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

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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.