Abstract
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.
Highlights
The contribution of cancer-derived cell lines to the understanding of molecular and cellular processes in the complex physiology of cancer has been immense
To produce A431 spheroids for solid-state NMR (ssNMR) experiments, spheroids were grown over a period of about 48 h until they reached a diameter of 600–700 μm (70–100.000 cells)
Nuclei (DNA) were stained using DAPI and actin filaments using phalloidin. This procedure confirmed that the spheroid contained tightly packed cells without the formation of cavities (Fig. 1b)
Summary
The contribution of cancer-derived cell lines to the understanding of molecular and cellular processes in the complex physiology of cancer has been immense. Development of novel methods using 3D cell culture techniques for protein structural studies will contribute to a better understanding of the effect of the microenvironment on the structure–function relationship of proteins. Progress has been made to produce isotope-labelled proteins in the context of ssNMR-based studies of human cells and their proteins (Kaplan et al 2016a; Albert et al 2018; Narasimhan et al 2019; Luo et al 2020). The latter advancements formed the conceptual basis for research described below. Our data highlight the potential of conducting ssNMR-based studies on 3D cell cultures that in the future may reveal structure–function relationship of proteins in their native 3D cell microenvironment
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