Development of new measurement methods of tumor oxygenation

众所周知，实体肿瘤中缺氧细胞的存在是放疗和一些化疗方案后失败的最重要原因之一。到目前为止，已经做出了许多努力来克服这个问题。然而，一个重要的问题是，我们没有有效的方法来常规测量患者的肿瘤内缺氧分数。以往的研究表明，不同肿瘤类型的低氧分数不同，但目前尚不清楚哪种肿瘤含有高的低氧分数，这对放疗策略是困难的。在这项研究中，我们测试了用两种方法鉴定肿瘤中缺氧部分的可能性：彗星法（单细胞电泳法）和用PET扫描检测缺氧细胞，以检测Cu(II)-二乙酰基-双（n4 -甲基硫代氨基脲）。彗星试验已发展成为一种测量辐照后单细胞DNA损伤的方法。我们开发了自己的方法和图像分析系统，用于彗星测定，以识别缺氧组分。在体外，我们用培养的肿瘤细胞系测试了我们的系统。在体内，我们用两种表现出不同类型缺氧的啮齿动物肿瘤，将这种方法检测到的缺氧成分与体内体外克隆生成法检测到的低氧成分进行了比较：急性和慢性。在体外，我们的方法可以利用尾矩参数区分缺氧细胞和缺氧细胞。在体内，在两种肿瘤中，彗星试验和克隆性试验测定的低氧组分之间存在良好的相关性。通过对两种方法在慢性缺氧和急性缺氧肿瘤中的比较，我们进一步证实了彗星测定法在临床上对估计实体肿瘤的缺氧部分是有用的。

It is well known that the presence of hypoxic cells in solid tumors is one of the most important cause of failure after radiation therapy and some chemotherapy regimens. Up to this time, many efforts have been made to overcome this problem. However, there is an important problem that we have no valid methods for the routine measurement of the intratumoral hypoxic fraction in patients. Previous study suggests that the hypoxic fraction differs with the tumor type, but it is not known what kind of tumor contains a high hypoxic fraction, that is refractory to strategy for radiotherapy.In this study, we have tested the possibilities to identify hypoxic fraction in tumors using two methods : the comet assay (single-cell electrophoresis assay) and detection of hypoxic cells using PET scan for Cu(II)-diacetyl-bis(N4-methylthiosemicarbazone).The comet assay has been developed as a method for measuring DNA damage in single cells after irradiation. We have developed our own methods and image analysis system for the comet assay to identify hypoxic fractions. In vitro, we tested our system using a cultured tumor cell line. In vivo, we compared the hypoxic fractions detected by this assay with those determined by the in vivo-in vitro clonogenic assay using two rodent tumors, which exhibit different types of hypoxia : acute and chronic.In vitro, our method could differentiate hypoxic cells from oxic cells, using the parameter of tail moment. In vivo, there were good correlations between the hypoxic fractions determined by the comet assay and by the clonogenic assay, in both tumors. By comparison of the two methods in chronically hypoxic and acutely hypoxic tumors, we further confirmed that the comet assay is clinically useful for estimation hypoxic fractions of solid tumors.