TWO-PHOTON IMAGING OF NAD(P)H IN TUMOR SPHEROIDS

我们建议使用双光子激光扫描显微镜（TPLSM）来测量 在多细胞肿瘤球体中的NADH和NADPH（NAD（P）H）荧光作为一种 PDT期间缺氧发展的指示，一种新的基于激光的 目前正在全球范围内进行临床试验的癌症疗法。 到 表明NAD（P）H的TPLSM可用于监测缺氧。 多细胞肿瘤球体，将有必要证明NAD（P）H， 荧光可以与细胞缺氧在亚群 细胞组成一个球状体。 在目标1中，我们将测量 正常和EMTS/Ro细胞单层的荧光平衡在各种 氧分压 此外，荧光的分数 来源于线粒体和荧光部分， 取决于氧分压，将确定。 多小区 在不同氧分压下平衡的肿瘤球体将 用于建立和控制该模型肿瘤中的缺氧程度 系统 在目标2中，TPLSM将与球体中的缺氧相关， 用尖端直径为5 μ m的氧敏感微电极测量 利用从目标1中获得的信息确定缺氧的程度， NAD（P）H荧光图像。 作为Aim的一部分进行的研究 3然后将监测FDT期间缺氧的发作和进展 在几个最佳通量下进行，已知会导致 不同程度。 最后，目标4将探索潜在的分析方法 利用NAD（P）H荧光作为直接的非侵入性诊断 作为该模型肿瘤系统体外治疗功效指标， 以及体内的肿瘤。 这项技术的发展将是 在治疗计划和监测对治疗的反应方面非常有用 例如癌症的放射和光动力疗法。

We propose to use two-photon laser scanning microscopy (TPLSM) to measure NADH and NADPH (NAD(P)H) fluorescence in the multicell tumor spheroid as an indication of the development of anoxia during PDT, a novel laser-based cancer therapy which is currently undergoing clinical trials worldwide. To demonstrate that TPLSM of NAD(P)H can be used to monitor anoxia in the multicell tumor spheroid, it will be necessary to demonstrate that NAD(P)H fluorescence can be correlated to cellular anoxia ina the subpopulations of cells which comprise a spheroid. In Aim 1, we will measure athe fluorescence of normal and EMTS/Ro cell monolayers equilibrated at various oxygen partial pressures. Additionally, the fraction of fluorescence originating from the mitochondria and the fraction of fluorescence which is dependent on the oxygen partial pressure will be determined. Multicell tumor spheroids equilibratfed at various oxygen partial pressures will then be used to establish and control the extent of anoxia in this model tumor system. In Aim 2, TPLSM will be correlated to anoxia in the spheroid as measured with an oxygen sensitive microelectrode with a 5 mum tip diameter using information obtained from Aim 1 to determine the extent of the anoxic region from NAD(P)H fluorescence images. Studies conducted as part of Aim 3 will then monitor the onset and progression of anoxia during FDT conducted at several optimal fluence raises known to cause anomia of various extent. Finally, Aim 4 will explore potential analytical methods to utilize NAD(P)H fluorescence as a direct non-invasive diagnostic indicator of therapeutic efficacy in this model tumor system in vitro as well as tumors in vivo. The development of this technique would be extremely useful in treatment planning and monitoring response to treatment interventions such as radiation and photodynamic therapy of cancer.