Damman Reinier, Lucini Paioni Alessandra, Xenaki Katerina T, Beltrán Hernández Irati, van Bergen En Henegouwen Paul M P, Baldus Marc
NMR Spectroscopy, Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands.
Cell Biology, Neurobiology and Biophysics, Department of Biology, Faculty of Science, Utrecht University, 3584 CH, Utrecht, The Netherlands.
J Biomol NMR. 2020 Sep;74(8-9):401-412. doi: 10.1007/s10858-020-00328-8. Epub 2020 Jun 19.
Recent advances in the field of in-cell NMR spectroscopy have made it possible to study proteins in the context of bacterial or mammalian cell extracts or even entire cells. As most mammalian cells are part of a multi-cellular complex, there is a need to develop novel NMR approaches enabling the study of proteins within the complexity of a 3D cellular environment. Here we investigate the use of the hanging drop method to grow spheroids which are homogenous in size and shape as a model system to study solid tumors using solid-state NMR (ssNMR) spectroscopy. We find that these spheroids are stable under magic-angle-spinning conditions and show a clear change in metabolic profile as compared to single cell preparations. Finally, we utilize dynamic nuclear polarization (DNP)-supported ssNMR measurements to show that low concentrations of labelled nanobodies targeting EGFR (7D12) can be detected inside the spheroids. These findings suggest that solid-state NMR can be used to directly examine proteins or other biomolecules in a 3D cellular microenvironment with potential applications in pharmacological research.
细胞内核磁共振光谱领域的最新进展使得在细菌或哺乳动物细胞提取物甚至整个细胞的背景下研究蛋白质成为可能。由于大多数哺乳动物细胞是多细胞复合体的一部分，因此需要开发新的核磁共振方法，以便在三维细胞环境的复杂性中研究蛋白质。在这里，我们研究了使用悬滴法培养大小和形状均一的球体，作为使用固态核磁共振（ssNMR）光谱研究实体瘤的模型系统。我们发现这些球体在魔角旋转条件下是稳定的，并且与单细胞制剂相比，代谢谱有明显变化。最后，我们利用动态核极化（DNP）支持的ssNMR测量表明，在球体内部可以检测到低浓度的靶向表皮生长因子受体（EGFR，7D12）的标记纳米抗体。这些发现表明，固态核磁共振可用于直接检测三维细胞微环境中的蛋白质或其他生物分子，在药理学研究中具有潜在应用价值。
