Development of in vitro-grown spheroids as a 3D tumor model system for solid-state NMR spectroscopy
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ARTICLE
Development of in vitro‑grown spheroids as a 3D tumor model system for solid‑state NMR spectroscopy Reinier Damman1,2 · Alessandra Lucini Paioni1 · Katerina T. Xenaki2 · Irati Beltrán Hernández2,3 · Paul M. P. van Bergen en Henegouwen2 · Marc Baldus1 Received: 23 March 2020 / Accepted: 9 June 2020 © The Author(s) 2020
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. Keywords Solid-state NMR · In-cell NMR · Spheroids · DNP · EGFR · Nanobodies
Introduction The contribution of cancer-derived cell lines to the understanding of molecular and cellular processes in the complex physiology of cancer has been immense. However, a full translation of the acquired knowledge towards patients can be challenging as the tumor microenvironment or tumor stroma is much more complex than an in vitro monolayer culture. Due to the globular shape of a tumor, concentration * Paul M. P. van Bergen en Henegouwen [email protected] * Marc Baldus [email protected] 1
NMR Spectroscopy, Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands
2
Cell Biology, Neurobiology and Biophysics, Department of Biology, Faculty of Science, Utrecht University, 3584 CH Utrecht, The Netherlands
3
Pharmaceutics, Department of Pharmaceutical Sciences, Utrecht University, Universiteitsweg 99, 3584 CG Utrecht, The Netherlands
gradients exist for oxygen and nutrients, resulting in a different availability for the proliferative outside layer as compared to the core (Kim and Wu 2012). Moreover, as a result of the Warburg effect, synthesis of lactate, the end product of glycolysis, induces an acidification of the tumor and consequently results in altered gene expression and changes in the proteome. Eventually, as a result of these conditions, cells in the inner core die and form a necrotic core. Furthermore, the extracellular matrix (ECM) within the stroma forms a dense network of fibrils in which the tumor is embedded, d
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