Abstract
Oncogenesis and tumour progression are supported by alterations in cell signalling. Using flow cytometry, it is now possible to track and analyse signalling events in individual cancer cells. Data from this type of analysis can be used to create a network map of signalling in each cell and to link specific signalling profiles with clinical outcomes. This form of 'single-cell proteomics' can identify pathways that are activated in therapy-resistant cells and can provide biomarkers for cancer diagnosis and for determining patient prognosis.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Buy this article
- Purchase on SpringerLink
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Perez, O. D., Krutzik, P. O. & Nolan, G. P. Flow cytometric analysis of kinase signaling cascades. Methods Mol. Biol. 263, 67–94 (2004).
Krutzik, P. O. & Nolan, G. P. Intracellular phospho-protein staining techniques for flow cytometry: monitoring single cell signaling events. Cytometry 55A, 61–70 (2003).
Perfetto, S. P., Chattopadhyay, P. K. & Roederer, M. Seventeen-colour flow cytometry: unravelling the immune system. Nature Rev. Immunol. 4, 648–655 (2004).
Irish, J. M. et al. Single cell profiling of potentiated phospho-protein networks in cancer cells. Cell 118, 217–228 (2004).
Timmerman, J. M. et al. Idiotype-pulsed dendritic cell vaccination for B-cell lymphoma: clinical and immune responses in 35 patients. Blood 99, 1517–1526 (2002).
Pruzlj, N., Jurisica, I. & Wigle, D. Knowledge Discovery in Proteomics: Graph Theory Analysis of Protein–Protein Interactions 129–198 (Chapman & Hall/CRC Mathematica Biology and Medicine, 2005).
Xia, Y. et al. Analyzing cellular biochemistry in terms of molecular networks. Annu. Rev. Biochem. 73, 1051–1087 (2004).
Barrios-Rodiles, M. et al. High-throughput mapping of a dynamic signaling network in mammalian cells. Science 307, 1621–1625 (2005).
Hanahan, D. & Weinberg, R. A. The hallmarks of cancer. Cell 100, 57–70 (2000).
Martin, G. S. Cell signaling and cancer. Cancer Cell 4, 167–174 (2003).
Druker, B. J. et al. Efficacy and safety of a specific inhibitor of the BCR–ABL tyrosine kinase in chronic myeloid leukemia. N. Engl. J. Med. 344, 1031–1037 (2001).
Klein, S., McCormick, F. & Levitzki, A. Killing time for cancer cells. Nature Rev. Cancer 5, 573–580 (2005).
Hardy, R. R. & Hayakawa, K. B cell development pathways. Annu. Rev. Immunol. 19, 595–621 (2001).
Engel, P. et al. Abnormal B lymphocyte development, activation, and differentiation in mice that lack or overexpress the CD19 signal transduction molecule. Immunity 3, 39–50 (1995).
Sachs, K., Perez, O., Pe'er, D., Lauffenburger, D. A. & Nolan, G. P. Causal protein-signaling networks derived from multiparameter single-cell data. Science 308, 523–529 (2005).
Lackner, M. R. et al. Chemical genetics identifies Rab geranylgeranyl transferase as an apoptotic target of farnesyl transferase inhibitors. Cancer Cell 7, 325–336 (2005).
Pardanani, A. & Tefferi, A. Imatinib targets other than bcr–abl and their clinical relevance in myeloid disorders. Blood 104, 1931–1939 (2004).
Buchdunger, E. et al. Abl protein-tyrosine kinase inhibitor STI571 inhibits in vitro signal transduction mediated by c-kit and platelet-derived growth factor receptors. J. Pharmacol. Exp. Ther. 295, 139–145 (2000).
Shankaran, V. et al. IFNγ and lymphocytes prevent primary tumour development and shape tumour immunogenicity. Nature 410, 1107–1111 (2001).
Calo, V. et al. STAT proteins: from normal control of cellular events to tumorigenesis. J. Cell. Physiol. 197, 157–168 (2003).
Townsend, P. A. et al. STAT-1 interacts with p53 to enhance DNA damage-induced apoptosis. J. Biol. Chem. 279, 5811–5820 (2003).
Druker, B. J. STI571 (Gleevec) as a paradigm for cancer therapy. Trends Mol. Med. 8, S14–S18 (2002).
Krutzik, P. O., Irish, J. M., Nolan, G. P. & Perez, O. D. Analysis of protein phosphorylation and cellular signaling events by flow cytometry: techniques and clinical applications. Clin. Immunol. 110, 206–221 (2004).
Chow, S. et al. Whole blood fixation and permeabilization protocol with red blood cell lysis for flow cytometry of intracellular phosphorylated epitopes in leukocyte subpopulations. Cytometry A 67, 4–17 (2005).
Dave, S. S. et al. Prediction of survival in follicular lymphoma based on molecular features of tumor-infiltrating immune cells. N. Engl. J. Med. 351, 2159–2169 (2004).
Orimo, A. et al. Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion. Cell 121, 335–348 (2005).
Podar, K. & Anderson, K. C. The pathophysiological role of VEGF in hematological malignancies: therapeutic implications. Blood 105, 1383–1395 (2004).
Dunn, G. P., Old, L. J. & Schreiber, R. D. The three Es of cancer immunoediting. Annu. Rev. Immunol. 22, 329–360 (2004).
Wozniak, J. & Kopec-Szlezak, J. c-Kit receptor (CD117) expression on myeloblasts and white blood cell counts in acute myeloid leukemia. Cytometry B Clin. Cytom. 58, 9–16 (2004).
Holyoake, T., Jiang, X., Eaves, C. & Eaves, A. Isolation of a highly quiescent subpopulation of primitive leukemic cells in chronic myeloid leukemia. Blood 94, 2056–2064 (1999).
Robillard, N., Pellat-Deceunynck, C. & Bataille, R. Phenotypic characterization of the human myeloma cell growth fraction. Blood 105, 4845–4848 (2005).
Mason, D. et al. CD antigens 2002. Blood 99, 3877–3880 (2002).
O'Brien, M. C. & Bolton, W. E. Comparison of cell viability probes compatible with fixation and permeabilization for combined surface and intracellular staining in flow cytometry. Cytometry 19, 243–255 (1995).
Juan, G. et al. Histone H3 phosphorylation and expression of cyclins A and B1 measured in individual cells during their progression through G2 and mitosis. Cytometry 32, 71–77 (1998).
Erlanson, M. & Landberg, G. Flow cytometric quantification of cyclin E in human cell lines and hematopoietic malignancies. Cytometry 32, 214–222 (1998).
Castillo, R. et al. Proliferative response of mantle cell lymphoma cells stimulated by CD40 ligation and IL-4. Leukemia 14, 292–298 (2000).
Cooperman, J., Neely, R., Teachey, D. T., Grupp, S. & Choi, J. K. Cell division rates of primary human precursor B cells in culture reflect in vivo rates. Stem Cells 22, 1111–1120 (2004).
Laane, E. et al. Flow cytometric immunophenotyping including Bcl-2 detection on fine needle aspirates in the diagnosis of reactive lymphadenopathy and non-Hodgkin's lymphoma. Cytometry B Clin. Cytom. 64, 34–42 (2005).
Morkve, O., Halvorsen, O. J., Stangeland, L., Gulsvik, A. & Laerum, O. D. Quantitation of biological tumor markers (p53, c-myc, Ki-67 and DNA ploidy) by multiparameter flow cytometry in non-small-cell lung cancer. Int. J. Cancer 52, 851–855 (1992).
Andreeff, M., Slater, D. E., Bressler, J. & Furth, M. E. Cellular ras oncogene expression and cell cycle measured by flow cytometry in hematopoietic cell lines. Blood 67, 676–681 (1986).
Zheng, A. et al. p53 status of newly established acute myeloid leukaemia cell lines. Br. J. Cancer 79, 407–415 (1999).
Carney, W. P. et al. Monoclonal antibody specific for an activated RAS protein. Proc. Natl Acad. Sci. USA 83, 7485–7489 (1986).
Ohtani, S. et al. Quantitative analysis of p53-targeted gene expression and visualization of p53 transcriptional activity following intratumoral administration of adenoviral p53 in vivo. Mol. Cancer Ther. 3, 93–100 (2004).
Belloc, F. et al. Flow cytometry detection of caspase 3 activation in preapoptotic leukemic cells. Cytometry 40, 151–160 (2000).
Armstrong, J. S. et al. Role of glutathione depletion and reactive oxygen species generation in apoptotic signaling in a human B lymphoma cell line. Cell Death Differ. 9, 252–263 (2002).
Chow, S. & Hedley, D. Flow cytometric determination of glutathione in clinical samples. Cytometry 21, 68–71 (1995).
Maecker, H. T. & Levy, R. Prevalence of antigen receptor variants in human T cell lines and tumors. J. Immunol. 142, 1395–1404 (1989).
Lee, P. P. et al. Characterization of circulating T cells specific for tumor-associated antigens in melanoma patients. Nature Med. 5, 677–685 (1999).
Trentin, L. et al. Homeostatic chemokines drive migration of malignant B cells in patients with non-Hodgkin lymphomas. Blood 104, 502–508 (2004).
Panoskaltsis, N., Reid, C. D. & Knight, S. C. Quantification and cytokine production of circulating lymphoid and myeloid cells in acute myelogenous leukaemia. Leukemia 17, 716–730 (2003).
Kottaridis, P. D. et al. The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy: analysis of 854 patients from the United Kingdom Medical Research Council AML 10 and 12 trials. Blood 98, 1752–1759 (2001).
Taniguchi, M. et al. Effect of c-kit mutation on prognosis of gastrointestinal stromal tumors. Cancer Res. 59, 4297–4300 (1999).
Davies, H. et al. Mutations of the BRAF gene in human cancer. Nature 417, 949–954 (2002).
Houben, R. et al. Constitutive activation of the Ras–Raf signaling pathway in metastatic melanoma is associated with poor prognosis. J. Carcinog. 3, 6 (2004).
Slebos, R. J. et al. K-ras oncogene activation as a prognostic marker in adenocarcinoma of the lung. N. Engl. J. Med. 323, 561–565 (1990).
Wan, P. T. et al. Mechanism of activation of the RAF–ERK signaling pathway by oncogenic mutations of B-RAF. Cell 116, 855–867 (2004).
Iida, M. et al. Lack of constitutive activation of MAP kinase pathway in human acute myeloid leukemia cells with N-Ras mutation. Leukemia 13, 585–589 (1999).
Lohrisch, C. & Piccart, M. HER2/neu as a predictive factor in breast cancer. Clin. Breast Cancer 2, 129–135 (2001).
Smith, B. L. et al. The efficacy of Herceptin therapies is influenced by the expression of other erbB receptors, their ligands and the activation of downstream signalling proteins. Br. J. Cancer 91, 1190–1194 (2004).
Yakes, F. M. et al. Herceptin-induced inhibition of phosphatidylinositol-3 kinase and Akt Is required for antibody-mediated effects on p27, cyclin D1, and antitumor action. Cancer Res. 62, 4132–4141 (2002).
Zhou, B. P. et al. HER-2/neu blocks tumor necrosis factor-induced apoptosis via the Akt–NF-κB pathway. J. Biol. Chem. 275, 8027–8031 (2000).
Shuai, K., Halpern, J., ten Hoeve, J., Rao, X. & Sawyers, C. L. Constitutive activation of STAT5 by the BCR–ABL oncogene in chronic myelogenous leukemia. Oncogene 13, 247–254 (1996).
Nieborowska-Skorska, M. et al. Signal transducer and activator of transcription (STAT)5 activation by BCR–ABL is dependent on intact Src homology (SH)3 and SH2 domains of BCR–ABL and is required for leukemogenesis. J. Exp. Med. 189, 1229–1242 (1999).
Gorre, M. E. et al. Clinical resistance to STI-571 cancer therapy caused by BCR–ABL gene mutation or amplification. Science 293, 876–880 (2001).
Barthe, C., Cony-Makhoul, P., Melo, J. V. & Mahon, J. R. Roots of clinical resistance to STI-571 cancer therapy. Science 293, 2163 (2001).
Benekli, M. et al. Constitutive activity of signal transducer and activator of transcription 3 protein in acute myeloid leukemia blasts is associated with short disease-free survival. Blood 99, 252–257 (2002).
Sattler, M. & Salgia, R. Targeting c-Kit mutations: basic science to novel therapies. Leuk. Res. 28 (Suppl.), S11–S20 (2004).
Pietras, K., Sjoblom, T., Rubin, K., Heldin, C. H. & Ostman, A. PDGF receptors as cancer drug targets. Cancer Cell 3, 439–443 (2003).
Stirewalt, D. L. & Radich, J. P. The role of FLT3 in haematopoietic malignancies. Nature Rev. Cancer 3, 650–665 (2003).
Küppers, R. Mechanisms of B-cell lymphoma pathogenesis. Nature Rev. Cancer 5, 251–262 (2005).
Mishra, L., Shetty, K., Tang, Y., Stuart, A. & Byers, S. W. The role of TGF-β and Wnt signaling in gastrointestinal stem cells and cancer. Oncogene 24, 5775–5789 (2005).
LeRoith, D. & Helman, L. The new kid on the block(ade) of the IGF-1 receptor. Cancer Cell 5, 201–202 (2004).
Pasca di Magliano, M. & Hebrok, M. Hedgehog signalling in cancer formation and maintenance. Nature Rev. Cancer 3, 903–911 (2003).
Ruiz i Altaba, A., Sanchez, P. & Dahmane, N. Gli and hedgehog in cancer: tumours, embryos and stem cells. Nature Rev. Cancer 2, 361–372 (2002).
Gschwind, A., Fischer, O. M. & Ullrich, A. The discovery of receptor tyrosine kinases: targets for cancer therapy. Nature Rev. Cancer 4, 361–370 (2004).
Harari, D. & Yarden, Y. Molecular mechanisms underlying ErbB2/HER2 action in breast cancer. Oncogene 19, 6102–6114 (2000).
Silva, C. M. Role of STATs as downstream signal transducers in Src family kinase-mediated tumorigenesis. Oncogene 23, 8017–8023 (2004).
Levy, D. E. & Gilliland, D. G. Divergent roles of STAT1 and STAT5 in malignancy as revealed by gene disruptions in mice. Oncogene 19, 2505–2510 (2000).
Yu, H. & Jove, R. The STATs of cancer — new molecular targets come of age. Nature Rev. Cancer 4, 97–105 (2004).
Cichowski, K. & Jacks, T. NF1 tumor suppressor gene function: narrowing the GAP. Cell 104, 593–604 (2001).
Downward, J. Targeting RAS signalling pathways in cancer therapy. Nature Rev. Cancer 3, 11–22 (2003).
Rassenti, L. Z. et al. ZAP-70 compared with immunoglobulin heavy-chain gene mutation status as a predictor of disease progression in chronic lymphocytic leukemia. N. Engl. J. Med. 351, 893–901 (2004).
Pelengaris, S., Khan, M. & Evan, G. c-MYC: more than just a matter of life and death. Nature Rev. Cancer 2, 764–776 (2002).
Vivanco, I. & Sawyers, C. L. The phosphatidylinositol 3-kinase AKT pathway in human cancer. Nature Rev. Cancer 2, 489–501 (2002).
Dougall, W. C. et al. The neu-oncogene: signal transduction pathways, transformation mechanisms and evolving therapies. Oncogene 9, 2109–2123 (1994).
Ashkenazi, A. Targeting death and decoy receptors of the tumour-necrosis factor superfamily. Nature Rev. Cancer 2, 420–430 (2002).
Karin, M. & Greten, F. R. NF-κB: linking inflammation and immunity to cancer development and progression. Nature Rev. Immunol. 5, 749–759 (2005).
Harris, S. L. & Levine, A. J. The p53 pathway: positive and negative feedback loops. Oncogene 24, 2899–2908 (2005).
Cory, S. & Adams, J. M. The Bcl2 family: regulators of the cellular life-or-death switch. Nature Rev. Cancer 2, 647–656 (2002).
Hood, J. D. & Cheresh, D. A. Role of integrins in cell invasion and migration. Nature Rev. Cancer 2, 91–100 (2002).
Reya, T. & Clevers, H. Wnt signalling in stem cells and cancer. Nature 434, 843–850 (2005).
Nelson, W. J. & Nusse, R. Convergence of Wnt, β-catenin, and cadherin pathways. Science 303, 1483–1487 (2004).
Shiloh, Y. ATM and related protein kinases: safeguarding genome integrity. Nature Rev. Cancer 3, 155–168 (2003).
Minamino, T., Miyauchi, H., Tateno, K., Kunieda, T. & Komuro, I. Akt-induced cellular senescence: implication for human disease. Cell Cycle 3, 449–451 (2004).
Acknowledgements
The authors would like to thank R. Ihrie for reviewing the manuscript and many useful discussions.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Competing interests
Garry Nolan is a paid consultant to vendors of reagents and flow cytometry products. He also consults with several pharmaceutical companies in the area of technologies discussed in this review.
Related links
Related links
DATABASES
National Cancer Institute
FURTHER INFORMATION
National Center for Biotechnology Information gene-expression omnibus
Rights and permissions
About this article
Cite this article
Irish, J., Kotecha, N. & Nolan, G. Mapping normal and cancer cell signalling networks: towards single-cell proteomics. Nat Rev Cancer 6, 146–155 (2006). https://doi.org/10.1038/nrc1804
Issue Date:
DOI: https://doi.org/10.1038/nrc1804
This article is cited by
-
Probing low-copy-number proteins in single living cells using single-cell plasmonic immunosandwich assays
Nature Protocols (2021)
-
Electrical properties characterization of single yeast cells by dielectrophoretic motion and electro-rotation
Biomedical Microdevices (2021)
-
Nanoinformatics and biomolecular nanomodeling: a novel move en route for effective cancer treatment
Environmental Science and Pollution Research (2020)
-
Isolation of single motile cells using a high-speed picoliter pipette
Microfluidics and Nanofluidics (2019)
-
Computational algorithms for in silico profiling of activating mutations in cancer
Cellular and Molecular Life Sciences (2019)