Enrichment of epithelial cells for molecular studies

Accurate molecular analysis of tumors and their precursor lesions requires the extraction of specific subpopulations of cells (normal, preneoplastic and tumor) from a composite background of multiple cell types. Attempts to obtain pure tumor cell samples have resulted in the genesis of several methods of cell enrichment, including the use of tumor cell lines, xenografted tumors, and microdissection of frozen or paraffin-embedded tissues. All methods now used have advantages and limitations. The first two methods provide self-replicating sources of high-quality reagents but are difficult, expensive and time consuming and the results may not be representative of the parent tumor. The development of laser capture microdissection (LCM) has revolutionized microdissection. However, this technique is limited by the poor quality of nucleic acids and proteins from archival samples and by the drawbacks of working with frozen sections. To enhance the potential of LCM and to overcome some of the limitations of other methods, we have developed a rapid, simple procedure for enrichment of normal and tumor cells called epithelial aggregate separation and isolation (EASI). Smears of tumor tissues are prepared on glass slides, rapidly fixed in methanol and stained. EASI preps yield nearly pure aggregates of epithelial cell populations (malignant, normal and preneoplastic), which are identified by microscopic examination. LCM is then done and the intermediate-quality nucleic acids thus obtained are suitable for many DNA and RNA assays, including allelotyping, mutation analysis, automatic sequencing, RT-PCR and library construction. The EASI method is applicable to most normal, preneoplastic and malignant epithelial tissues, requires minimal expertise and can be conveniently integrated into standard surgical pathology practice. For tumor or epithelial cell enrichment, the method of choice should be based on its relative advantages and limitations, as well as on the available facilities.