RESUMEN
Circulating tumour cells (CTCs) are recognized as important markers for cancer research. Nonetheless, the extreme rarity of CTCs in blood samples limits their availability for multiple characterization. The cultivation of CTCs is still technically challenging due to the lack of information of CTC proliferation, and it is difficult for conventional microscopy to monitor CTC cultivation owing to low throughput. In addition, for precise monitoring, CTCs need to be distinguished from the blood cells which co-exist with CTCs. Lensless imaging is an emerging technique to visualize micro-objects over a wide field of view, and has been applied for various cytometry analyses including blood tests. However, discrimination between tumour cells and blood cells was not well studied. In this study, we evaluated the potential of the lensless imaging system as a tool for monitoring CTC cultivation. Cell division of model tumour cells was examined using the lensless imaging system composed of a simple setup. Subsequently, we confirmed that tumour cells, JM cells (model lymphocytes), and erythrocytes exhibited cell line-specific patterns on the lensless images. After several discriminative parameters were extracted, discrimination between the tumour cells and other blood cells was demonstrated based on linear discriminant analysis. We also combined the highly efficient CTC recovery device, termed microcavity array, with the lensless-imaging to demonstrate recovery, monitoring and discrimination of the tumour cells spiked into whole blood samples. This study indicates that lensless imaging can be a powerful tool to investigate CTC proliferation and cultivation.
Asunto(s)
Células Neoplásicas Circulantes , Células Sanguíneas , Recuento de Células , Diagnóstico por Imagen , HumanosRESUMEN
Genetic analysis of single-cells is widely recognized as a powerful tool for understanding cellular heterogeneity and obtaining genetic information from rare populations. Recently, many kinds of single-cell isolation systems have been developed to facilitate single-cell genetic analysis. However, these systems mainly target non-adherent cells or cells in a cell suspension. Thus, it is still challenging to isolate single-adherent cells of interest from a culture dish using a microscope. We had previously developed a single-cell isolation technique termed "gel-based cell manipulation" (GCM). In GCM, single-cells could be visualized by photopolymerizable-hydrogel encapsulation that made it easier to isolate the single-cells. In this study, GCM-based isolation of single-adherent cancer cells from a culture dish was demonstrated. Single-adherent cells were encapsulated in a photopolymerizable hydrogel using a microscope and isolated with high efficiency. Furthermore, whole genome amplification and sequencing for the isolated single-adherent cell could be achieved. We propose that the GCM-based approach demonstrated in this study has the potential for efficient analysis of single-adherent cells at the genetic level.
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Adhesión Celular , Separación Celular/métodos , ADN de Neoplasias/análisis , Genoma Humano , Hidrogeles/química , Análisis de la Célula Individual/métodos , Células A549 , Genotipo , Células HeLa , HumanosRESUMEN
Circulating tumor cells (CTCs) are potential precursors of metastatic cancer, and genomic information obtained from CTCs have the potential to provide new insights into the biology of cancer metastasis. We previously developed a technique for single-cell manipulation based on the encapsulation of a single cell in a photopolymerized hydrogel that can be used for subsequent genetic analysis. However, this technique has limitations in terms of throughput because light irradiation must be performed on each individual cell from the confocal laser-scanning microscopy. Here, we present a high-throughput cell manipulation technique using a multiple single-cell encapsulation system with a digital micromirror device. This system enables rapid cell imaging within a microcavity array, a microfilter for the recovery of CTCs from blood samples, as well as the simultaneous encapsulation of several CTCs with hydrogels photopolymerized using a multiple light-irradiation system. Furthermore, single-cell labeling using two differently shaped hydrogels was examined to distinguish between NCI-H1975 cells and A549 cells, demonstrating the utility of the system for single-cell gene mutation analysis. In addition to CTCs, our system can be widely applied for analyses of mammalian cells and microorganisms.
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A cell entrapment device consisting of a microcavity array was used to analyze the deformability of MCF-10 human breast epithelial and MCF-7 human breast cancer cell lines by confocal laser scanning microscopy. Entrapment of up to 8 × 103 cells was achieved within 3 min. Protrusions were formed at the bottom surface of the array with a pore size of 3 µm. Protrusion length increased at higher filtration pressures and could be used to distinguish between MCF-7 and MCF-10 cells. These results indicate that our system is useful for high-throughput deformability analysis of cancer cells, which can provide insight into the mechanisms underlying tumor cell malignancy.
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Forma de la Célula/fisiología , Línea Celular Tumoral , Núcleo Celular/fisiología , Fluoresceínas/química , Humanos , Células MCF-7 , Microscopía Confocal , Níquel/química , Análisis de Matrices TisularesRESUMEN
Underpotential photodeposition of Pb yields an ultrathin shell layer on the Au(111) surface of Au nanoparticle(NP)-loaded TiO2 (Au/TiO2 ) with heteroepitaxial nanojunctions. The localized surface plasmon resonance of Au/TiO2 undergoes no damping with the Pb-shell formation, and the Pb shell offers resistance to aerobic oxidation. Mesoporous films comprising the Au(core)-Pb(shell) NP-loaded TiO2 and unmodified Au/TiO2 were formed on fluorine-doped tin oxide (FTO) electrode. Using them as the photoanode, photoelectrochemical cells were fabricated, and the photocurrent was measured under illumination of simulated sunlight. The photocurrent for water splitting is dramatically enhanced by the Pb-shell formation. The photoelectrochemical measurements of the hot-electron lifetime and density functional theory calculations for model clusters indicate that the Pb-shell effect originates from the charge separation enhancement.
RESUMEN
Genetic characterization of circulating tumor cells (CTCs) could guide the choice of therapies for individual patients and also facilitate the development of new drugs. We previously developed a CTC recovery system using a microcavity array, which demonstrated highly efficient CTC recovery based on differences in cell size and deformability. However, the CTC recovery system lacked an efficient cell manipulation tool suitable for subsequent genetic analysis. Here, we resolve this issue and present a simple and rapid manipulation method for single CTCs using a photopolymerized hydrogel, polyethylene glycol diacrylate (PEGDA), which is useful for subsequent genetic analysis. First, PEGDA was introduced into the cells entrapped on the microcavity array. Then, excitation light was projected onto the target single cells for encapsulation of each CTC by confocal laser-scanning microscopy. The encapsulated single CTCs could be visualized by the naked eye and easily handled with tweezers. The single CTCs were only partially encapsulated on the PEGDA hydrogel, which allowed for sufficient whole-genome amplification and accurate genotyping. Our proposed methodology is a valuable tool for the rapid and simple manipulation of single CTCs and is expected to become widely utilized for analyses of mammalian cells and microorganisms in addition to CTCs.
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Genoma Humano , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Microscopía Confocal , Células Neoplásicas Circulantes/química , Línea Celular Tumoral , ADN de Neoplasias/química , ADN de Neoplasias/aislamiento & purificación , ADN de Neoplasias/metabolismo , Receptores ErbB/química , Receptores ErbB/genética , Receptores ErbB/metabolismo , Genotipo , Humanos , Luz , Células Neoplásicas Circulantes/metabolismo , Células Neoplásicas Circulantes/patología , Polimerizacion/efectos de la radiación , Análisis de Secuencia de ADN , Análisis de la Célula IndividualRESUMEN
Adsorption experiments and density functional theory (DFT) simulations indicated that Cu(acac)2 is chemisorbed on the monoclinic sheelite (ms)-BiVO4 surface to form an O2-bridged binuclear complex (OBBC/BiVO4) like hemocyanin. Multi-electron reduction of O2 is induced by the visible-light irradiation of the OBBC/BiVO4 in the same manner as a blue Cu enzyme. The drastic enhancement of the O2 reduction renders ms-BiVO4 to work as a good visible-light photocatalyst without any sacrificial reagents. As a model reaction, we show that this biomimetic hybrid photocatalyst exhibits a high level of activity for the aerobic oxidation of amines to aldehydes in aqueous solution and imines in THF solution at 25 °C giving selectivities above 99% under visible-light irradiation.
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Biomimética , Electrones , Luz , Metales/química , Oxígeno/química , Semiconductores , Catálisis , Oxidación-ReducciónRESUMEN
Extracellular vesicles (EVs) are small cellular organelles involved in intracellular signaling and cell-to-cell interactions. Recent studies suggested that exosomes may have potential applications in the diagnosis and treatment of cancer and neurodegenerative diseases. In this study, extracellular vesicles of the human nonsmall cell lung cancer cell line H1299 and the unlabeled antiCD63 antibody were imaged using a new label-free terahertz chemical microscopy (TCM) technique to detect changes in the terahertz wave amplitude. To verify the high specificity of the protein biomarkers and the sensitivity of the biosensor surface, we also confirmed the selective binding of the antibody to the antigen, bovine serum albumin, and cancer cells. We also performed real-time measurements of the interaction between EVs from the H1299 cell and the antiCD63 antibody, which showed that the amount of change in the terahertz intensity increased with increasing concentration and the time to saturation decreased. Finally, to reuse the used biosensors (sensing plates), plasma-oxygen cleaning was used, and the activity of the biosensor surface was confirmed by terahertz microscopy and atomic force microscopy and was found to be reusable after less than 3 min of cleaning. Consequently, terahertz chemical microscopy was able to detect the presence or absence of antigen-antibody binding and its reaction rate and binding strength.
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Técnicas Biosensibles , Vesículas Extracelulares , Tetraspanina 30 , Humanos , Tetraspanina 30/metabolismo , Vesículas Extracelulares/química , Vesículas Extracelulares/metabolismo , Línea Celular Tumoral , Técnicas Biosensibles/métodos , Microscopía/métodos , Albúmina Sérica Bovina/químicaRESUMEN
In this study, we present a method for efficient enrichment of small-sized circulating tumor cells (CTCs) such as those found in the blood of small-cell lung cancer (SCLC) patients using a microcavity array (MCA) system. To enrich CTCs from whole blood, a microfabricated nickel filter with a rectangular MCA (10(4) cavities/filter) was integrated with a miniaturized device, allowing for the isolation of tumor cells based on differences in size and deformability between tumor and blood cells. The shape and porosity of the MCA were optimized to efficiently capture small tumor cells on the microcavities under low flow resistance conditions, while allowing other blood cells to effectively pass through. Under optimized conditions, approximately 80% of SCLC (NCI-H69 and NCI-H82) cells spiked in 1 mL of whole blood were successfully recovered. In clinical samples, CTCs were detectable in 16 of 16 SCLC patients. In addition, the number of leukocytes captured on the rectangular MCA was significantly lower than that on the circular MCA (p < 0.001), suggesting that the use of the rectangular MCA diminishes a considerable number of carryover leukocytes. Therefore, our system has potential as a tool for the detection of CTCs in small cell-type tumors and detailed molecular analyses of CTCs.
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Separación Celular/métodos , Tamaño de la Célula , Neoplasias Pulmonares/sangre , Células Neoplásicas Circulantes/metabolismo , Carcinoma Pulmonar de Células Pequeñas/sangre , Línea Celular Tumoral , Humanos , Neoplasias Pulmonares/diagnóstico , Células Neoplásicas Circulantes/química , Células Neoplásicas Circulantes/patología , Carcinoma Pulmonar de Células Pequeñas/diagnósticoRESUMEN
Transcriptome analysis of circulating tumor cells (CTCs), which migrate into blood vessels from primary tumor tissues, at the single-cell level offers critical insights into the biology of metastasis and contributes to drug discovery. However, transcriptome analysis of single CTCs has only been reported for a limited number of cancer types, such as multiple myeloma, breast, hepatocellular, and prostate cancer. Herein, we report the transcriptome analysis of gastric cancer single-CTCs. We utilized an antigen-independent strategy for CTC isolation from metastatic gastric cancer patients involving a size-dependent recovery of CTCs and a single cell isolation technique. The transcriptomic profile of single-CTCs revealed that a majority of gastric CTCs had undergone epithelial-mesenchymal transition (EMT), and indicated the contribution of platelet adhesion toward EMT progression and acquisition of chemoresistance. Taken together, this study serves to employ CTC characterization to elucidate the mechanisms of chemoresistance and metastasis in gastric cancer.
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Células Neoplásicas Circulantes , Neoplasias Gástricas , Transcriptoma/genética , Línea Celular Tumoral , Transición Epitelial-Mesenquimal/genética , Humanos , Células Neoplásicas Circulantes/metabolismo , Células Neoplásicas Circulantes/patología , Análisis de la Célula Individual , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologíaRESUMEN
A comprehensive understanding of phytoplankton diversity is valuable for assessing an environment of interest as phytoplankton are primary producers in various aquatic food webs. Microscopic analyses are useful for diversity assessment based on characteristic cell morphologies. However, phylogenetic classification based solely on morphology requires an extremely high level of expertise. The genetic approach is another option for evaluating phytoplankton diversity; however, it cannot reveal morphological information. To integrate these two approaches, an original technology was developed, that is referred to as microcavity array (MCA)/gel-based cell manipulation (GCM). The model experiments using monocultures of various phytoplankton indicated that the efficiencies of cell recovery and isolation of single-cell plankton were dependent on cell size and shape. Cells with widths larger than the cavity width showed high level of recovery and isolation efficiency. Subsequent whole-genome amplification (WGA) of isolated single-cell plankton provided a sufficient amount (≈30 µg) of WGA products for genetic analyses. Furthermore, it is showed that MCA/GCM could directly analyze phytoplankton in ocean water obtained from Suruga Bay, Japan, without any cumbersome pretreatment. These results indicate that MCA/GCM technology is a powerful tool for elucidating the phytoplankton diversity in marine environment.
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Fitoplancton , Agua , Genotipo , Océanos y Mares , Filogenia , Fitoplancton/genética , Plancton/genéticaRESUMEN
Circulating tumor cells (CTCs) are widely known as useful biomarkers in the liquid biopsies of cancer patients. Although single-cell genetic analysis of CTCs is a promising diagnostic tool that can provide detailed clinical information for precision medicine, the capacity of single-CTC isolation for genetic analysis requires improvement. To overcome this problem, we previously developed a multiple single-cell encapsulation system for CTCs using hydrogel-encapsulation, which allowed for the high-throughput isolation of single CTCs. However, isolation of a single cell from adjacent cells remained difficult and often resulted in contamination by neighboring cells due to the limited resolution of the generated hydrogel. We developed a novel multiple single-cell encapsulation system equipped with a high magnification lens for high throughput and a more accurate single-cell encapsulation. The multiple single-cell encapsulation system has sufficient sensitivity to detect immune-stained CTCs, and could also generate a micro-scaled hydrogel that can isolate a single cell from adjacent cells within 10 µm, with high efficiency. The proposed system enables high throughput and accurate single-cell manipulation and genome amplification without contamination from neighboring cells.
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Chitons are herbivorous invertebrates that use rows of ultrahard magnetite-based teeth connected to a flexible belt (radula) to rasp away algal deposits growing on and within rocky outcrops along coastlines around the world. Each tooth is attached to the radula by an organic structure (stylus) that provides mechanical support during feeding. However, the underlying structures within the stylus, and their subsequent function within the chiton have yet to be investigated. Here, we investigate the macrostructural architecture, the regional material and elemental distribution and subsequent nano-mechanical properties of the stylus from the Northern Pacific dwelling Cryptochiton stelleri. Using a combination of µ-CT imaging, optical and electron microscopy, as well as elemental analysis, we reveal that the stylus is a highly contoured tube, mainly composed of alpha-chitin fibers, with a complex density distribution. Nanoindentation reveals regiospecific and graded mechanical properties that can be correlated with both the elemental composition and material distribution. Finite element modeling shows that the unique macroscale architecture, material distribution and elemental gradients have been optimized to preserve the structural stability of this flexible, yet robust functionally-graded fiber-reinforced composite tube, providing effective function during rasping. Understanding these complex fiber-based structures offers promising blueprints for lightweight, multifunctional and integrated materials.
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Poliplacóforos , Diente , Animales , Óxido Ferrosoférrico , Microscopía ElectrónicaRESUMEN
Circulating tumor cells (CTCs) provide potentially accessible in vivo sources of metastatic cancer cells. As such, considerable focus has been placed on analyzing the genetics of single-CTCs. Prior to these analyses, however, CTCs must first be detected within the blood samples of cancer patients. Current methods for detection of CTCs by fluorescence microscopy require the analysis of hundreds of images per blood sample, making this a time-consuming process that creates a bottleneck in CTC analysis. In this study, we therefore developed a wide-field fluorescence imaging system for rapid CTC detection. For these analyses, CTCs were first isolated using the microcavity array (MCA), a micro-sized filter for CTC recovery that separates cells based on differences in cell size and deformability. Notably, the proposed imaging system enabled rapid (â¼10 s) visualization of all stained cells within the 6 mm × 6 mm MCA field via one-shot imaging. Furthermore, the morphology of the cells in the resulting images accurately reflected that observed by conventional microscopy. In total, isolation and detection of CTCs using the MCA combined with our novel wide-field fluorescence imaging system was achieved within 1 h. Thus, our proposed system will provide rapid CTC detection system.
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Microscopía Fluorescente , Células Neoplásicas Circulantes , Imagen Óptica , HumanosRESUMEN
Copper(II) meso-tetra(4-carboxyphenyl)porphyrin surface-modified monoclinic scheelite bismuth vanadate (CuTCPP/BiVO4) has been synthesized via a two-step route involving chemisorption of TCPP on BiVO4 and successive Cu(II) ion incorporation into the TCPP, and the surface modification drastically enhances the water oxidation to oxygen (O2) under visible-light irradiation (λ > 430 nm).
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Au and Pt nanoparticles with varying mean particle size and comparable loading amounts were loaded on the surface of TiO2 (Au/TiO2 and Pt/TiO2, respectively). The photocatalytic activities of Au/TiO2 and Pt/TiO2 for the oxygen reduction reaction (ORR) in an aerated aqueous solution containing 4% ethanol were compared under ultraviolet-light irradiation at 298 K. The initial rate of H2O2 generation (or H2O2 formation rate) in the Au/TiO2 system is much greater than that in the Pt/TiO2 system regardless of the metal particle size. To clarify the origin for the striking difference in the activity, the photocatalytic ORR on the model slabs (M28/(TiO2)32 and M50/(TiO2)96, M = Au and Pt) was simulated by density functional theory (DFT) calculations taking the solvation effect into consideration. The DFT calculations clearly show that regardless of the cluster size, H2O2 formation more easily occurs structurally and energetically for the Au/TiO2 system, whereas H2O is generated with the O-O bond cleavage in the Pt/TiO2 system.
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Circulating tumor cells (CTCs) are well recognized as useful biomarker for cancer diagnosis and potential target of drug discovery for metastatic cancer. Efficient and precise recovery of extremely low concentrations of CTCs from blood has been required to increase the detection sensitivity. Here, an automated system equipped with a microcavity array (MCA) was demonstrated for highly efficient and reproducible CTC recovery. The use of MCA allows selective recovery of cancer cells from whole blood on the basis of differences in size between tumor and blood cells. Intra- and inter-assays revealed that the automated system achieved high efficiency and reproducibility equal to the assay manually performed by well-trained operator. Under optimized assay workflow, the automated system allows efficient and precise cell recovery for non-small cell lung cancer cells spiked in whole blood. The automated CTC recovery system will contribute to high-throughput analysis in the further clinical studies on large cohort of cancer patients.