Corver W E, Koopman L A, van der Aa J, Regensburg M, Fleuren G J, Cornelisse C J
Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands.
Cytometry. 2000 Feb 1;39(2):96-107.
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.
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.
Overall, PI produced better CVs than did TP3. The optimal concentration of PI was 50-100 microM 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 microM. 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.
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.
使用单激光台式流式细胞仪进行多参数DNA流式细胞术一直局限于三种不同颜色。最近推出的双激光FACSCalibur流式细胞仪可实现四色分析。因此，我们将三色法（Corver等人，1994年，Corver等人，1996年）进行优化并扩展，以使用台式流式细胞仪对肿瘤内表型异质性进行四色分析。
首先，使用多聚甲醛固定、溶血卵磷脂通透处理的外周血淋巴细胞（PBL）以及SiHa和HeLa宫颈癌细胞，测量一系列不同浓度的碘化丙啶（PI）和碘化TO-PRO-3（TP3）对DNA直方图变异系数（CV）的影响。其次，用抗角蛋白抗体混合物优化对实体瘤中新鲜分离的宫颈癌的标记。第三，对FACSCalibur硬件进行改造，从而能够同时测量别藻蓝蛋白（APC）荧光（FL4）并结合FL3脉冲处理（FL3-W与FL3-A）。然后将优化后的程序应用于来自四种不同人类宫颈癌的细胞悬液，以研究肿瘤内表型异质性。细胞悬液同时用DNA（PI，荧光）和三种细胞抗原进行染色：（a）上皮细胞黏附分子（Ep-CAM；APC荧光），（b）角蛋白（藻红蛋白[RPE]荧光）以识别上皮部分，以及（c）波形蛋白（异硫氰酸荧光素[FITC]荧光）以标记基质细胞。
总体而言，PI产生的CV比TP3更好。对于所有测试细胞，PI的最佳浓度为50 - 100 microM。平均CV分别为1.76%（PBL）、3.16%（HeLa）和2.50%（SiHa）。TP3的最佳浓度为0.25 - 2.0 microM。平均CV分别为2.58%（PBL）·5.16%（HeLa）和3.96%（SiHa）。在角蛋白阳性部分观察到Ep-CAM表达的DNA干细胞系间或系内异质性。波形蛋白阳性、角蛋白阴性细胞局限于DNA二倍体部分。
在使用完整的正常PBL和人类癌细胞时，PI是比TP3更优的DNA染色剂。四色高分辨率多参数DNA流式细胞术能够以PI作为DNA染色剂，FITC、RPE和APC作为报告分子来识别肿瘤内亚群。FACSCalibur台式流式细胞仪可用于此目的，使得该技术能够应用于临床实验室。
