Laser capture microdissection

Abstract: Deciphering the cellular and molecular interactions that drive disease within the tissue microenvironment holds promise for discovering drug targets of the future. In order to recapitulate the in vivo interactions thorough molecular analysis, one must be able to analyze specific cell populations within the context of their heterogeneous tissue microecology. Laser-capture microdissection (LCM) is a method to procure subpopulations of tissue cells under direct microscopic visualization. LCM technology can harvest the cells of interest directly or can isolate specific cells by cutting away unwanted cells to give histologically pure enriched cell populations. A variety of downstream applications exist: DNA genotyping and loss-of-heterozygosity (LOH) analysis, RNA transcript profiling, cDNA library generation, proteomics discovery and signal-pathway profiling. Herein we provide a thorough description of LCM techniques, with an emphasis on tips and troubleshooting advice derived from LCM users. The total time required to carry out this protocol is typically 1-1.5 h.

Abstract: We have developed a new assay, ISET (isolation by size of epithelial tumor cells), which allows the counting and the immunomorphological and molecular characterization of circulating tumor cells in patients with carcinoma, using peripheral blood sample volumes as small as 1 ml. Using this assay, epithelial tumor cells can be isolated individually by filtration because of their larger size when compared to peripheral blood leukocytes. ISET parameters were defined using peripheral blood spiked with tumor cell lines (HepG2, Hep3B, MCF-7, HeLa, and LNCaP). ISET can detect a single, micropipetted tumor cell, added to 1 ml of blood. We also demonstrate that fluorescence in situ hybridization can be used to perform chromosomal analyses on tumor cells collected using ISET. Polymerase chain reaction-based genetic analyses can be applied to ISET-isolated cells, and, as an example, we demonstrate homozygous p53 deletion in single Hep3B cells after filtration and laser microdissection. Finally, we provide evidence for the in vivo feasibility of ISET in patients with hepatocellular carcinoma undergoing tumor resection. ISET, but not reverse transcriptase-polymerase chain reaction, allowed analysis of cell morphology, counting of tumor cells, and demonstration of tumor microemboli spread into peripheral blood during surgery. Overall, ISET constitutes a novel approach that should open new perpectives in molecular medicine.

Abstract: Using the URL at the end of this item, readers can immediately offer feedback and suggestions on this topic. A s the list of expressed human genes expands, a major scientific and medical challenge is to understand the molecular events that drive normal tissue morphogenesis and the progression of

Abstract: Laser capture microdissection (LCM) is a technique for isolating pure cell populations from a heterogeneous tissue section or cytological preparation via direct visualization of the cells. This technique is applicable to molecular profiling of diseased and disease-free tissue, permitting correlation of cellular molecular signatures with specific cell populations. DNA, RNA, or protein analysis can be performed with the microdissected tissue by any method with adequate sensitivity. The principle components of LCM technology are (1) visualization of the cells of interest via microscopy, (2) transfer of laser energy to a thermolabile polymer with formation of a polymer-cell composite, and (3) removal of the cells of interest from the heterogeneous tissue section. LCM is compatible with a variety of tissue types, cellular staining methods, and tissue-preservation protocols that allow microdissection of fresh or archival specimens. LCM platforms are available as a manual system (PixCell; Arcturus Bioscience) or as an automated system (AutoPix).