Development of an all-in-one technology for glycan profiling targeting formalin-embedded tissue sections

https://doi.org/10.1016/j.bbrc.2008.03.090Get rights and content

Abstract

An ultra-sensitive method for glycan analysis targeting small tissue sections (1.5 mm in diameter) is described as an application of a recently-established lectin microarray technology. The developed system achieved a high level of detection of a tissue section consisting of approximately 500 cells for differential profiling, where both N- and O-glycans attached to a pool of glycoproteins are subjected to multiplex analysis with 43 lectins. By using an optimized protocol for differential glycan analysis, sections of adenocarcinoma (n = 28) and normal epithelia (n = 12) of the colon were analyzed in an all-in-one manner. As a result, Wisteria floribunda agglutinin (WFA) was found to clearly differentiate cancerous from normal epithelia with P < 0.0001. The obtained results correlated well with the subsequent histochemical study using biotinylated WFA. Thus, the developed technology proved to be valid for expanding the lectin microarray applications to tissue-based glycomics, and hence, should accelerate a discovery phase of glycan-related biomarkers.

Section snippets

Materials and methods

Protein extraction from tissue sections. Formalin-fixed paraffine-embedded tissue microarrays (each section with 1.5 mm diameter and 5 μm thickness) were purchased from Cybrdi, Inc. (Frederick, MD). They consisted of brain glioblastoma (21 cases of individual patients, each in triplicate), colon adenocarcinoma (grade III; 23 cases of individual patients, each in triplicate), colon adenocarcinoma (grades I + II; 21 cases of individual patients, each in triplicate; for the term “grade”, see ref. [16]

Construction of a method for multiplex glycan analysis targeting one-dot tissue sections

Since alteration of glycans is often associated with tumor progression, searching disease-associated glyco-markers to visualize changes in glycan structure using tissue specimens should provide useful information. A multiplex glycan analysis, targeting a relatively homogeneous area of a tissue section comprising heterogeneous carcinoma cells by means of lectins and antibodies, is a methodology with the potential to allow discovery of useful glyco-biomarkers. Although a lectin microarray

Acknowledgments

We thank N. Uchiyama, Y. Kubo, J. Murakami, and S. Koizumi for technical assistance. We also thank Y. Ikehara, T. Sato, and Y. Itakura for helpful discussion. This study was supported by New Energy and Industrial Technology Development Organization (NEDO) in Japan.

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