Laboratory of Molecular Oncology and Innovative Therapies, Military Institute of Medicine, Warsaw, Poland.
Postgraduate School of Molecular Medicine, Medical University of Warsaw, Warsaw, Poland.
In Vitro Cell Dev Biol Anim. 2022 May;58(5):349-364. doi: 10.1007/s11626-022-00685-8. Epub 2022 May 10.
Tumor development studies should adapt to cancer cells' specific mechanisms in connection with their microenvironment. Standard two-dimensional cultures and gas composition are not relevant to the real cancer environment. Existing three-dimensional models are often requiring sophisticated conditions. Here, we propose and characterize, in two cancer models, melanoma (B16F10) and kidney cancer (RenCa), a three-dimensional culture method, reporting the presence of hypoxia-related genes/proteins and aggressiveness mechanisms (epithelial mesenchymal transition and cancer stem cells). We validate the designed three-dimensional method by comparing it with in vivo growing tumors. The developed method brings simplicity and data reproducibility. Melanoma spheroid-growing cells reached a cell cycle arrest at the G0/G1 phase and showed induction of hypoxia. Spheroid-recovered RenCa cells were enriched in proliferating cells and displayed delayed hypoxia. Moreover, the responses to hypoxia observed in spheroids were validated by in vivo tumor studies for both lines. Three-dimensional shapes induced cancer stem cells in renal cancer, whereas epithelial to mesenchymal transition occurred in the melanoma model. Such distinction in the use of different aggressiveness-leading pathways was observed in in vivo melanoma vs kidney tumors. Thus, this 3D culture model approach is adequate to uncover crucial molecular pathways using distinct mechanisms to reach aggressiveness; i.e., B16F10 cells perform epithelial to mesenchymal transition while RenCa cells dedifferentiate into cancer stem cells. Such three-dimensional models help mimic the in vivo tumor features, i.e., hypoxia and aggressiveness mechanisms as validated here by next-generation sequencing analysis, and are proposed for further alternative methods to in vivo studies.
肿瘤发展研究应适应与癌细胞微环境相关的特定机制。标准的二维培养和气体组成与真实的癌症环境不相关。现有的三维模型通常需要复杂的条件。在这里，我们在两种癌症模型（黑色素瘤（B16F10）和肾癌（RenCa））中提出并描述了一种三维培养方法，报告了缺氧相关基因/蛋白和侵袭机制（上皮间质转化和癌症干细胞）的存在。我们通过将其与体内生长的肿瘤进行比较来验证设计的三维方法。所开发的方法具有简单性和数据重现性。黑色素瘤球体生长细胞在 G0/G1 期停滞，并显示出缺氧诱导。球体恢复的 RenCa 细胞富含增殖细胞，并表现出延迟缺氧。此外，两种细胞系的体内肿瘤研究验证了球体中观察到的缺氧反应。三维形状在肾癌中诱导癌症干细胞，而上皮间质转化发生在黑色素瘤模型中。在体内黑色素瘤与肾肿瘤中观察到对不同侵袭性主导途径的这种区分。因此，这种 3D 培养模型方法适合利用不同的侵袭性机制来揭示关键的分子途径；即，B16F10 细胞进行上皮间质转化，而 RenCa 细胞分化为癌症干细胞。这种三维模型有助于模拟体内肿瘤特征，例如这里通过下一代测序分析验证的缺氧和侵袭机制，并被提议用于替代体内研究的进一步方法。
