DETECTION OF HYPOXIC CELLS IN A MURINE TUMOR WITH THE USE OF THE COMET ASSAY
DETECTION OF HYPOXIC CELLS IN A MURINE TUMOR WITH THE USE OF THE COMET ASSAY
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DOI：
10.1093/jnci/84.9.707
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发表时间：
1992-05-06
期刊：
影响因子：
--
通讯作者：
DURAND, RE
中科院分区：
文献类型：
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作者：
OLIVE, PL;DURAND, RE
背景：实体瘤中的低分化细胞可能限制放射治疗和某些化疗药物对肿瘤的治愈。目的：为了量化实体瘤中的缺氧分数，我们开发了一种方法，该方法测量单个肿瘤细胞中辐射诱导的DNA单链断裂，并利用有氧细胞中产生的链断裂比缺氧细胞中产生的链断裂多三倍的事实。研究方法：在用4-20戈伊剂量照射后，立即取出在C3 H小鼠中生长的SCCVII鳞状细胞癌并冷却，制备单细胞悬浮液。然后将这些细胞包埋在琼脂糖中，在碱性溶液中裂解，进行电泳，并用荧光DNA结合染料染色。通过使用荧光图像处理系统对来自单个细胞的受损DNA的量和迁移距离进行评分，其中差异辐射敏感的需氧和缺氧细胞群体导致双峰损伤分布。曲线拟合程序提供了缺氧细胞的分数的定量估计。结果如下：在小鼠暴露于10-20戈伊后，SCCVII肿瘤（450-600 mg）显示出具有18.5% +/-10.6%（11个肿瘤的平均值+/- SD）的缺氧分数，其与使用配对存活曲线方法观察到的11.6%的值相当。结论：我们的研究结果表明，这种方法，它只需要几千个细胞，是一种快速和敏感的方法来检测实体动物肿瘤中的缺氧细胞。含义：如果该方法的灵敏度可以提高到允许在2戈伊的剂量后检测到缺氧细胞，则可以估计接受放射治疗的可接近的人类肿瘤中的缺氧。
Background: Hypoxic cells within solid tumors are likely to limit tumor curability by radiation therapy and some chemotherapeutic agents. Purpose: To quantify a hypoxic fraction in solid tumors, we developed a method which measures radiation-induced DNA single-strand breaks in individual tumor cells and makes use of the fact that three times more strand breaks are produced in aerobic than in hypoxic cells. Methods: Immediately after irradiation with doses of 4-20 Gy, SCCVII squamous cell carcinomas growing in C3H mice were removed and cooled, and a single-cell suspension was prepared. These cells were then embedded in agarose, lysed in an alkaline solution, subjected to electrophoresis, and stained with a fluorescent DNA-binding dye. The amount and migration distance of damaged DNA from individual cells were scored by using a fluorescence image processing system, where differentially radiosensitive aerobic and hypoxic cell populations resulted in bimodal damage distributions. Curve-fitting routines provided quantitative estimates of the fraction of hypoxic cells. Results: After the mice were exposed to 10-20 Gy, the SCCVII tumors (450-600 mg) were shown to have a hypoxic fraction of 18.5% +/- 10.6% (mean +/- SD for 11 tumors), which compares well with the value of 11.6% observed using the paired survival curve method. Conclusions: Our results indicate that this method, which requires only a few thousand cells, is a rapid and sensitive way to detect hypoxic cells in solid animal tumors. Implications: Estimating hypoxia in accessible human tumors undergoing radiotherapy may be possible if the sensitivity of the method can be improved to allow detection of hypoxic cells after a dose of 2 Gy.