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J Proteome Res. 2009 Nov;8(11):5048-59. doi: 10.1021/pr9004635.

Scanning the cell surface proteome of cancer cells and identification of metastasis-associated proteins using a subtractive immunization strategy.

Author information

1
Medical Biotechnology Center, Institute of Medical Biology, University of Southern Denmark, J. B. Winsloewsvej 25, DK-5000 Odense C, Denmark.

Abstract

Identification of the cell surface proteome and comparison of their expression between cells with different phenotypic characteristics is crucial to the discovery of novel cancer drug targets as well as elucidating the basic biologic processes of cancer. However, cell surface proteomics are complex and technologically challenging, and no ideal method is currently available. Here, we describe a strategy that allows scanning of the entire cell surface and identification of molecules that exhibit altered expression between two cell types. Concurrently, this method gives rise to valuable reagents for further characterization of the identified proteins. The strategy is based on subtractive immunization of mice, and we used the two isogenic cell lines, NM-2C5 and M-4A4, derived from the MDA-MB-435 cancer cell line, as a model system. Although the two cell lines are equally tumorigenic, only M-4A4 has metastatic capabilities. Our results yielded a large panel of monoclonal antibodies (mAbs) that recognized cell surface markers preferentially or exclusively expressed on metastatic vs nonmetastatic cancer cells. Four mAbs and their corresponding antigens were further characterized. Importantly, analysis on an extended panel of breast cancer cell lines demonstrated that the four mAbs bound preferentially to cell lines known to be metastatic in vivo, suggesting that these markers have general applications. Immunohistochemical analysis showed that mAb 11E6 reacted preferentially with neuroendocrine tumors while exhibiting no or very weak reactivity with normal tissues. mAb 15C7 stained a variety of cancers as well as some normal lymphoid organs and was subsequently identified to react with HLA-DR-beta. A third mAb, 31D7, that also specifically recognized HLA-DR-beta was capable of inhibiting the growth of MZ2 melanoma cells in vitro. Further, we found that the reduced expression of HLA-DR antigens in nonmetastatic cells of this model was not regulated by class II transactivator, but by posttranscriptional mechanisms. The study demonstrates the advantage of using the exquisitely discriminating recognition system of the immune system itself to scan the cell surface proteome for differentially expressed proteins. The subtractive immunization strategy should be broadly applicable as a quantitative and comparative proteomic approach for screening the cell surface and also allow generation of mAbs to study these cell surface antigens in more detail.

PMID:
19764822
DOI:
10.1021/pr9004635
[Indexed for MEDLINE]

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