We thank staff at the Anatomical Pathology Laboratories of the Royal Prince Alfred and Concord Repatriation Hospitals for collecting fresh samples of CRC and normal intestinal mucosa. The work was funded by a Cancer Institute New South Wales Translational Program Grant.

<img src="/files/ftp_upload/3322/3322fig1.jpg" alt="Figure 1" /> 
Figure 1. Work flow for preparation of a suspension of live cells from a surgical sample of CRC.
1. Clinical sample disaggregation 
All samples were collected from the Royal Prince Alfred Hospital (Camperdown, NSW, Australia) and Concord Repatriation Hospital (Concord West, NSW, Australia) with informed consent under Protocol No. X08-164. 
<ol>
 <li>Collect fresh colorectal can...

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	<li>Steinert, R., Buschmann, T., vander Linden, M., Fels, L. M., Lippert, H., Reymond, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+proteomics+in+the+diagnosis+and+outcome+prediction+in+colorectal+cancer.">The role of proteomics in the diagnosis and outcome prediction in colorectal cancer.</a> Technol. Cancer. Res. Treat. 1, 297 (2002).</li><li>Eifel, P., Axelson, J. A., Costa, J., Crowley, J., Curran, W. J., Deshler, A., Fulton, S., Hendricks, C. B., Kemeny, M., Kornblith, A. B., Louis, T. A., Markman, M., Mayer, R., Roter, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=National+Institutes+of+Health+Consensus+Development+Conference+Statement:+adjuvant+therapy+for+breast+cancer.">National Institutes of Health Consensus Development Conference Statement: adjuvant therapy for breast cancer.</a> J. Natl. Canc. Inst. 93, 979 (2001).</li><li>Swerdlow, S. H., Campo, E., Harris, H. L., Jaffe, E. S., Pileri, S. A., Stein, H., Thiele, J., Vardiman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=WHO+classification+of+tumour+of+haematopoietic+and+lymphoid+tissues.">WHO classification of tumour of haematopoietic and lymphoid tissues.</a> IARC WHO Classification of Tumours. 2, (2008).</li><li>Xiao, G. G., Recker, R. R., Deng, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+proteomics+and+cancer+biomarker+discovery.">Recent advances in proteomics and cancer biomarker discovery.</a> Clin. Med. Oncol. , (2008).</li><li>Belov, L., Mulligan, S. P., Barber, N., Woolfson, A., Scott, M., Stoner, K., Chrisp, J. S., Sewell, W. A., Bradstock, K. F., Bandall, L., Pascovici, D. S., Thomas, M., Erber, W., Huang, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+human+leukaemias+and+lymphomas+using+extensive+immunophenotypes+from+an+antibody+microarray.">Analysis of human leukaemias and lymphomas using extensive immunophenotypes from an antibody microarray.</a> Br. J. Haematol. 135, 184 (2006).</li><li>Belov, L., Huang, P., Barber, N., Mulligan, S. P., Christopherson, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+repertories+of+surface+antigens+on+leukemias+using+an+antibody+microarray.">Identification of repertories of surface antigens on leukemias using an antibody microarray.</a> Proteomics. 3, 2147 (2003).</li><li>Zhou, J., Belov, L., Huang, P. Y., Shin, J., Solomon, M. J., Chapuis, P. H., Bokey, L., Chan, C., Clarke, C., Clarke, S. J., Christopherson, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+antigen+profiling+of+colorectal+cancer+using+antibody+microarrays+with+fluorescence+multiplexing.">Surface antigen profiling of colorectal cancer using antibody microarrays with fluorescence multiplexing.</a> J. Immunol. Methods. 355 (1-2), 40-51 (2010).</li><li>Ellmark, P., Belov, L., Huang, P., Lee, C. S., Solomon, M. J., Morgan, D. K., Christopherson, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+detection+of+surface+molecules+on+colorectal+cancers.">Multiplex detection of surface molecules on colorectal cancers.</a> Proteomics. 6, 1791 (2006).</li><li>Pearson, J. P., Allen, A., Hutton, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rheology+of+mucin.">Rheology of mucin.</a> Methods Mol. Biol. 125, 99 (2000).</li><li>Yang, Y. H., Dudoit, S., Luu, P., Lin, D. M., Peng, V., Ngai, J., Speed, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normalization+for+cDNA+microarray+data:+a+robust+composite+method+addressing+single+and+multiple+slide+systematic+variation.">Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation.</a> Nucleic Acids Res. 30, 15 (2002).</li><li>Al Saeed, ., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TMA:+A+free,+open-source+system+for+microarray+data+management+and+analysis.">TMA: A free, open-source system for microarray data management and analysis.</a> BioTechniques. 34, 374-378 (2003).</li></ol>

No conflicts of interest declared.

In this video, we demonstrate how the DotScan antibody microarray can be used in a simple, semi-quantitative way to study surface antigen profiles for cell populations from CRC tissue.
Obtaining a viable single cell suspension from tissue is critical to the success of the experiment, because energy-dependent processes (eg., antigen capping and/or pseudopodia formation) appear to be required for firm binding of whole cells to antibody dots during incubation, while dead cells are subsequently wa...

The author's email has been corrected in the publication of <a href="http://www.jove.com/video/3322">Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing</a>. There was an error with the author, Jerry Zhou's, email. The author's email has been updated to:
j.zhou@uws.edu.au
from:
jzho7551@mail.usyd.edu.au

Erratum: Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing

<table border="1">
 <tbody>
 <tr>
 <th>Name of reagent or equipment</th>
 <th>Company</th>
 <th>Catalogue number</th>
 <th>Comments</th>
 </tr>
 <tr>
 <td>Hanks' balanced salt solution</td>
 <td>Sigma-Aldrich</td>
 <td>H6136-10X1L</td>
 <td>Buffered with 25 mM Hepes (Sigma #H3375)</td>
 </tr>
 <tr>
 <td>Airpure biological safety cabinet class II</td>
 <td>Westinghouse</td>
 <td>1687-2340/612</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Surgical blades</td>
 <td>Livingstone</td>
 <td>090609</td>
 <td>Pack of 100</td>
 </tr>
 <tr>
 <td>RPMI 1640 with 2 mM Hepes</td>
 <td>Sigma-Aldrich</td>
 <td>R4130-10X1L</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Collagenase type 4</td>
 <td>Worthington</td>
 <td>4188</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Deoxyribonuclease 1</td>
 <td>Sigma-Aldrich</td>
 <td>DN25-1G</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Terumo Syringe (10 mL)</td>
 <td>Terumo</td>
 <td>SS+10L</td>
 <td>Box of 100</td>
 </tr>
 <tr>
 <td>Filcon filter (200 &mu;m)</td>
 <td>BD Biosciences</td>
 <td>340615</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Filcon filter (50 &mu;m)</td>
 <td>Filcon filter (50 &mu;m)</td>
 <td>Filcon filter (50 &mu;m) Filcon filter (50 &mu;m) 340603 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Fetal calf serum</td>
 <td>Gibco/Invitrogen</td>
 <td>10099-141</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Centrifuge 5810 R</td>
 <td>Eppendorf</td>
 <td>7017</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Dimethyl sulphoxide</td>
 <td>Sigma-Aldrich</td>
 <td>D2650</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Trypan blue</td>
 <td>Sigma-Aldrich</td>
 <td>T8154</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Hemocymeter Technocolor Neubar</td>
 <td>Hirschmann</td>
 <td>not available</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Light microscope</td>
 <td>Nikon</td>
 <td>Nikon TMS</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Cyrovial tubes</td>
 <td>Greiner bio-one</td>
 <td>121278</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Cryo freezing contrainer</td>
 <td>Nalgene</td>
 <td>5100-0001</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>DotScan antibody microarray kit</td>
 <td>Medsaic</td>
 <td>not available</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>DotScan microarray wash tray</td>
 <td>Medsaic</td>
 <td>not available</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>KimWipes</td>
 <td>Kimberly-Clark</td>
 <td>4103</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Formaldehyde 37%</td>
 <td>Sigma-Aldrich</td>
 <td>F1635-500ML</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>DotReaderTM</td>
 <td>Medsaic</td>
 <td>not available</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Bovine serum albumin</td>
 <td>Sigma-Aldrich</td>
 <td>A9418-10G</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Heat-inactivated AB serum 2%</td>
 <td>Invitrogen</td>
 <td>34005100</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Phycoerythrin-conjugated CD3</td>
 <td>Beckman Coulter</td>
 <td>ET386</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>AlexaFluor647-conjugated EpCAM</td>
 <td>BioLegend</td>
 <td>324212</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Typhoon FLA 9000</td>
 <td>GE Healthcare</td>
 <td>28-9558-08</td>
 <td>532 nm laser, 580 BP30 emission filter for PE. 633 nm laser and 670 BP30 emission filter for Alexa647</td>
 </tr>
 <tr>
 <td>MultiExperiment Viewer v4.4</td>
 <td>TM4 Microarray Software Suite</td>
 <td>Open &#x2013; source software (Ref 11)</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

colorectal cancer cell surface protein profiling using an antibody microarray and fluorescence multiplexing

The current prognosis and classification of CRC relies on staging systems that integrate histopathologic and clinical findings. However, in the majority of CRC cases, cell dysfunction is the result of numerous mutations that modify protein expression and post-translational modification1.
A number of cell surface antigens, including cluster of differentiation (CD) antigens, have been identified as potential prognostic or metastatic biomarkers in CRC. These antigens make ideal biomarkers as their expression often changes with tumour progression or interactions with other cell types, such as tumour-infiltrating lymphocytes (TILs) and tumour-associated macrophages (TAMs).
The use of immunohistochemistry (IHC) for cancer sub-classification and prognostication is well established for some tumour types2,3. However, no single &#x2018;marker&#x2019; has shown prognostic significance greater than clinico-pathological staging or gained wide acceptance for use in routine pathology reporting of all CRC cases. 
A more recent approach to prognostic stratification of disease phenotypes relies on surface protein profiles using multiple 'markers'. While expression profiling of tumours using proteomic techniques such as iTRAQ is a powerful tool for the discovery of biomarkers4, it is not optimal for routine use in diagnostic laboratories and cannot distinguish different cell types in a mixed population. In addition, large amounts of tumour tissue are required for the profiling of purified plasma membrane glycoproteins by these methods. 
In this video we described a simple method for surface proteome profiling of viable cells from disaggregated CRC samples using a DotScan CRC antibody microarray. The 122-antibody microarray consists of a standard 82-antibody region recognizing a range of lineage-specific leukocyte markers, adhesion molecules, receptors and markers of inflammation and immune response5, together with a satellite region for detection of 40 potentially prognostic markers for CRC. Cells are captured only on antibodies for which they express the corresponding antigen. The cell density per dot, determined by optical scanning, reflects the proportion of cells expressing that antigen, the level of expression of the antigen and affinity of the antibody6. 
For CRC tissue or normal intestinal mucosa, optical scans reflect the immunophenotype of mixed populations of cells. Fluorescence multiplexing can then be used to profile selected sub-populations of cells of interest captured on the array. For example, Alexa 647-anti-epithelial cell adhesion molecule (EpCAM; CD326), is a pan-epithelial differentiation antigen that was used to detect CRC cells and also epithelial cells of normal intestinal mucosa, while Phycoerythrin-anti-CD3, was used to detect infiltrating T-cells7. The DotScan CRC microarray should be the prototype for a diagnostic alternative to the anatomically-based CRC staging system.

Watch this Scientific Journal Video about Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing at JoVE.com

Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing

We described a procedure for the disaggregation of colorectal cancer (CRC) to produce viable single cells, which are then captured on customized antibody microarrays recognizing surface antigens (DotScan CRC microarray). Sub-populations of cells bound to the microarray can be profiled by fluorescence multiplexing using monoclonal antibodies tagged with fluorescent dyes.

The current prognosis and classification of CRC relies on staging systems that integrate histopathologic and clinical findings. However, in the majority ...

colorectal-cancer-cell-surface-protein-profiling-using-an-antibody

Research

JoVE Journal

Immunology and Infection

13.9K Görüntüleme.  University of Sydney. Kolorektal kanser (CRC) uygulanabilir tek bir hücre üretmek için, daha sonra yüzey antijenleri (DotScan ÇHS mikroarray) tanıyan özelleştirilmiş antikor mikrodizinlerinde yakalanır ayrıştırılmasıyla için bir prosedür nitelendirdi. Mikroarray bağlı hücrelerin alt popülasyonlarda floresan boya ile monoklonal antikorlar kullanılarak floresans çoklama profilli olabilir.