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Nanofluidic proteomic assay for serial analysis of oncoprotein activation in clinical specimens

Nature Medicine volume 15, pages 566–571 (2009)Cite this article

Abstract

Current methods of protein detection are insensitive to detecting subtle changes in oncoprotein activation that underlie key cancer signaling processes. The requirement for large numbers of cells precludes serial tumor sampling for assessing a response to therapeutics. Therefore, we have developed a nanofluidic proteomic immunoassay (NIA) to quantify total and low-abundance protein isoforms in nanoliter volumes. Our method can quantify amounts of MYC oncoprotein and B cell lymphoma protein-2 (BCL2) in Burkitt's and follicular lymphoma; identify changes in activation of extracellular signal–related kinases-1 (ERK1) and ERK2, mitogen-activated kinase-1 (MEK), signal transducer and activator of transcription protein-3 (STAT3) and STAT5, c-Jun N-terminal kinase (JNK) and caspase-3 in imatinib-treated chronic myelogeneous leukemia (CML) cells; measure an unanticipated change in the phosphorylation of an ERK2 isomer in individuals with CML who responded to imatinib; and detect a decrease in STAT3 and STAT5 phosphorylation in individuals with lymphoma who were treated with atorvastatin. Therefore, we have described a new and highly sensitive method for determining oncoprotein expression and phosphorylation in clinical specimens for the development of new therapeutics for cancer.

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Figure 1: NIA for the quantitative analysis of oncoproteins.
Figure 2: NIA for the detection of oncoproteins in human cancer specimens.
Figure 3: NIA detection of changes in oncoprotein activation in CML cells treated with imatinib.
Figure 4: NIA detected changes in pERK in individuals with CML who responded to imatinib treatment.
Figure 5: NIA detected decrease in oncoproteins upon treatment with biologic response modifying therapeutic agent.

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Acknowledgements

This manuscript is dedicated to the memory of Roger O'Neill. This work was supported, in part, by the US National Cancer Institute grants CA89305, CA034233, P01 CA034233, NIH/NCI ICMIC P50, Burroughs Welcome Fund, the Damon Runyon Foundation (to D.W.F.), and the Leukemia and Lymphoma Society (to D.W.F. and A.C.F.). We thank the members of the Felsher laboratory for their helpful suggestions. We thank W.-K. Weng (Stanford Bone Marrow Transplantation Division) for providing access to previously banked tumor samples and the Stanford Hematology Division Tissue Bank for the use of samples.

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Authors and Affiliations

  1. Division of Oncology, Departments of Medicine and Pathology, Stanford University, School of Medicine, Stanford, California, USA

    Alice C Fan, Mathias W Orban, Liwen Xu, Amy E Shirer, Shelly Beer, Ada X Yee & Dean W Felsher

  2. Cell Biosciences, Palo Alto, California, USA

    Debabrita Deb-Basu, Roger O'Neill, Daryl Taketa & David W Voehringer

  3. Division of Hematology, Department of Medicine, Stanford University, School of Medicine, Stanford Cancer Center, Stanford, California, USA

    Jason R Gotlib

  4. Department of Pathology, Stanford University, School of Medicine, Stanford, California, USA

    Yasodha Natkunam

  5. Institut Universitaire d'Hematologie, Institut National de la Santé et de la Recherche Médicale U718, Hopital Saint Louis, Paris, France

    Rose-Ann Padua

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Correspondence to Dean W Felsher.

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Competing interests

D.W.F. is a consultant for Cell Biosciences, and D.W.V., D.D.-B., R. O. and D.T. were employees of Cell Biosciences at the time the experiments were performed.

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Fan, A., Deb-Basu, D., Orban, M. et al. Nanofluidic proteomic assay for serial analysis of oncoprotein activation in clinical specimens. Nat Med 15, 566–571 (2009). https://doi.org/10.1038/nm.1903

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