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NMR光谱学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，因此有必要开发新颖的NMR方法，从而能够在3D细胞环境的复杂性内研究蛋白质。在这里，我们研究使用悬滴法生长大小和形状均一的球状体作为模型系统，以使用固态NMR（ssNMR）光谱研究实体瘤。我们发现这些球体在魔角旋转条件下是稳定的，并且与单细胞制备相比显示出代谢特征的明显变化。最后，我们利用动态核极化（DNP）支持的ssNMR测量来显示可在球体内检测到低浓度的靶向EGFR（7D12）的标记纳米抗体。这些发现表明，固态NMR可用于直接检查3D细胞微环境中的蛋白质或其他生物分子，并在药理研究中具有潜在的应用。