We propose to use flow cytometry techniques developed in this laboratory and elsewhere to quantitate changes in nucleic acid content, chromatin structure and protein of intact nuclei and whole cells from colonic epithelium of normal mucosa, inflammatory and neoplastic polyps, and primary and metastatic adenocarcinoma. Deviations from normal in DNA content, known to occur in colon carcinoma, and deviations in the other features measured as well as calculated values such as cell cycle distribution will be correlated with conventional histophathology and clinical course to trace the sequence of changes during carcinogenesis of the colon, and to identify any features of prognostic importance. Needle aspiration or other biopsy samples of tumor also will be examined before and during chemotherapy, and the drug effect on cell viability and cell cycle kinetics correlated with clinical responses. Modifications of sampling and cell preparatory techniques will be evaluated, making use of the known mechanical enzymatic and ionic methods for dissociation of epithelial cells, to select the best combination of methods for producing suspensions of intact, whole cells from colonic epithelium. This will facilitate measurement and correlative studies, as above, of cytoplasmic organelles such as mitrochondria (using rhodamine 123), total cellular vs. nuclear RNA and protein, and cell classification by monoclonal antibodies as they become available for cellular antigens and particularly for the tumor associated antigens. Improvements in our interactive computer data analysis program will be implemented to deal with the multiparameter data from large numbers of cells of mixed populations in each sample. These multiparameter flow cytometry analyses of colonic cell suspensions are expected to yield biochemical and functional information about the cells that cannot be obtained in any other way. They provide a powerful new tool for quantitative examinations of colonic tumors that will be of value for prognosis and for monitoring drug treatment effect, and will also yield basic information on changes in DNA, cell cycle kinetics, RNA (transcription) and protein (translation) during carcinogenesis.
| Stephenson, R A; Gay, H; Fair, W R et al. (1986) Effect of section thickness on quality of flow cytometric DNA content determinations in paraffin-embedded tissues. Cytometry 7:41-4 |
