoln., this has not translated to imaging of biol. specimens, because of the large size of conventional affinity reagents. Here we introduce slow off-rate modified aptamers (SOMAmers) as small and specific labeling reagents for use with DNA points accumulation in nanoscale topog. (DNA-PAINT). To demonstrate the achievable resoln., specificity, and multiplexing capability of SOMAmers, we labeled and imaged both transmembrane and intracellular targets in fixed and live cells.
- 19Nerreter, T.; Letschert, S.; Götz, R.; Doose, S.; Danhof, S.; Einsele, H.; Sauer, M.; Hudecek, M. Super-Resolution Microscopy Reveals Ultra-Low CD19 Expression on Myeloma Cells That Triggers Elimination by CD19 CAR-T. Nat. Commun. 2019, 10, 3137, DOI: 10.1038/s41467-019-10948-wGoogle Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3MzotlOnug%253D%253D&md5=7ba7da4f6a44018192ae96237bac7e95Super-resolution microscopy reveals ultra-low CD19 expression on myeloma cells that triggers elimination by CD19 CAR-TNerreter Thomas; Danhof Sophia; Einsele Hermann; Hudecek Michael; Letschert Sebastian; Gotz Ralph; Doose Soren; Sauer MarkusNature communications (2019), 10 (1), 3137 ISSN:.Immunotherapy with chimeric antigen receptor-engineered T-cells (CAR-T) is under investigation in multiple myeloma. There are reports of myeloma remission after CD19 CAR-T therapy, although CD19 is hardly detectable on myeloma cells by flow cytometry (FC). We apply single molecule-sensitive direct stochastic optical reconstruction microscopy (dSTORM), and demonstrate CD19 expression on a fraction of myeloma cells (10.3-80%) in 10 out of 14 patients (density: 13-5,000 molecules per cell). In contrast, FC detects CD19 in only 2 of these 10 patients, on a smaller fraction of cells. Treatment with CD19 CAR-T in vitro results in elimination of CD19-positive myeloma cells, including those with <100 CD19 molecules per cell. Similar data are obtained by dSTORM analyses of CD20 expression on myeloma cells and CD20 CAR-T. These data establish a sensitivity threshold for CAR-T and illustrate how super-resolution microscopy can guide patient selection in immunotherapy to exploit ultra-low density antigens.
- 20Tobin, S. J.; Wakefield, D. L.; Jones, V.; Liu, X.; Schmolze, D.; Jovanović-Talisman, T. Single Molecule Localization Microscopy Coupled with Touch Preparation for the Quantification of Trastuzumab-Bound HER2. Sci. Rep. 2018, 8, 15154, DOI: 10.1038/s41598-018-33225-0Google Scholar20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3czot1elsw%253D%253D&md5=8ff31d6fe7b0b09a17359ec90b04c1c9Single molecule localization microscopy coupled with touch preparation for the quantification of trastuzumab-bound HER2Tobin Steven J; Wakefield Devin L; Jovanovic-Talisman Tijana; Jones Veronica; Liu Xueli; Schmolze DanielScientific reports (2018), 8 (1), 15154 ISSN:.All breast cancers are assessed for levels of human epidermal growth factor receptor 2 (HER2). Fluorescence in situ hybridization (FISH) and immunohistochemistry are currently used to determine if a patient is eligible for anti-HER2 therapy. Limitations of both tests include variability and relatively long processing times. Additionally, neither test determines whether HER2 contains the extracellular domain. While truncated in some tumors, this domain is required for binding of the therapeutic antibody trastuzumab. Here, trastuzumab was used to directly detect HER2 with quantitative single molecule localization microscopy (qSMLM). In proof of concept studies, our new method rapidly quantified both HER2 density and features of nano-organization. In cultured cells, the method was sensitive to subtle variations in HER2 expression. To assess patient samples, we combined qSMLM with tissue touch preparation (touch prep-qSMLM) and examined large areas of intact membranes. For cell lines and patient samples, HER2 copy numbers from FISH showed a significant positive correlation with detected densities from qSMLM and trended with HER2 cluster occupancy.
- 21Chen, C.; Zong, S.; Liu, Y.; Wang, Z.; Zhang, Y.; Chen, B.; Cui, Y. Profiling of Exosomal Biomarkers for Accurate Cancer Identification: Combining DNA-PAINT with Machine- Learning-Based Classification. Small 2019, 15, 1901014, DOI: 10.1002/smll.201901014Google Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhslWjtrjP&md5=8417cadf49b5a80ce1d61dad81e29e7dProfiling of Exosomal Biomarkers for Accurate Cancer Identification: Combining DNA-PAINT with Machine- Learning-Based ClassificationChen, Chen; Zong, Shenfei; Liu, Yun; Wang, Zhuyuan; Zhang, Yizhi; Chen, Baoan; Cui, YipingSmall (2019), 15 (43), 1901014CODEN: SMALBC; ISSN:1613-6810. (Wiley-VCH Verlag GmbH & Co. KGaA)Exosomes are endosome-derived vesicles enriched in body fluids such as urine, blood, and saliva. So far, they have been recognized as potential biomarkers for cancer diagnostics. However, the present single-variate anal. of exosomes has greatly limited the accuracy and specificity of diagnoses. Besides, most diagnostic approaches focus on bulk anal. using lots of exosomes and tend to be less accurate because they are vulnerable to impure extn. and concn. differences of exosomes. To address these challenges, a quant. anal. platform is developed to implement a sequential quantification anal. of multiple exosomal surface biomarkers at the single-exosome level, which uses DNA-PAINT and a machine learning algorithm to automatically analyze the results. As a proof of concept, the profiling of four exosomal surface biomarkers (HER2, GPC-1, EpCAM, EGFR) is developed to identify exosomes from cancer-derived blood samples. Then, this technique is further applied to detect pancreatic cancer and breast cancer from unknown samples with 100% accuracy.
- 22Lennon, K. M.; Wakefield, D. L.; Maddox, A. L.; Brehove, M. S.; Willner, A. N.; Garcia-Mansfield, K.; Meechoovet, B.; Reiman, R.; Hutchins, E.; Miller, M. M.; Goel, A.; Pirrotte, P.; Van Keuren-Jensen, K.; Jovanovic-Talisman, T. Single Molecule Characterization of Individual Extracellular Vesicles from Pancreatic Cancer. J Extracell Vesicles 2019, 8, 1685634, DOI: 10.1080/20013078.2019.1685634Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXosFGqsLw%253D&md5=4e7972689b99a646ab2c8098edb4760aSingle molecule characterization of individual extracellular vesicles from pancreatic cancerLennon, Kathleen M.; Wakefield, Devin L.; Maddox, Adam L.; Brehove, Matthew S.; Willner, Ari N.; Garcia-Mansfield, Krystine; Meechoovet, Bessie; Reiman, Rebecca; Hutchins, Elizabeth; Miller, Marcia M.; Goel, Ajay; Pirrotte, Patrick; Van Keuren-Jensen, Kendall; Jovanovic-Talisman, TijanaJournal of Extracellular Vesicles (2019), 8 (1), 1685634CODEN: JEVOA4; ISSN:2001-3078. (Taylor & Francis Ltd.)Biofluid-accessible extracellular vesicles (EVs) may represent a new means to improve the sensitivity and specificity of detecting disease. However, current methods to isolate EVs encounter challenges when they are used to select specific populations. Moreover, it has been difficult to comprehensively characterize heterogeneous EV populations at the single vesicle level. Here, we robustly assessed heterogeneous EV populations from cultured cell lines via nanoparticle tracking anal., proteomics, transcriptomics, transmission electron microscopy, and quant. single mol. localization microscopy (qSMLM). Using qSMLM, we quantified the size and biomarker content of individual EVs. We applied qSMLM to patient plasma samples and identified a pancreatic cancer-enriched EV population. Our goal is to advance single mol. characterization of EVs for early disease detection.
- 23Schnitzbauer, J.; Strauss, M. T.; Schlichthaerle, T.; Schueder, F.; Jungmann, R. Super-Resolution Microscopy with DNA-PAINT. Nat. Protoc. 2017, 12, 1198– 1228, DOI: 10.1038/nprot.2017.024Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXnvF2rsLo%253D&md5=83998c799df527fa89eb74000aee4889Super-resolution microscopy with DNA-PAINTSchnitzbauer, Joerg; Strauss, Maximilian T.; Schlichthaerle, Thomas; Schueder, Florian; Jungmann, RalfNature Protocols (2017), 12 (6), 1198-1228CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)Super-resoln. techniques have begun to transform biol. and biomedical research by allowing researchers to observe structures well below the classic diffraction limit of light. DNA points accumulation for imaging in nanoscale topog. (DNA-PAINT) offers an easy-to-implement approach to localization-based super-resoln. microscopy, owing to the use of DNA probes. In DNA-PAINT, transient binding of short dye-labeled ('imager') oligonucleotides to their complementary target ('docking') strands creates the necessary 'blinking' to enable stochastic super-resoln. microscopy. Using the programmability and specificity of DNA mols. as imaging and labeling probes allows researchers to decouple blinking from dye photophysics, alleviating limitations of current super-resoln. techniques, making them compatible with virtually any single-mol.-compatible dye. Recent developments in DNA-PAINT have enabled spectrally unlimited multiplexing, precise mol. counting and ultra-high, mol.-scale (sub-5-nm) spatial resoln., reaching ∼1-nm localization precision. DNA-PAINT can be applied to a multitude of in vitro and cellular applications by linking docking strands to antibodies. Here, we present a protocol for the key aspects of the DNA-PAINT framework for both novice and expert users. This protocol describes the creation of DNA origami test samples, in situ sample prepn., multiplexed data acquisition, data simulation, super-resoln. image reconstruction and post-processing such as drift correction, mol. counting (qPAINT) and particle averaging. Moreover, we provide an integrated software package, named Picasso, for the computational steps involved. The protocol is designed to be modular, so that individual components can be chosen and implemented per requirements of a specific application. The procedure can be completed in 1-2 d.
- 24Jungmann, R.; Steinhauer, C.; Scheible, M.; Kuzyk, A.; Tinnefeld, P.; Simmel, F. C. Single-Molecule Kinetics and Super-Resolution Microscopy by Fluorescence Imaging of Transient Binding on DNA Origami. Nano Lett. 2010, 10, 4756– 4761, DOI: 10.1021/nl103427wGoogle Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhtlWiu7rK&md5=e93210e6c0d8347498b003e120ca8248Single-Molecule Kinetics and Super-Resolution Microscopy by Fluorescence Imaging of Transient Binding on DNA OrigamiJungmann, Ralf; Steinhauer, Christian; Scheible, Max; Kuzyk, Anton; Tinnefeld, Philip; Simmel, Friedrich C.Nano Letters (2010), 10 (11), 4756-4761CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)DNA origami is a powerful method for the programmable assembly of nanoscale mol. structures. For applications of these structures as functional biomaterials, the study of reaction kinetics and dynamic processes in real time and with high spatial resoln. becomes increasingly important. We present a single-mol. assay for the study of binding and unbinding kinetics on DNA origami. The authors find that the kinetics of hybridization to single-stranded extensions on DNA origami is similar to isolated substrate-immobilized DNA with a slight position dependence on the origami. On the basis of the knowledge of the kinetics, the authors exploit reversible specific binding of labeled oligonucleotides to DNA nanostructures for PAINT (points accumulation for imaging in nanoscale topog.) imaging with <30 nm resoln. The method is demonstrated for flat monomeric DNA structures as well as multimeric, ribbon-like DNA structures.
- 25Jungmann, R.; Avendaño, M. S.; Dai, M.; Woehrstein, J. B.; Agasti, S. S.; Feiger, Z.; Rodal, A.; Yin, P. Quantitative Super-Resolution Imaging with QPAINT. Nat. Methods 2016, 13, 439– 442, DOI: 10.1038/nmeth.3804Google Scholar25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XkvV2jtLk%253D&md5=1ce6b7fdb5e94a4309c88942f751a172Quantitative super-resolution imaging with qPAINTJungmann, Ralf; Avendano, Maier S.; Dai, Mingjie; Woehrstein, Johannes B.; Agasti, Sarit S.; Feiger, Zachary; Rodal, Avital; Yin, PengNature Methods (2016), 13 (5), 439-442CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)Counting mols. in complexes is challenging, even with super-resoln. microscopy. Here, we use the programmable and specific binding of dye-labeled DNA probes to count integer nos. of targets. This method, called quant. points accumulation in nanoscale topog. (qPAINT), works independently of dye photophysics for robust counting with high precision and accuracy over a wide dynamic range. qPAINT was benchmarked on DNA nanostructures and demonstrated for cellular applications by quantifying proteins in situ and the no. of single-mol. FISH probes bound to an mRNA target.
- 26Mücksch, J.; Blumhardt, P.; Strauss, M. T.; Petrov, E. P.; Jungmann, R.; Schwille, P. Quantifying Reversible Surface Binding via Surface-Integrated Fluorescence Correlation Spectroscopy. Nano Lett. 2018, 18, 3185– 3192, DOI: 10.1021/acs.nanolett.8b00875Google Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXns1Sjsb0%253D&md5=3c1bdd82250f137e36b22741e9656c8fQuantifying Reversible Surface Binding via Surface-Integrated Fluorescence Correlation SpectroscopyMuecksch, Jonas; Blumhardt, Philipp; Strauss, Maximilian T.; Petrov, Eugene P.; Jungmann, Ralf; Schwille, PetraNano Letters (2018), 18 (5), 3185-3192CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)We present a simple and versatile single-mol.-based method for the accurate detn. of binding rates to surfaces or surface bound receptors. To quantify the reversible surface attachment of fluorescently labeled mols., we have modified previous schemes for fluorescence correlation spectroscopy with total internal reflection illumination (TIR-FCS) and camera-based detection. In contrast to most modern applications of TIR-FCS, we completely disregard spatial information in the lateral direction. Instead, we perform correlation anal. on a spatially integrated signal, effectively converting the illuminated surface area into the measurement vol. In addn. to providing a high surface selectivity, our new approach resolves assocn. and dissocn. rates in equil. over a wide range of time scales. We chose the transient hybridization of fluorescently labeled single-stranded DNA to the complementary handles of surface-immobilized DNA origami structures as a reliable and well-characterized test system. We varied the no. of base pairs in the duplex, yielding different binding times in the range of hundreds of milliseconds to tens of seconds, allowing us to quantify the resp. surface affinities and binding rates.
- 27Civitci, F.; Shangguan, J.; Zheng, T.; Tao, K.; Rames, M.; Kenison, J.; Zhang, Y.; Wu, L.; Phelps, C.; Esener, S.; Nan, X. Fast and Multiplexed Superresolution Imaging with DNA-PAINT-ERS. Nat. Commun. 2020, 11, 4339, DOI: 10.1038/s41467-020-18181-6Google Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhslejtLjM&md5=3ca7982cb134412d8445cbc9b31fd53eFast and multiplexed superresolution imaging with DNA-PAINT-ERSCivitci, Fehmi; Shangguan, Julia; Zheng, Ting; Tao, Kai; Rames, Matthew; Kenison, John; Zhang, Ying; Wu, Lei; Phelps, Carey; Esener, Sadik; Nan, XiaolinNature Communications (2020), 11 (1), 4339CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)DNA points accumulation for imaging in nanoscale topog. (DNA-PAINT) facilitates multiplexing in superresoln. microscopy but is practically limited by slow imaging speed. To address this issue, we propose the addns. of ethylene carbonate (EC) to the imaging buffer, sequence repeats to the docking strand, and a spacer between the docking strand and the affinity agent. Collectively termed DNA-PAINT-ERS (E = EC, R = Repeating sequence, and S = Spacer), these strategies can be easily integrated into current DNA-PAINT workflows for both accelerated imaging speed and improved image quality through optimized DNA hybridization kinetics and efficiency. We demonstrate the general applicability of DNA-PAINT-ERS for fast, multiplexed superresoln. imaging using previously validated oligonucleotide constructs with slight modifications.
- 28Khater, I. M.; Nabi, I. R.; Hamarneh, G. A Review of Super-Resolution Single-Molecule Localization Microscopy Cluster Analysis and Quantification Methods. Patterns (N Y) 2020, 1, 100038, DOI: 10.1016/j.patter.2020.100038Google Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3s3mt1OmtA%253D%253D&md5=b08d6544181cc88888cc3f869b25cb32A Review of Super-Resolution Single-Molecule Localization Microscopy Cluster Analysis and Quantification MethodsKhater Ismail M; Hamarneh Ghassan; Nabi Ivan RobertPatterns (New York, N.Y.) (2020), 1 (3), 100038 ISSN:.Single-molecule localization microscopy (SMLM) is a relatively new imaging modality, winning the 2014 Nobel Prize in Chemistry, and considered as one of the key super-resolution techniques. SMLM resolution goes beyond the diffraction limit of light microscopy and achieves resolution on the order of 10-20 nm. SMLM thus enables imaging single molecules and study of the low-level molecular interactions at the subcellular level. In contrast to standard microscopy imaging that produces 2D pixel or 3D voxel grid data, SMLM generates big data of 2D or 3D point clouds with millions of localizations and associated uncertainties. This unprecedented breakthrough in imaging helps researchers employ SMLM in many fields within biology and medicine, such as studying cancerous cells and cell-mediated immunity and accelerating drug discovery. However, SMLM data quantification and interpretation methods have yet to keep pace with the rapid advancement of SMLM imaging. Researchers have been actively exploring new computational methods for SMLM data analysis to extract biosignatures of various biological structures and functions. In this survey, we describe the state-of-the-art clustering methods adopted to analyze and quantify SMLM data and examine the capabilities and shortcomings of the surveyed methods. We classify the methods according to (1) the biological application (i.e., the imaged molecules/structures), (2) the data acquisition (such as imaging modality, dimension, resolution, and number of localizations), and (3) the analysis details (2D versus 3D, field of view versus region of interest, use of machine-learning and multi-scale analysis, biosignature extraction, etc.). We observe that the majority of methods that are based on second-order statistics are sensitive to noise and imaging artifacts, have not been applied to 3D data, do not leverage machine-learning formulations, and are not scalable for big-data analysis. Finally, we summarize state-of-the-art methodology, discuss some key open challenges, and identify future opportunities for better modeling and design of an integrated computational pipeline to address the key challenges.
- 29Strauss, M. T.; Schueder, F.; Haas, D.; Nickels, P. C.; Jungmann, R. Quantifying Absolute Addressability in DNA Origami with Molecular Resolution. Nat. Commun. 2018, 9, 1600, DOI: 10.1038/s41467-018-04031-zGoogle Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1Mjlslyksw%253D%253D&md5=b9a83f6cace855a6ebf73db91ef12e5cQuantifying absolute addressability in DNA origami with molecular resolutionStrauss Maximilian T; Schueder Florian; Haas Daniel; Nickels Philipp C; Jungmann Ralf; Strauss Maximilian T; Schueder Florian; Haas Daniel; Nickels Philipp C; Jungmann RalfNature communications (2018), 9 (1), 1600 ISSN:.Self-assembled DNA nanostructures feature an unprecedented addressability with sub-nanometer precision and accuracy. This addressability relies on the ability to attach functional entities to single DNA strands in these structures. The efficiency of this attachment depends on two factors: incorporation of the strand of interest and accessibility of this strand for downstream modification. Here we use DNA-PAINT super-resolution microscopy to quantify both incorporation and accessibility of all individual strands in DNA origami with molecular resolution. We find that strand incorporation strongly correlates with the position in the structure, ranging from a minimum of 48% on the edges to a maximum of 95% in the center. Our method offers a direct feedback for the rational refinement of the design and assembly process of DNA nanostructures and provides a long sought-after quantitative explanation for efficiencies of DNA-based nanomachines.
- 30Feiner-Gracia, N.; Beck, M.; Pujals, S.; Tosi, S.; Mandal, T.; Buske, C.; Linden, M.; Albertazzi, L. Super-Resolution Microscopy Unveils Dynamic Heterogeneities in Nanoparticle Protein Corona. Small 2017, 13, 1701631, DOI: 10.1002/smll.201701631Google ScholarThere is no corresponding record for this reference.
- 31Strauss, S.; Jungmann, R. Up to 100-Fold Speed-up and Multiplexing in Optimized DNA-PAINT. Nat. Methods 2020, 17, 789– 791, DOI: 10.1038/s41592-020-0869-xGoogle Scholar31https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXht1yjtbfO&md5=14599350694abb5936e9b187d8ce7c15Up to 100-fold speed-up and multiplexing in optimized DNA-PAINTStrauss, Sebastian; Jungmann, RalfNature Methods (2020), 17 (8), 789-791CODEN: NMAEA3; ISSN:1548-7091. (Nature Research)DNA-PAINT's imaging speed has recently been significantly enhanced by optimized sequence design and buffer conditions. However, this implementation has not reached an ultimate speed limit and is only applicable to imaging of single targets. To further improve acquisition speed, we introduce concatenated, periodic DNA sequence motifs, yielding up to 100-fold-faster sampling in comparison to traditional DNA-PAINT. We extend this approach to six orthogonal sequence motifs, now enabling speed-optimized multiplexed imaging.
- 32Auer, A.; Strauss, M. T.; Schlichthaerle, T.; Jungmann, R. Fast, Background-Free DNA-PAINT Imaging Using FRET-Based Probes. Nano Lett. 2017, 17, 6428– 6434, DOI: 10.1021/acs.nanolett.7b03425Google Scholar32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhsVeksrjI&md5=df6aec1a08359e6d9f378bbd86954f8aFast, Background-Free DNA-PAINT Imaging Using FRET-Based ProbesAuer, Alexander; Strauss, Maximilian T.; Schlichthaerle, Thomas; Jungmann, RalfNano Letters (2017), 17 (10), 6428-6434CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)DNA points accumulation in nanoscale topog. (DNA-PAINT) enables super-resoln. microscopy by harnessing the predictable, transient hybridization between short dye-labeled imager and complementary target-bound docking strands. DNA-PAINT microscopy allows sub-5-nm spatial resoln., spectrally unlimited multiplexing and quant. image anal. However, these abilities come at the cost of non-fluorogenic imager strands, also emitting fluorescence when not bound to their docking strands. This has thus far prevented rapid image acquisition with DNA-PAINT, as the blinking rate of probes is limited by an upper-bound of imager strand concns., which in turn is dictated by the necessity to facilitate detection of single-mol. binding events over the background of unbound, freely diffusion probes. To overcome this limitation and enable fast, background-free DNA-PAINT microscopy, the authors here introduce FRET-based imaging probes, alleviating the concn.-limit of imager strands and speeding up image acquisition by several orders of magnitude. The authors assay two approaches for FRET-based DNA-PAINT (or FRET-PAINT) using either fixed or transient acceptor dyes in combination with transiently binding donor-labeled DNA strands and achieve high-quality super-resoln. imaging on DNA origami structures in a few tens of seconds. Finally, the authors also demonstrate the applicability of FRET-PAINT in a cellular environment by performing super-resoln. imaging of microtubules in under 30 s. FRET-PAINT combines the advantages of conventional DNA-PAINT with fast image acquisition times, facilitating the potential study of dynamic processes.
- 33Schueder, F.; Stein, J.; Stehr, F.; Auer, A.; Sperl, B.; Strauss, M. T.; Schwille, P.; Jungmann, R. An Order of Magnitude Faster DNA-PAINT Imaging by Optimized Sequence Design and Buffer Conditions. Nat. Methods 2019, 16, 1101– 1104, DOI: 10.1038/s41592-019-0584-7Google Scholar33https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhvFejs7%252FP&md5=06f54e8b078705956baa0db8c75f460aAn order of magnitude faster DNA-PAINT imaging by optimized sequence design and buffer conditionsSchueder, Florian; Stein, Johannes; Stehr, Florian; Auer, Alexander; Sperl, Bianca; Strauss, Maximilian T.; Schwille, Petra; Jungmann, RalfNature Methods (2019), 16 (11), 1101-1104CODEN: NMAEA3; ISSN:1548-7091. (Nature Research)DNA points accumulation in nanoscale topog. (DNA-PAINT) is a relatively easy-to-implement super-resoln. technique. However, image acquisition is slow compared to most other approaches. Here, we overcome this limitation by designing optimized DNA sequences and buffer conditions. We demonstrate our approach in vitro with DNA origami and in situ using cell samples, and achieve an order of magnitude faster imaging speeds without compromising image quality or spatial resoln. This improvement now makes DNA-PAINT applicable to high-throughput studies.
- 34Brennan, M.; Lim, B. The Actual Role of Receptors as Cancer Markers, Biochemical and Clinical Aspects: Receptors in Breast Cancer. Adv. Exp. Med. Biol. 2015, 867, 327– 337, DOI: 10.1007/978-94-017-7215-0_20Google Scholar34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXlvVOnur4%253D&md5=146124f18bea349e146c509f19a75b93The actual role of receptors as cancer markers, biochemical and clinical aspects: receptors in breast cancerBrennan, Matthew; Lim, BoraAdvances in Experimental Medicine and Biology (2015), 867 (Advances in Cancer Biomarkers), 327-337CODEN: AEMBAP; ISSN:2214-8019. (Springer)A biomarker is a characteristic that is objectively measured and evaluated as an indicator of normal biol. processes, pathogenic processes, or pharmacol. responses to a therapeutic intervention. The discovery and development of proper biomarkers is a crit. part of modern oncol. Among the many different types of biomarkers, cell receptors have demonstrated important roles as diagnostic, prognostic, and predictive biomarkers in cancer research and therapy, leading to their integration into drug development trials. In breast cancer, Estrogen/Progesterone receptors and HER2/neu receptors are two good examples of biomarkers that are prognostic of outcomes, as well as predictive of response to certain therapies. Limitations exist, however, such as the invasive procedures required obtaining tissue, and the difficulty measuring the actual distribution of the receptors. Thus, continued efforts to develop receptors as comprehensive cancer biomarkers with novel approaches is mandated to further advance the modern oncol.
- 35Henry, N. L.; Hayes, D. F. Cancer Biomarkers. Molecular Oncology 2012, 6, 140– 146, DOI: 10.1016/j.molonc.2012.01.010Google Scholar35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XmslynsLk%253D&md5=6fa4df49b95f15508c542f28689b5916Cancer biomarkersHenry, N. Lynn; Hayes, Daniel F.Molecular Oncology (2012), 6 (2), 140-146CODEN: MOONC3; ISSN:1574-7891. (Elsevier B.V.)A review. Biomarkers have many potential applications in oncol., including risk assessment, screening, differential diagnosis, detn. of prognosis, prediction of response to treatment, and monitoring of progression of disease. Because of the crit. role that biomarkers play at all stages of disease, it is important that they undergo rigorous evaluation, including anal. validation, clin. validation, and assessment of clin. utility, prior to incorporation into routine clin. care. In this review we address key steps in the development of biomarkers, including ways to avoid introducing bias and guidelines to follow when reporting results of biomarker studies.
- 36Cremers, G. A. O.; Rosier, B. J. H. M.; Riera Brillas, R.; Albertazzi, L.; de Greef, T. F. A. Efficient Small-Scale Conjugation of DNA to Primary Antibodies for Multiplexed Cellular Targeting. Bioconjugate Chem. 2019, 30, 2384– 2392, DOI: 10.1021/acs.bioconjchem.9b00490Google Scholar36https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhs1egu73E&md5=37e7a98c0473bc62cefb6dc7fe7f5ae7Efficient Small-Scale Conjugation of DNA to Primary Antibodies for Multiplexed Cellular TargetingCremers, Glenn A. O.; Rosier, Bas J. H. M.; Riera Brillas, Roger; Albertazzi, Lorenzo; de Greef, Tom F. A.Bioconjugate Chemistry (2019), 30 (9), 2384-2392CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)The combination of the specificity of antibodies and the programmability of DNA nanotechnol. has provided the scientific community with a powerful tool to label and unambiguously distinguish a large no. of subcellular targets using fluorescence-based read-out methods. Whereas primary antibodies are com. available for a large class of targets, a general stoichiometric site-selective DNA labeling strategy for this affinity reagent is lacking. Here we present a universal, site-selective conjugation method using a small photo-cross-linkable protein G adaptor that allows labeling of antibodies of different host species with a controlled no. of short oligonucleotides (ODNs). Importantly, we illustrate that this conjugation method can be directly performed on com. available primary antibodies on a small scale and without cross-reactivity towards bovine serum albumin. In addn., we present a general benchtop-compatible strategy to purify DNA-labeled antibodies without a loss of function. The application of protein G-ODN-labeled primary antibodies is demonstrated by employing three well-known methods for detecting subcellular targets using fluorescence read-out, including flow cytometry, DNA-PAINT, and dSTORM. This work thus establishes a general and efficient platform for the synthesis of a library of unique ODN-antibody conjugates, facilitating the broader use of DNA-based programmable tags for multiplexed labeling to identify subcellular features with nanometer precision and improving our understanding of cellular structure and function.
- 37Jin, S.; Xu, B.; Yu, L.; Fu, Y.; Wu, H.; Fan, X.; Liu, B.; Wei, J. The PD-1, PD-L1 Expression and CD3+ T Cell Infiltration in Relation to Outcome in Advanced Gastric Signet-Ring Cell Carcinoma, Representing a Potential Biomarker for Immunotherapy. JCO 2017, 35, e15609 DOI: 10.1200/JCO.2017.35.15_suppl.e15609Google ScholarThere is no corresponding record for this reference.
- 38Milanezi, F.; Carvalho, S.; Schmitt, F. C. EGFR/HER2 in Breast Cancer: A Biological Approach for Molecular Diagnosis and Therapy. Expert Review of Molecular Diagnostics 2008, 8, 417– 34, DOI: 10.1586/14737159.8.4.417Google Scholar38https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXotFOqt78%253D&md5=a13759c1e74226053e20e895a6d34fbbEGFR/HER2 in breast cancer: a biological approach for molecular diagnosis and therapyMilanezi, Fernanda; Carvalho, Silvia; Schmitt, Fernando C.Expert Review of Molecular Diagnostics (2008), 8 (4), 417-434CODEN: ERMDCW; ISSN:1473-7159. (Expert Reviews Ltd.)A review. Novel cancer therapies have focused on specific mol. markers present in malignant tumors. The rationale of targeted therapy relies on the knowledge of mol. mechanisms involved in carcinogenesis and their influence in clin. outcome allied to a more specific and less toxic treatment. Activation of EGF receptor and HER2 is an important factor for initiation and progression of malignancies, including breast cancer where the status of HER2 is an essential step in the diagnostic workup; EGFR overexpression has been assocd. to the so-called basal-like breast carcinomas, which opens a new avenue for diagnosis and therapeutic approach in these tumors. This review will focus on mechanisms of HER2 and EGF receptor upregulation, the targeted therapies that are currently in use for these receptors, possible combined therapies, as well as the approach for mol. diagnosis from the pathologist's point of view.
- 39van Waarde, A.; Rybczynska, A. A.; Ramakrishnan, N. K.; Ishiwata, K.; Elsinga, P. H.; Dierckx, R. A. J. O. Potential Applications for Sigma Receptor Ligands in Cancer Diagnosis and Therapy. Biochimica et Biophysica Acta (BBA) - Biomembranes 2015, 1848, 2703– 2714, DOI: 10.1016/j.bbamem.2014.08.022Google ScholarThere is no corresponding record for this reference.
- 40Ledermann, J. A.; Canevari, S.; Thigpen, T. Targeting the Folate Receptor: Diagnostic and Therapeutic Approaches to Personalize Cancer Treatments. Annals of Oncology 2015, 26, 2034– 2043, DOI: 10.1093/annonc/mdv250Google Scholar40https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2Mbis1ekuw%253D%253D&md5=0790b619d365bec194745e5c9c23ffbfTargeting the folate receptor: diagnostic and therapeutic approaches to personalize cancer treatmentsLedermann J A; Canevari S; Thigpen TAnnals of oncology : official journal of the European Society for Medical Oncology (2015), 26 (10), 2034-43 ISSN:.BACKGROUND: In cancer therapy, molecularly targeted agents have the potential to maximize antitumor efficacy while minimizing treatment-related toxicity. However, these agents may only be effective in specific tumor subtypes with defined genomic profiles. This emphasizes the importance of developing personalized cancer therapeutic strategies (i.e. through the use of companion diagnostic tests) to appropriately select and treat patients who are likely to benefit from specific targeted therapies, thus leading to improvements in clinical and safety outcomes. A potential biological target is the folate receptor (FR), which has been shown to be overexpressed on the surface of many cancers, including tumors of the lungs and ovaries. DESIGN: We carried out a literature search to identify how the FR can be a potential target for selected tumors, and how the FR expression can be exploited by targeted therapies. RESULTS: The two main therapeutic strategies for targeting the FR are based on the use of: (i) an anti-FR antibody (e.g. farletuzumab) and (ii) folate conjugates of folate-targeted chemotherapies and companion radiodiagnostic imaging agents (e.g. vintafolide and (99m)technetium-etarfolatide). Both of these strategies are being assessed in phase III trials. CONCLUSIONS: The important role that the FR plays in cancer development and progression has led to the development of FR-targeted therapeutic approaches. To date, the promising data observed in phase II clinical trials have not been confirmed in phase III studies. Accordingly, there is a need for further research in the refinement of patient selection and identification of new therapeutic combinations. In particular, the development of these targeted therapies requires reliable methods to be developed to detect FR-positive tumors in order to help select patients who may benefit from treatment.
- 41Cremers, G. A. O.; Rosier, B. J. H. M.; Meijs, A.; Tito, N. B.; van Duijnhoven, S. M. J.; van Eenennaam, H.; Albertazzi, L.; de Greef, T. F. A. Determinants of Ligand-Functionalized DNA Nanostructure–Cell Interactions. J. Am. Chem. Soc. 2021, 143, 10131– 10142, DOI: 10.1021/jacs.1c02298Google Scholar41https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhsVSru7zK&md5=9da63ce736c38e8dfbcccde3c9363c4aDeterminants of Ligand-Functionalized DNA Nanostructure-Cell InteractionsCremers, Glenn A. O.; Rosier, Bas J. H. M.; Meijs, Ab; Tito, Nicholas B.; van Duijnhoven, Sander M. J.; van Eenennaam, Hans; Albertazzi, Lorenzo; de Greef, Tom F. A.Journal of the American Chemical Society (2021), 143 (27), 10131-10142CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Synthesis of ligand-functionalized nanomaterials with control over size, shape, and ligand orientation facilitates the design of targeted nanomedicines for therapeutic purposes. DNA nanotechnol. has emerged as a powerful tool to rationally construct two- and three-dimensional nanostructures, enabling site-specific incorporation of protein ligands with control over stoichiometry and orientation. To efficiently target cell surface receptors, exploration of the parameters that modulate cellular accessibility of these nanostructures is essential. In this study, we systematically investigate tunable design parameters of antibody-functionalized DNA nanostructures binding to therapeutically relevant receptors, including the programmed cell death protein 1, the epidermal growth factor receptor, and the human epidermal growth factor receptor 2. We show that, although the native affinity of antibody-functionalized DNA nanostructures remains unaltered, the abs. no. of bound surface receptors is lower compared to sol. antibodies due to receptor accessibility by the nanostructure. We explore structural determinants of this phenomenon to improve efficiency, revealing that receptor binding is mainly governed by nanostructure size and DNA handle location. The obtained results provide key insights in the ability of ligand-functionalized DNA nanostructures to bind surface receptors and yields design rules for optimal cellular targeting.
- 42Vasilyev, F. F.; Lopatnikova, J. A.; Sennikov, S. V. Optimized Flow Cytometry Protocol for Analysis of Surface Expression of Interleukin-1 Receptor Types I and II. Cytotechnology 2013, 65, 795– 802, DOI: 10.1007/s10616-013-9546-6Google Scholar42https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhs12rtb7L&md5=df7761b2621674014b653435a42576ceOptimized flow cytometry protocol for analysis of surface expression of interleukin-1 receptor types I and IIVasilyev, Filipp Filippovich; Lopatnikova, Julia Anatolievna; Sennikov, Sergey VitalievichCytotechnology (2013), 65 (5), 795-802CODEN: CYTOER; ISSN:0920-9069. (Springer)The biol. effects of interleukin (IL)-1 are realized through binding to specific membrane-bound receptors. The efficiency of IL-1 action depends on the no. of receptors on the cell. We detd. the percentage of cells that express IL-1 receptor type I (IL-1RI) and IL-1 receptor type II (IL-1RII) by flow cytometry using phycoerythrin (PE)-labeled antibodies to the IL-1Rs, and the mean abs. no. of membrane-bound IL-1Rs per cell using QuantiBRITE PE calibration beads. We showed that different subpopulations of immunocompetent cells expressed different nos. of mols. of membrane-bound IL-1RI and IL-1RII. We also established that when cells were stimulated with bacterial lipopolysaccharide, there was a significant increase in the no. of IL-1RI expressed, and a significant decrease in the mean no. of IL-1RII mols. per cell. Detn. of the mean no. of membrane-bound IL-1R mols. using this protocol enables us to obtain precise and reproducible data that are necessary for full evaluation of expression levels.
- 43Anselmo, A.; Mazzon, C.; Borroni, E. M.; Bonecchi, R.; Graham, G. J.; Locati, M. Flow Cytometry Applications for the Analysis of Chemokine Receptor Expression and Function. Cytometry Part A 2014, 85, 292– 301, DOI: 10.1002/cyto.a.22439Google Scholar43https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXltFemtLk%253D&md5=74abfe7b8d0493da28f3991856b658aeFlow cytometry applications for the analysis of chemokine receptor expression and functionAnselmo, Achille; Mazzon, Cristina; Borroni, Elena Monica; Bonecchi, Raffaella; Graham, Gerard J.; Locati, MassimoCytometry, Part A (2014), 85A (4), 292-301CODEN: CPAYAV; ISSN:1552-4922. (Wiley-Blackwell)Chemokine receptors play an important role in leukocyte migration, both in physiol. and pathol. conditions, and the interest in new methodologies for their detection is increasing. In this review, we focused on chemokine receptors detection through flow cytometric approaches, including the use of specific antibodies and fluorescent chemokines, and on approaches aimed at the anal. of their functions, from intracellular trafficking to signaling activities. © 2014 International Society for Advancement of Cytometry.
- 44Kužílková, D.; Puñet-Ortiz, J.; Aui, P. M.; Fernández, J.; Fišer, K.; Engel, P.; van Zelm, M. C.; Kalina, T. Standardization of Workflow and Flow Cytometry Panels for Quantitative Expression Profiling of Surface Antigens on Blood Leukocyte Subsets: An HCDM CDMaps Initiative. Frontiers in Immunology 2022, 13, 827898, DOI: 10.3389/fimmu.2022.827898Google Scholar44https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB2M3nsVShuw%253D%253D&md5=b1282eafb1743d839d5da199dc36bd32Standardization of Workflow and Flow Cytometry Panels for Quantitative Expression Profiling of Surface Antigens on Blood Leukocyte Subsets: An HCDM CDMaps InitiativeKuzilkova Daniela; Fiser Karel; Kalina Tomas; Punet-Ortiz Joan; Fernandez Javier; Engel Pablo; Aui Pei M; van Zelm Menno C; van Zelm Menno CFrontiers in immunology (2022), 13 (), 827898 ISSN:.Background: The Human Cell Differentiation Molecules (HCDM) organizes Human Leukocyte Differentiation Antigen (HLDA) workshops to test and name clusters of antibodies that react with a specific antigen. These cluster of differentiation (CD) markers have provided the scientific community with validated antibody clones, consistent naming of targets and reproducible identification of leukocyte subsets. Still, quantitative CD marker expression profiles and benchmarking of reagents at the single-cell level are currently lacking. Objective: To develop a flow cytometric procedure for quantitative expression profiling of surface antigens on blood leukocyte subsets that is standardized across multiple research laboratories. Methods: A high content framework to evaluate the titration and reactivity of Phycoerythrin (PE)-conjugated monoclonal antibodies (mAbs) was created. Two flow cytometry panels were designed: an innate cell tube for granulocytes, dendritic cells, monocytes, NK cells and innate lymphoid cells (12-color) and an adaptive lymphocyte tube for naive and memory B and T cells, including TCRγδ(+), regulatory-T and follicular helper T cells (11-color). The potential of these 2 panels was demonstrated via expression profiling of selected CD markers detected by PE-conjugated antibodies and evaluated using 561 nm excitation. Results: Using automated data annotation and dried backbone reagents, we reached a robust workflow amenable to processing hundreds of measurements in each experiment in a 96-well plate format. The immunophenotyping panels enabled discrimination of 27 leukocyte subsets and quantitative detection of the expression of PE-conjugated CD markers of interest that could quantify protein expression above 400 units of antibody binding capacity. Expression profiling of 4 selected CD markers (CD11b, CD31, CD38, CD40) showed high reproducibility across centers, as well as the capacity to benchmark unique clones directed toward the same CD3 antigen. Conclusion: We optimized a procedure for quantitative expression profiling of surface antigens on blood leukocyte subsets. The workflow, bioinformatics pipeline and optimized flow panels enable the following: 1) mapping the expression patterns of HLDA-approved mAb clones to CD markers; 2) benchmarking new antibody clones to established CD markers; 3) defining new clusters of differentiation in future HLDA workshops.
- 45Sharma, M.; Yang, Z.; Miyamoto, H. Immunohistochemistry of Immune Checkpoint Markers PD-1 and PD-L1 in Prostate Cancer. Medicine (Baltimore) 2019, 98, e17257 DOI: 10.1097/MD.0000000000017257Google ScholarThere is no corresponding record for this reference.
- 46Hutchinson, R. A.; Adams, R. A.; McArt, D. G.; Salto-Tellez, M.; Jasani, B.; Hamilton, P. W. Epidermal Growth Factor Receptor Immunohistochemistry: New Opportunities in Metastatic Colorectal Cancer. J Transl Med 2015, 13, 217, DOI: 10.1186/s12967-015-0531-zGoogle Scholar46https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC28%252FgvFGhsQ%253D%253D&md5=bc1088230200c5f5c02a1f1449c4af03Epidermal growth factor receptor immunohistochemistry: new opportunities in metastatic colorectal cancerHutchinson Ryan A; McArt Darragh G; Salto-Tellez Manuel; Hamilton Peter W; Hutchinson Ryan A; Adams Richard A; Jasani BharatJournal of translational medicine (2015), 13 (), 217 ISSN:.The treatment of cancer is becoming more precise, targeting specific oncogenic drivers with targeted molecular therapies. The epidermal growth factor receptor has been found to be over-expressed in a multitude of solid tumours. Immunohistochemistry is widely used in the fields of diagnostic and personalised medicine to localise and visualise disease specific proteins. To date the clinical utility of epidermal growth factor receptor immunohistochemistry in determining monoclonal antibody efficacy has remained somewhat inconclusive. The lack of an agreed reproducible scoring criteria for epidermal growth factor receptor immunohistochemistry has, in various clinical trials yielded conflicting results as to the use of epidermal growth factor receptor immunohistochemistry assay as a companion diagnostic. This has resulted in this test being removed from the licence for the drug panitumumab and not performed in clinical practice for cetuximab. In this review we explore the reasons behind this with a particular emphasis on colorectal cancer, and to suggest a way of resolving the situation through improving the precision of epidermal growth factor receptor immunohistochemistry with quantitative image analysis of digitised images complemented with companion molecular morphological techniques such as in situ hybridisation and section based gene mutation analysis.
- 47Gaber, R.; Goldmann, T. Mini Review: Immunohistochemistry Using EGFR-Mutant Specific Antibodies in Non-Small Cell Lung Carcinoma: Accuracy and Reliability. Journal of Cancer Treatment and Diagnosis 2018, 2().Google ScholarThere is no corresponding record for this reference.
- 48Queiroga, F. L.; Perez-Alenza, M. D.; González-Gil, A.; Silván, G.; Peña, L.; Illera, J. C. Quantification of Epidermal Growth Factor Receptor (EGFR) in Canine Mammary Tumours by ELISA Assay: Clinical and Prognostic Implications. Vet Comp Oncol 2017, 15, 383– 390, DOI: 10.1111/vco.12174Google Scholar48https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXnsV2jtr8%253D&md5=c9f45014ddfef5659894701da5b0311fQuantification of epidermal growth factor receptor (EGFR) in canine mammary tumours by ELISA assay: clinical and prognostic implicationsQueiroga, F. L.; Perez-Alenza, M. D.; Gonzalez-Gil, A.; Silvan, G.; Pena, L.; Illera, J. C.Veterinary and Comparative Oncology (2017), 15 (2), 383-390CODEN: VCOEAN; ISSN:1476-5810. (Wiley-Blackwell)The involvement of epidermal growth factor receptor (EGFR) is well established in human breast cancer, however, in canine mammary tumors (CMT), including inflammatory mammary carcinomas (IMC), still needs to be clarified. Enzyme immune assay techniques were used for EGFR detns. in tumor tissue from 45 bitches with CMT and in normal mammary glands from eight control dogs. Higher tissue EGFR levels were found in CMT compared with controls (P < 0.05). In malignant CMT, tissue EGFR elevated concns. were statistically significantly assocd. with tumor relapse and/or distant metastasis during follow-up and with reduced disease-free and overall survival times. The IMC cases had the highest tissue EGFR levels compared with other malignant non-IMC tumors (P < 0.001). The results support the hypothesis that EGFR levels influence prognosis in malignant CMT, suggesting that EGFR may represent a therapeutic target in cases of high histol. aggressiveness and esp. in cases of metastatic phenotype and poor prognosis.
- 49Takeuchi, M.; Doi, T.; Obayashi, K.; Hirai, A.; Yoneda, K.; Tanaka, F.; Iwai, Y. Soluble PD-L1 with PD-1-Binding Capacity Exists in the Plasma of Patients with Non-Small Cell Lung Cancer. Immunol. Lett. 2018, 196, 155– 160, DOI: 10.1016/j.imlet.2018.01.007Google Scholar49https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXitFKitr8%253D&md5=097cf14a36e6b02e1a77fe95bce6d1d3Soluble PD-L1 with PD-1-binding capacity exists in the plasma of patients with non-small cell lung cancerTakeuchi, Masahiro; Doi, Tomomitsu; Obayashi, Kunie; Hirai, Ayako; Yoneda, Kazue; Tanaka, Fumihiro; Iwai, YoshikoImmunology Letters (2018), 196 (), 155-160CODEN: IMLED6; ISSN:0165-2478. (Elsevier B.V.)PD-L1 is one of the important immune checkpoint mols. that can be targeted by cancer immunotherapies. PD-L1 has a sol. form (sPD-L1) and a membrane-bound form (mPD-L1). Conventional ELISA (ELISA) systems can detect sPD-L1 using anti-PD-L1 capture antibody through the antigen-antibody reaction, but cannot evaluate the quality and function of sPD-L1. In this study, we developed a novel ELISA system for the detection and quantification of sPD-L1 with PD-1-binding capacity (bsPD-L1). To capture bsPD-L1 through the ligand-receptor reaction, the anti-PD-L1 capture antibody in the conventional ELISA was replaced with PD-1-Ig fusion protein in the new ELISA. The new ELISA could detect bsPD-L1 in 29 out of 75 plasma samples from patients with non-small cell lung cancer (NSCLC), with higher sensitivity and frequency than the conventional ELISA. The western blot anal. showed that sPD-L1 in the plasma was glycosylated. Treatment of the samples with glycosidase reduced the absorbance detd. by the new ELISA but had no effect on the absorbance detd. by the conventional ELISA. These results suggest that glycosylation of sPD-L1 is important for its binding to the immobilized PD-1 in the new ELISA. Our new ELISA system may be useful for the evaluation of functional sPD-L1 with PD-1-binding capacity in cancer patients.
- 50Zhang, X.; Zhang, Y.; Tang, H.; He, J. EGFR Gene Copy Number as a Predictive/Biomarker for Patients with Non-Small-Cell Lung Cancer Receiving Tyrosine Kinase Inhibitor Treatment: A Systematic Review and Meta-Analysis. J Investig Med 2017, 65, 72– 81, DOI: 10.1136/jim-2016-000252Google Scholar50https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2svjsFSjtw%253D%253D&md5=4e0170759ae2e5272fef4112a3ec8986EGFR gene copy number as a predictive/biomarker for patients with non-small-cell lung cancer receiving tyrosine kinase inhibitor treatment: a systematic review and meta-analysisZhang Xin; He Jianxing; Zhang Yiwen; Tang HailingJournal of investigative medicine : the official publication of the American Federation for Clinical Research (2017), 65 (1), 72-81 ISSN:.Epidermal growth factor receptor (EGFR) gene copy number has been proposed as a candidate biomarker for predicting treatment response to EGFR tyrosine kinase inhibitors (EGFR-TKIs) in patients with advanced non-small-cell lung cancer (NSCLC). MEDLINE, PubMed, Cochrane, and Google Scholar databases were searched until October 21, 2015 using the following search terms: lung neoplasms/lung cancer/non-small cell lung cancer/NSCLC, EGFR, gene amplification, copy number, erlotinib, gefitinib, tyrosine-kinase inhibitor/TKI, predictor. 17 studies were included in the analysis with a total of 2047 patients. The overall analysis found that increased EGFR gene copy number was associated with higher overall response rate (ORR), overall survival (OS) and progression-free survival (PFS; p values ≤0.008) compared with patients without a high EGFR gene copy number. Subgroup analysis found that in a population of patients who were primarily Caucasian, a higher EGFR gene copy number was also associated with increased ORR, OS, and PFS (p values ≤0.018). The results were similar in a population of Asian patients, except that a higher EGFR gene copy number was not associated with improved OS (p=0.248). Sensitivity analysis indicated that no one study overly influenced the results and that the findings are robust. The result of the analysis found that EGFR gene copy number was associated with increased OS and PFS, supporting the idea that EGFR gene copy number is a biomarker for response to EGFR-TKI therapy in patients with advanced NSCLC.
- 51Gaber, R.; Watermann, I.; Kugler, C.; Reinmuth, N.; Huber, R. M.; Schnabel, P. A.; Vollmer, E.; Reck, M.; Goldmann, T. Correlation of EGFR Expression, Gene Copy Number and Clinicopathological Status in NSCLC. Diagnostic Pathology 2014, 9, 165, DOI: 10.1186/s13000-014-0165-0Google Scholar51https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXitFGns7fP&md5=57fd452df93ceebc6504222b3a0c2de0Correlation of EGFR expression, gene copy number and clinicopathological status in NSCLCGaber, Rania; Watermann, Iris; Kugler, Christian; Reinmuth, Nils; Huber, Rudolf M.; Schnabel, Philipp A.; Vollmer, Ekkehard; Reck, Martin; Goldmann, TorstenDiagnostic Pathology (2014), 9 (), 165/1-165/26, 26 pp.CODEN: DPIAAA; ISSN:1746-1596. (BioMed Central Ltd.)Background: Epidermal Growth Factor Receptor (EGFR) targeting therapies are currently of great relevance for the treatment of lung cancer. For this reason, in addn. to mutational anal. immunohistochem. (IHC) of EGFR in lung cancer has been discussed for the decision making of according therapeutic strategies. The aim of this study was to obtain standardization of EGFR-expression methods for the selection of patients who might benefit of EGFR targeting therapies. Methods: As a starting point of a broad investigation, aimed at elucidating the expression of EGFR on different biol. levels, four EGFR specific antibodies were analyzed concerning potential differences in expression levels by Immunohistochem. (IHC) and correlated with fluorescence in situ hybridization (FISH) anal. and clinicopathol. data. 206 tumor tissues were analyzed in a tissue microarray format employing immunohistochem. with four different antibodies including Dako PharmDx kit (clone 2-18C9), clone 31G7, clone 2.1E1 and clone SP84 using three different scoring methods. Protein expression was compared to FISH utilizing two different probes. Results: EGFR protein expression detd. by IHC with Dako PharmDx kit, clone 31G7 and clone 2.1E1 (p ≤ 0.05) correlated significantly with both FISH probes independently of the three scoring methods; best correlation is shown for 31G7 using the scoring method that defined EGFR positivity when ≥10% of the tumor cells show membranous staining of moderate and severe intensity (p = 0.001). Conclusion: Overall, our data show differences in EGFR expression detd. by IHC, due to the applied antibody. Highest concordance with FISH is shown for antibody clone 31G7, evaluated with score B (p = 0,001). On this account, this antibody clone might by utilized for std. evaluation of EGFR expression by IHC.
- 52Zhang, F.; Wang, S.; Yin, L.; Yang, Y.; Guan, Y.; Wang, W.; Xu, H.; Tao, N. Quantification of Epidermal Growth Factor Receptor Expression Level and Binding Kinetics on Cell Surfaces by Surface Plasmon Resonance Imaging. Anal. Chem. 2015, 87, 9960– 9965, DOI: 10.1021/acs.analchem.5b02572Google Scholar52https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhsVyksbrO&md5=8d291df500a3ec460d5ef57aa0912a38Quantification of Epidermal Growth Factor Receptor Expression Level and Binding Kinetics on Cell Surfaces by Surface Plasmon Resonance ImagingZhang, Fenni; Wang, Shaopeng; Yin, Linliang; Yang, Yunze; Guan, Yan; Wang, Wei; Xu, Han; Tao, NongjianAnalytical Chemistry (Washington, DC, United States) (2015), 87 (19), 9960-9965CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Epidermal growth factor receptor (EGFR, also known as ErbB-1 or HER-1) is a membrane bound protein that has been assocd. with a variety of solid tumors and the control of cell survival, proliferation, and metab. Quantification of the EGFR expression level in cell membranes and the interaction kinetics with drugs are thus important for cancer diagnosis and treatment. Here we report mapping of the distribution and interaction kinetics of EGFR in their native environment with the surface plasmon resonance imaging (SPRi) technique. The monoclonal anti-EGFR antibody was used as a model drug in this study. The binding of the antibody to EGFR overexpressed A431 cells was monitored in real time, which was found to follow the first-order kinetics with an assocn. rate const. (ka) and dissocn. rate const. (kd) of (2.7 ± 0.6) × 105 M-1s-1 and (1.4 ± 0.5) × 10-4 s-1, resp. The dissocn. const. (KD) was detd. to be 0.53 ± 0.26 nM with up to seven-fold variation among different individual A431 cells. In addn., the averaged A431 cell surface EGFR d. was found to be 636/μm2 with an estn. of 5 × 105 EGFR per cell. Addnl. measurement also revealed that different EGFR pos. cell lines (A431, HeLa, and A549) show receptor d. dependent anti-EGFR binding kinetics. The results demonstrate that SPRi is a valuable tool for direct quantification of membrane protein expression level and ligand binding kinetics at single cell resoln. Our findings show that the local environment affects the drug-receptor interactions, and in situ measurement of membrane protein binding kinetics is important.
- 53Ma, H.; Liu, Y. Super-Resolution Localization Microscopy: Toward High Throughput, High Quality, and Low Cost. APL Photonics 2020, 5, 060902, DOI: 10.1063/5.0011731Google Scholar53https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhsVKmt7jO&md5=d3f68aca6cc4050b1bdfd5e50a5d67b5Super-resolution localization microscopy: Toward high throughput, high quality, and low costMa, Hongqiang; Liu, YangAPL Photonics (2020), 5 (6), 060902CODEN: APPHD2; ISSN:2378-0967. (American Institute of Physics)A review. After nearly 15 years since its initial debut, super-resoln. localization microscopy that surpasses the diffraction-limited resoln. barrier of optical microscopy has rapidly gotten out of the ivory tower and entered a new phase to address various challenging biomedical questions. Recent advances in this technol. greatly increased the imaging throughput, improved the imaging quality, simplified the sample prepn., and reduced the system cost, making this technol. suitable for routine biomedical research. We will provide our perspective on the recent tech. advances and their implications in serving the community of biomedical research. (c) 2020 American Institute of Physics.
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- Wei Shan Tan, Arthur M. de Jong, Menno W. J. Prins. Revealing Spatial Molecular Heterogeneity of High-Density Biofunctionalized Surfaces Using DNA-PAINT. ACS Applied Materials & Interfaces 2024, 16
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- 3Hughes, L. D.; Rawle, R. J.; Boxer, S. G. Choose Your Label Wisely: Water-Soluble Fluorophores Often Interact with Lipid Bilayers. PLOS ONE 2014, 9, e87649 DOI: 10.1371/journal.pone.00876493https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhsVWgsLbL&md5=f7c048612b19ac808915dc3c6999d5d3Choose your label wisely: water-soluble fluorophores often interact with lipid bilayersHughes, Laura D.; Rawle, Robert J.; Boxer, Steven G.PLoS One (2014), 9 (2), e87649/1-e87649/8, 8 pp.CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)Water-sol. org. fluorophores are widely used as labels in biol. systems. However, in many cases these fluorophores can interact strongly with lipid bilayers, influencing the interaction of the target with the bilayer and/or leading to misleading fluorescent signals. Here, we quantify the interaction of 32 common water-sol. dyes with model lipid bilayers to serve as an addnl. criterion when selecting a dye label.
- 4Visser, E. W. A.; Yan, J.; van IJzendoorn, L. J.; Prins, M. W. J. Continuous Biomarker Monitoring by Particle Mobility Sensing with Single Molecule Resolution. Nat. Commun. 2018, 9, 2541, DOI: 10.1038/s41467-018-04802-84https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3c%252FjsVKrtg%253D%253D&md5=f67f9191dad47c1ba9fde81361b95006Continuous biomarker monitoring by particle mobility sensing with single molecule resolutionVisser Emiel W A; van IJzendoorn Leo J; Prins Menno W J; Visser Emiel W A; Yan Junhong; van IJzendoorn Leo J; Prins Menno W J; Yan Junhong; Prins Menno W JNature communications (2018), 9 (1), 2541 ISSN:.Healthcare is in demand of technologies for real-time sensing in order to continuously guard the state of patients. Here we present biomarker-monitoring based on the sensing of particle mobility, a concept wherein particles are coupled to a substrate via a flexible molecular tether, with both the particles and substrate provided with affinity molecules for effectuating specific and reversible interactions. Single-molecular binding and unbinding events modulate the Brownian particle motion and the state changes are recorded using optical scattering microscopy. The technology is demonstrated with DNA and protein as model biomarkers, in buffer and in blood plasma, showing sensitivity to picomolar and nanomolar concentrations. The sensing principle is direct and self-contained, without consuming or producing any reactants. With its basis in reversible interactions and single-molecule resolution, we envisage that the presented technology will enable biosensors for continuous biomarker monitoring with high sensitivity, specificity, and accuracy.
- 5Hong, R.; Hu, Y.; Huang, H. Biomarkers for Chimeric Antigen Receptor T Cell Therapy in Acute Lymphoblastic Leukemia: Prospects for Personalized Management and Prognostic Prediction. Frontiers in Immunology 2021, 12, 627764, DOI: 10.3389/fimmu.2021.6277645https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXpvVyht7c%253D&md5=3e3e58b3abf0f6b6e1db416940134cefBiomarkers for chimeric antigen receptor t cell therapy in acute lymphoblastic leukemia: prospects for personalized management and prognostic predictionHong, Ruimin; Hu, Yongxian; Huang, HeFrontiers in Immunology (2021), 12 (), 627764CODEN: FIRMCW; ISSN:1664-3224. (Frontiers Media S.A.)A review. Chimeric antigen receptor (CAR) T cell therapy represents a breakthrough in immunotherapy with the potential of ushering in a new era in cancer treatment. Remarkable therapeutic response and complete remission of this innovative management have been obsd. in patients with relapse/refractory acute lymphoblastic leukemia. With CAR-T cell therapy becoming widely used both in multicenter clin. trials and as a com. treatment, therapeutic efficacy monitoring and management of toxicities will be indispensable for ensuring safety and improving overall survival. Biomarkers can act not only as effective indicators reflecting patients baseline characteristics, CAR-T cell potency, and the immune microenvironment, but can also assess side effects during treatment. In this review, we will elaborate on a series of biomarkers assocd. with therapeutic response as well as treatment-related toxicities, and present their current condition and latent value with respect to the clin. utility. The combination of biomarker research and CAR-T cell therapy will contribute to establishing a safer and more powerful monitoring system and prolonging the event-free survival of patients.
- 6Martínez-Pérez, C.; Turnbull, A. K.; Dixon, J. M. The Evolving Role of Receptors as Predictive Biomarkers for Metastatic Breast Cancer. Expert Review of Anticancer Therapy 2019, 19, 121– 138, DOI: 10.1080/14737140.2019.15521386https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXisVGnurfO&md5=727a51a76cc869db40f6c7ab31ec0a94The evolving role of receptors as predictive biomarkers for metastatic breast cancerMartinez-Perez, Carlos; Turnbull, Arran K.; Dixon, J. MichaelExpert Review of Anticancer Therapy (2019), 19 (2), 121-138CODEN: ERATBJ; ISSN:1473-7140. (Taylor & Francis Ltd.)A review. In breast cancer, estrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2) are essential biomarkers to predict response to endocrine and anti-HER2 therapies, resp. In metastatic breast cancer, the use of these receptors and targeted therapies present addnl. challenges: temporal heterogeneity, together with limited sampling methodologies, hinders receptor status assessment, and the const. evolution of the disease invariably leads to resistance to treatment. This review summarizes the genomic abnormalities in ER and HER2, such as mutations, amplifications, translocations, and alternative splicing, emerging as novel biomarkers that provide an insight into underlying mechanisms of resistance and hold potential predictive value to inform treatment selection. We also describe how liq. biopsies for sampling of circulating markers and ultrasensitive detection technologies have emerged which complement ongoing efforts for biomarker discovery and anal. While evidence suggests that genomic aberrations in ER and HER2 could contribute to meeting the pressing need for better predictive biomarkers, efforts need to be made to standardize assessment methods and better understand the resistance mechanisms these markers denote. Taking advantage of emerging technologies, research in upcoming years should include prospective trials incorporating these predictors into the study design to validate their potential clin. value.
- 7Cox, G.; Sheppard, C. J. R. Practical Limits of Resolution in Confocal and Non-Linear Microscopy. Microscopy Research and Technique 2004, 63, 18– 22, DOI: 10.1002/jemt.104237https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD3srpsFWhtQ%253D%253D&md5=03bb4376bb113c6df1c147e6ba2cc93dPractical limits of resolution in confocal and non-linear microscopyCox Guy; Sheppard Colin J RMicroscopy research and technique (2004), 63 (1), 18-22 ISSN:1059-910X.Calculated and measured resolution figures are presented for confocal microscopes with different pinhole sizes and for nonlinear (2-photon and second harmonic) microscopes. A modest degree of super-resolution is predicted for a confocal microscope but in practice this is not achievable and confocal fluorescence gives little resolution improvement over widefield. However, practical non-linear microscopes do approach their theoretical resolution and therefore show no resolution disadvantage relative to confocal microscopes in spite of the longer excitation wavelength.
- 8Schermelleh, L.; Ferrand, A.; Huser, T.; Eggeling, C.; Sauer, M.; Biehlmaier, O.; Drummen, G. P. C. Super-Resolution Microscopy Demystified. Nat. Cell Biol. 2019, 21, 72– 84, DOI: 10.1038/s41556-018-0251-88https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXmvVOhsL4%253D&md5=8b2f987dc08e1ad2407538f702df3b70Super-resolution microscopy demystifiedSchermelleh, Lothar; Ferrand, Alexia; Huser, Thomas; Eggeling, Christian; Sauer, Markus; Biehlmaier, Oliver; Drummen, Gregor P. C.Nature Cell Biology (2019), 21 (1), 72-84CODEN: NCBIFN; ISSN:1465-7392. (Nature Research)Super-resoln. microscopy (SRM) bypasses the diffraction limit, a phys. barrier that restricts the optical resoln. to roughly 250 nm and was previously thought to be impenetrable. SRM techniques allow the visualization of subcellular organization with unprecedented detail, but also confront biologists with the challenge of selecting the best-suited approach for their particular research question. Here, we provide guidance on how to use SRM techniques advantageously for investigating cellular structures and dynamics to promote new discoveries.
- 9Nicovich, P. R.; Owen, D. M.; Gaus, K. Turning Single-Molecule Localization Microscopy into a Quantitative Bioanalytical Tool. Nat. Protoc. 2017, 12, 453– 460, DOI: 10.1038/nprot.2016.1669https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXitVWnt7w%253D&md5=56491eb69975910566567e70e596893eTurning single-molecule localization microscopy into a quantitative bioanalytical toolNicovich, Philip R.; Owen, Dylan M.; Gaus, KatharinaNature Protocols (2017), 12 (3), 453-460CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)A review. Single-mol. localization microscopy (SMLM) generates super-resoln. images by serially detecting individual fluorescent mols. The power of SMLM, however, goes beyond images: biol. relevant information can be extd. from the math. relationships between the positions of the fluorophores in space and time. Here we review the history of SMLM and how recent progress in methods for spatial point anal. has enabled quant. measurement of SMLM data, providing insights into biomol. patterning, clustering and oligomerization in biol. systems.
- 10Hummert, J.; Yserentant, K.; Fink, T.; Euchner, J.; Herten, D.-P. Photobleaching Step Analysis for Robust Determination of Protein Complex Stoichiometries. bioRxiv 2020, 26, 268086, DOI: 10.1101/2020.08.26.268086There is no corresponding record for this reference.
- 11Belfiore, L.; Spenkelink, L. M.; Ranson, M.; van Oijen, A. M.; Vine, K. L. Quantification of Ligand Density and Stoichiometry on the Surface of Liposomes Using Single-Molecule Fluorescence Imaging. J. Controlled Release 2018, 278, 80– 86, DOI: 10.1016/j.jconrel.2018.03.02211https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXnsVemsrc%253D&md5=7f39de86a16f519c078c2997f7819595Quantification of ligand density and stoichiometry on the surface of liposomes using single-molecule fluorescence imagingBelfiore, Lisa; Spenkelink, Lisanne M.; Ranson, Marie; van Oijen, Antoine M.; Vine, Kara L.Journal of Controlled Release (2018), 278 (), 80-86CODEN: JCREEC; ISSN:0168-3659. (Elsevier B.V.)Despite the longstanding existence of liposome technol. in drug delivery applications, there have been no ligand-directed liposome formulations approved for clin. use to date. This lack of translation is due to several factors, one of which is the absence of mol. tools for the robust quantification of ligand d. on the surface of liposomes. We report here for the first time the quantification of proteins attached to the surface of small unilamellar liposomes using single-mol. fluorescence imaging. Liposomes were surface-functionalized with fluorescently labeled human proteins previously validated to target the cancer cell surface biomarkers plasminogen activator inhibitor-2 (PAI-2) and trastuzumab (TZ, Herceptin). These protein-conjugated liposomes were visualized using a custom-built wide-field fluorescence microscope with single-mol. sensitivity. By counting the photobleaching steps of the fluorescently labeled proteins, we calcd. the no. of attached proteins per liposome, which was 11±4 proteins for single-ligand liposomes. Imaging of dual-ligand liposomes revealed stoichiometries of the two attached proteins in accordance with the molar ratios of protein added during prepn. Prepn. of PAI-2/TZ dual-ligand liposomes via two different methods revealed that the post-insertion method generated liposomes with a more equal representation of the two differently sized proteins, demonstrating the ability of this prepn.
- 12Geissbuehler, S.; Bocchio, N. L.; Dellagiacoma, C.; Berclaz, C.; Leutenegger, M.; Lasser, T. Mapping Molecular Statistics with Balanced Super-Resolution Optical Fluctuation Imaging (BSOFI). Optical Nanoscopy 2012, 1, 4, DOI: 10.1186/2192-2853-1-4There is no corresponding record for this reference.
- 13Gruβmayer, K. S.; Yserentant, K.; Herten, D.-P. Photons in - Numbers out: Perspectives in Quantitative Fluorescence Microscopy for in Situ Protein Counting. Methods Appl. Fluoresc. 2019, 7, 012003, DOI: 10.1088/2050-6120/aaf2ebThere is no corresponding record for this reference.
- 14Pujals, S.; Feiner-Gracia, N.; Delcanale, P.; Voets, I.; Albertazzi, L. Super-Resolution Microscopy as a Powerful Tool to Study Complex Synthetic Materials. Nature Reviews Chemistry 2019, 3, 68– 84, DOI: 10.1038/s41570-018-0070-2There is no corresponding record for this reference.
- 15Lubken, R. M.; de Jong, A. M.; Prins, M. W. J. How Reactivity Variability of Biofunctionalized Particles Is Determined by Superpositional Heterogeneities. ACS Nano 2021, 15, 1331– 1341, DOI: 10.1021/acsnano.0c0857815https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhsFKgsg%253D%253D&md5=fde4a71d9b491536c9c245de7bc3ab09How Reactivity Variability of Biofunctionalized Particles Is Determined by Superpositional HeterogeneitiesLubken, Rafiq M.; de Jong, Arthur M.; Prins, Menno W. J.ACS Nano (2021), 15 (1), 1331-1341CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)The biofunctionalization of particles with specific targeting moieties forms the foundation for mol. recognition in biomedical applications such as targeted nanomedicine and particle-based biosensing. To achieve a high precision of targeting for nanomedicine and high precision of sensing for biosensing, it is important to understand the consequences of heterogeneities of particle properties. Here, we present a comprehensive methodol. to study with expts. and simulations the collective consequences of particle heterogeneities on multiple length scales, called superpositional heterogeneities, in generating reactivity variability per particle. Single-mol. techniques are used to quantify stochastic, interparticle, and intraparticle variabilities, in order to show how these variabilities collectively contribute to reactivity variability per particle, and how the influence of each contributor changes as a function of the system parameters such as particle interaction area, the particle size, the targeting moiety d., and the no. of particles. The results give insights into the consequences of superpositional heterogeneities for the reactivity variability in biomedical applications and give guidelines on how the precision can be optimized in the presence of multiple independent sources of variability.
- 16Horáček, M.; Engels, D. J.; Zijlstra, P. Dynamic Single-Molecule Counting for the Quantification and Optimization of Nanoparticle Functionalization Protocols. Nanoscale 2020, 12, 4128– 4136, DOI: 10.1039/C9NR10218C16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhslWhurk%253D&md5=3eaa15c54c9acf68dc470e3ab8eb6115Dynamic single-molecule counting for the quantification and optimization of nanoparticle functionalization protocolsHoracek, Matej; Engels, Dion J.; Zijlstra, PeterNanoscale (2020), 12 (6), 4128-4136CODEN: NANOHL; ISSN:2040-3372. (Royal Society of Chemistry)Applications of colloidal particles in the fields of i.e. biosensors, mol. targeting, or drug-delivery require their functionalization with biol. active and specific mol. ligands. Functionalization protocols often result in a heterogeneous population of particles with a varying d., spatial distribution and orientation of the functional groups on the particle surface. A lack of methods to directly resolve these mol. properties of the particle's surface hampers optimization of functionalization protocols and applications. Here quant. single-mol. interaction kinetics is used to count the no. of ligands on the surface of hundreds of individual nanoparticles simultaneously. By analyzing the waiting-time between single-mol. binding events we quantify the particle functionalization both accurately and precisely for a large range of ligand densities. We observe significant particle-to-particle differences in functionalization which are dominated by the particle-size distribution for high mol. densities, but are substantially broadened for sparsely functionalized particles. From time-dependent studies we find that ligand reorganization on long timescales drastically reduces this heterogeneity, a process that has remained hidden up to now in ensemble-averaged studies. The quant. single-mol. counting therefore provides a direct route to quantification and optimization of coupling protocols towards molecularly controlled colloidal interfaces.
- 17Delcanale, P.; Miret-Ontiveros, B.; Arista-Romero, M.; Pujals, S.; Albertazzi, L. Nanoscale Mapping Functional Sites on Nanoparticles by Points Accumulation for Imaging in Nanoscale Topography (PAINT). ACS Nano 2018, 12, 7629– 7637, DOI: 10.1021/acsnano.7b0906317https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtl2js7vJ&md5=c04b4297511e06ae763f8aa53441f3ffNanoscale Mapping Functional Sites on Nanoparticles by Points Accumulation for Imaging in Nanoscale Topography (PAINT)Delcanale, Pietro; Miret-Ontiveros, Bernat; Arista-Romero, Maria; Pujals, Silvia; Albertazzi, LorenzoACS Nano (2018), 12 (8), 7629-7637CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)The ability of nanoparticles to selectively recognize a mol. target constitutes the key toward nanomedicine applications such as drug delivery and diagnostics. The activity of such devices is mediated by the presence of multiple copies of functional mols. on the nanostructure surface. Therefore, understanding the no. and the distribution of nanoparticle functional groups is of utmost importance for the rational design of effective materials. Anal. methods are available, but to obtain quant. information at the level of single particles and single functional sites, i.e., going beyond the ensemble, remains highly challenging. Here the authors introduce the use of an optical nanoscopy technique, DNA points accumulation for imaging in nanoscale topog. (DNA-PAINT), to address this issue. Combining subdiffraction spatial resoln. with mol. selectivity and sensitivity, DNA-PAINT provides both geometrical and functional information at the level of a single nanostructure. DNA-PAINT can be used to image and quantify relevant functional proteins such as antibodies and streptavidin on nanoparticles and microparticles with nanometric accuracy in 3D and multiple colors. The generality and the applicability of the authors' method without the need for fluorescent labeling hold great promise for the robust quant. nanocharacterization of functional nanomaterials.
- 18Strauss, S.; Nickels, P. C.; Strauss, M. T.; Jimenez Sabinina, V. J.; Ellenberg, J.; Carter, J. D.; Gupta, S.; Janjic, N.; Jungmann, R. Modified Aptamers Enable Quantitative Sub-10-Nm Cellular DNA-PAINT Imaging. Nat. Methods 2018, 15, 685, DOI: 10.1038/s41592-018-0105-018https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhsFGjur3K&md5=11808272f7077ba2e2e6e157a01bea3aModified aptamers enable quantitative sub-10-nm cellular DNA-PAINT imagingStrauss, Sebastian; Nickels, Philipp C.; Strauss, Maximilian T.; Jimenez Sabinina, Vilma; Ellenberg, Jan; Carter, Jeffrey D.; Gupta, Shashi; Janjic, Nebojsa; Jungmann, RalfNature Methods (2018), 15 (9), 685-688CODEN: NMAEA3; ISSN:1548-7091. (Nature Research)Although current implementations of super-resoln. microscopy are tech. approaching true mol.-scale resoln., this has not translated to imaging of biol. specimens, because of the large size of conventional affinity reagents. Here we introduce slow off-rate modified aptamers (SOMAmers) as small and specific labeling reagents for use with DNA points accumulation in nanoscale topog. (DNA-PAINT). To demonstrate the achievable resoln., specificity, and multiplexing capability of SOMAmers, we labeled and imaged both transmembrane and intracellular targets in fixed and live cells.
- 19Nerreter, T.; Letschert, S.; Götz, R.; Doose, S.; Danhof, S.; Einsele, H.; Sauer, M.; Hudecek, M. Super-Resolution Microscopy Reveals Ultra-Low CD19 Expression on Myeloma Cells That Triggers Elimination by CD19 CAR-T. Nat. Commun. 2019, 10, 3137, DOI: 10.1038/s41467-019-10948-w19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3MzotlOnug%253D%253D&md5=7ba7da4f6a44018192ae96237bac7e95Super-resolution microscopy reveals ultra-low CD19 expression on myeloma cells that triggers elimination by CD19 CAR-TNerreter Thomas; Danhof Sophia; Einsele Hermann; Hudecek Michael; Letschert Sebastian; Gotz Ralph; Doose Soren; Sauer MarkusNature communications (2019), 10 (1), 3137 ISSN:.Immunotherapy with chimeric antigen receptor-engineered T-cells (CAR-T) is under investigation in multiple myeloma. There are reports of myeloma remission after CD19 CAR-T therapy, although CD19 is hardly detectable on myeloma cells by flow cytometry (FC). We apply single molecule-sensitive direct stochastic optical reconstruction microscopy (dSTORM), and demonstrate CD19 expression on a fraction of myeloma cells (10.3-80%) in 10 out of 14 patients (density: 13-5,000 molecules per cell). In contrast, FC detects CD19 in only 2 of these 10 patients, on a smaller fraction of cells. Treatment with CD19 CAR-T in vitro results in elimination of CD19-positive myeloma cells, including those with <100 CD19 molecules per cell. Similar data are obtained by dSTORM analyses of CD20 expression on myeloma cells and CD20 CAR-T. These data establish a sensitivity threshold for CAR-T and illustrate how super-resolution microscopy can guide patient selection in immunotherapy to exploit ultra-low density antigens.
- 20Tobin, S. J.; Wakefield, D. L.; Jones, V.; Liu, X.; Schmolze, D.; Jovanović-Talisman, T. Single Molecule Localization Microscopy Coupled with Touch Preparation for the Quantification of Trastuzumab-Bound HER2. Sci. Rep. 2018, 8, 15154, DOI: 10.1038/s41598-018-33225-020https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3czot1elsw%253D%253D&md5=8ff31d6fe7b0b09a17359ec90b04c1c9Single molecule localization microscopy coupled with touch preparation for the quantification of trastuzumab-bound HER2Tobin Steven J; Wakefield Devin L; Jovanovic-Talisman Tijana; Jones Veronica; Liu Xueli; Schmolze DanielScientific reports (2018), 8 (1), 15154 ISSN:.All breast cancers are assessed for levels of human epidermal growth factor receptor 2 (HER2). Fluorescence in situ hybridization (FISH) and immunohistochemistry are currently used to determine if a patient is eligible for anti-HER2 therapy. Limitations of both tests include variability and relatively long processing times. Additionally, neither test determines whether HER2 contains the extracellular domain. While truncated in some tumors, this domain is required for binding of the therapeutic antibody trastuzumab. Here, trastuzumab was used to directly detect HER2 with quantitative single molecule localization microscopy (qSMLM). In proof of concept studies, our new method rapidly quantified both HER2 density and features of nano-organization. In cultured cells, the method was sensitive to subtle variations in HER2 expression. To assess patient samples, we combined qSMLM with tissue touch preparation (touch prep-qSMLM) and examined large areas of intact membranes. For cell lines and patient samples, HER2 copy numbers from FISH showed a significant positive correlation with detected densities from qSMLM and trended with HER2 cluster occupancy.
- 21Chen, C.; Zong, S.; Liu, Y.; Wang, Z.; Zhang, Y.; Chen, B.; Cui, Y. Profiling of Exosomal Biomarkers for Accurate Cancer Identification: Combining DNA-PAINT with Machine- Learning-Based Classification. Small 2019, 15, 1901014, DOI: 10.1002/smll.20190101421https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhslWjtrjP&md5=8417cadf49b5a80ce1d61dad81e29e7dProfiling of Exosomal Biomarkers for Accurate Cancer Identification: Combining DNA-PAINT with Machine- Learning-Based ClassificationChen, Chen; Zong, Shenfei; Liu, Yun; Wang, Zhuyuan; Zhang, Yizhi; Chen, Baoan; Cui, YipingSmall (2019), 15 (43), 1901014CODEN: SMALBC; ISSN:1613-6810. (Wiley-VCH Verlag GmbH & Co. KGaA)Exosomes are endosome-derived vesicles enriched in body fluids such as urine, blood, and saliva. So far, they have been recognized as potential biomarkers for cancer diagnostics. However, the present single-variate anal. of exosomes has greatly limited the accuracy and specificity of diagnoses. Besides, most diagnostic approaches focus on bulk anal. using lots of exosomes and tend to be less accurate because they are vulnerable to impure extn. and concn. differences of exosomes. To address these challenges, a quant. anal. platform is developed to implement a sequential quantification anal. of multiple exosomal surface biomarkers at the single-exosome level, which uses DNA-PAINT and a machine learning algorithm to automatically analyze the results. As a proof of concept, the profiling of four exosomal surface biomarkers (HER2, GPC-1, EpCAM, EGFR) is developed to identify exosomes from cancer-derived blood samples. Then, this technique is further applied to detect pancreatic cancer and breast cancer from unknown samples with 100% accuracy.
- 22Lennon, K. M.; Wakefield, D. L.; Maddox, A. L.; Brehove, M. S.; Willner, A. N.; Garcia-Mansfield, K.; Meechoovet, B.; Reiman, R.; Hutchins, E.; Miller, M. M.; Goel, A.; Pirrotte, P.; Van Keuren-Jensen, K.; Jovanovic-Talisman, T. Single Molecule Characterization of Individual Extracellular Vesicles from Pancreatic Cancer. J Extracell Vesicles 2019, 8, 1685634, DOI: 10.1080/20013078.2019.168563422https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXosFGqsLw%253D&md5=4e7972689b99a646ab2c8098edb4760aSingle molecule characterization of individual extracellular vesicles from pancreatic cancerLennon, Kathleen M.; Wakefield, Devin L.; Maddox, Adam L.; Brehove, Matthew S.; Willner, Ari N.; Garcia-Mansfield, Krystine; Meechoovet, Bessie; Reiman, Rebecca; Hutchins, Elizabeth; Miller, Marcia M.; Goel, Ajay; Pirrotte, Patrick; Van Keuren-Jensen, Kendall; Jovanovic-Talisman, TijanaJournal of Extracellular Vesicles (2019), 8 (1), 1685634CODEN: JEVOA4; ISSN:2001-3078. (Taylor & Francis Ltd.)Biofluid-accessible extracellular vesicles (EVs) may represent a new means to improve the sensitivity and specificity of detecting disease. However, current methods to isolate EVs encounter challenges when they are used to select specific populations. Moreover, it has been difficult to comprehensively characterize heterogeneous EV populations at the single vesicle level. Here, we robustly assessed heterogeneous EV populations from cultured cell lines via nanoparticle tracking anal., proteomics, transcriptomics, transmission electron microscopy, and quant. single mol. localization microscopy (qSMLM). Using qSMLM, we quantified the size and biomarker content of individual EVs. We applied qSMLM to patient plasma samples and identified a pancreatic cancer-enriched EV population. Our goal is to advance single mol. characterization of EVs for early disease detection.
- 23Schnitzbauer, J.; Strauss, M. T.; Schlichthaerle, T.; Schueder, F.; Jungmann, R. Super-Resolution Microscopy with DNA-PAINT. Nat. Protoc. 2017, 12, 1198– 1228, DOI: 10.1038/nprot.2017.02423https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXnvF2rsLo%253D&md5=83998c799df527fa89eb74000aee4889Super-resolution microscopy with DNA-PAINTSchnitzbauer, Joerg; Strauss, Maximilian T.; Schlichthaerle, Thomas; Schueder, Florian; Jungmann, RalfNature Protocols (2017), 12 (6), 1198-1228CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)Super-resoln. techniques have begun to transform biol. and biomedical research by allowing researchers to observe structures well below the classic diffraction limit of light. DNA points accumulation for imaging in nanoscale topog. (DNA-PAINT) offers an easy-to-implement approach to localization-based super-resoln. microscopy, owing to the use of DNA probes. In DNA-PAINT, transient binding of short dye-labeled ('imager') oligonucleotides to their complementary target ('docking') strands creates the necessary 'blinking' to enable stochastic super-resoln. microscopy. Using the programmability and specificity of DNA mols. as imaging and labeling probes allows researchers to decouple blinking from dye photophysics, alleviating limitations of current super-resoln. techniques, making them compatible with virtually any single-mol.-compatible dye. Recent developments in DNA-PAINT have enabled spectrally unlimited multiplexing, precise mol. counting and ultra-high, mol.-scale (sub-5-nm) spatial resoln., reaching ∼1-nm localization precision. DNA-PAINT can be applied to a multitude of in vitro and cellular applications by linking docking strands to antibodies. Here, we present a protocol for the key aspects of the DNA-PAINT framework for both novice and expert users. This protocol describes the creation of DNA origami test samples, in situ sample prepn., multiplexed data acquisition, data simulation, super-resoln. image reconstruction and post-processing such as drift correction, mol. counting (qPAINT) and particle averaging. Moreover, we provide an integrated software package, named Picasso, for the computational steps involved. The protocol is designed to be modular, so that individual components can be chosen and implemented per requirements of a specific application. The procedure can be completed in 1-2 d.
- 24Jungmann, R.; Steinhauer, C.; Scheible, M.; Kuzyk, A.; Tinnefeld, P.; Simmel, F. C. Single-Molecule Kinetics and Super-Resolution Microscopy by Fluorescence Imaging of Transient Binding on DNA Origami. Nano Lett. 2010, 10, 4756– 4761, DOI: 10.1021/nl103427w24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhtlWiu7rK&md5=e93210e6c0d8347498b003e120ca8248Single-Molecule Kinetics and Super-Resolution Microscopy by Fluorescence Imaging of Transient Binding on DNA OrigamiJungmann, Ralf; Steinhauer, Christian; Scheible, Max; Kuzyk, Anton; Tinnefeld, Philip; Simmel, Friedrich C.Nano Letters (2010), 10 (11), 4756-4761CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)DNA origami is a powerful method for the programmable assembly of nanoscale mol. structures. For applications of these structures as functional biomaterials, the study of reaction kinetics and dynamic processes in real time and with high spatial resoln. becomes increasingly important. We present a single-mol. assay for the study of binding and unbinding kinetics on DNA origami. The authors find that the kinetics of hybridization to single-stranded extensions on DNA origami is similar to isolated substrate-immobilized DNA with a slight position dependence on the origami. On the basis of the knowledge of the kinetics, the authors exploit reversible specific binding of labeled oligonucleotides to DNA nanostructures for PAINT (points accumulation for imaging in nanoscale topog.) imaging with <30 nm resoln. The method is demonstrated for flat monomeric DNA structures as well as multimeric, ribbon-like DNA structures.
- 25Jungmann, R.; Avendaño, M. S.; Dai, M.; Woehrstein, J. B.; Agasti, S. S.; Feiger, Z.; Rodal, A.; Yin, P. Quantitative Super-Resolution Imaging with QPAINT. Nat. Methods 2016, 13, 439– 442, DOI: 10.1038/nmeth.380425https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XkvV2jtLk%253D&md5=1ce6b7fdb5e94a4309c88942f751a172Quantitative super-resolution imaging with qPAINTJungmann, Ralf; Avendano, Maier S.; Dai, Mingjie; Woehrstein, Johannes B.; Agasti, Sarit S.; Feiger, Zachary; Rodal, Avital; Yin, PengNature Methods (2016), 13 (5), 439-442CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)Counting mols. in complexes is challenging, even with super-resoln. microscopy. Here, we use the programmable and specific binding of dye-labeled DNA probes to count integer nos. of targets. This method, called quant. points accumulation in nanoscale topog. (qPAINT), works independently of dye photophysics for robust counting with high precision and accuracy over a wide dynamic range. qPAINT was benchmarked on DNA nanostructures and demonstrated for cellular applications by quantifying proteins in situ and the no. of single-mol. FISH probes bound to an mRNA target.
- 26Mücksch, J.; Blumhardt, P.; Strauss, M. T.; Petrov, E. P.; Jungmann, R.; Schwille, P. Quantifying Reversible Surface Binding via Surface-Integrated Fluorescence Correlation Spectroscopy. Nano Lett. 2018, 18, 3185– 3192, DOI: 10.1021/acs.nanolett.8b0087526https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXns1Sjsb0%253D&md5=3c1bdd82250f137e36b22741e9656c8fQuantifying Reversible Surface Binding via Surface-Integrated Fluorescence Correlation SpectroscopyMuecksch, Jonas; Blumhardt, Philipp; Strauss, Maximilian T.; Petrov, Eugene P.; Jungmann, Ralf; Schwille, PetraNano Letters (2018), 18 (5), 3185-3192CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)We present a simple and versatile single-mol.-based method for the accurate detn. of binding rates to surfaces or surface bound receptors. To quantify the reversible surface attachment of fluorescently labeled mols., we have modified previous schemes for fluorescence correlation spectroscopy with total internal reflection illumination (TIR-FCS) and camera-based detection. In contrast to most modern applications of TIR-FCS, we completely disregard spatial information in the lateral direction. Instead, we perform correlation anal. on a spatially integrated signal, effectively converting the illuminated surface area into the measurement vol. In addn. to providing a high surface selectivity, our new approach resolves assocn. and dissocn. rates in equil. over a wide range of time scales. We chose the transient hybridization of fluorescently labeled single-stranded DNA to the complementary handles of surface-immobilized DNA origami structures as a reliable and well-characterized test system. We varied the no. of base pairs in the duplex, yielding different binding times in the range of hundreds of milliseconds to tens of seconds, allowing us to quantify the resp. surface affinities and binding rates.
- 27Civitci, F.; Shangguan, J.; Zheng, T.; Tao, K.; Rames, M.; Kenison, J.; Zhang, Y.; Wu, L.; Phelps, C.; Esener, S.; Nan, X. Fast and Multiplexed Superresolution Imaging with DNA-PAINT-ERS. Nat. Commun. 2020, 11, 4339, DOI: 10.1038/s41467-020-18181-627https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhslejtLjM&md5=3ca7982cb134412d8445cbc9b31fd53eFast and multiplexed superresolution imaging with DNA-PAINT-ERSCivitci, Fehmi; Shangguan, Julia; Zheng, Ting; Tao, Kai; Rames, Matthew; Kenison, John; Zhang, Ying; Wu, Lei; Phelps, Carey; Esener, Sadik; Nan, XiaolinNature Communications (2020), 11 (1), 4339CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)DNA points accumulation for imaging in nanoscale topog. (DNA-PAINT) facilitates multiplexing in superresoln. microscopy but is practically limited by slow imaging speed. To address this issue, we propose the addns. of ethylene carbonate (EC) to the imaging buffer, sequence repeats to the docking strand, and a spacer between the docking strand and the affinity agent. Collectively termed DNA-PAINT-ERS (E = EC, R = Repeating sequence, and S = Spacer), these strategies can be easily integrated into current DNA-PAINT workflows for both accelerated imaging speed and improved image quality through optimized DNA hybridization kinetics and efficiency. We demonstrate the general applicability of DNA-PAINT-ERS for fast, multiplexed superresoln. imaging using previously validated oligonucleotide constructs with slight modifications.
- 28Khater, I. M.; Nabi, I. R.; Hamarneh, G. A Review of Super-Resolution Single-Molecule Localization Microscopy Cluster Analysis and Quantification Methods. Patterns (N Y) 2020, 1, 100038, DOI: 10.1016/j.patter.2020.10003828https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3s3mt1OmtA%253D%253D&md5=b08d6544181cc88888cc3f869b25cb32A Review of Super-Resolution Single-Molecule Localization Microscopy Cluster Analysis and Quantification MethodsKhater Ismail M; Hamarneh Ghassan; Nabi Ivan RobertPatterns (New York, N.Y.) (2020), 1 (3), 100038 ISSN:.Single-molecule localization microscopy (SMLM) is a relatively new imaging modality, winning the 2014 Nobel Prize in Chemistry, and considered as one of the key super-resolution techniques. SMLM resolution goes beyond the diffraction limit of light microscopy and achieves resolution on the order of 10-20 nm. SMLM thus enables imaging single molecules and study of the low-level molecular interactions at the subcellular level. In contrast to standard microscopy imaging that produces 2D pixel or 3D voxel grid data, SMLM generates big data of 2D or 3D point clouds with millions of localizations and associated uncertainties. This unprecedented breakthrough in imaging helps researchers employ SMLM in many fields within biology and medicine, such as studying cancerous cells and cell-mediated immunity and accelerating drug discovery. However, SMLM data quantification and interpretation methods have yet to keep pace with the rapid advancement of SMLM imaging. Researchers have been actively exploring new computational methods for SMLM data analysis to extract biosignatures of various biological structures and functions. In this survey, we describe the state-of-the-art clustering methods adopted to analyze and quantify SMLM data and examine the capabilities and shortcomings of the surveyed methods. We classify the methods according to (1) the biological application (i.e., the imaged molecules/structures), (2) the data acquisition (such as imaging modality, dimension, resolution, and number of localizations), and (3) the analysis details (2D versus 3D, field of view versus region of interest, use of machine-learning and multi-scale analysis, biosignature extraction, etc.). We observe that the majority of methods that are based on second-order statistics are sensitive to noise and imaging artifacts, have not been applied to 3D data, do not leverage machine-learning formulations, and are not scalable for big-data analysis. Finally, we summarize state-of-the-art methodology, discuss some key open challenges, and identify future opportunities for better modeling and design of an integrated computational pipeline to address the key challenges.
- 29Strauss, M. T.; Schueder, F.; Haas, D.; Nickels, P. C.; Jungmann, R. Quantifying Absolute Addressability in DNA Origami with Molecular Resolution. Nat. Commun. 2018, 9, 1600, DOI: 10.1038/s41467-018-04031-z29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1Mjlslyksw%253D%253D&md5=b9a83f6cace855a6ebf73db91ef12e5cQuantifying absolute addressability in DNA origami with molecular resolutionStrauss Maximilian T; Schueder Florian; Haas Daniel; Nickels Philipp C; Jungmann Ralf; Strauss Maximilian T; Schueder Florian; Haas Daniel; Nickels Philipp C; Jungmann RalfNature communications (2018), 9 (1), 1600 ISSN:.Self-assembled DNA nanostructures feature an unprecedented addressability with sub-nanometer precision and accuracy. This addressability relies on the ability to attach functional entities to single DNA strands in these structures. The efficiency of this attachment depends on two factors: incorporation of the strand of interest and accessibility of this strand for downstream modification. Here we use DNA-PAINT super-resolution microscopy to quantify both incorporation and accessibility of all individual strands in DNA origami with molecular resolution. We find that strand incorporation strongly correlates with the position in the structure, ranging from a minimum of 48% on the edges to a maximum of 95% in the center. Our method offers a direct feedback for the rational refinement of the design and assembly process of DNA nanostructures and provides a long sought-after quantitative explanation for efficiencies of DNA-based nanomachines.
- 30Feiner-Gracia, N.; Beck, M.; Pujals, S.; Tosi, S.; Mandal, T.; Buske, C.; Linden, M.; Albertazzi, L. Super-Resolution Microscopy Unveils Dynamic Heterogeneities in Nanoparticle Protein Corona. Small 2017, 13, 1701631, DOI: 10.1002/smll.201701631There is no corresponding record for this reference.
- 31Strauss, S.; Jungmann, R. Up to 100-Fold Speed-up and Multiplexing in Optimized DNA-PAINT. Nat. Methods 2020, 17, 789– 791, DOI: 10.1038/s41592-020-0869-x31https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXht1yjtbfO&md5=14599350694abb5936e9b187d8ce7c15Up to 100-fold speed-up and multiplexing in optimized DNA-PAINTStrauss, Sebastian; Jungmann, RalfNature Methods (2020), 17 (8), 789-791CODEN: NMAEA3; ISSN:1548-7091. (Nature Research)DNA-PAINT's imaging speed has recently been significantly enhanced by optimized sequence design and buffer conditions. However, this implementation has not reached an ultimate speed limit and is only applicable to imaging of single targets. To further improve acquisition speed, we introduce concatenated, periodic DNA sequence motifs, yielding up to 100-fold-faster sampling in comparison to traditional DNA-PAINT. We extend this approach to six orthogonal sequence motifs, now enabling speed-optimized multiplexed imaging.
- 32Auer, A.; Strauss, M. T.; Schlichthaerle, T.; Jungmann, R. Fast, Background-Free DNA-PAINT Imaging Using FRET-Based Probes. Nano Lett. 2017, 17, 6428– 6434, DOI: 10.1021/acs.nanolett.7b0342532https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhsVeksrjI&md5=df6aec1a08359e6d9f378bbd86954f8aFast, Background-Free DNA-PAINT Imaging Using FRET-Based ProbesAuer, Alexander; Strauss, Maximilian T.; Schlichthaerle, Thomas; Jungmann, RalfNano Letters (2017), 17 (10), 6428-6434CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)DNA points accumulation in nanoscale topog. (DNA-PAINT) enables super-resoln. microscopy by harnessing the predictable, transient hybridization between short dye-labeled imager and complementary target-bound docking strands. DNA-PAINT microscopy allows sub-5-nm spatial resoln., spectrally unlimited multiplexing and quant. image anal. However, these abilities come at the cost of non-fluorogenic imager strands, also emitting fluorescence when not bound to their docking strands. This has thus far prevented rapid image acquisition with DNA-PAINT, as the blinking rate of probes is limited by an upper-bound of imager strand concns., which in turn is dictated by the necessity to facilitate detection of single-mol. binding events over the background of unbound, freely diffusion probes. To overcome this limitation and enable fast, background-free DNA-PAINT microscopy, the authors here introduce FRET-based imaging probes, alleviating the concn.-limit of imager strands and speeding up image acquisition by several orders of magnitude. The authors assay two approaches for FRET-based DNA-PAINT (or FRET-PAINT) using either fixed or transient acceptor dyes in combination with transiently binding donor-labeled DNA strands and achieve high-quality super-resoln. imaging on DNA origami structures in a few tens of seconds. Finally, the authors also demonstrate the applicability of FRET-PAINT in a cellular environment by performing super-resoln. imaging of microtubules in under 30 s. FRET-PAINT combines the advantages of conventional DNA-PAINT with fast image acquisition times, facilitating the potential study of dynamic processes.
- 33Schueder, F.; Stein, J.; Stehr, F.; Auer, A.; Sperl, B.; Strauss, M. T.; Schwille, P.; Jungmann, R. An Order of Magnitude Faster DNA-PAINT Imaging by Optimized Sequence Design and Buffer Conditions. Nat. Methods 2019, 16, 1101– 1104, DOI: 10.1038/s41592-019-0584-733https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhvFejs7%252FP&md5=06f54e8b078705956baa0db8c75f460aAn order of magnitude faster DNA-PAINT imaging by optimized sequence design and buffer conditionsSchueder, Florian; Stein, Johannes; Stehr, Florian; Auer, Alexander; Sperl, Bianca; Strauss, Maximilian T.; Schwille, Petra; Jungmann, RalfNature Methods (2019), 16 (11), 1101-1104CODEN: NMAEA3; ISSN:1548-7091. (Nature Research)DNA points accumulation in nanoscale topog. (DNA-PAINT) is a relatively easy-to-implement super-resoln. technique. However, image acquisition is slow compared to most other approaches. Here, we overcome this limitation by designing optimized DNA sequences and buffer conditions. We demonstrate our approach in vitro with DNA origami and in situ using cell samples, and achieve an order of magnitude faster imaging speeds without compromising image quality or spatial resoln. This improvement now makes DNA-PAINT applicable to high-throughput studies.
- 34Brennan, M.; Lim, B. The Actual Role of Receptors as Cancer Markers, Biochemical and Clinical Aspects: Receptors in Breast Cancer. Adv. Exp. Med. Biol. 2015, 867, 327– 337, DOI: 10.1007/978-94-017-7215-0_2034https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXlvVOnur4%253D&md5=146124f18bea349e146c509f19a75b93The actual role of receptors as cancer markers, biochemical and clinical aspects: receptors in breast cancerBrennan, Matthew; Lim, BoraAdvances in Experimental Medicine and Biology (2015), 867 (Advances in Cancer Biomarkers), 327-337CODEN: AEMBAP; ISSN:2214-8019. (Springer)A biomarker is a characteristic that is objectively measured and evaluated as an indicator of normal biol. processes, pathogenic processes, or pharmacol. responses to a therapeutic intervention. The discovery and development of proper biomarkers is a crit. part of modern oncol. Among the many different types of biomarkers, cell receptors have demonstrated important roles as diagnostic, prognostic, and predictive biomarkers in cancer research and therapy, leading to their integration into drug development trials. In breast cancer, Estrogen/Progesterone receptors and HER2/neu receptors are two good examples of biomarkers that are prognostic of outcomes, as well as predictive of response to certain therapies. Limitations exist, however, such as the invasive procedures required obtaining tissue, and the difficulty measuring the actual distribution of the receptors. Thus, continued efforts to develop receptors as comprehensive cancer biomarkers with novel approaches is mandated to further advance the modern oncol.
- 35Henry, N. L.; Hayes, D. F. Cancer Biomarkers. Molecular Oncology 2012, 6, 140– 146, DOI: 10.1016/j.molonc.2012.01.01035https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XmslynsLk%253D&md5=6fa4df49b95f15508c542f28689b5916Cancer biomarkersHenry, N. Lynn; Hayes, Daniel F.Molecular Oncology (2012), 6 (2), 140-146CODEN: MOONC3; ISSN:1574-7891. (Elsevier B.V.)A review. Biomarkers have many potential applications in oncol., including risk assessment, screening, differential diagnosis, detn. of prognosis, prediction of response to treatment, and monitoring of progression of disease. Because of the crit. role that biomarkers play at all stages of disease, it is important that they undergo rigorous evaluation, including anal. validation, clin. validation, and assessment of clin. utility, prior to incorporation into routine clin. care. In this review we address key steps in the development of biomarkers, including ways to avoid introducing bias and guidelines to follow when reporting results of biomarker studies.
- 36Cremers, G. A. O.; Rosier, B. J. H. M.; Riera Brillas, R.; Albertazzi, L.; de Greef, T. F. A. Efficient Small-Scale Conjugation of DNA to Primary Antibodies for Multiplexed Cellular Targeting. Bioconjugate Chem. 2019, 30, 2384– 2392, DOI: 10.1021/acs.bioconjchem.9b0049036https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhs1egu73E&md5=37e7a98c0473bc62cefb6dc7fe7f5ae7Efficient Small-Scale Conjugation of DNA to Primary Antibodies for Multiplexed Cellular TargetingCremers, Glenn A. O.; Rosier, Bas J. H. M.; Riera Brillas, Roger; Albertazzi, Lorenzo; de Greef, Tom F. A.Bioconjugate Chemistry (2019), 30 (9), 2384-2392CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)The combination of the specificity of antibodies and the programmability of DNA nanotechnol. has provided the scientific community with a powerful tool to label and unambiguously distinguish a large no. of subcellular targets using fluorescence-based read-out methods. Whereas primary antibodies are com. available for a large class of targets, a general stoichiometric site-selective DNA labeling strategy for this affinity reagent is lacking. Here we present a universal, site-selective conjugation method using a small photo-cross-linkable protein G adaptor that allows labeling of antibodies of different host species with a controlled no. of short oligonucleotides (ODNs). Importantly, we illustrate that this conjugation method can be directly performed on com. available primary antibodies on a small scale and without cross-reactivity towards bovine serum albumin. In addn., we present a general benchtop-compatible strategy to purify DNA-labeled antibodies without a loss of function. The application of protein G-ODN-labeled primary antibodies is demonstrated by employing three well-known methods for detecting subcellular targets using fluorescence read-out, including flow cytometry, DNA-PAINT, and dSTORM. This work thus establishes a general and efficient platform for the synthesis of a library of unique ODN-antibody conjugates, facilitating the broader use of DNA-based programmable tags for multiplexed labeling to identify subcellular features with nanometer precision and improving our understanding of cellular structure and function.
- 37Jin, S.; Xu, B.; Yu, L.; Fu, Y.; Wu, H.; Fan, X.; Liu, B.; Wei, J. The PD-1, PD-L1 Expression and CD3+ T Cell Infiltration in Relation to Outcome in Advanced Gastric Signet-Ring Cell Carcinoma, Representing a Potential Biomarker for Immunotherapy. JCO 2017, 35, e15609 DOI: 10.1200/JCO.2017.35.15_suppl.e15609There is no corresponding record for this reference.
- 38Milanezi, F.; Carvalho, S.; Schmitt, F. C. EGFR/HER2 in Breast Cancer: A Biological Approach for Molecular Diagnosis and Therapy. Expert Review of Molecular Diagnostics 2008, 8, 417– 34, DOI: 10.1586/14737159.8.4.41738https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXotFOqt78%253D&md5=a13759c1e74226053e20e895a6d34fbbEGFR/HER2 in breast cancer: a biological approach for molecular diagnosis and therapyMilanezi, Fernanda; Carvalho, Silvia; Schmitt, Fernando C.Expert Review of Molecular Diagnostics (2008), 8 (4), 417-434CODEN: ERMDCW; ISSN:1473-7159. (Expert Reviews Ltd.)A review. Novel cancer therapies have focused on specific mol. markers present in malignant tumors. The rationale of targeted therapy relies on the knowledge of mol. mechanisms involved in carcinogenesis and their influence in clin. outcome allied to a more specific and less toxic treatment. Activation of EGF receptor and HER2 is an important factor for initiation and progression of malignancies, including breast cancer where the status of HER2 is an essential step in the diagnostic workup; EGFR overexpression has been assocd. to the so-called basal-like breast carcinomas, which opens a new avenue for diagnosis and therapeutic approach in these tumors. This review will focus on mechanisms of HER2 and EGF receptor upregulation, the targeted therapies that are currently in use for these receptors, possible combined therapies, as well as the approach for mol. diagnosis from the pathologist's point of view.
- 39van Waarde, A.; Rybczynska, A. A.; Ramakrishnan, N. K.; Ishiwata, K.; Elsinga, P. H.; Dierckx, R. A. J. O. Potential Applications for Sigma Receptor Ligands in Cancer Diagnosis and Therapy. Biochimica et Biophysica Acta (BBA) - Biomembranes 2015, 1848, 2703– 2714, DOI: 10.1016/j.bbamem.2014.08.022There is no corresponding record for this reference.
- 40Ledermann, J. A.; Canevari, S.; Thigpen, T. Targeting the Folate Receptor: Diagnostic and Therapeutic Approaches to Personalize Cancer Treatments. Annals of Oncology 2015, 26, 2034– 2043, DOI: 10.1093/annonc/mdv25040https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2Mbis1ekuw%253D%253D&md5=0790b619d365bec194745e5c9c23ffbfTargeting the folate receptor: diagnostic and therapeutic approaches to personalize cancer treatmentsLedermann J A; Canevari S; Thigpen TAnnals of oncology : official journal of the European Society for Medical Oncology (2015), 26 (10), 2034-43 ISSN:.BACKGROUND: In cancer therapy, molecularly targeted agents have the potential to maximize antitumor efficacy while minimizing treatment-related toxicity. However, these agents may only be effective in specific tumor subtypes with defined genomic profiles. This emphasizes the importance of developing personalized cancer therapeutic strategies (i.e. through the use of companion diagnostic tests) to appropriately select and treat patients who are likely to benefit from specific targeted therapies, thus leading to improvements in clinical and safety outcomes. A potential biological target is the folate receptor (FR), which has been shown to be overexpressed on the surface of many cancers, including tumors of the lungs and ovaries. DESIGN: We carried out a literature search to identify how the FR can be a potential target for selected tumors, and how the FR expression can be exploited by targeted therapies. RESULTS: The two main therapeutic strategies for targeting the FR are based on the use of: (i) an anti-FR antibody (e.g. farletuzumab) and (ii) folate conjugates of folate-targeted chemotherapies and companion radiodiagnostic imaging agents (e.g. vintafolide and (99m)technetium-etarfolatide). Both of these strategies are being assessed in phase III trials. CONCLUSIONS: The important role that the FR plays in cancer development and progression has led to the development of FR-targeted therapeutic approaches. To date, the promising data observed in phase II clinical trials have not been confirmed in phase III studies. Accordingly, there is a need for further research in the refinement of patient selection and identification of new therapeutic combinations. In particular, the development of these targeted therapies requires reliable methods to be developed to detect FR-positive tumors in order to help select patients who may benefit from treatment.
- 41Cremers, G. A. O.; Rosier, B. J. H. M.; Meijs, A.; Tito, N. B.; van Duijnhoven, S. M. J.; van Eenennaam, H.; Albertazzi, L.; de Greef, T. F. A. Determinants of Ligand-Functionalized DNA Nanostructure–Cell Interactions. J. Am. Chem. Soc. 2021, 143, 10131– 10142, DOI: 10.1021/jacs.1c0229841https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhsVSru7zK&md5=9da63ce736c38e8dfbcccde3c9363c4aDeterminants of Ligand-Functionalized DNA Nanostructure-Cell InteractionsCremers, Glenn A. O.; Rosier, Bas J. H. M.; Meijs, Ab; Tito, Nicholas B.; van Duijnhoven, Sander M. J.; van Eenennaam, Hans; Albertazzi, Lorenzo; de Greef, Tom F. A.Journal of the American Chemical Society (2021), 143 (27), 10131-10142CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Synthesis of ligand-functionalized nanomaterials with control over size, shape, and ligand orientation facilitates the design of targeted nanomedicines for therapeutic purposes. DNA nanotechnol. has emerged as a powerful tool to rationally construct two- and three-dimensional nanostructures, enabling site-specific incorporation of protein ligands with control over stoichiometry and orientation. To efficiently target cell surface receptors, exploration of the parameters that modulate cellular accessibility of these nanostructures is essential. In this study, we systematically investigate tunable design parameters of antibody-functionalized DNA nanostructures binding to therapeutically relevant receptors, including the programmed cell death protein 1, the epidermal growth factor receptor, and the human epidermal growth factor receptor 2. We show that, although the native affinity of antibody-functionalized DNA nanostructures remains unaltered, the abs. no. of bound surface receptors is lower compared to sol. antibodies due to receptor accessibility by the nanostructure. We explore structural determinants of this phenomenon to improve efficiency, revealing that receptor binding is mainly governed by nanostructure size and DNA handle location. The obtained results provide key insights in the ability of ligand-functionalized DNA nanostructures to bind surface receptors and yields design rules for optimal cellular targeting.
- 42Vasilyev, F. F.; Lopatnikova, J. A.; Sennikov, S. V. Optimized Flow Cytometry Protocol for Analysis of Surface Expression of Interleukin-1 Receptor Types I and II. Cytotechnology 2013, 65, 795– 802, DOI: 10.1007/s10616-013-9546-642https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhs12rtb7L&md5=df7761b2621674014b653435a42576ceOptimized flow cytometry protocol for analysis of surface expression of interleukin-1 receptor types I and IIVasilyev, Filipp Filippovich; Lopatnikova, Julia Anatolievna; Sennikov, Sergey VitalievichCytotechnology (2013), 65 (5), 795-802CODEN: CYTOER; ISSN:0920-9069. (Springer)The biol. effects of interleukin (IL)-1 are realized through binding to specific membrane-bound receptors. The efficiency of IL-1 action depends on the no. of receptors on the cell. We detd. the percentage of cells that express IL-1 receptor type I (IL-1RI) and IL-1 receptor type II (IL-1RII) by flow cytometry using phycoerythrin (PE)-labeled antibodies to the IL-1Rs, and the mean abs. no. of membrane-bound IL-1Rs per cell using QuantiBRITE PE calibration beads. We showed that different subpopulations of immunocompetent cells expressed different nos. of mols. of membrane-bound IL-1RI and IL-1RII. We also established that when cells were stimulated with bacterial lipopolysaccharide, there was a significant increase in the no. of IL-1RI expressed, and a significant decrease in the mean no. of IL-1RII mols. per cell. Detn. of the mean no. of membrane-bound IL-1R mols. using this protocol enables us to obtain precise and reproducible data that are necessary for full evaluation of expression levels.
- 43Anselmo, A.; Mazzon, C.; Borroni, E. M.; Bonecchi, R.; Graham, G. J.; Locati, M. Flow Cytometry Applications for the Analysis of Chemokine Receptor Expression and Function. Cytometry Part A 2014, 85, 292– 301, DOI: 10.1002/cyto.a.2243943https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXltFemtLk%253D&md5=74abfe7b8d0493da28f3991856b658aeFlow cytometry applications for the analysis of chemokine receptor expression and functionAnselmo, Achille; Mazzon, Cristina; Borroni, Elena Monica; Bonecchi, Raffaella; Graham, Gerard J.; Locati, MassimoCytometry, Part A (2014), 85A (4), 292-301CODEN: CPAYAV; ISSN:1552-4922. (Wiley-Blackwell)Chemokine receptors play an important role in leukocyte migration, both in physiol. and pathol. conditions, and the interest in new methodologies for their detection is increasing. In this review, we focused on chemokine receptors detection through flow cytometric approaches, including the use of specific antibodies and fluorescent chemokines, and on approaches aimed at the anal. of their functions, from intracellular trafficking to signaling activities. © 2014 International Society for Advancement of Cytometry.
- 44Kužílková, D.; Puñet-Ortiz, J.; Aui, P. M.; Fernández, J.; Fišer, K.; Engel, P.; van Zelm, M. C.; Kalina, T. Standardization of Workflow and Flow Cytometry Panels for Quantitative Expression Profiling of Surface Antigens on Blood Leukocyte Subsets: An HCDM CDMaps Initiative. Frontiers in Immunology 2022, 13, 827898, DOI: 10.3389/fimmu.2022.82789844https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB2M3nsVShuw%253D%253D&md5=b1282eafb1743d839d5da199dc36bd32Standardization of Workflow and Flow Cytometry Panels for Quantitative Expression Profiling of Surface Antigens on Blood Leukocyte Subsets: An HCDM CDMaps InitiativeKuzilkova Daniela; Fiser Karel; Kalina Tomas; Punet-Ortiz Joan; Fernandez Javier; Engel Pablo; Aui Pei M; van Zelm Menno C; van Zelm Menno CFrontiers in immunology (2022), 13 (), 827898 ISSN:.Background: The Human Cell Differentiation Molecules (HCDM) organizes Human Leukocyte Differentiation Antigen (HLDA) workshops to test and name clusters of antibodies that react with a specific antigen. These cluster of differentiation (CD) markers have provided the scientific community with validated antibody clones, consistent naming of targets and reproducible identification of leukocyte subsets. Still, quantitative CD marker expression profiles and benchmarking of reagents at the single-cell level are currently lacking. Objective: To develop a flow cytometric procedure for quantitative expression profiling of surface antigens on blood leukocyte subsets that is standardized across multiple research laboratories. Methods: A high content framework to evaluate the titration and reactivity of Phycoerythrin (PE)-conjugated monoclonal antibodies (mAbs) was created. Two flow cytometry panels were designed: an innate cell tube for granulocytes, dendritic cells, monocytes, NK cells and innate lymphoid cells (12-color) and an adaptive lymphocyte tube for naive and memory B and T cells, including TCRγδ(+), regulatory-T and follicular helper T cells (11-color). The potential of these 2 panels was demonstrated via expression profiling of selected CD markers detected by PE-conjugated antibodies and evaluated using 561 nm excitation. Results: Using automated data annotation and dried backbone reagents, we reached a robust workflow amenable to processing hundreds of measurements in each experiment in a 96-well plate format. The immunophenotyping panels enabled discrimination of 27 leukocyte subsets and quantitative detection of the expression of PE-conjugated CD markers of interest that could quantify protein expression above 400 units of antibody binding capacity. Expression profiling of 4 selected CD markers (CD11b, CD31, CD38, CD40) showed high reproducibility across centers, as well as the capacity to benchmark unique clones directed toward the same CD3 antigen. Conclusion: We optimized a procedure for quantitative expression profiling of surface antigens on blood leukocyte subsets. The workflow, bioinformatics pipeline and optimized flow panels enable the following: 1) mapping the expression patterns of HLDA-approved mAb clones to CD markers; 2) benchmarking new antibody clones to established CD markers; 3) defining new clusters of differentiation in future HLDA workshops.
- 45Sharma, M.; Yang, Z.; Miyamoto, H. Immunohistochemistry of Immune Checkpoint Markers PD-1 and PD-L1 in Prostate Cancer. Medicine (Baltimore) 2019, 98, e17257 DOI: 10.1097/MD.0000000000017257There is no corresponding record for this reference.
- 46Hutchinson, R. A.; Adams, R. A.; McArt, D. G.; Salto-Tellez, M.; Jasani, B.; Hamilton, P. W. Epidermal Growth Factor Receptor Immunohistochemistry: New Opportunities in Metastatic Colorectal Cancer. J Transl Med 2015, 13, 217, DOI: 10.1186/s12967-015-0531-z46https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC28%252FgvFGhsQ%253D%253D&md5=bc1088230200c5f5c02a1f1449c4af03Epidermal growth factor receptor immunohistochemistry: new opportunities in metastatic colorectal cancerHutchinson Ryan A; McArt Darragh G; Salto-Tellez Manuel; Hamilton Peter W; Hutchinson Ryan A; Adams Richard A; Jasani BharatJournal of translational medicine (2015), 13 (), 217 ISSN:.The treatment of cancer is becoming more precise, targeting specific oncogenic drivers with targeted molecular therapies. The epidermal growth factor receptor has been found to be over-expressed in a multitude of solid tumours. Immunohistochemistry is widely used in the fields of diagnostic and personalised medicine to localise and visualise disease specific proteins. To date the clinical utility of epidermal growth factor receptor immunohistochemistry in determining monoclonal antibody efficacy has remained somewhat inconclusive. The lack of an agreed reproducible scoring criteria for epidermal growth factor receptor immunohistochemistry has, in various clinical trials yielded conflicting results as to the use of epidermal growth factor receptor immunohistochemistry assay as a companion diagnostic. This has resulted in this test being removed from the licence for the drug panitumumab and not performed in clinical practice for cetuximab. In this review we explore the reasons behind this with a particular emphasis on colorectal cancer, and to suggest a way of resolving the situation through improving the precision of epidermal growth factor receptor immunohistochemistry with quantitative image analysis of digitised images complemented with companion molecular morphological techniques such as in situ hybridisation and section based gene mutation analysis.
- 47Gaber, R.; Goldmann, T. Mini Review: Immunohistochemistry Using EGFR-Mutant Specific Antibodies in Non-Small Cell Lung Carcinoma: Accuracy and Reliability. Journal of Cancer Treatment and Diagnosis 2018, 2().There is no corresponding record for this reference.
- 48Queiroga, F. L.; Perez-Alenza, M. D.; González-Gil, A.; Silván, G.; Peña, L.; Illera, J. C. Quantification of Epidermal Growth Factor Receptor (EGFR) in Canine Mammary Tumours by ELISA Assay: Clinical and Prognostic Implications. Vet Comp Oncol 2017, 15, 383– 390, DOI: 10.1111/vco.1217448https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXnsV2jtr8%253D&md5=c9f45014ddfef5659894701da5b0311fQuantification of epidermal growth factor receptor (EGFR) in canine mammary tumours by ELISA assay: clinical and prognostic implicationsQueiroga, F. L.; Perez-Alenza, M. D.; Gonzalez-Gil, A.; Silvan, G.; Pena, L.; Illera, J. C.Veterinary and Comparative Oncology (2017), 15 (2), 383-390CODEN: VCOEAN; ISSN:1476-5810. (Wiley-Blackwell)The involvement of epidermal growth factor receptor (EGFR) is well established in human breast cancer, however, in canine mammary tumors (CMT), including inflammatory mammary carcinomas (IMC), still needs to be clarified. Enzyme immune assay techniques were used for EGFR detns. in tumor tissue from 45 bitches with CMT and in normal mammary glands from eight control dogs. Higher tissue EGFR levels were found in CMT compared with controls (P < 0.05). In malignant CMT, tissue EGFR elevated concns. were statistically significantly assocd. with tumor relapse and/or distant metastasis during follow-up and with reduced disease-free and overall survival times. The IMC cases had the highest tissue EGFR levels compared with other malignant non-IMC tumors (P < 0.001). The results support the hypothesis that EGFR levels influence prognosis in malignant CMT, suggesting that EGFR may represent a therapeutic target in cases of high histol. aggressiveness and esp. in cases of metastatic phenotype and poor prognosis.
- 49Takeuchi, M.; Doi, T.; Obayashi, K.; Hirai, A.; Yoneda, K.; Tanaka, F.; Iwai, Y. Soluble PD-L1 with PD-1-Binding Capacity Exists in the Plasma of Patients with Non-Small Cell Lung Cancer. Immunol. Lett. 2018, 196, 155– 160, DOI: 10.1016/j.imlet.2018.01.00749https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXitFKitr8%253D&md5=097cf14a36e6b02e1a77fe95bce6d1d3Soluble PD-L1 with PD-1-binding capacity exists in the plasma of patients with non-small cell lung cancerTakeuchi, Masahiro; Doi, Tomomitsu; Obayashi, Kunie; Hirai, Ayako; Yoneda, Kazue; Tanaka, Fumihiro; Iwai, YoshikoImmunology Letters (2018), 196 (), 155-160CODEN: IMLED6; ISSN:0165-2478. (Elsevier B.V.)PD-L1 is one of the important immune checkpoint mols. that can be targeted by cancer immunotherapies. PD-L1 has a sol. form (sPD-L1) and a membrane-bound form (mPD-L1). Conventional ELISA (ELISA) systems can detect sPD-L1 using anti-PD-L1 capture antibody through the antigen-antibody reaction, but cannot evaluate the quality and function of sPD-L1. In this study, we developed a novel ELISA system for the detection and quantification of sPD-L1 with PD-1-binding capacity (bsPD-L1). To capture bsPD-L1 through the ligand-receptor reaction, the anti-PD-L1 capture antibody in the conventional ELISA was replaced with PD-1-Ig fusion protein in the new ELISA. The new ELISA could detect bsPD-L1 in 29 out of 75 plasma samples from patients with non-small cell lung cancer (NSCLC), with higher sensitivity and frequency than the conventional ELISA. The western blot anal. showed that sPD-L1 in the plasma was glycosylated. Treatment of the samples with glycosidase reduced the absorbance detd. by the new ELISA but had no effect on the absorbance detd. by the conventional ELISA. These results suggest that glycosylation of sPD-L1 is important for its binding to the immobilized PD-1 in the new ELISA. Our new ELISA system may be useful for the evaluation of functional sPD-L1 with PD-1-binding capacity in cancer patients.
- 50Zhang, X.; Zhang, Y.; Tang, H.; He, J. EGFR Gene Copy Number as a Predictive/Biomarker for Patients with Non-Small-Cell Lung Cancer Receiving Tyrosine Kinase Inhibitor Treatment: A Systematic Review and Meta-Analysis. J Investig Med 2017, 65, 72– 81, DOI: 10.1136/jim-2016-00025250https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2svjsFSjtw%253D%253D&md5=4e0170759ae2e5272fef4112a3ec8986EGFR gene copy number as a predictive/biomarker for patients with non-small-cell lung cancer receiving tyrosine kinase inhibitor treatment: a systematic review and meta-analysisZhang Xin; He Jianxing; Zhang Yiwen; Tang HailingJournal of investigative medicine : the official publication of the American Federation for Clinical Research (2017), 65 (1), 72-81 ISSN:.Epidermal growth factor receptor (EGFR) gene copy number has been proposed as a candidate biomarker for predicting treatment response to EGFR tyrosine kinase inhibitors (EGFR-TKIs) in patients with advanced non-small-cell lung cancer (NSCLC). MEDLINE, PubMed, Cochrane, and Google Scholar databases were searched until October 21, 2015 using the following search terms: lung neoplasms/lung cancer/non-small cell lung cancer/NSCLC, EGFR, gene amplification, copy number, erlotinib, gefitinib, tyrosine-kinase inhibitor/TKI, predictor. 17 studies were included in the analysis with a total of 2047 patients. The overall analysis found that increased EGFR gene copy number was associated with higher overall response rate (ORR), overall survival (OS) and progression-free survival (PFS; p values ≤0.008) compared with patients without a high EGFR gene copy number. Subgroup analysis found that in a population of patients who were primarily Caucasian, a higher EGFR gene copy number was also associated with increased ORR, OS, and PFS (p values ≤0.018). The results were similar in a population of Asian patients, except that a higher EGFR gene copy number was not associated with improved OS (p=0.248). Sensitivity analysis indicated that no one study overly influenced the results and that the findings are robust. The result of the analysis found that EGFR gene copy number was associated with increased OS and PFS, supporting the idea that EGFR gene copy number is a biomarker for response to EGFR-TKI therapy in patients with advanced NSCLC.
- 51Gaber, R.; Watermann, I.; Kugler, C.; Reinmuth, N.; Huber, R. M.; Schnabel, P. A.; Vollmer, E.; Reck, M.; Goldmann, T. Correlation of EGFR Expression, Gene Copy Number and Clinicopathological Status in NSCLC. Diagnostic Pathology 2014, 9, 165, DOI: 10.1186/s13000-014-0165-051https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXitFGns7fP&md5=57fd452df93ceebc6504222b3a0c2de0Correlation of EGFR expression, gene copy number and clinicopathological status in NSCLCGaber, Rania; Watermann, Iris; Kugler, Christian; Reinmuth, Nils; Huber, Rudolf M.; Schnabel, Philipp A.; Vollmer, Ekkehard; Reck, Martin; Goldmann, TorstenDiagnostic Pathology (2014), 9 (), 165/1-165/26, 26 pp.CODEN: DPIAAA; ISSN:1746-1596. (BioMed Central Ltd.)Background: Epidermal Growth Factor Receptor (EGFR) targeting therapies are currently of great relevance for the treatment of lung cancer. For this reason, in addn. to mutational anal. immunohistochem. (IHC) of EGFR in lung cancer has been discussed for the decision making of according therapeutic strategies. The aim of this study was to obtain standardization of EGFR-expression methods for the selection of patients who might benefit of EGFR targeting therapies. Methods: As a starting point of a broad investigation, aimed at elucidating the expression of EGFR on different biol. levels, four EGFR specific antibodies were analyzed concerning potential differences in expression levels by Immunohistochem. (IHC) and correlated with fluorescence in situ hybridization (FISH) anal. and clinicopathol. data. 206 tumor tissues were analyzed in a tissue microarray format employing immunohistochem. with four different antibodies including Dako PharmDx kit (clone 2-18C9), clone 31G7, clone 2.1E1 and clone SP84 using three different scoring methods. Protein expression was compared to FISH utilizing two different probes. Results: EGFR protein expression detd. by IHC with Dako PharmDx kit, clone 31G7 and clone 2.1E1 (p ≤ 0.05) correlated significantly with both FISH probes independently of the three scoring methods; best correlation is shown for 31G7 using the scoring method that defined EGFR positivity when ≥10% of the tumor cells show membranous staining of moderate and severe intensity (p = 0.001). Conclusion: Overall, our data show differences in EGFR expression detd. by IHC, due to the applied antibody. Highest concordance with FISH is shown for antibody clone 31G7, evaluated with score B (p = 0,001). On this account, this antibody clone might by utilized for std. evaluation of EGFR expression by IHC.
- 52Zhang, F.; Wang, S.; Yin, L.; Yang, Y.; Guan, Y.; Wang, W.; Xu, H.; Tao, N. Quantification of Epidermal Growth Factor Receptor Expression Level and Binding Kinetics on Cell Surfaces by Surface Plasmon Resonance Imaging. Anal. Chem. 2015, 87, 9960– 9965, DOI: 10.1021/acs.analchem.5b0257252https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhsVyksbrO&md5=8d291df500a3ec460d5ef57aa0912a38Quantification of Epidermal Growth Factor Receptor Expression Level and Binding Kinetics on Cell Surfaces by Surface Plasmon Resonance ImagingZhang, Fenni; Wang, Shaopeng; Yin, Linliang; Yang, Yunze; Guan, Yan; Wang, Wei; Xu, Han; Tao, NongjianAnalytical Chemistry (Washington, DC, United States) (2015), 87 (19), 9960-9965CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Epidermal growth factor receptor (EGFR, also known as ErbB-1 or HER-1) is a membrane bound protein that has been assocd. with a variety of solid tumors and the control of cell survival, proliferation, and metab. Quantification of the EGFR expression level in cell membranes and the interaction kinetics with drugs are thus important for cancer diagnosis and treatment. Here we report mapping of the distribution and interaction kinetics of EGFR in their native environment with the surface plasmon resonance imaging (SPRi) technique. The monoclonal anti-EGFR antibody was used as a model drug in this study. The binding of the antibody to EGFR overexpressed A431 cells was monitored in real time, which was found to follow the first-order kinetics with an assocn. rate const. (ka) and dissocn. rate const. (kd) of (2.7 ± 0.6) × 105 M-1s-1 and (1.4 ± 0.5) × 10-4 s-1, resp. The dissocn. const. (KD) was detd. to be 0.53 ± 0.26 nM with up to seven-fold variation among different individual A431 cells. In addn., the averaged A431 cell surface EGFR d. was found to be 636/μm2 with an estn. of 5 × 105 EGFR per cell. Addnl. measurement also revealed that different EGFR pos. cell lines (A431, HeLa, and A549) show receptor d. dependent anti-EGFR binding kinetics. The results demonstrate that SPRi is a valuable tool for direct quantification of membrane protein expression level and ligand binding kinetics at single cell resoln. Our findings show that the local environment affects the drug-receptor interactions, and in situ measurement of membrane protein binding kinetics is important.
- 53Ma, H.; Liu, Y. Super-Resolution Localization Microscopy: Toward High Throughput, High Quality, and Low Cost. APL Photonics 2020, 5, 060902, DOI: 10.1063/5.001173153https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhsVKmt7jO&md5=d3f68aca6cc4050b1bdfd5e50a5d67b5Super-resolution localization microscopy: Toward high throughput, high quality, and low costMa, Hongqiang; Liu, YangAPL Photonics (2020), 5 (6), 060902CODEN: APPHD2; ISSN:2378-0967. (American Institute of Physics)A review. After nearly 15 years since its initial debut, super-resoln. localization microscopy that surpasses the diffraction-limited resoln. barrier of optical microscopy has rapidly gotten out of the ivory tower and entered a new phase to address various challenging biomedical questions. Recent advances in this technol. greatly increased the imaging throughput, improved the imaging quality, simplified the sample prepn., and reduced the system cost, making this technol. suitable for routine biomedical research. We will provide our perspective on the recent tech. advances and their implications in serving the community of biomedical research. (c) 2020 American Institute of Physics.
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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acssensors.2c01736.
Typical field of view of single-molecule fluorescence events, histograms of dwell times (dark-and-bright) of binding events, simulation of the direct and kinetic counting performance, control experiments, cumulative distribution functions and calibration, time traces, and number of events on cell membranes (PDF)
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