Olive P L, Vikse C, Trotter M J
Medical Biophysics Unit, British Columbia Cancer Research Centre, Vancouver, Canada.
Int J Radiat Oncol Biol Phys. 1992;22(3):397-402. doi: 10.1016/0360-3016(92)90840-e.
Oxygen diffusion distance was measured in solid tumor "cubes" prepared by excising the tumor from the mouse and incubating 1-2 mm sided tumor cubes in spinner culture flasks with fluorescent drugs (AF-2 or DM113) which bind to hypoxic cells. After incubation, frozen sections were prepared and examined for AF-2 or DM113 binding using a fluorescence image processing system. Alternatively, cubes were stained with a slowly penetrating fluorescent dye, Hoechst 33342, prior to disaggregation and measurement of AF-2 binding using flow cytometry. The distance from the cube surface to the AF-2 stained region (i.e., the thickness of the unstained rim) was used as an indication of oxygen diffusion distance, which depends on the square root of the tumor oxygen consumption rate. Oxygen diffusion distance was dependent on tumor type, external oxygen concentration, and temperature during incubation with AF-2, but was unaffected by tumor size up to 1.2 gm or position of the cube within the tumor. Oxygen diffusion distances (in planar geometry) measured for cubes prepared from SCCVII, RIF-1, Lewis lung or WiDr tumors were 107, 123, 153 and 193 microns, respectively. For SCCVII and WiDr tumors, the percentage of blood vessels separated by a distance greater than double the mean oxygen diffusion distance was used as an indication of the hypoxic fraction.
通过从小鼠体内切除肿瘤并将边长为1 - 2毫米的肿瘤“方块”置于含有与缺氧细胞结合的荧光药物（AF - 2或DM113）的旋转培养瓶中培养，来测量实体瘤“方块”中的氧扩散距离。培养后，制备冰冻切片，并使用荧光图像处理系统检查AF - 2或DM113的结合情况。或者，在解离并使用流式细胞术测量AF - 2结合之前，用一种穿透缓慢的荧光染料Hoechst 33342对“方块”进行染色。从“方块”表面到AF - 2染色区域的距离（即未染色边缘的厚度）被用作氧扩散距离的指标，氧扩散距离取决于肿瘤氧消耗率的平方根。氧扩散距离取决于肿瘤类型、外部氧浓度以及与AF - 2孵育期间的温度，但不受肿瘤大小（最大达1.2克）或“方块”在肿瘤内位置的影响。从SCCVII、RIF - 1、Lewis肺癌或WiDr肿瘤制备的“方块”所测量的氧扩散距离（在平面几何中）分别为107、123、153和193微米。对于SCCVII和WiDr肿瘤，将被大于平均氧扩散距离两倍的距离分隔开的血管百分比用作缺氧分数的指标。
