Coculture system with an organotypic brain slice and 3D spheroid of carcinoma cells.
Coculture system with an organotypic brain slice and 3D spheroid of carcinoma cells.
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DOI：
10.3791/50881
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发表时间：
2013-10-09
期刊：
影响因子：
--
通讯作者：
Pukrop T
中科院分区：
文献类型：
--
作者：
Chuang HN;Lohaus R;Hanisch UK;Binder C;Dehghani F;Pukrop T
肿瘤脑转移患者预后较差。然而，转移部位的过程几乎没有被研究，特别是常驻(间质)细胞的作用。对原发癌的研究表明，微环境对转移的影响，甚至对预后的影响1，2。尤其是肿瘤相关巨噬细胞()支持迁移、侵袭和增殖。有趣的是，转移的主要靶点具有组织特异性巨噬细胞，如肝脏中的Kupffer细胞或中枢神经系统中的小胶质细胞。此外，转移部位还拥有其他组织特异性细胞，如星形胶质细胞。最近，星形胶质细胞被证明可以促进癌细胞的增殖和持续存在。因此，这些组织特异性细胞类型的功能在脑转移过程中似乎非常重要。然而，尽管有这些观察，到目前为止，还没有合适的体内/体外模型来直接观察脑转移形成过程中的胶质反应，特别是通过明亮的视野显微镜。最近对癌细胞的活体成像显示了它们的脑定植行为8。然而，这种方法费时费力，成本高，技术复杂。此外，这些动物实验仅限于小系列，对动物来说有很大的压力(通过植入玻璃板、注射肿瘤细胞、重复麻醉和长期固定)。此外，到目前为止，体内成像仅限于癌细胞的可视化，而与常驻细胞的相互作用尚未被阐明。最后，在具有免疫能力的动物体内研究人类癌细胞是不可能的。为此，我们建立了一个由器官型小鼠脑片和镶嵌在基质(3D细胞球)中的上皮细胞组成的共培养系统。将3D癌细胞球直接放置在脑切片边缘，以观察邻近脑组织的侵袭情况。这使我们能够通过荧光甚至通过明亮的视野显微镜来观察胶质细胞和癌细胞之间的形态变化和相互作用。共培养实验结束后，可以收集脑组织或3D细胞球体，用于进一步的分子分析(如qRT-PCR、IHC或免疫印迹)以及用于共聚焦显微镜的研究。这种方法可以应用于监测活的脑组织内的事件数天，而不会对脑片造成有害影响。该模型还允许捐赠者组织中的细胞选择性地抑制和替换常驻细胞，以确定给定基因的不同影响。最后，当测试靶向药物操作时，共培养模型是体内方法的一个可行的替代方法。
Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in particular the role of the resident (stromal) cells. Studies in primary carcinomas demonstrate the influence of the microenvironment on metastasis, even on prognosis1,2. Especially the tumor associated macrophages (TAM) support migration, invasion and proliferation3. Interestingly, the major target sites of metastasis possess tissue-specific macrophages, such as Kupffer cells in the liver or microglia in the CNS. Moreover, the metastatic sites also possess other tissue-specific cells, like astrocytes. Recently, astrocytes were demonstrated to foster proliferation and persistence of cancer cells4,5. Therefore, functions of these tissue-specific cell types seem to be very important in the process of brain metastasis6,7. Despite these observations, however, up to now there is no suitable in vivo/in vitro model available to directly visualize glial reactions during cerebral metastasis formation, in particular by bright field microscopy. Recent in vivo live imaging of carcinoma cells demonstrated their cerebral colonization behavior8. However, this method is very laborious, costly and technically complex. In addition, these kinds of animal experiments are restricted to small series and come with a substantial stress for the animals (by implantation of the glass plate, injection of tumor cells, repetitive anaesthesia and long-term fixation). Furthermore, in vivo imaging is thus far limited to the visualization of the carcinoma cells, whereas interactions with resident cells have not yet been illustrated. Finally, investigations of human carcinoma cells within immunocompetent animals are impossible8. For these reasons, we established a coculture system consisting of an organotypic mouse brain slice and epithelial cells embedded in matrigel (3D cell sphere). The 3D carcinoma cell spheres were placed directly next to the brain slice edge in order to investigate the invasion of the neighboring brain tissue. This enables us to visualize morphological changes and interactions between the glial cells and carcinoma cells by fluorescence and even by bright field microscopy. After the coculture experiment, the brain tissue or the 3D cell spheroids can be collected and used for further molecular analyses (e.g. qRT-PCR, IHC, or immunoblot) as well as for investigations by confocal microscopy. This method can be applied to monitor the events within a living brain tissue for days without deleterious effects to the brain slices. The model also allows selective suppression and replacement of resident cells by cells from a donor tissue to determine the distinct impact of a given genotype. Finally, the coculture model is a practicable alternative to in vivo approaches when testing targeted pharmacological manipulations.
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影响因子：
6.2
作者：
Pukrop, Tobias;Dehghani, Faramarz;Binder, Claudia
通讯作者：
Binder, Claudia
DOI：
10.1038/nrclinonc.2011.58
发表时间：
2011-06
期刊：
Nature reviews. Clinical oncology
影响因子：
--
作者：
通讯作者：
--
DOI：
10.4049/jimmunol.1100421
发表时间：
2012-01-01
期刊：
Journal of immunology (Baltimore, Md. : 1950)
影响因子：
--
作者：
Mizutani M;Pino PA;Saederup N;Charo IF;Ransohoff RM;Cardona AE
通讯作者：
Cardona AE
影响因子：
14.8
作者：
De Simoni, Anna;Yu, Lily My
通讯作者：
Yu, Lily My
DOI：
10.1006/bbrc.2000.2332
发表时间：
2000-03-16
影响因子：
3.1
作者：
Matsumura, H;Ohnishi, T;Yoshimine, T
通讯作者：
Yoshimine, T