Laser-capture microdissection in prostate cancer research: establishment and validation of a powerful tool for the assessment of tumour-stroma interactions.
OBJECTIVE
To describe our experience with the optimization and validation of laser-capture microdissection (LCM) for biomarker analysis in prostate tissues. As LCM allows the separation of benign and malignant epithelial structures and stromal elements, it not only allows identification of the source of the biomarker, but might also accentuate gene or protein expression changes by reducing contamination by other cellular elements.


METHODS
In all, 19 fresh-frozen prostate tissue samples were subjected to LCM, with the cDNA being analysed using quantitative polymerase chain reaction for several genes, to identify the optimum number of cells for capture, as well as gene markers assessing for the purity of the captured cells. The localization was further confirmed by in situ hybridization.


RESULTS
Prostate-specific antigen (PSA) and cytokeratin 8, were expressed solely by epithelial cells, whereas hepatocyte growth factor (HGF) and tissue inhibitor of metalloproteinases-3 (TIMP3) were expressed only by stromal cells, and the levels of transcripts of these genes were unaltered between benign and malignant tissues.


CONCLUSIONS
These data suggest that PSA, cytokeratin 8, HGF and TIMP3 are reliable gene markers of purity of epithelial and stromal compartments for LCM of prostate tumours. Although this technique is not new and is increasingly used in laboratories, it needs optimization and stringent validation criteria before data analysis. This applies to all tissue types subjected to LCM.
Shukla
Chitranjan J
CJ
Norfolk and Norwich University Hospital NHS Trust and School of Biological Sciences, University of East Anglia, Norwich, UK.
Pennington
Caroline J
CJ
Riddick
Antony C P
AC
Sethia
Krishna K
KK
Ball
Richard Y
RY
Edwards
Dylan R W
DR
D004847:Epithelial Cells / Q000473:pathology; D015870:Gene Expression; D017228:Hepatocyte Growth Factor / Q000378:metabolism; D006801:Humans; D017403:In Situ Hybridization; D053533:Keratin-8 / Q000378:metabolism; D007834:Lasers; D008297:Male; D042282:Microdissection / Q000379:methods* / Q000592:standards; D011467:Prostate / Q000473:pathology*; D017430:Prostate-Specific Antigen / Q000378:metabolism; D011471:Prostatic Neoplasms / Q000473:pathology*; D012334:RNA, Neoplasm / Q000032:analysis*; D017154:Stromal Cells / Q000473:pathology; D019717:Tissue Inhibitor of Metalloproteinase-3 / Q000378:metabolism
D053533:Keratin-8; D012334:RNA, Neoplasm; D019717:Tissue Inhibitor of Metalloproteinase-3; D017228:Hepatocyte Growth Factor; D017430:Prostate-Specific Antigen