Dean Smith

Method for physiologic phenotype characterization at the single-cell level in non-interacting and interacting cells

Intercellular heterogeneity is a key factor in a variety of core cellular processes including proliferation, stimulus response, carcinogenesis, and drug resistance. However, cell-to-cell variability studies at the single-cell level have been hampered by the lack of enabling experimental techniques. We present a measurement platform that features the capability to quantify oxygen consumption rates of individual, non-interacting and interacting cells under normoxic and hypoxic conditions. It is based on real-time concentration measurements of metabolites of interest by means of extracellular optical sensors in cell-isolating microwells of subnanoliter volume. We present the results of a series of measurements of oxygen consumption rates (OCRs) of individual non-interacting and interacting human epithelial cells. We measured the effects of cell-to-cell interactions by using the systems capability to isolate two and three cells in a single well. The major advantages of the approach are: 1. ratiometric, intensity-based characterization of the metabolic phenotype at the single-cell level, 2. minimal invasiveness due to the distant positioning of sensors, and 3. ability to study the effects of cell-cell interactions on cellular respiration rates.

Cohort analysis of FISH testing of CD138(+) cells in relapsed multiple myeloma: implications for prognosis and choice of therapy.

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A Minimally Invasive Method for Retrieving Single Adherent Cells of Different Types from Cultures

The field of single-cell analysis has gained a significant momentum over the last decade. Separation and isolation of individual cells is an indispensable step in almost all currently available single-cell analysis technologies. However, stress levels introduced by such manipulations remain largely unstudied. We present a method for minimally invasive retrieval of selected individual adherent cells of different types from cell cultures. The method is based on a combination of mechanical (shear flow) force and biochemical (trypsin digestion) treatment. We quantified alterations in the transcription levels of stress response genes in individual cells exposed to varying levels of shear flow and trypsinization. We report optimal temperature, RNA preservation reagents, shear force and trypsinization conditions necessary to minimize changes in the stress-related gene expression levels. The method and experimental findings are broadly applicable and can be used by a broad research community working in the field of single cell analysis.

Fish freshness sensor

An array of semiconducting metal oxide (SMO) chemiresistive sensor can quantitatively measure the freshness of Atlantic salmon. A variety of SMO films were tested, including films containing oxides of tungsten and tin. Experiments were performed with these films to determine if they were sensitive to trimethylamine and/or dimethylamine, known Atlantic salmon degradation products. The electrical resistance of the films before and during exposures to concentrations of the amines was monitored. The sensor temperatures were varied to determine the operating conditions for sensitivity and selectivity to one or both gases. The most promising films were then used to monitor the degradation of Atlantic salmon is conjunction with a sensory evaluation panel and tests using gas chromatography mass spectrometry and total volatile base nitrogen.

Automated platform for multiparameter stimulus response studies of metabolic activity at the single-cell level

We have developed a fully automated platform for multiparameter characterization of physiological response of individual and small numbers of interacting cells. The platform allows for minimally invasive monitoring of cell phenotypes while administering a variety of physiological insults and stimuli by means of precisely controlled microfluidic subsystems. It features the capability to integrate a variety of sensitive intra- and extra-cellular fluorescent probes for monitoring minute intra- and extra-cellular physiological changes. The platform allows for performance of other, post- measurement analyses of individual cells such as transcriptomics. Our method is based on the measurement of extracellular metabolite concentrations in hermetically sealed ~200-pL microchambers, each containing a single cell or a small number of cells. The major components of the system are a) a confocal laser scan head to excite and detect with single photon sensitivity the emitted photons from sensors; b) a microfluidic cassette to confine and incubate individual cells, providing for dynamic application of external stimuli, and c) an integration module consisting of software and hardware for automated cassette manipulation, environmental control and data collection. The custom-built confocal scan head allows for fluorescence intensity detection with high sensitivity and spatial confinement of the excitation light to individual pixels of the sensor area, thus minimizing any phototoxic effects. The platform is designed to permit incorporation of multiple optical sensors for simultaneous detection of various metabolites of interest. The modular detector structure allows for several imaging modalities, including high resolution intracellular probe imaging and extracellular sensor readout. The integrated system allows for simulation of physiologically relevant microenvironmental stimuli and simultaneous measurement of the elicited phenotypes. We present details of system design, system characterization and metabolic response analysis of individual eukaryotic cells.

Abstract 1766: Persistence of drug-induced DNA interstrand cross-links distinguishes bendamustine from conventional DNA cross-linking agents

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Bendamustine has demonstrated substantial clinical efficacy in the treatment of hematologic malignancies and continues to distinguish itself from other alkylating agents. The mechanistic and clinical differences associated with bendamustine may be directly related to its unique structural features, although the precise mechanism of action is still poorly understood. We have undertaken a detailed study of the drug-DNA interactions of bendamustine. It alkylates DNA primarily at guanine-N7 positions with a sequence selectivity similar to other nitrogen mustards. It produces DNA interstrand cross-links (ICLs) in both solid tumor and hematological tumor cells at doses that cause growth inhibition. ICLs peak at 8h following a 1h treatment and persist over 48h in tumor cells including those that are proficient at repairing (unhooking) ICLs formed by melphalan or cisplatin. This persistence of ICLs is also observed in multiple myeloma cells from patients who have relapsed on melphalan therapy and which efficiently repair melphalan-induced ICLs compared to cells from treatment naive patients. The peak γ-H2AX response follows the ICL peak and also persists beyond 48h. Cells defective in ICL unhooking proteins ERCC1 and XPF, or defective in homologous recombination repair, show increased sensitivity to bendamustine, but at a level less than observed for other nitrogen mustards or cisplatin. Real time PCR profiling showed that at equivalent peak levels of DNA ICLs bendamustine induced fewer changes in DNA damage signaling and DNA repair gene expression compared to either cisplatin or melphalan. These data suggest that the critical DNA damage produced by bendamustine induces a different signaling and repair response to conventional cross-linking agents, including other nitrogen mustards, which may be an important contribution to its clinical efficacy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1766. doi:1538-7445.AM2012-1766

Transcriptional regulation by normal epithelium of premalignant to malignant progression in Barrett’s esophagus

In carcinogenesis, intercellular interactions within and between cell types are critical but remain poorly understood. We present a study on intercellular interactions between normal and premalignant epithelial cells and their functional relevance in the context of premalignant to malignant progression in Barrett’s esophagus. Using whole transcriptome profiling we found that in the presence of normal epithelial cells, dysplastic cells but not normal cells, exhibit marked down-regulation of a number of key signaling pathways, including the transforming growth factor beta (TGFβ) and epithelial growth factor (EGF). Functional assays revealed both cell types showed repressed proliferation and significant changes in motility (speed, displacement and directionality) as a result of interactions between the two cell types. Cellular interactions appear to be mediated through both direct cell-cell contact and secreted ligands. The findings of this study are important in that they reveal, for the first time, the effects of cellular communication on gene expression and cellular function between premalignant (dysplastic) epithelial cells and their normal counterparts.

A novel method for multiparameter physiological phenotype characterization at the single-cell level

Non-genetic intercellular heterogeneity has been increasingly recognized as one of the key factors in a variety of core cellular processes including proliferation, stimulus response, carcinogenesis and drug resistance. Many diseases, including cancer, originate in a single or a few cells. Early detection and characterization of these abnormal cells can provide new insights into the pathogenesis and serve as a tool for better disease diagnosis and treatment. We report on a novel technology for multiparameter physiological phenotype characterization at the single-cell level. It is based on real-time measurements of concentrations of several metabolites by means of extracellular optical sensors in microchambers of sub-nL volume containing single cells. In its current configuration, the measurement platform features the capability to detect oxygen consumption rate and pH changes under normoxic and hypoxic conditions at the single-cell level. We have conceived, designed and developed a semi-automated method for single-cell manipulation and loading into microwells utilizing custom, high-precision fluid handling at the nanoliter scale. We present the results of a series of measurements of oxygen consumption rates (OCRs) of single human metaplastic esophageal epithelial cells. In addition, to assess the effects of cell-to-cell interactions, we have measured OCRs of two and three cells placed in a single well. The major advantages of the approach are a) multiplexed characterization of cell phenotype at the single-cell level, b) minimal invasiveness due to the distant positioning of sensors, and c) flexibility in terms of accommodating measurements of other metabolites or biomolecules of interest.

Integrated surface acoustic-wave and semiconducting-metal-oxide sensor array

Gas sensor development traditionally focuses on a single sensing platform that is optimized for a specific task. An ideal sensor is one that is completely sensitive as well as selective to the target gas of interest while being non- sensitive to interferent gases. Unfortunately, ideal sensors do not exist. Sensor arrays expand on the single sensor concept by incorporating a number of sensor utilizing the same sensing technology to provide a fingerprint of the particular measurand. Multiple sensor response are then analyzed with pattern recognition techniques such as neural networks. The limitation with single technology design methods is that single sensor limitations propagate. A sensor technology effective in detecting specific measurands may not be sensitive to related measurands. A novel sensor approach is underway that incorporates two sensing techniques, surface acoustic wave (SAW) and SMO, into an array of arrays. This integrated sensor array can provide marked improvements over either array alone in the increased bandwidth of measurands, the capability of cross verifying result with complimentary sensor technology responses, and the performance in the presence of interferents. Difficulties arise in the management, coordination and signal processing of the two methods. This is due to the fact that SAW_based sensor responded via a change in frequency, while SMO-based sensor response via a change in resistance. Each of these changes vary in magnitude proportional to the amount of analyte present. This paper will focus on aspects of: sensor selection, sensor data collection, manipulation, management, and processing for an integrated SAW and SMO sensor array.

Optical computed tomography for spatially isotropic four-dimensional imaging of live single cells

We describe a method based on principles of computed tomography for 4D imaging of live cells with isotropic spatial resolution. Quantitative three-dimensional (3D) computed tomography (CT) imaging of living single cells enables orientation-independent morphometric analysis of the intricacies of cellular physiology. Since its invention, x-ray CT has become indispensable in the clinic for diagnostic and prognostic purposes due to its quantitative absorption-based imaging in true 3D that allows objects of interest to be viewed and measured from any orientation. However, x-ray CT has not been useful at the level of single cells because there is insufficient contrast to form an image. Recently, optical CT has been developed successfully for fixed cells, but this technology called Cell-CT is incompatible with live-cell imaging due to the use of stains, such as hematoxylin, that are not compatible with cell viability. We present a novel development of optical CT for quantitative, multispectral functional 4D (three spatial + one spectral dimension) imaging of living single cells. The method applied to immune system cells offers truly isotropic 3D spatial resolution and enables time-resolved imaging studies of cells suspended in aqueous medium. Using live-cell optical CT, we found a heterogeneous response to mitochondrial fission inhibition in mouse macrophages and differential basal remodeling of small (0.1 to 1 fl) and large (1 to 20 fl) nuclear and mitochondrial structures on a 20- to 30-s time scale in human myelogenous leukemia cells. Because of its robust 3D measurement capabilities, live-cell optical CT represents a powerful new tool in the biomedical research field.