Institute of Microbiology AS CR, Videnska 1083, 142 00 Prague 4, Czech Republic.
Anticancer Res. 2009 Nov;29(11):4513-7.
Cancer research is focused on processes which influence in vivo tumor growth dynamics, tumor microenvironment and antitumor immune responses. Recently, it was documented that some cytostatics, including their polymeric derivatives, were able to trigger an anticancer immune response. Such interactions are studied mainly in vitro but relevant in vivo studies are necessary and were only recently started. Whole-body imaging of fluorescently labeled tumors, which enables visualization of the whole-body down to single cell-cell interactions, is therefore a promising tool in understanding these processes.
EL-4 T-cell lymphoma cells were transfected with plasmid containing either fusion construct of enhanced green fluorecent protein (EGFP) with H2B histone or pure EGFP gene under cytomegalovirus promotor and resistance to neomycin. Stability of expression was determined by flow cytometry and cellular localization of green fluorescence signal was tested using fluorescent microscopy. An in vivo whole-body imaging system was used to evaluate growth in vivo.
EL-4 cells were successfully transfected and established stable transfectants with a proliferation rate comparable to that of wild-type EL-4. Clone 12, with very strong whole-cell expression, enables tracking of metastatic spreading, whereas clone 3, with EGFP within the cell nucleus, allows frozen section analysis and observing of interaction with immunocompetent cells.
Established imageable EL-4-EGFP(+) cell lines are a magnificent tool for the study of tumor growth and the tumor microenvironment.
癌症研究集中在影响体内肿瘤生长动态、肿瘤微环境和抗肿瘤免疫反应的过程上。最近有文献记载，一些细胞抑制剂，包括它们的聚合物衍生物，能够引发抗肿瘤免疫反应。这些相互作用主要在体外进行研究，但相关的体内研究是必要的，并且最近才开始。对荧光标记肿瘤进行全身成像，可以可视化整个身体，甚至可以观察到单细胞间的相互作用，因此是理解这些过程的一种很有前途的工具。
EL-4 T 细胞淋巴瘤细胞用含有增强型绿色荧光蛋白（EGFP）与 H2B 组蛋白融合构建体或纯 EGFP 基因的质粒进行转染，该基因在巨细胞病毒启动子和新霉素抗性下表达。通过流式细胞术确定表达的稳定性，并使用荧光显微镜测试绿色荧光信号的细胞定位。使用体内全身成像系统评估体内生长情况。
EL-4 细胞成功转染并建立了稳定的转染子，其增殖率与野生型 EL-4 相当。具有非常强的全细胞表达的克隆 12 能够跟踪转移的扩散，而 EGFP 位于细胞核内的克隆 3 允许进行冷冻切片分析并观察与免疫活性细胞的相互作用。
建立的可成像的 EL-4-EGFP(+)细胞系是研究肿瘤生长和肿瘤微环境的极好工具。
