Webster L, Hodgkiss R J, Wilson G D
Cancer Research Campaign Gray Laboratory, Mount Vernon Hospital, Northwood, Middlesex, United Kingdom.
Cytometry. 1995 Dec 1;21(4):344-51. doi: 10.1002/cyto.990210406.
Hypoxia and proliferation rate are two important biological factors influencing the outcome of radiotherapy regimes for solid tumours. Hypoxic cells are more resistant to radiation than aerobic cells and a rapidly dividing tumour may repopulate faster during treatment. Clinical trials are underway to assess the importance of both these parameters. In this article we describe a method to simultaneously measure hypoxia and proliferation using multiparameter flow cytometry. Hypoxic cells were detected using a bioreductively bound marker with an immuno-recognisable side-chain, NITP and proliferation was measured by bromodeoxyuridine (BrdUrd) incorporation. These parameters were related to cell cycle position by measuring total DNA content with 7-aminoactinomycin D. The data were analysed using single laser excitation on a bench top flow cytometer. Simultaneous measurement of the three parameters shows the presence of cells which have incorporated BrdUrd and are also hypoxic by the criterion of NITP binding. The murine SaF tumour has a relatively constant aneuploid labelling index of 24%. However, the level of aneuploid hypoxia was variable ranging from 0.8 to 40.9% with a mean value of 15.6%. Within the BrdUrd labelled population there is a range of hypoxia from 2.8 to 28.5% (mean 15.1%); this represents 0.7 to 6.6% of the total tumour population. There are approximately twice as many oxygenated cells than hypoxic cells actively in the cell cycle. In vivo tumours contain cells with S phase DNA content which do not incorporate BrdUrd. This cell population has equivalent proportions of hypoxic and oxic cells. However, there are up to 12-fold more hypoxic cells in the unlabelled S than the BrdUrd labelled population. These data show that proliferation and hypoxia can be measured simultaneously using flow cytometry and the technique may form the basis of a predictive assay for these two important biological determinants of radiotherapy outcome.
缺氧和增殖率是影响实体瘤放射治疗效果的两个重要生物学因素。缺氧细胞比有氧细胞对辐射更具抗性，且快速分裂的肿瘤在治疗期间可能重新增殖得更快。目前正在进行临床试验以评估这两个参数的重要性。在本文中，我们描述了一种使用多参数流式细胞术同时测量缺氧和增殖的方法。使用具有免疫可识别侧链的生物还原结合标记物NITP检测缺氧细胞，并通过溴脱氧尿苷（BrdUrd）掺入来测量增殖。通过用7-氨基放线菌素D测量总DNA含量，将这些参数与细胞周期位置相关联。数据在台式流式细胞仪上使用单激光激发进行分析。三个参数的同时测量显示存在已掺入BrdUrd且根据NITP结合标准也处于缺氧状态的细胞。小鼠SaF肿瘤具有相对恒定的非整倍体标记指数，为24%。然而，非整倍体缺氧水平变化范围为0.8%至40.9%，平均值为15.6%。在BrdUrd标记的群体中，缺氧范围为2.8%至28.5%（平均值为15.1%）；这占肿瘤总群体的0.7%至6.6%。在细胞周期中，活跃的含氧细胞数量大约是缺氧细胞的两倍。体内肿瘤含有具有S期DNA含量但未掺入BrdUrd的细胞。这个细胞群体中缺氧细胞和含氧细胞的比例相当。然而，未标记的S期细胞中的缺氧细胞比BrdUrd标记的群体中多高达12倍。这些数据表明，可以使用流式细胞术同时测量增殖和缺氧，并且该技术可能构成针对放射治疗结果的这两个重要生物学决定因素的预测性检测的基础。
