Name: Breakthrough Cell-CT for Early Diagnosis of Lung Cancer from Sputum and Blood
Uploaded: 2013-10-22T00:00:00
Duration: 58 min 23 s
Description: Breakthrough Cell-CT for Early Diagnosis of Lung Cancer from Sputum and Blood

SELECTBIO

Single Cell Analysis Summit 2013

Breakthrough Cell-CT for Early Diagnosis of Lung Cancer from Sputum and Blood

Single cell genetic analysis offers an unprecedented view of the behaviors of cell populations that are masked by the ensemble average.  In many cancers, small subpopulations of distinct cell types cause or drive the disease but are difficult to study using conventional biomolecular methods. Using emulsion PCR and RT-PCR, we are able to determine the sequences and frequencies of genetic mutations and changes in gene expression at the single cell level.  The microfluidic droplet generators developed by my group facilitate rapid analysis of the thousands of single cells required for meaningful statistical analysis.  Specifically, we have developed a method for high-throughput single cell DNA purification and detection of the t(14;18) translocation, an approach for investigating the frequency of rare mutation events within individuals prior to the onset of clinical symptoms, and an assay for telomerase splice variants and expression levels in single cells that are associated with more severe cancer cases.  These techniques open cellular behaviors to be quantitatively studied with unprecedented sensitivity in order to develop a greater mechanistic understanding of cellular processes of cancer onset and progression.


High Throughput Technologies for Single Cell Genetic Analysis

SELECTBIO has been tracking the various market segments that constitute the single cell analysis space and have been collecting market data relating to several active segments within this field. &nbsp;In this presentation, I will provide a snapshot of the market landscape, SWOT, and competitive analysis of this fast-moving field.


Market Analysis of Single Cell Analysis: Segmentation, Sizing, and Opportunities

Mass spectrometry (MS) is an indispensable technique in food safety, forensics, pharmaceuticals development, and in the detection of hazardous substances; and its use within the biological sciences is growing exponentially. Whereas traditional MS studies began with sample homogenization, recent technological advances have enabled imaging by mass spectrometry (MSI). Mass spectrometry requires the ionization (charging) of a sample, and this talk presents methods for improving the spatial resolution for ionization that employs lasers and a chemical “matrix” (Matrix-Assisted Laser Desorption Ionization-MALDI). Two novel techniques, both of which increase spatial resolution of MALDI imaging by ~10-fold and enabled the first analysis of single mammalian cells (neurons), will be presented. The first technique
involves the simultaneous chemical fixation of tissues and deposition of matrix. The second technique involves the injection of a solution of MALDI matrix only upon cells of interest. Both techniques are compatible with fluorescence microscopy. The application of these techniques to the study of a mouse model of the neurodegenerative disease, Amyotrophic Lateral Sclerosis (ALS) is presented.


Mass Spectrometry Imaging of Single Cells in Mouse Models of ALS

Methods from engineering and the physical sciences are now being used to create novel tools to study basic biological phenomena in order to elucidate the complex relationships that underlie the behaviors of living cells with a focus on individual cells and cellular microenvironments.  Much of my research is focused on the development and testing of bioanalytical platforms to enable the study of how single cells receive and process information. We are currently developing integrated microanalytical platforms in both array and microfluidic formats to address critical preclinical and clinical needs in accurately monitoring drug action and identifying the patient who will respond to new therapies aimed at modulating signal transduction pathways.  In order to manipulate individual cells, the lab has also pioneered the development of novel microfabricated devices to enable the analysis and isolation of cells while they remain in culture.  These technologies possess a number of advantages over current cell separation methods as cells can be monitored over time and selected based on a wide range of characteristics.  Numerous applications of these microfabricated devices are being pursued, including efficient cloning of mouse stem cells, purification of cancer stem cells from patient samples and isolation of tumor-targeted lymphocytes for cancer immunotherapy.


Microengineered Devices for Single Cell Analysis in Preclinical and Clinical Research

Redox signaling is important in determining cell fate decisions and plays a pivotal role in the pathology of diseases such as cancer and neurodegeneration. There are significant challenges in quantitatively measuring intracellular redox potentials, and in this presentation, I will introduce a new approach. This approach is based on the use of nanosensors which comprise molecules that sense the local redox potential, assembled on a gold nanoshell. Since the Raman spectrum of the sensor molecule changes depending on its oxidation state and since the nanoshell allows a huge enhancement of the Raman spectrum, intracellular potential can be calculated by a simple optical measurement. The nanosensors can be controllably delivered cells, without any toxic effects, allowing redox potential to be monitored in a reversible, non-invasive manner over a previously unattainable potential range encompassing both superphysiological and physiological oxidative stress. In this presentation, the concept, the reporter molecules and the technique will be introduced. Furthermore, I will discuss how we have used this approach to study the regulation of redox potential in apoptosis and have correlated redox potential changes with changes in caspase activity.


Optical Sensors for Quantification of Intracellular Redox Potentials

While metastic disease causes 90% of all cancer deaths, determining the role of various cancer cells in the metastatic process has been difficult due to the rare nature of these cells and the continuum of phenotypes they possess. To assist in studies based on the use of rare CTCs that may be involved in metastasis, an integrated system was designed, fabricated and evaluated. The system consisted of a cell selection unit, an electrical detector for enumeration, an imaging unit for phenotyping single cells and a molecular analysis unit for detecting mutations. The integrated system could correlate a cell’s phenotype to its genotype. The cell selection unit contained a series of 190 nL fluidic channels. The walls of the channels were decorated with antibodies used to recognize and select various sub-populations of CTCs. The cell selection unit could process 7.5 mL of whole blood in ~20 min with a selection efficiency of 97% and purity &gt;80%. The CTCs were subsequently released from the surface-immobilized antibodies and enumerated by measuring impedance signatures of single cells that traversed through a pair of electrodes. The CTCs were then stained and phenotyped with the appropriate cells selected for molecular analysis. The CTCs were molecularly profiled by expression profiling certain genes from selected single cells. The use of the system for determining the molecular characteristics of CTC sub-populations involved in an epithelial-to-mesenchymal transition were evaluated using pancreatic cancer as the example disease. 


Selection and Molecular Profiling of Circulating Tumor Cell (CTC) Sub-populations: Single-Cell Studies of Epithelial-to-Mesenchymal Transitions

As we age, our cells undergo a process where they enter a senescent state as a potent tumor suppression mechanism, but this cellular change may also contribute to the aging pathology seen within tissues including inflammation and bioenergetic compromise. In this talk the use of single cell analysis is used to dissect the cellular transition from a quiescent to senescent state.   


Single Cell Analysis of Senescent Cells

Development begins with the fertilized egg, which gives rise to an adult with amazing cellular variety and multiple organ systems. Underlying the enormous complexity of this process are simplifying strategies that make repeated use of individual genes and signaling systems to drive distinct developmental decisions in different contexts. At many stages in development we observe histologically uniform groups of cells that produce varied progeny. We know that morphogen gradients, flanking cell crosstalk, and even stochastic gene expression can regulate decisions that generate diversity from apparent uniformity. Nevertheless, to date we have not created a detailed blueprint of this process, at single cell resolution, for any developing organ system. Studies of population pools cannot define early events marking distinct developmental destinies in flanking cells that appear identical. Single cell analysis is required. To this end we have been using the Fludigm C1 microfluidic/robotic system that facilitates high throughput single cell RNA-Seq gene expression profiling. We have applied this technology to the analysis of lung and kidney development. Many hundreds of single cells were examined with RNA-Seq. The challenges of this approach, including technical and biological noise, will be discussed. This strategy can be applied to the analysis of any system with interesting cellular heterogeneity present. 


Breakthrough Cell-CT for Early Diagnosis of Lung Cancer from Sputum and Blood

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SELECTBIO

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October 22, 2013

Single Cell Analysis Summit 2013

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