阻断线粒体经隧道纳米管转运抑制膀胱癌细胞获得侵袭能力

Bladder cancer is a highly heterogeneous malignant lesion. Subtypes of bladder cancer cells with different invasive abilities could transfer inter-cellular information, in order to adapt to the same tumor microenvironment. Tunneling-nanotubes (TNTs) are a novel kind of cell to cell communication, which could transfer inter-cellular compartments, such as mitochondria, endoplasmic reticulum (ER), Golgi and endosomes. In previous studies, the applicants observed the presence of TNTs-like structures between RT4 and T24 bladder cancer cells with different invasive ability. In addition, literature review confirmed that mitochondrial function closely related to cancer cell invasive ability. According to phenomenon above, we hypothesize that bladder cancer cells with divergent invasive ability could form TNTs. Bladder cancer cells could acquire invasive ability by mitochondrial transportation, mediated by TNTs. To verify this hypothesis, in this application, with different invasive abilities, RT4 and T24 bladder cancer cell membrane and mitochondrial are labeled by fluorescence marks, and co-cultured in vitro and in vivo. The phenomenon of TNTs formation, transportation of T24 cells mitochondrion to RT4 cells and enhance of invasive ability of RT4 cells acquired T24 cells mitochondria are observed by FACS, Laser cofocal assay and Transwell assay et al. The role of lactic acid and ATP gradient played in mechanisms of phenomenon aboved are also discussed. Whether blocking the formation of TNTs, composed by F-actin, could inhibit non invasive bladder cancer cell acquiring invasive ability are evaluated by F-actin polymerase inhibitor Latrunculin B. This study could provide a novel therapeutic target for inhibiting progress of bladder cancer.

膀胱癌是具有高度异质性的恶性肿瘤，瘤体内不同侵袭能力的膀胱癌细胞可进行信息传递，以适应相同的肿瘤微环境。隧道纳米管是最近发现的细胞间信息传递通道，细胞能藉此通道进行跨细胞成分转运。前期研究中，申请者观察到，不同侵袭能力的膀胱癌细胞间存在隧道纳米管样结构。此外，大量研究已证实，线粒体功能与肿瘤侵袭能力密切相关。有鉴于此，申请者提出不同侵袭能力膀胱癌细胞间能通过形成隧道纳米管进行线粒体转运，进而改变细胞侵袭能力的假说。本项目拟对RT4和T24膀胱癌细胞进行体内外共培养，观察两者间能否形成隧道纳米管；T24细胞线粒体能否经该通道转运至RT4细胞内；获取T24细胞线粒体能否改变RT4细胞的侵袭能力；探讨上述现象是否与细胞代谢异常引起的乳酸及ATP水平差异相关；阻断隧道纳米管的形成，能否抑制非侵袭性膀胱癌细胞获得侵袭能力等问题，对该假说进行验证。希望为抑制膀胱癌的进展提供新的治疗途径。

本项目发现，高侵袭性膀胱肿瘤细胞T24与低侵袭性膀胱肿瘤细胞RT4共同培养后，在光镜、荧光显微镜和电镜下均能观察到两种细胞之间形成隧道纳米管（Tunneling nanotubes，TNTs）。该管道内径约为100 nm到200 nm；长度约为20 μm到1 mm，最长可跨越数个细胞。通过免疫荧光染色证实，该结构主要由细胞骨架F-actin构成，具有一定的光稳定性，但长时间暴露在机械震动、强光源和化学溶剂下可引起管道断裂。电镜下可见TNTs由强侵袭性T24细胞形成，并以“伪足”形式延伸至弱侵袭性RT4细胞。荧光显微镜和流式细胞筛选发现，T24细胞内线粒体能借助TNTs主动单向转运至RT4细胞内；而RT4细胞内线粒体不能转运至T24细胞内。采用F-actin抑制剂红海海绵素B（LatB）处理细胞后，LatB能抑制T24细胞与RT4细胞之间形成TNTs；同时，线粒体跨细胞转运也受到阻断。因此，TNTs是线粒体跨细胞转运的重要途径之一。通过流式细胞分选技术筛选出融合了T24细胞线粒体的子代RT4细胞（即RT4-Mito-T24细胞），与母代RT4细胞相比，RT4-Mito-T24细胞获得了较强的体内外侵袭能力。检测RT4、T24、RT4-Mito-T24三种细胞线粒体及细胞骨架（F-actin）的亚细胞分布发现，RT4细胞线粒体主要集中在细胞核周边分布，其细胞骨架沿细胞膜内侧分布；T24细胞线粒体主要在细胞浆内弥散分布，细胞骨架在细胞膜及细胞浆内呈网状分布；融合后的RT4-Mito-T24细胞则表现出线粒体和细胞骨架分布重构，向T24细胞的分布形态转化，该转化可能诱导融合细胞侵袭能力增强。Akt和mTOR蛋白可定位于细胞线粒体膜，其下游通道4EBP1和p70S6K蛋白是调节细胞侵袭能力重要的信号通路。对比T24、RT4和RT4-Mito-T24三种细胞上述细胞信号通路蛋白的表达水平发现，Akt/p-Akt、mTOR/p-mTOR和4EBP1表达水平和磷酸化水平升高，信号活性增强，这可能是导致融合细胞侵袭能力增强的另一重要机制。因此，低级别膀胱癌细胞能借助TNTs获取外源性线粒体，获得高侵袭能力，表现出“趋同效应”。选择性抑制F-actin聚合，能阻断线粒体藉TNTs跨细胞转运，继而抑制低级别膀胱癌细胞获得高侵袭能力。