A major scientific challenge is to understand the molecular events that drive the evolution of premalignant lesions in actual tissue. Laser capture microdissection (LCM) was originated to provide a reliable method to procure pure populations of cells from specific microscopic regions of tissue sections; in one step, under direct visualization. The cells of interest are transferred to a polymer film that is activated by laser pulses. The exact morphology of the procured cells (with intact DNA, RNA and proteins) is retained and held on the transfer film. LCM technology has been successfully applied to DNA, and RNA analysis from frozen and fixed embedded tissue. In the past it has not been possible to extract, quantify and characterize the functional state of specific proteins expressed by individual subpopulations of cells in actual tissue. Consequently, an important ongoing and future goal is to extend our microdissection technology to include the molecular profiling of cancer progression into the realm of quantitative proteomics: characterization of known proteins, as well as discovery of new proteins associated with progression. Quantitative protein analysis using 3rd generation immunoassays, has been successfully performed on stained tissue cells selected and LCM microdissected under direct microscopic visualization. The sensitivity is 5 cells. Proteins from LCM microdissected normal prostate epithelium, and invasive carcinoma, normal esophageal squamous epithelium, and squamous carcinoma, have been extracted and subjected to 2-D gel electrophoresis. Approximately 800 spots could be discriminated by silver staining using a starting material of 7500 laser shots. Highly reproduceable protein differences were identified in carcinoma versus normal epithelium. LCM was also applied to high throughput screening of microdissected protein profiles using solid phase protein bait binding arrays. Proteins which bind to the bait can be detected using the Ciphergen SELDI time of flight mass mapping. A mass mapping spectrum of protein peaks was successfully produced with a sensitivity of 20 cells. Specific differences in protein patterns (reproduced between and among different patients) could be detected between microdissected normal, PIN, and invasive carcinoma. We envision the ultimate protein assay chip which reads out the functional state of several or all proteins in signal transduction pathway. This will be accomplished using a microarray of protein bait capture ligands which recognize different functional states of specific pathway members (e.g. phosphorylation, cleaved, activated, bound to partner proteins, or unoccupied binding sites for protein partners or DNA regulatory domains).

Agency
National Institute of Health (NIH)
Institute
Division of Clinical Sciences - NCI (NCI)
Type
Intramural Research (Z01)
Project #
1Z01SC009185-13
Application #
6756871
Study Section
(LP)
Project Start
Project End
Budget Start
Budget End
Support Year
13
Fiscal Year
2002
Total Cost
Indirect Cost
Name
Clinical Sciences
Department
Type
DUNS #
City
State
Country
United States
Zip Code
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