Follow-on Funding Microscale Life Sciences Center
1) Live-Cell Microarray for High-Throughput Observation of Metabolic Signatures
(NIH U01, Deirdre Meldrum, PI)
The Cellarium project is developing a platform for dynamic, multiparameter sensing of single-cell metabolic phenotypes in a high throughput live-cell microarray format. Funded in 2011, the project supports data collection efforts like the Library of Integrated Network-Based Cellular Signatures (LINCS). A sandwich microarray called the "Cellarium" will be developed and used to analyze individual live cells. This microarray is an extensible tool for deriving a standardized multiparameter set of data that can be integrated in a coordinated way into LINCS.
The Cellarium project leverages the advancements of the NIH Center of Excellence in Genomic Sciences called the Microscale Life Sciences Center, also part of the Center for Biosignatures Discovery Automation. Single-cell analysis which directly reveals heterogeneity in metabolic response to perturbations within an isogenic cell population is critical to biological inference.
2) Live-Cell Computed Tomography Instrumentation Development
(W.M. Keck Foundation Award, Deirdre Meldrum, PI)
In 2010 Dr. Meldrum received a $1M grant from the Keck Foundation to develop a 3D cell tomograph. This project was motivated by the work we have been pursuing under our CEGS funding, and our desire to develop additional tools to assess the functional state of individual cells.
This four-year project aims to build a novel, live-cell imaging instrument for basic and clinical science application. The 3D microscope we call the “cell CT scanner” will provide functional images, revealing the molecular mechanisms underlying important metabolic and disease processes. Cell CT is analogous to diagnostic radiology CT in that we will reconstruct 3D images with isotropic, submicron resolution from hundreds of projections acquired from many angles as the cell is rotated. A key component of the research is to determine the best method for rotating cells, which must be done extremely precisely without harming the cell. We will investigate one method that rotates an optically trapped cell in a microfluidic vortex and another that uses an asymmetrical infrared light beam. We will use fluorescent antibody probes and fusion-protein constructs specific to the proteins we are interested in to label cells for emission CT scanning. We will validate the technology studying cells from immortalized epithelial cell lines representative of various stages of esophageal cancer, and disaggregated from human biopsies spanning the same disease spectrum. Cell CT will enable for the first time rapid estimation of local protein concentrations in cellular compartments and microdomains, providing powerful insights concerning relationships between cell structure, function and disease.
3) In Situ Single Cell Laser Lysis Downstream qRT-PCR Profiling
(NIH R21 Deirdre Meldrum, PI)
With funding from the NIH Single Cell Analysis Program, we are developing an innovative, microfluidic-based tool for clinical and scientific users to analyze in situ gene expression heterogeneity using single-cell mRNA expression analysis. The device uses a two-photon laser to lyse a sequence of individual cells at known coordinates within a 3D tissue. The lysate is immediately transported to an emulsion-based (oil-droplet) qRT-PCR module to profile mRNA expression. This research could lead ultimately to a highly multiplexed platform capable of detecting dozens of mRNA sequences for each initial droplet eluted from the sample.
4) Water Soluble Nanoarrays for Single-Cell Proteomics
(NIH R01, Hao Yan, PI, in collaboration with Meldrum lab)
In 2009, Deirdre Meldrum’s Center for Biosignatures Discovery Automation joined with Professor Hao Yan of the Center for Single-Molecule Biophysics, also in ASU’s Biodesign Institute, on a project to perform few- and single-cell proteomics using functionalized DNA nanoarrays. Our contribution (the fourth technology mentioned below) was made possible by the technologies and protocols developed under our CEGS funding.
What if protein-capture arrays could be synthesized on a molecular scale? If this could be done, the arrays themselves become reagents capable of being incubated with the contents of even a single cell. As reagents, the arrays themselves could be used in titrations, giving a potentially enormous dynamic detection range. Thus, for example, one could read out the protein content and protein modifications for each generation of the progeny of a single parent cell. Nanotechnology already provides the components for such a system, and we propose to integrate four new technologies to develop self-assembled nanoarrays for protein analysis (Fig. 1).
- The first is construction of an array of single molecule probes arranged at nanometer density using nucleic acid self-assembly. The arrays are themselves giant molecules and are used like a reagent in a solution analysis, only being deposited onto a surface for a final readout.
- The second is a nanoscale readout using atomic force microscopy (AFM). The AFM can scan these giant molecules with nm-resolution, reading out the state of each probe on the array. It is capable of reading out >30,000 sites per minute with current technology.
- The third is a molecular recognition development approach to engineer aptamers and multivalent aptamers to have the affinity and specificity for proteins and posttranslationally-modified protein molecules. Because aptamers are synthetic and also nucleic acid sequences, individual aptamers can bind to a unique position on the array through nucleic acid hybridization and require no printing or lithography.
- The fourth is microfluidic technology to allow the nanoarray to interact with proteins produced from single-cell lysate and to deliver the arrays to predefined locations for readout.
Our goal is to fill a unique niche in proteomics: parallel analysis of minute amounts of protein taken from single cells. Can we make suitable ligands and will they work on arrays? Can we read the arrays accurately with AFM? Can we synthesize ligands that are highly selective for post-translational modifications? What factors cause biodegradation of the arrays, and how can we control them without losing sensitivity and selectivity? What conditions are required to exploit the arrays for proteomics in a microfluidic system? What methodologies can be used to deposit nanoliter solutions of reacted arrays at precise locations for AFM readout? Our specific aims are focused on these questions. We address the broader question of why this technology should be developed in the ‘Significance’ section of the proposal.
5) Center for the Convergence of Physical Sciences and Cancer Biology
(NIH U54, Paul Davies, PI, in collaboration with Meldrum lab)
In a series of workshops held at NCI in 2008, participants addressed the question: Could progress in understanding and controlling malignancy be made by scientists from outside the established cancer research community? These workshops culminated in the issuance of an FOA for Center proposals to bring eminent physical scientists into the cancer research community. Twelve Physical Sciences in Oncology Centers (PS-OCs) were established around the U.S. with funding commencing in 2009. ASU was the recipient of one such award, led by Paul Davies, a theoretical cosmologist (cancer-insights.asu.edu). Deirdre Meldrum’s lab, the Center for Biosignatures Discovery Automation, is the home to one of three research projects in the ASU PS-OC. Our project, “Single-cell Physiology and 3D Tomography” was made possible by the single-cell physiological analysis methods developed under our CEGS funding, with 3D single-cell imaging added to the suite of tools we are using to probe cells’ physical and functional properties.
Recent breakthroughs in microfabricated systems for single-cell analysis and in high-resolution optical CT imaging enable physiological measurements and isotropic 3D images to be obtained from single cells. This research project aims to conduct the single-cell equivalents of clinical protocols for metabolic rate measurements and CT and PET scanning. The overall objective of the project is to apply new technologies for single-cell measurements to test existing hypotheses and new hypotheses advanced by the Cancer Forum core with respect to the physics of cancer cells. An example of an existing hypothesis we will test is that nuclear morphometry and chromatin patterns we can quantify with exquisite sensitivity using 3D cell imaging are progression phase-specific signatures of cancer.
The Center for Biosignatures Discovery Automation has demonstrated success in cell manipulation and placement, and in single-cell physiological monitoring using sophisticated microfluidic devices and optical sensing of key analytes including oxygen and pH. We have fine tuned single-cell transcriptomic protocols, and are developing capabilities to quantify calcium, potassium and sodium fluxes and ATP concentration. We are building a new single-cell analysis platform which implements confocal optical sensor and molecular probe data acquisition, and provides the single-photon sensitivity required to quantify target-specific probes within or on the cell.
We have recently acquired the first optical microtomograph capable of 3D cell imaging at isotropic, 100-nm resolution. The cell computerized tomography (cell CT) instrument produces cell imagery based on hundreds of angular projections acquired using broad-band illumination of epithelial cancer cells fixed and stained with absorbing dyes. This structural imaging mode has demonstrated utility in elucidating gross nuclear architecture when chromatin is the primary absorbing species. Results using narrow-band excitation in conjunction with fluorescent probes and full-field projection acquisition are promising.
We propose to apply single-cell physiological and transcriptomic measurements and 3D tomography to quantify the physical and structural properties of cancer cells and to employ this data to investigate hypotheses regarding cancer cells, their heterogeneity and disease progression and arrest. We will measure respiration rates and ion fluxes on individual cells from cultured cancer cell lines and disaggregated cells from tissue biopsies, and correlate them with RNA expression levels. Using absorption-mode cell CT imagery we will define and extract nuclear architectural and textural features and search for correlations between these and transcriptomic and physiological measurements. In years 3-5, using fluorescence emission imaging capabilities developed and refined in years 1 and 2, we will measure the concentrations and spatial distributions of selected proteins within and on the cells using both the single-cell analysis platform and cell CT, and correlate these functional measurements between the two modalities and with the structural and physiological data. We will seek to extract, describe and begin to understand relationships among the measurements and physical parameters obtained from the same types of cells and their nuclei by our sister projects on elasticity and chromatin.