From a (Successful) Grant Application by Tanner Et Al

From a (successful) grant application by Tanner et al.:

This is an extract from a successful grant that Scott wrote in late 2009. I do not know how much of this has already been adapted directly into other people’s grant applications. I have omitted certain sections that are heavily biased towards the particular governmental group that sponsored the grant, and deleted certain particularly proprietary parts. This grant was reviewed by a few US referees, but I suspect that the wordmanship will not be recognized widely: therefore, I authorize to use what you can from this (but be careful that the wording supports your particular direction). This grant was constructed as a collaboration of international laboratories that would evaluate and advise on the continuing development of the mass cytometry technology.

Layperson:

Mass cytometry is a transformational new technologythat allows the identification and investigation of rare, diseased cells.

... is a transformational technology that enables the detection and characterization of rare and heterogeneous cell populations, such as cancer stem cells, at the individual cell level. It marries the resolution, specificity and sensitivity of atomic mass spectrometry to the high throughput, single cell analytical advantages currently provided by flow cytometry. Using stable isotope (non-radioactive metal) tags, many proteins can be identified and quantified simultaneously in single cells, yielding a biomarker signature that distinguishes diseased cells at an early stage when therapies can be less aggressive and more effective.

Technical:

The “mass cytometer” instrument addresses the analytical challenges that are normally encountered using flow cytometry, by using “element-tagged” immunological staining combined with atomic mass spectrometry that provides many more independent and quantitative detection channels. The technology has ... been described as potentially transformational for medical research into the genesis of disease and for clinical diagnostics. Mass cytometry provides, for the first time, the ability to simultaneously determine at least 30, and potentially as many as 100, biomarkers in individual cells. This portends the ability to recognize extremely rare diseased cells in patient’s samples, and offers a potentially transformative step towards personalized healthcare characterized by early and correct diagnosis that improves the efficacy of less-aggressive therapeutic intervention.

More detail:

Mass cytometry is an innovative new technology that facilitates massively multi-parameter assay of single cells and particles at high throughput [1, 2, 4, 5]. It is poised to transform medical research into the genesis of disease, enabling rational drug discovery and development, and offers a real opportunity for personalized clinical diagnosis and prognosis. The ability to simultaneously measure more than 30 (potentially up to 100) biomarkers should allow the confident identification of rare cells (e.g., cancer stem cells) in complex patients’ samples, the subclassification of rare cell distributions that indicate the progression of disease and response to therapy, and provide an information-rich snapshot of a wide distribution of protein translational modifications resulting from stimulation or suppression.

The technology addresses applications that are typically run by flow cytometer analyzers,but extends the capability to many simultaneous parameters. The detector is based on atomic mass spectrometry that provides exquisite resolution of many mass channels while bringing unique quantitative capabilities to the biological arena. The breakthrough realization lay in recognizing the potential of element-tagged immunoassay linked with atomic mass spectrometry. According to this empowering method, antibodies are tagged with metal atoms (elements of the periodic table, or preferably their enriched stable isotopes) such that a given antibody is associated with a unique isotope label. Incubation with a sample in the normal manner associates the isotope tag with the target antigen. The tag is quantitatively determined by Inductively Coupled Plasma Mass Spectrometry (ICP-MS). The method can be extended to other affinity assays, including those using oligonucleotides (genes), lectins, aptamers, etc.

ICP-MS is the preferred means of determining the elemental composition, especially ultra-trace components, of materials. It has found acceptance in various applications including environmental (e.g., drinking, river, sea and waste water analyses), geological (e.g., trace element patterning), clinical (e.g., determination of trace metals in blood, serum and urine) and high purity materials (e.g., semiconductor reagents and components) analysis. With few exceptions, it is a diagnostic tool unknown in the biological arena.

Briefly, a sample, most commonly an aerosol produced by nebulisation, is injected into a high- temperature plasma obtained by heating a flowing argon gas stream with radio frequency (RF) energy. Under conditions approximating those at the surface of the sun, the sample is promptly vaporized, atomised and ionized as it flows through the plasma. High speed mass analysis provides a "mass fingerprint" that identifies the elements contained in the sample. The particular attributes of the method of note include: wide linear dynamic range (up to 9 orders of magnitude), exceptional sensitivity (sub-part per trillion, or attomole/microlitre, detection), enormous abundance sensitivity (<10-4 overlap between adjacent isotopes), counting-statistics-limited precision, absolute quantification, and tolerance of concomitant matrix [7].

The method is amenable to a variety of analytical formats for cellular analysis. The common characteristic is the elemental labelling of the target analytes (proteins, nucleic acids, polysaccharides; generically “biomarkers”) in the whole cell, or its lysate. A facile means for immunological staining, using a metal chelating polymer or copolymer (MCP), has been developed [19]. These polymers are designed with a functional group at one end to enable the polymer to be covalently attached to a monoclonal antibody (mAb) or other biological macromolecule. In addition, the polymer has metal-chelating ligands to bind multiple copies of a lanthanide (Ln) metal or Ln isotope. In this way, the MCP-labeledmAb or biological molecule can carry many copies of aLn metal or isotope, or other metal ion, for a particular biological application. Simultaneous analysis of many antigens is enabled by incubating a cell sample with a cocktail of antibodies, each type labeled with a different metal or isotope. After stringent washing, or other separation, the different isotopes are indicative of their respective antigens (or biomarkers). In each analytical format, the signal intensity of each isotope, when measured by ICP-MS, provides quantitative information about the number of copies of each antigen present.

Different analytical challenges can then be addressed by: {I’ve deleted 4 or 5 rather proprietary applications...}

(1)Solution (or bulk) analysis: the stained sample is digested in acid to form a homogeneous solution that can be analyzed by conventional ICP-MS [8]. This quantifies the biomarker signature averaged over the cell ensemble. It may have diagnostic value when the patient is in the blast stage and the average signature over the sample is sufficient as a prognostic indicator. It also answers the need for a multi-parameter ELISA assay. In the present proposal, the method will be frequently used to characterize, qualify and quantify binding characteristics of different tagging constructs and affinity materials [8, 9, 10, 11].

(2)Mass cytometry: a suspension of whole stained cells is nebulised in a manner to stochastically introduce individual cells into the ICP whereupon a full multi-parameter analysis of each cell is effected. Since each cell generates a transient signal of only 200-400 microseconds duration, a purpose-specific instrument configuration that provides high-speed data collection and interpretation is demanded. The method can be considered a massively multi-parameter analog of fluorescent flow cytometry. It offers the unique ability to distinguish, identify and interrogate rare cells in complex (patients’) samples [1].

(3)Multiplexed Mass Cytometry: while mass cytometry is inherently multi-parameter, it can simultaneously be used for multiplexed analysis, wherein different cell samples are distinguished through uptake of a metal-labeling solution or endocytosis of metal-encoded beads. An admixture of samples that have been separately encoded and stained allows high-throughput assay with deconvolution via the encoding signals.

References for the above section:

References:

1. Mass Cytometry: A Novel Technique for Real Time Single Cell Multi-target Immunoassay based on Inductively Coupled Plasma Time-of-Flight Mass-Spectrometry, D. R. Bandura, V.I. Baranov, O.I. Ornatsky, A. Antonov, R. Kinach, X. Lou, S. Pavlov, S. Vorobiev, J. E. Dick and S.D. Tanner, Analytical Chemistry (accepted 2009 | doi: 10.1021/ac901049w)

2. Elemental Analysis of Tagged Biologically Active Materials, Vladimir I. Baranov, Scott D. Tanner, Dmitry R. Bandura and Zoe Quinn, US patent number 7,135,296 (issued November 14, 2006).

3. Method and Apparatus for Flow Cytometry Linked with Elemental Analysis, Vladimir I. Baranov, Dmitry R. Bandura and Scott D. Tanner. US patent number 7,479,630 (issued January 20, 2009).

4. Flow Cytometer with ICP-MS Detection for Massively Multiplexed Single Cell Biomarker Assay, S.D. Tanner, D.R. Bandura, O. Ornatsky, V.I. Baranov, M. Nitz and M.A. Winnik, Pure and Applied Chemistry, 80, 2627-2641 (2008).

5. Multiplex Bio-Assay with Inductively Coupled Plasma Mass Spectrometry: Towards a Massively Multivariate Single Cell Technology, S.D. Tanner, O. Ornatsky, D.R. Bandura and V.I. Baranov, SpectrochimicaActa Part B, 62, 188-195 (2007).

6. A Sensitive and Quantitative Element-Tagged Immunoassay with ICP-MS Detection, V.I. Baranov, Z. Quinn, D.R. Bandura and S.D. Tanner, Analytical Chemistry, 74,1629-1636 (2002).

7. The Potential for Elemental Analysis in Biotechnology, V.I. Baranov, Z.A. Quinn D.R. Bandura and S.D. Tanner, Journal of Analytical Atomic Spectrometry, 17, 1148-1152 (2002).

8. Development of Analytical Methods for Multiplex Bio-assay with Inductively Coupled Plasma Mass Spectrometry, O.I. Ornatsky, R. Kinach, D.R. Bandura, X. Lou, S.D. Tanner, V.I. Baranov, M. Nitz and M.A. Winnik, Journal of Analytical Atomic Spectrometry, 23, 463-469 (2008).

9. Multiple Cellular Antigen Detection by ICP-MS, O. Ornatsky, V.I. Baranov, D.R. Bandura, S.D. Tanner, and J. Dick, Journal of Immunological Methods, 308, 68-76 (2006).

10. Element-tagged immunoassay with ICP-MS detection: evaluation and comparison to conventional Immunoassays, E. Razumienko, O. Ornatsky, R. Kinach, M. Milyavsky, E. Lechman,M.A. Winnik and S.D. Tanner, Journal of Immunological Methods, 336, 56-63 (2008).

11. Lectins Conjugated to Lanthanide-Chelating Polymers, M.D. Leipold, I. Herrera, O. Ornatsky, V. Baranov and M. Nitz, Journal of Proteome Research,8, 443-449 (2009).

12. “Combination of immunohistochemistry and laser ablation ICP mass spectrometry for imaging of cancer biomarkers”, J. Seuma, J. Bunch, A. Cox, C. McLeod, J. Bell and C. Murray, Proteomics, 8, 3775-3784 (2008).

13. “Labelling of proteins by use of iodination and detection by ICP-MS”, N. Jakubowski, J. Messerschmidt, M. GarijoAnorbe, L. Waentig, H. Hayen, P. H. Roos, Journal of Analytical Atomic Spectrometry, 23, 1487 (2008).

14. “Labelling of proteins with 2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid and lanthanides and detection by ICP-MS”, N. Jakubowski, L. Waentig, H. Hayen, A. Venkatachalam, A. von Bohlen, P. H. Roos, A. Manz, Journal of Analytical Atomic Spectrometry,23, 1497 (2008).

15. “Labelling of antibodies and detection by laser ablation inductively coupled plasma mass spectrometry. PART III. Optimization of antibody labelling for application in a Western blot procedure”, L. Waentig, P.H. Roos and N. Jakubowski, Journal of Analytical Atomic Spectrometry, 24, 924-933 (2009).

16. D. L. Costantini, C. Chan, Z. Cai, K. A. Vallis, and R. M. Reilly, J. Nucl. Med., 2007, 48, 1357.

17. D. L. Costantini, M. Hu, and R. M. Reilly, Cancer Biother. Radiopharm., 2008, 23, 3.

18. Messenger RNA Detection in Leukemia Cell Lines by Novel Metal-Tagged in situ Hybridization Using Inductively Coupled Plasma Mass Spectrometry, O. Ornatsky, V.I. Baranov, D.R. Bandura, S.D. Tanner, and J. Dick, Translational Oncogenomics1, 1-9 (2006).

19. Polymer-Based Elemental Tags for Sensitive Bioassay, X. Lou, G. Zhang, I. Herrera, R. Kinach, O. Ornatsky, V. Baranov, M. Nitz, M.A. Winnik, AngewandteChemie International Edition, 46, 6111-6114 (2007).

20. Antibody-Dendrimer Conjugates: The Number, Not the Size of the Dendrimers, Determines the Immunoreactivity, C. Wängler, G. Moldenhauer, M. Eisenhut, U. Haberkorn, and W. Mier, Bioconjugate Chem., 19, 813–820 (2008).

21. “Controlled Synthesis and Water Dispersibility of Hexagonal Phase NaGdF4:Ho3+/Yb3+ Nanoparticles”, R. Naccache, F. Vetrone, V. Mahalingam, L. A. Cuccia, J. A. Capobianco, Chemistry of Materials, 21, 717 (2009).

22. “From Trifluoroacetate Complex Precursors to Monodisperse Rare-Earth Fluoride and OxyfluorideNanocrystals with Diverse Shapes through Controlled Fluorination in Solution Phase”, X. Sun, Y. W. Zhang, Y. P. Du, Z. G. Yan, R. Si, L. P. You, C. H. Yan, Chemistry-a European Journal, 13, 2320 (2007).

23. “Biocompatible Hybrid Nanogels”, A. Pich, F. Zhang, L. Shen. S. Berger, O. Ornatsky, V. Baranov, M. A. Winnik, SMALL,4,2171-2175 (2008).

24. “Lanthanide-Containing Polymer Microspheres by Multiple-Stage Dispersion Polymerization for Highly Multiplexed Bioassays”, A. I. Abdelrahman, S. Dai, S. C. Thickett, O. Ornatsky, D. Bandura, V. Baranov and M. A. Winnik J. Am. Chem. Soc, (submitted for publication, May 2009).

25. “A metal-coded affinity tag approach to quantitative proteomics”, R. Ahrends, S. Pieper, A. Kühn, H. Weisshoff, M. Hamester, T. Lindemann, C. Scheler, K. Lehmann, K. Taubner, M.W. Linscheid, Mol. Cell. Proteomics, 6, 1907 (2007).

26. “Causal protein-signaling networks derived from multiparameter single-cell data”, K. Sachs, O. Perez, D. Pe'er, D.A. Lauffenburger, and G. Nolan, Science308, 523-529 (2005).

27. "Learning signaling network structures from sparsely distributed data", K. Sachs, S. Itani, J. Carlisle, G. P. Nolan, D. Pe'er and Douglas A. Lauffenburger, RECOMB proceedings and Journal of Computational Biology16(2), 201-20 (2009).

28. “Learning cyclic signaling pathway structures while minimizing data requirements“,K. Sachs, S. Itani, J. Fitzgerald, L. Wille, B. Schoeberl, M.A. Dahleh and G.P. Nolan, Proceedings of the Pacific Symposium on Biocomputing, 2009.

29. “Formalism and structure learning for cyclic networks”, S. Itani, M. Ohannessian, K. Sachs, G. P. Nolan and M. A. Dahleh, Journal of Machine Learning Research (JMLR), (in revision).

30. “Characterization of patient specific signaling via augmentation of Bayesian networks with disease and patient state nodes”, K. Sachs, A. J. Gentles, R. Youland, S. Itani, J. Irish, G. P. Nolan and S. K. Plevritis, 31st Annual International Conference of the IEEE Engineering in Medicine and Biology Society (accepted, to appear)

31.”Seventeen-colour flow cytometry: unravelling the immune system”, S.P. Perfetto, P.K. Chattopadhyay and M. Roederer,Nat Rev Immunol4, 648–55 (2004).

Figures for above section:

Figures:

Figure 1: Left: emission spectra for 8 commonly use fluorophores. Right: mass spectra of 30 enriched stable lanthanide isotopes.

Figure 2: Experimental protocol for tagging antibodies with metal-chelating polymers. The antibody of interest is subjected to selective reduction of –S-S-groups to produce reactive -SH groups, which are reacted with the terminal maleimide groups of a polymer bearing metal-chelating ligands along its backbone. The polymer-bearing antibodies are purified, treated with a given lanthanide ion, and then purified again. Each type of antibody is labeled with a different element. (from reference 19).

Figure 3: Schematic of the prototype Mass Cytometer, comprising a novel configuration of ICP-TOF-MS (from reference 1).

Figure 4: A PBMC (Peripheral Blood Mononuclear Cell) sample was probed with antibodies against 15 cell surface proteins (antigens), each antibody being labeled with a different stable isotope, and the DNA was stained with an Ir intercalator. The data is displayed here in the 162-1= N two-dimensional plots that are theformat of conventional flow cytometry (using FlowJo software).

Figure 5: Polar diagrams of median intensity values for surface antigens measured using metal-tagged antibodies on leukemia patient samples, monoblastic M5 AML(A) and monocytic M5 AML (B). The monoblastic phenotype (A) shows high CD33 and HLA-DR and low CD34, CD13, CD14, and CD64 expression levels. The more differentiated monocytic M5 (B) type displays increased CD13, CD14, and CD64 and lower HLA-DR levels. Data were collected on the mass cytometer and processed with FlowJo software. The typical population size used for averaging was 15 000-20 000 cells. Each of the 22 axes represents an antibody (or contrast reagent, CR, or Ir-DNA intercalator) measured by detecting the isotopic tags indicated in Table 2 per individual cell event. Samples were generously provided by the Quebec Leukemia Cell Bank. (from reference 1).