Experimental and Clinical Research Center, Charité University Medicine Berlin, at the Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Straße 10, 13125, Berlin, Germany.
Clin Exp Metastasis. 2012 Aug;29(6):573-83. doi: 10.1007/s10585-012-9472-6. Epub 2012 Apr 7.
We previously identified the gene metastasis-associated in colon cancer-1 (MACC1) and demonstrated its important role for metastasis prediction in colorectal cancer. MACC1 induces cell motility and proliferation in vitro as well as metastasis in several mouse models. Here we report non-invasive real time imaging of inhibition of colorectal tumor progression and metastasis in xenografted mice by MACC1 shRNA. First, we demonstrated reduction of tumors and liver metastases by endpoint imaging of mice transplanted with MACC1 endogenously high expressing colorectal cancer cells and treated with shRNAs acting on MACC1 or Met. Next, we generated a novel bicistronic IRES vector simultaneously expressing the reporter gene firefly luciferase and MACC1 to ensure a direct correlation of bioluminescence signal with MACC1 expression. We transfected MACC1 endogenously low expressing colorectal cancer cells with this luciferase-IRES-MACC1 construct, transplanted them intrasplenically, and monitored MACC1 induced tumor growth and metastasis by in vivo imaging over time. Transfection of an IRES construct harboring the firefly luciferase reporter gene together with MACC1 lacking the SH3-domain reduced tumor growth and metastasis. Finally, we counteracted the luciferase-IRES-MACC1 induced effects by shRNA targeting MACC1 and monitored reduced tumor growth and metastasis by in vivo imaging over weeks. In summary, the new bicistronic luciferase-IRES-MACC1 construct is suitable for in vivo imaging of tumor progression and metastasis, and moreover, for imaging of therapy response such as treatment with MACC1 shRNA. Thereby, we provide proof-of-concept for employment of this MACC1-based in vivo model for evaluating therapeutic intervention strategies aiming at inhibition of tumor growth and metastasis.
我们之前鉴定了结肠癌转移相关基因-1（MACC1），并证明其在结直肠癌转移预测中具有重要作用。MACC1 在体外诱导细胞迁移和增殖，并在几种小鼠模型中诱导转移。在这里，我们报告了通过 MACC1 shRNA 对异种移植小鼠的结直肠肿瘤进展和转移进行非侵入性实时成像。首先，我们通过对内源性高表达 MACC1 的结直肠癌细胞移植的小鼠进行终点成像，证明了肿瘤和肝转移的减少，并用针对 MACC1 或 Met 的 shRNA 进行治疗。接下来，我们生成了一种新型双顺反子 IRES 载体，同时表达报告基因萤火虫荧光素酶和 MACC1，以确保生物发光信号与 MACC1 表达直接相关。我们用这种荧光素酶-IRES-MACC1 构建体转染内源性低表达 MACC1 的结直肠癌细胞，将其脾内移植，并通过体内成像随时间监测 MACC1 诱导的肿瘤生长和转移。转染缺失 SH3 结构域的荧光素酶-IRES-MACC1 构建体可降低肿瘤生长和转移。最后，我们通过针对 MACC1 的 shRNA 来抵消荧光素酶-IRES-MACC1 诱导的作用，并通过数周的体内成像来监测肿瘤生长和转移的减少。总之，新型双顺反子荧光素酶-IRES-MACC1 构建体适合用于肿瘤进展和转移的体内成像，并且还可以用于成像治疗反应，例如用 MACC1 shRNA 治疗。因此，我们提供了基于 MACC1 的体内模型用于评估旨在抑制肿瘤生长和转移的治疗干预策略的概念验证。
