Application of laser capture microdissection and proteomics in colon cancer

Abstract

Aims-Laser capture microdissection is a recent development that enables the isolation of specific cell types for subsequent molecular analysis. This study describes a method for obtaining proteome information from laser capture microdissected tissue using colon cancer as a model. Methods-Laser capture microdissection was performed on toluidine blue stained frozen sections of colon cancer. Tumour cells were selectively microdissected. Conditions were established for solubilising proteins from laser microdissected samples and these proteins were separated by two dimensional gel electrophoresis. Individual protein spots were cut from the gel, characterised by mass spectrometry, and identified by database searching. These results were compared with protein expression patterns and mass spectroscopic data obtained from bulk tumour samples run in parallel. Results-Proteins could be recovered from laser capture microdissected tissue in a form suitable for two dimensional gel electrophoresis. The solubilised proteins retained their expected electrophoretic mobility in two dimensional gels as compared with bulk samples, and mass spectrometric analysis was also unaVected. Conclusion-A method for performing two dimensional gel electrophoresis and mass spectrometry using laser capture microdissected tissue has been developed. (J Clin Pathol: Mol Pathol 2001;54:253-258)

Low expression of chloride channel accessory 1 predicts a poor prognosis in colorectal cancer: The question is still open

“…[3][4][5] Bonferroni correction is a method with which to counteract the problem of multiple comparisons. We applied the Bonferroni critical value (P <.0017) to examine the expression level of CLCA1 and CRC status in patients.…”

“…Because of the tumor heterogeneity within tissues, the enrichment of CRC cells from tissue biopsies, even within the same clinical subgroup of patients, using laser microdissection would help to isolate a relatively pure population and decrease false-positive, tumor cell-specific signals. 5,6 Moreover, the evaluation of tumor heterogeneity and possible evolution of cancer cells after disease recurrence should be documented within multiple samples of a single tumor and repeated biopsies performed to decrease the risk of avoidable mistakes. [5][6][7] In addition, immunohistochemistry is a sensitive and empirical method and outcomes depend on a pathologist's visual expertise and the antibody used.…”

“…5,6 Moreover, the evaluation of tumor heterogeneity and possible evolution of cancer cells after disease recurrence should be documented within multiple samples of a single tumor and repeated biopsies performed to decrease the risk of avoidable mistakes. [5][6][7] In addition, immunohistochemistry is a sensitive and empirical method and outcomes depend on a pathologist's visual expertise and the antibody used. Thus, validation of the results of the study by Yang et al 1 and reevaluation of the slides by at least 2 independent pathologists may decrease the risk of error, 7 although it is beneficial to know whether those who performed the data analysis and interpretation were also blinded to the experimental part.…”

Proteomics in pathology research

“…Thus, protein extraction protocols entail; the homogenization of cells and tissues followed by application of detergents such as 3-(dimethylammonio])-1 propane sulfate (CHAPS), 7 Tween and sodium dodecyl sulfate (SDS), which help to dissolve the proteins and separate them from the lipid components, reducing agents such as dithiothreitol (DTT); denaturing agents such as urea which disrupt the bonds that are responsible for the formation of secondary and tertiary conformational structure; and enzymes which degrade nucleic acids such as DNAses and RNAses. Tissues obtained from laser capture microdissection of human colon cancer 8 and hepatocellular carcinoma have been successfully used for 2D gel electrophoresis followed by MALDI-TOF mass spectrometry analysis and 2D liquid chromatography tandem mass spectrometry. 9 Separation of complex proteins into simpler components Several techniques are utilized for the analytical separation of proteins.…”

Intact‐protein based sample preparation strategies for proteome analysis in combination with mass spectrometry

“…The results showed that half of the observed peaks in the mass range between 1,000 and 4,000 Da were indicative of either adenocarcinoma or squamous cell carcinoma, and may be used as a fingerprint for those cancer types. Lawrie et al (2001) used LCM to selectively microdissect tumor cells from colon cancer tissues. The protein mixtures recovered from the captured cells were separated and characterized by 2D PAGE/Bio-MS. A method that utilized an adjacent stained section to guide the dissection of an unstained region of interest was used to overcome problems that resulted from an immuno-histochemical marking of the dissected cells; that staining has detrimental effects on protein analysis by 2D PAGE (Wong et al, 2000).…”