A versatile ex vivo technique for assaying tumor angiogenesis and microglia in the brain.
A versatile ex vivo technique for assaying tumor angiogenesis and microglia in the brain.
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DOI：
10.18632/oncotarget.6550
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发表时间：
2016-01-12
期刊：
影响因子：
--
通讯作者：
Savaskan NE
中科院分区：
文献类型：
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作者：
Ghoochani A;Yakubov E;Sehm T;Fan Z;Hock S;Buchfelder M;Eyüpoglu IY;Savaskan NE
原发性脑肿瘤的特点是其对微环境和血管系统的破坏性活动。然而，很少有实验技术能够在保持细胞和细胞外成分的情况下，在解剖完整的条件下获得肿瘤微环境。在这里，我们详细介绍了一种体外血管胶质瘤冲击方法(VOGIM)，在与体内情况非常相似的条件下，研究胶质瘤和化疗药物对肿瘤微环境和血管生成的影响。我们从大鼠和转基因小鼠中获得了器官型脑片培养物，并将表达荧光报告蛋白的胶质瘤细胞植入体内。在VOGIM中，肿瘤诱导的血管呈现了与人类原发脑瘤标本相同的血管病理和肿瘤区的全部范围。相比之下，未转化的细胞，如原代星形胶质细胞，不会改变血管的结构。血管分支、连接和血管长度的血管特征以及瘤周区域都是可以定量评估的。特别是，VOGIM类似于脑肿瘤的微环境，改变了神经元、小胶质细胞和细胞存活。因此，该方法允许对几乎任何表达荧光报告的细胞进行活细胞监测。我们进一步分析了肿瘤细胞和替莫唑胺(Temodal/Temcad®)等化疗药物影响下的血管和小胶质细胞。值得注意的是，替莫唑胺使植入肿瘤的大脑中的血管连接和分支以及小胶质细胞的分布正常化。此外，VOGIM还可以方便地在实验中实现3R。综上所述，VOGIM是一种通用和稳健的技术，它允许在形态和定量水平上使用血管生成、神经细胞死亡和小胶质细胞活性等参数来评估脑肿瘤微环境。
Primary brain tumors are hallmarked for their destructive activity on the microenvironment and vasculature. However, solely few experimental techniques exist to access the tumor microenvironment under anatomical intact conditions with remaining cellular and extracellular composition. Here, we detail an ex vivo vascular glioma impact method (VOGIM) to investigate the influence of gliomas and chemotherapeutics on the tumor microenvironment and angiogenesis under conditions that closely resemble the in vivo situation. We generated organotypic brain slice cultures from rats and transgenic mice and implanted glioma cells expressing fluorescent reporter proteins. In the VOGIM, tumor-induced vessels presented the whole range of vascular pathologies and tumor zones as found in human primary brain tumor specimens. In contrast, non-transformed cells such as primary astrocytes do not alter the vessel architecture. Vascular characteristics with vessel branching, junctions and vessel length are quantitatively assessable as well as the peritumoral zone. In particular, the VOGIM resembles the brain tumor microenvironment with alterations of neurons, microglia and cell survival. Hence, this method allows live cell monitoring of virtually any fluorescence-reporter expressing cell. We further analyzed the vasculature and microglia under the influence of tumor cells and chemotherapeutics such as Temozolamide (Temodal/Temcad®). Noteworthy, temozolomide normalized vasculare junctions and branches as well as microglial distribution in tumor-implanted brains. Moreover, VOGIM can be facilitated for implementing the 3Rs in experimentations. In summary, the VOGIM represents a versatile and robust technique which allows the assessment of the brain tumor microenvironment with parameters such as angiogenesis, neuronal cell death and microglial activity at the morphological and quantitative level.