Wartenberg M, Hescheler J, Acker H, Diedershagen H, Sauer H
Department of Neurophysiology, University of Cologne, Germany.
Cytometry. 1998 Feb 1;31(2):137-45. doi: 10.1002/(sici)1097-0320(19980201)31:2<137::aid-cyto9>3.0.co;2-j.
Multicell-mediated drug resistance is a major impediment for the effectiveness of chemotherapeutic approaches and has been shown to be a feature of many solid tumors. We used confocal laser scanning microscopy to evaluate the depth distribution of the fluorescent cytostatic drug doxorubicin (Dox) in two size classes of multicellular cancer spheroids (MCS) (psi150+/-50 microm and 350+/-50 microm). MCS (psi150+/-50 microm) solely consist of proliferating cells, whereas in MCS (psi350+/-50 microm) peripheral proliferating cell layers are followed in the depth of the tissue by drug resistant quiescent cell areas. A technique was developed which allows noninvasively to trace fluorescence distributions down to a depth of approximately 180 microm in living MCS. This was achieved by confocal radial recordings of the mean Dox fluorescence in 600 microm2 regions of interest (ROI), equidistantly spaced (10 microm) from the center of MCS towards their periphery. The resulting fluorescence intensity profiles were subsequently corrected for absorbtion and light scattering in the depth of the tissue by a convenient algorithm. A 10 min incubation of MCS (psi150+/-50 microm) with Dox (10 microM) led to a peripheral accumulation, after 2 h Dox was homogeneously distributed within the whole MCS. In contrast, after Dox treatment of MCS (psi350+/-50 microm) for 2 h, the drug was accumulated within the peripheral proliferating cell rim of 78+/-8 microm, whereas deeper, quiescent cell layers remained unstained. When MCS were incubated with verapamil, cyclosporin A, orthovanadate, and quinidine, which are known to reverse P-glycoprotein (Pgp)-mediated multidrug resistance (MDR), Dox accumulated also in deeper cell layers. Genistein and indometacin which reverse multidrug resistance mediated by the multidrug resistance-associated protein (MRP) were without effects. The optical probe technique proved to be well suited to study MDR in a living three dimensional tissue context.
多细胞介导的耐药性是化疗方法有效性的主要障碍，并且已被证明是许多实体瘤的一个特征。我们使用共聚焦激光扫描显微镜来评估荧光细胞抑制药物阿霉素（Dox）在两种大小类别的多细胞癌球体（MCS）（直径150±50微米和350±50微米）中的深度分布。直径150±50微米的MCS仅由增殖细胞组成，而在直径350±50微米的MCS中，外周增殖细胞层在组织深度上之后是耐药性静止细胞区域。开发了一种技术，该技术允许在活的MCS中无创地追踪荧光分布至约180微米的深度。这是通过在600平方微米的感兴趣区域（ROI）中对平均Dox荧光进行共聚焦径向记录来实现的，这些区域从MCS的中心向其周边等距间隔（10微米）。随后通过一种简便的算法对所得的荧光强度分布进行组织深度中的吸收和光散射校正。直径150±50微米的MCS与Dox（10微摩尔）孵育10分钟导致外周积累，2小时后Dox在整个MCS内均匀分布。相比之下，直径350±50微米的MCS用Dox处理2小时后，药物积聚在78±8微米的外周增殖细胞边缘内，而更深的静止细胞层仍未染色。当MCS与已知可逆转P - 糖蛋白（Pgp）介导的多药耐药性（MDR）的维拉帕米、环孢菌素A、原钒酸盐和奎尼丁一起孵育时，Dox也积聚在更深的细胞层中。逆转多药耐药相关蛋白（MRP）介导的多药耐药性的染料木黄酮和吲哚美辛则没有效果。光学探针技术被证明非常适合在活的三维组织环境中研究多药耐药性。
