Solid tumor preparation for flow cytometry using a standard murine model

Abstract

The application of flow cytometry (FCM) to solid human tumors has been hindered by the difficulty in producing high yield, viable, unaltered single cell suspensions. Carcinomas containing a high desmosomal content, such as well-differentiated squamous cell (SCC) cancers of the head and neck (H&N) region, are particularly difficult to prepare. The desire to employ FCM to study cellular DNA parameters of these tumors led to the use of a 3-methylcholanthrene induced murine SCC for the comparative testing of preparative techniques. Dissociation techniques, including mechanical, enucleation, chemical, single and combination enzymes methods, were comparatively tested. Of these, the combination enzyme treatment employing trypsin and collagenase produced the highest cell yields in the shortest time with the highest dye exclusion viability and the least expense. Several fixation systems including glutaraldehyde, paraformaldehyde, acetic acid, and ethanol were comparatively tested using percent of cell loss and quality of the DNA histograms produced as end points. Ethanolwater systems with added fetal calf serum provided minimal cell loss and high quality histograms which were stable for extended periods of time. A murine tumor, closely mimicking the histology of the human tumor of interest, may be used as a model for the determination of optimum techniques of solid tumor preparation for flow cytometric analysis.

The clinical application of DNA content and kinetic parameters in the treatment of patients with squamous cell carcinomas of the head and neck

“…In 1989 we reported a prospective study of whole cell DNA content parameters performed on 237 flesh SCCHN specimens [19] following thorough development and validation of methodology [20,21]. Neither DNA ploidy status or % SPF correlated with conventional stage categories (I-IV), or primary tumor size (T stage) but did correlate with degree of lymph node involvement (N stage), Conventional grade categories were not associated with differences in DNA content parameters but in a separate study, important individual features of histopathology as defined by Jacobson [5] and refined by Crissman inflammatory response, pattern of invasion, and mitotic indices) were strongly associated with DNA ploidy and associated % SPE…”

Variations in DNA aneuploid cell content during tumor dissociation in human colon and head and neck cancers analyzed by flow cytometry

“…A relatively high desmosome content is probably responsible for the inefficient release of DNA aneuploid subpopulations from SCCHN following mechanical dissociation (15). DNA aneuploid subpopulations of human colon cancer are released by both mechanical and enzymatic techniques, but appear more vulnerable to proteolytic digestion.…”

“…Experimental techniques including flow cytometry (15,16,18,52), clonigenic assays (26, 49), and cytogenetic and oncogenetic analysis (53) are dependent on the quality of such preparations. Validation of such methodology is often lacking, and large numbers of specimens from widely divergent tissue types are commonly processed by one preparative method (3,4,21, 51,56 49), and cytogenetic analysis (53).…”

“…Validation of such methodology is often lacking, and large numbers of specimens from widely divergent tissue types are commonly processed by one preparative method (3,4,21, 51,56 49), and cytogenetic analysis (53). We have reported that mechanical techniques were inferior to enzymatic methods in terms of cellular yields and viability for squamous cell carcinomas of the head and neck (SCCHN) (15,16 . Comparative studies to determine optimum cellular yields, viabilities, and the preservation of DNA aneuploid subpopulations have not been reported for this tumor.…”

Quality control study of the italian group of cytometry on flow cytometry cellular DNA content measurements

“…A major obstacle to the implementation of FCM DNA content measurements in clinical settings has been the disagreement among different institutions concerning type and adequacy of tumor samples (1,9,10,14,19), sampling modalities (3,6,12,22,23), monocellular suspension preparation (7,8), and staining, instrument calibration, use of external and internal standards, data analysis and interpretation, and quality control (1 lJ3).…”