The Goal

In the CGAP initiative we seek to understand the molecular forces driving the evolution of specific normal epithelial cells to become premalignant populations and then further transform into invasive and metastatic cancer. A major approach is to sample the normal appearing epithelium, the premalignant cells, and the frank invasive carcinoma all from the same tissue sample, from one patient. In this way we can compare the fluctuation of expressed genes or alterations in the cellular DNA that correlate with the transition from one disease stage to the next. In order to accomplish this goal, a means is required to sample pure cells in different stages of cancer progression without contamination by the wrong cells.

Microdissection

Microdissection is a method for procuring pure cells from specific microscopic regions of tissue sections. Under the microscope, tissues are heterogeneous complicated structures with hundreds of different cell types locked in morphologic units in dense adhesive interactions with adjacent cells, connective stroma, blood vessels, glandular and muscle components, adipose cells, and inflammatory or immune cells. The diseased cells of interest, such as precancerous cells or invading groups of cancer cells, are surrounded by these heterogeneous tissue elements. Epithelial cells, precancerous cells or even invading cancer cells may constitute less than 5% of the volume of the tissue biopsy sample. Therefore, microdissection is essential to apply molecular analysis methods to study evolving disease lesions in actual tissue. Two types of cDNA libraries are being generated for the CGAP: microdissected libraries and bulk tissue libraries. The microdissected libraries are designed to approximate the true pattern of gene expression of the pure cell subpopulations in their actual tissue context.

Laser Capture Microdissection (LCM)

Laser capture microdissection (LCM) is a new technology originated by NIH (Emmert-Buck et. al., Science 274,998-1001,1996), and commercially developed through a Collaborative Research and Development Agreement (CRADA) partnership with Arcturus Engineering Inc. (650-962-3020). LCM was developed to automate and standardize microdissection and enable any researcher or pathologist to conduct microdissection under a microscope with the push of a button.

The Principle of LCM

The principle of LCM is very simple. A laser beam and special transfer film are used to lift the desired cells out of the tissue section, leaving all of the contaminating or unwanted cells behind. The transparent transfer film is applied to the surface of the tissue section. Under the microscope, the diagnostic pathologist or researcher views the tissue through the film and chooses microscopic clusters of cells to study. When the cells of choice are in the center of the field of view, the operator pushes a button that activates a laser diode integral with the microscope optics. The pulsed laser beam activates a precise spot on the transfer film immediately above the cells of interest. At this precise location the film melts and fuses with the underlying cells of choice. When the film is removed, the chosen cell(s) remain stuck to its undersurface, while the rest of the tissue is left behind.

Arcturus Engineering Inc. has simplified the process of handling the transfer film. In the Arcturus system the film is bonded to the underside of a vial cap. The cap is set on the surface of the tissue and the operator views the tissue through the transparent cap and pulses the laser. The size of the laser pulse diameter can be focused by the operator. Two suggested settings are 30 and 60 micron diameters. Selected cells are transferred to the undersurface of the cap which is lifted off the tissue and placed directly onto a vial for molecular processing. The cells adherent to the film retain their morphologic features, and the operator can verify that the correct cells have been procured. NIH has developed computer software to store images of the microdissected cells before and after LCM, thus serving as a diagnostic record.

LCM in Practice

Example transfers verify the precision and accuracy of LCM. Below is the microscopic field of tissue with rows of vacancies left by successive laser shots 30 microns in diameter. On the right are the spots of cells transferred to the undersurface of the film. The laser transfer spots can be overlapped to capture complicated microstructures such as neoplastic clusters or an elongated breast duct embedded in dense connective tissue.

LCM can be applied to the procurement of DNA, RNA or protein from selected pure cells. The capture process does not damage these macromolecules because the laser energy is absorbed by the film. The starting tissue can be fixed (formalin or ethanol), embedded in paraffin, or frozen. The tissue section can be stained with a variety of standard stains in order to highlight the cell population of interest.