Four-color multiparameter DNA flow cytometric method to study phenotypic intratumor heterogeneity in cervical cancer

Abstract

BACKGROUND Multiparameter DNA flow cytometry using a one‐laser bench‐top flow cytometer has been restricted to three different colors. The two laser FACSCalibur has recently been introduced, allowing four‐color analysis. Therefore, we optimized and extended our three‐color method (Corver et al., 1994, Corver et al. 1996) to a four‐color analysis of phenotypic intra‐tumor heterogeneity using a bench‐top flow cytometer. METHODS First, the effect of a range of different propidium iodide (PI) and TO‐PRO‐3 iodide (TP3) concentrations on the coefficient of variation (CV) of the DNA histograms was measured using paraformaldehyde‐fixed lysolecithin‐permeabilized peripheral blood lymphocytes (PBLs) and SiHa and HeLa cervical cancer cells. Second, labeling freshly isolated cervical cancers from solid tumors was optimized with a mixture of anti‐keratin antibodies. Third, the FACSCalibur hardware was modified, thereby allowing the simultaneous measurement of allophycocyanin (APC) fluorescence (FL4) in combination with FL3 pulse processing (FL3‐W vs. FL3‐A). The optimized procedure was then applied to cell suspensions from four different human cervical cancers to study phenotypic intratumor heterogeneity. Cell suspensions were simultaneously stained for DNA (PI, fluorescence) and three cellular antigens: (a) the epithelial cell‐adhesion molecule (Ep‐CAM; APC fluorescence), (b) keratin (R‐phycoerythrin [RPE] fluorescence) to identify the epithelial fraction, and (c) vimentin (fluorescein‐isothiocyanate [FITC] fluorescence) to label stromal cells. RESULTS Overall, PI produced better CVs than did TP3. The optimal concentration of PI was 50–100 μM for all cells tested. Average CVs were 1.76% (PBL), 3.16% (HeLa), and 2.50% (SiHa). Optimal TP3 concentrations were 0.25–2.0 μM. Average CVs were 2.58% (PBL), 5.16% (HeLa), and 3.96% (SiHa). Inter‐ or intra‐DNA stem line heterogeneity of Ep‐CAM expression was observed in the keratin‐positive fractions. Vimentin‐positive, keratin‐negative cells were restricted to the DNA diploid fraction. CONCLUSIONS PI is a superior DNA stain to TP3 when using intact normal PBL and human cancer cells. Four‐color high‐resolution multiparameter DNA flow cytometry allows the identification of intratumor subpopulations using PI as DNA stain and FITC, RPE, and APC as reporter molecules. The FACSCalibur bench‐top flow cytometer can be used for this purpose, allowing the application of this technique in clinical laboratories. Cytometry 39:96–107, 2000 © 2000 Wiley‐Liss, Inc.

DRAQ5‐based DNA content analysis of hematolymphoid cell subpopulations discriminated by surface antigens and light scatter properties

“…Moreover, although DRAQ5 fluorescence is optimally excited at 647 nm, it is sufficiently energized at 488 nm, and is thus suitable for excitation with argon-ion lasers that are found in most clinical instruments. Because no fixation or permeabilization is needed, DRAQ5 is technically advantageous over other dyes that do require these extra steps, such as PI, Topro-3, and 7-AAD (4,7,8,15), which have previously been used in multiparameter DNA and surface antigen analysis. Hoechst 33342 is a DNAbinding fluorochrome that can be used without cell permeabilization since it enters viable cells by active uptake FIG.…”

“…These results likely represent minor differences in chromatin packing and/or DNA saturation by DRAQ5 staining within each distinct cell subpopulation. Thus, like Topro-3 (15) or Hoechst 33342 (3) stains that result in comparable wider CVs, DRAQ5 staining should be acceptable for identifying aneuploidy in a sample with a large change in DNA content, but may not be adequate for detection of minor changes in DNA content or chromatin condensation.…”

Genome-wide Allelic State Analysis on Flow-Sorted Tumor Fractions Provides an Accurate Measure of Chromosomal Aberrations

“…For analysis, data from 20,000 single cell events were collected using a standard FACScalibur (BD Biosciences) flow cytometer, equipped with a 15 mW Argon-ion laser (488 nm) and a 12 mW diode laser (635 nm; ref. 30). The FL3-A versus FL3-W pulse-processor was used to enrich for single cell events during acquisition and analysis.…”

The importance of the age factor in cancer vaccination at older age

“…As discussed later in this article, vaccination based on TAA could circumvent both problems. Even then, however, loss of TAA expression will allow tumor cells to escape vaccine-induced T cell responses [13, 14]. This underlines the need of multi-antigen vaccines.…”