Circulating tumor cells (CTCs) have been shown in many studies as a possible biomarker for metastasis and may be instrumental for the spread of the disease. Despite advances in CTC capturing ...technologies, the low frequency of CTCs in cancer patients and the heterogeneity of the CTCs have limited the wide application of the technology in clinic. In this study, we investigated a novel microfluidic technology that uses a size- and deformability-based capture system to characterize CTCs. This unique platform not only allows flexibility in the selection of antibody markers but also segregates the CTCs in their own chambers, thus, enabling morphological, immunological and genetic characterization of each CTC at the single cell level. In this study, different breast cancer cell lines including MCF7, MDA-MB-231 and SKBR3, as well as a panel of breast cancer biomarkers were used to test the device. The technology can capture a wide range of cells with high reproducibility. The capturing efficiency of the cells is greater than 80%. In addition, the background of leukocytes is minimized because individual cells are segregated in their own chambers. The device captured both epithelial cancer cells such as MCF7 and SKBR3 and mesenchymal cells such as MDA-MB-231. Immunostaining of the captured cells on the microchannel device suggests that a panel of breast cancer biomarkers can be used to further characterize differential expression of the captured cells.
Current analysis of circulating tumor cells (CTCs) is hindered by sub-optimal sensitivity and specificity of devices or assays as well as lack of capability of characterization of CTCs with clinical ...biomarkers. Here, we validate a novel technology to enrich and characterize CTCs from blood samples of patients with metastatic breast, prostate and colorectal cancers using a microfluidic chip which is processed by using an automated staining and scanning system from sample preparation to image processing. The Celsee system allowed for the detection of CTCs with apparent high sensitivity and specificity (94% sensitivity and 100% specificity). Moreover, the system facilitated rapid capture of CTCs from blood samples and also allowed for downstream characterization of the captured cells by immunohistochemistry, DNA and mRNA fluorescence in-situ hybridization (FISH). In a subset of patients with prostate cancer we compared the technology with a FDA-approved CTC device, CellSearch and found a higher degree of sensitivity with the Celsee instrument. In conclusion, the integrated Celsee system represents a promising CTC technology for enumeration and molecular characterization.
•CTCs in NSCLC correlate with the presence of Treg cells expressing checkpoint markers in the tumor microenvironment.•Treg cells in NSCLC express CCR5, a new class of drug targets in inhibiting tumor ...growth and metastasis.•NSCLC tumors have a signature that is predictive of the presence of CTCs.•Highly multiplexed immune profiling reveals different patterns of expression in diploid compared to aneuploidy tumor cells from the same tumor.•Highly sensitive CTC enumeration methodology.
Non-small cell lung cancer (NSCLC) has a poor prognosis. Targeted therapy and immunotherapy in recent years has significantly improved NSCLC patient outcome. In this study, we employed cell-by-cell immune and cancer marker profiling of the primary tumor cells to investigate possible signatures that might predict the presence or absence of circulating tumor cells (CTCs). We performed a comprehensive study on 10 NSCLC patient tissue samples with paired blood samples. The solid tissue biopsy samples were dissociated into single cells by non-enzymatic tissue homogenization and stained with a total 25 immune, cancer markers and DNA content dye and analyzed with high-parameter flow cytometry. CTCs were isolated and analyzed from the paired peripheral blood. We investigated a total of 74 biomarkers for their correlation with CTC number. Strong correlations were observed between CTC number and the frequency of immune checkpoint marker expressing lymphocytes (CTLA-4, LAG3, TIM3, PD-1), within the CD103+CD4+ T lymphocyte subset. CTC number is also correlated with the frequency of PD-L1 expressing cancer cells and cancer cell DNA content. In contrast, CTC number inversely correlated to the frequency of CD44+E-cadherin− cancer cells. Unsupervised clustering analysis based on the biomarker analysis separated the CTC negative patients from the CTC positive patients. Profiling multiple immune and cancer markers on cancer samples with multi-parametric flow cytometry allowed us to obtain protein expression information at the single cell level. Clustering analysis of the proteomic data revealed a signature driven by checkpoint marker expression on CD103+CD4+ T cells that could potentially be predictive of CTCs and targets of therapy.
An Integrated Nanoliter DNA Analysis Device Burns, Mark A.; Johnson, Brian N.; Brahmasandra, Sundaresh N. ...
Science (American Association for the Advancement of Science),
10/1998, Letnik:
282, Številka:
5388
Journal Article
Recenzirano
A device was developed that uses microfabricated fluidic channels, heaters, temperature sensors, and fluorescence detectors to analyze nanoliter-size DNA samples. The device is capable of measuring ...aqueous reagent and DNA-containing solutions, mixing the solutions together, amplifying or digesting the DNA to form discrete products, and separating and detecting those products. No external lenses, heaters, or mechanical pumps are necessary for complete sample processing and analysis. Because all of the components are made using conventional photolithographic production techniques, they operate as a single closed system. The components have the potential for assembly into complex, low-power, integrated analysis systems at low unit cost. The availability of portable, reliable instruments may facilitate the use of DNA analysis in applications such as rapid medical diagnostics and point-of-use agricultural testing.
Molecular analysis of circulating tumor cells (CTCs) is hindered by low sensitivity and high level of background leukocytes of currently available CTC enrichment technologies. We have developed a ...novel device to enrich and retrieve CTCs from blood samples by using a microfluidic chip. The Celsee PREP100 device captures CTCs with high sensitivity and allows the captured CTCs to be retrieved for molecular analysis. It uses the microfluidic chip which has approximately 56,320 capture chambers. Based on differences in cell size and deformability, each chamber ensures that small blood escape while larger CTCs of varying sizes are trapped and isolated in the chambers. In this report, we used the Celsee PREP100 to capture cancer cells spiked into normal donor blood samples. We were able to show that the device can capture as low as 10 cells with high reproducibility. The captured CTCs were retrieved from the microfluidic chip. The cell recovery rate of this back-flow procedure is 100% and the level of remaining background leukocytes is very low (about 300-400 cells). RNA from the retrieved cells are extracted and converted to cDNA, and gene expression analysis of selected cancer markers can be carried out by using RT-PCR assays. The sensitive and easy-to-use Celsee PREP100 system represents a promising technology for capturing and molecular characterization of CTCs.
A modeling approach is developed to account for the important effects of intermicellar exchange on the ultrafine particle formation in reverse micelles. A set of fusion−fission schemes is ...specifically proposed and used for modeling intermicellar metal exchange. The dynamic effects largely differing in their time scales are decoupled using a two-staged approach. Growth of metal particles is simulated to occur through intramicellar attachment of free metal atoms from an instantaneous reduction reaction and as well as by transfer and attachment through intermicellar exchange. Simulation results predict increase in particle size with aqueous core size and total aqueous phase volume, increase in particle number density with surfactant concentration at fixed aqueous phase to surfactant molar ratio, a minimum in average particle size as a function of salt concentration, and increase in particle size polydispersity with salt concentration and aqueous core size. Low values of the order of 10-1 are found for the number ratio of particles to micelles. All these effects have been observed experimentally. The mean core size and the critical metal nucleation number are found to be the primary influencing parameters for the final particle sizes. It is further found that the polydispersity can be controlled through proper choice of starting mixture conditions.
This research concentrates on developing an integrated nanoliter fluidic device for chemical and biochemical analysis. Individual components (metering unit, pump and mixer) necessary for manipulating ...nano liter-volume discrete liquid drops in a microchannel network are microfabricated and integrated into a self-contained device. Pre-determined volumes of liquid ranging from 1 to 1000 nanoliters are metered using a combination of hydrophobic surface treatment and air pressures. The pressures required for metering and pumping of discrete drops are generated on-chip by heating of air trapped in chambers. Pressures up to 10 kN/m2 are generated by heating the trapped air volume (100–400 nanoliters) by 30–40°C. The flow rate of the discrete drop (∼10 nl/s) is monitored by controlling the rate of air heating (∼5°C/s). Mixing of discrete drops is achieved in a few seconds by merging the drops and moving the combined drop by three or more drop lengths to interlayer the liquid across the channel depth (20–50 μm). In this dissertation, the theory and experimental results are described for each of the individual components as well as the integration of the individual components to form a self-contained integrated device.
Xanthomonas oryzae
pv.
oryzae
(Xoo) is a destructive pathogen that causes bacterial blight disease of rice worldwide. Xoo uses T3SS (type III secretion system) effectors to subvert rice innate ...immunity. However, the comprehensive knowledge of rice genes involved in T3SS effectors-mediated interaction remains unclear. In this study, the transcriptome profiles of rice infected with a virulent Xoo strain from North-eastern region of India relatives to its avirulent strain (that lacks functional T3SS) were analyzed at early (2–6 hpi) and late (16–24 hpi) hours of infection. Out of total 255 differentially expressed genes (DEGs), during early infection, 62 and 70 genes were upregulated and downregulated, respectively. At late infection, 70 and 53 genes were upregulated and downregulated, respectively. The transcriptomic data identified many differentially expressed resistant genes, transposons, transcription factors, serine/threonine protein kinase, cytochrome P450 and peroxidase genes that are involved in plant defense. Pathway analysis revealed that these DEGs are involved in hormone signaling, plant defense, cellular metabolism, growth and development processes. DEGs associated with plant defense were also validated through quantitative real-time PCR. Our study brings a comprehensive picture of the rice genes that are being differentially expressed during bacterial blight infection. Nevertheless, the DEG-associated pathways would provide sensible targets for developing resistance to bacterial blight.
The incidence of bronchoesophageal fistula in presence of benign pathology of tracheal tree or oesophagus is rare. It is encountered in thoracic diseases like tuberculosis, syphilis or histoplasmosis ...due to erosion by infected lymph node or abscess to adjoining structures. The source of primary pathology has to be eliminated followed by appropriate steps of fistula tract closure is essential for optimal result. We report a 25-year-old patient with left sided bronchoesophageal fistula. He had a past history of pulmonary tuberculosis. A left lower lobectomy followed by repair of oesophageal fistula opening was performed by primary closure and reinforcement with an intercostal muscle flap based on posterior intercostal artery. Postoperative oesophagogram showed short diverticula, which was occluded with n-butyl cyanoacrylate (NBCA) glue under radiological guidance. Feeding was started one week after application of glue without further complication. Reports on intercostals muscle flap repair and intervention of residual oesophageal diverticula with n-butyl cyanoacrylate (NBCA) glue under radiological guidance are scanty.
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