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- Research
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Lou X, Zhang G, Herrera I, Kinach R, Ornatsky O, Baranov V, Nitz M, Winnik MA. Polymer-based elemental tags for sensitive bioassays, Angew Chem Int Ed Engl. 2007 May 29;46(32):6111-6114.
Tanner SD, Ornatsky O, Bandura DR, Baranov VI. Multiplex bio-assay with inductively coupled plasma mass spectrometry: Towards a massively multivariate single-cell technology, Spectrochimica Acta Part B: Atomic Spectroscopy, 2007 Mar;62(3):188-195.
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“If cancer stem cells lie at the heart of some cancers, then being able to predict the behavior of tumors and providing effective therapies against them means understanding the abnormal growth pathways within the stem cells themselves.”
- John Dick, Project Leader (UHN)
“The only reason that I'm willing to take a risk on this project is my confidence in the quality and camaraderie of my colleagues.”
- Scott Tanner, Principal Investigator (University of Toronto)
“We're examining DNA not for its well-recognized role as the genetic material to store and transmit genetic information, but rather for its less-known potential to act as a novel cancer diagnostic tool.”
- Yingfu Li, Principal Investigator (McMaster University)
“We're gaining more understanding of cell defects and how to modify them, so that treatment is no longer a shotgun approach, but more like an arrow aimed at a specific target.”
- Mark Minden, Principal Investigator (OCI)
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The University of Toronto group is responsible for the construction of the Inductively Coupled Plasma Mass Spectrometry (ICP-MS) based Flow Cytometer, and for the development of a suite of affinity products able to detect targets of interest by ICP-MS.
In previous installments (see links at bottom of page), we described the development of the instrumentation and reagents, and presented examples of data obtained using our system. We continue to refine our methods and improve the power of our technology.
Here, we provide an overview of our workflow and examples of recent proof-of-principle experiments in bulk format showing multiplexed protein detection. We also demonstrate the ability of our instrument to perform multiplexed analysis of single cells.
Click here to view a recent poster describing our work on elemental tags.
The Tags: Labelling Antibodies with Elements
The key to our method of simultaneously measuring multiple biomarkers lies in our ability to produce element-tagged affinity products, known as the MAXPARTM reagent system. Because of the high resolution of detection channels in ICP-MS, multiplex assays of the order of 50-100 are possible. This technology allows for rapid protein expression profiling of both cell surface and intracellular biomarkers.
Figure 1: Synthesis of element-labeled tags. Lanthanide chelates produced in the lab of Prof. Mark Nitz (Dept. of Chemistry, U of T) replace functional groups on polymers that have been produced in collaboration with the lab of Prof. Mitch Winnik (Dept. of Chemistry, U of T). The tags are attached to antibodies through a linker. Metal atoms are incorporated in the final step. These structures form the basis of the MAXPARTM system of affinity reagents.
Figure 2: Protocol for tagging antibodies with metals. Antibodies are partially reduced and reacted with polymer-ligand conjugate. Metal atoms are then added to the tagged antibodies.
Figure 3: Protocol for labeling cells (in bulk) with metal-tagged antibodies. Metal-tagged antibodies are incubated with the cell samples. The samples are washed and Rh and Ir are added for signal normalization. Concentrated HCl is added, and the samples are measured by ICP-MS.
The Instrument: Flow Cytometer with Mass Spectrometer Detector
The MAXPARTM element-tagged reagents are most powerful when used with a novel elemental mass spectrometry based detection technology known as the CyTOFTM. This technology, also developed at the University of Toronto, provides the means to perform massively multiplexed bioanalytical assays for single cells and particles.
Figure 4: The Phase 0 prototype of the CyTOFTM.
Figure 5: Screen capture of multiplex detection of a single cell. Using the research prototype of the CyTOFTM instrument, 6 markers were detected on a single cell. (Iridium was used for normalization.)
Figure 6: Seven-plex surface marker analysis of individual KG1a cells. KG1a cells were probed for seven surface markers, as indicated, and analyzed using the research prototype CyTOFTM. Two-dimensional projections (pair-wise plots) of data obtained for individual KG1a cells, combined with distribution histogram of intensity of individual markers, are shown. Antibodies against selected surface markers were labeled with stable enriched isotopes of lanthanides as indicated. In addition to surface markers, iridium intercalator was used to quantify total cell DNA content.
The Application: Multiplex Analysis of Cells Using Element Tags
The overall goal of this project is to employ the MAXPARTM element tag technology in the CyTOFTM flow cytometer with mass spectrometer detector to perform multiplexed assays at the level of a single cell. We have been successful in illustrating the power of our technology in several proof-of-principle experiments.
Figure 7: Cell surface protein 10-plex assay in bulk (solution) format to examine cell differentiation. THP-1 cells (representative of promonocytic AML-M5) and KG1a (early myeloblasts, AML-M0) were treated with PMA for 72 hr to induce differentation. After treatment, cells were washed and immunolabeled with a solution containing 10 element-tagged antibodies or with element-tagged mouse IgG (for background control) for 30 min at room temperature. Washed cells were then fixed in formaldehyde and DNA was stained with Rh(III)-containing metallointercalator (which serves as an internal control to ensure equivalent numbers of cells are analyzed). Finally, cells were washed several times and dissolved in HCl. Polar diagrams in logarithmic scale represent data from triplicate samples normalized to internal standard (1 ppb Ir). In the case of THP-1 differentiation, the cells change morphology and increase cell surface attachment (A), which is reflected (in C) by upregulation in expression of adhesion receptors: a 10-fold increase in ICAM-1 (intercellular adhesion molecule, CD54), and 2-fold increase in the hyaluronan (CD44) and vitronectin receptors (CD51). Tyrosine phosphatase content (CD45), CD33 (myeloid precursor marker), and receptor for fibrinogen (CD41) are unchanged, while expression of CD38 (NAD glycohydrolase and ADP ribosylcyclase) is decreased. THP-1 cells do not express significant levels of CD34 (as evidenced by similar low levels in DMSO- and PMA-treated cells). KG1a cells (B) are known to be unresponsive to PMA stimulation, as reflected in the polar diagram by coincidence of black (DMSO) and white (PMA) lines.
Figure 8: A 20-plex assay in bulk (solution) format to compare cell surface marker expression. Twenty monoclonal antibodies were conjugated to polymer tags with different lanthanide isotopes. Four replicates of live cells (~80,000 cells/well) were distributed into wells of a 96-well filter plate and incubated with a mixture of the 20 element-tagged antibodies. As a control for non-specific binding, cell samples were incubated with a mixture of mouse IgG similarly labeled with elemental tags. Washed cells were fixed in 2% formaldehyde and DNA was labeled with Ir-containing intercalator for cell number normalization. Finally, washed cells were dissolved in concentrated HCl and analyzed by solution ICP-MS. Clear differences in surface protein expression could be detected in the different cell lines using element-tagged antibodies.
Figure 9: A 20-plex assay in bulk (solution) format to compare cell surface marker expression. The expression of 20 cell surface markers in 3 different cell lines was examined using element tags and ICP-MS as described above. Data is depicted in polar diagram format. Different protein expression signatures that are characteristic of each cell line are apparent.
Figure 10: Cell surface and intracellular protein 13-plex assay in bulk (solution) format to examine cell differentiation. Cells were treated with DMSO or PMA, and then fixed in 1% paraformaldehyde, blocked with 100 mM glycine, and permeabilized with 70% methanol. Cells (1 x 106 cells/sample) were immunolabeled with antibodies as indicated (intracellular antigens are shown in bold). Values were normalized by using Rh(III)-containing metallointercalator (an internal control for cell number) and background values for IgG-Ln were subtracted. Differences in PMA-responsiveness of the cell lines can be observed. KG1a cells do not respond to PMA. THP-1 cells upregulate expression of CD54 and CD44 when treated with PMA. Kas-3 cells treated with PMA demonstrate upregulated CD44, but decreased expression of CD54.
Figure 11: A 20-plex assay in bulk (solution) format to examine cell surface marker expression in two leukemic cells lines and one patient sample. The expression of 20 cell surface markers was examined in the KG1a and THP-1 cell lines, as well as in a patient sample obtained from the Quebec Leukemia Cell Bank (BCLQ). KG1a is a primitive cell line (characterized by expression of CD34), THP-1 is a differentiated cell line, and BCLQ is an AML patient (M5a subtype) sample obtained from the Quebec Leukemia Cell Bank. Antigens in black were probed with specific antibodies labeled with the stable isotope indicated in red.
Figure 12: Element-tagged assay as an alternative to colorimetric assay for activated p53. The principles of element-tagged immunoassay were applied to analysis of activated p53. This assay is similar to a standard ELISA-based assay for activated trancription factors. Microplate wells were coated with a double strand DNA consensus binding sequence for activated p53. In order for p53 to be activated it must be phosphorylated and arranged as a tetramer. Therefore, only phosphorylated p53 in the tetramer configuration can bind to the microplate wells. Nuclear lysate of MCF-7 breast cancer cells were added to the microplate. Following incubation and washing, levels of bound p53 were measured by indirect ELISA using a colorimetric reaction (left), or by ICP-MS immunoassay using Tm-labeled secondary antibodies (right). When a free consensus oligonucleotide (con-oligo) was added together with the nuclear extract, low binding was seen, indicating competition for activated p53 protein. Levels of p53 were similar to non-treated wells when a scrambled (scr-oligo) non-binding oligonucleotide was used. This shows the specificity of the assay for activated p53 for both the colorimetric and ICP-MS methods.
Click here for the first installment of this page
Click here for the second installment of this page
Click here for the third installment of this page
Click here for the fourth installment of this page
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