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Development of in vitro-grown spheroids as a 3D tumor model system for solid-state NMR spectroscopy.

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.

SUBMITTER: Damman R

PROVIDER: S-EPMC7508937 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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Development of in vitro-grown spheroids as a 3D tumor model system for solid-state NMR spectroscopy.

Damman Reinier R Lucini Paioni Alessandra A Xenaki Katerina T KT Beltrán Hernández Irati I van Bergen En Henegouwen Paul M P PMP Baldus Marc M

Journal of biomolecular NMR 20200619 8-9

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 ...[more]

PMID: 32562030

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Project description:Quadrupolar solid-state NMR carries a wealth of structural information, including insights about chemical environments arising through the determination of local coupling parameters. Current methods can successfully resolve these parameters for individual sites using sample-spinning methods techniques applicable to quadrupolar I ≥ 1 nuclei, provided second-order central transition broadenings do not exceed by much the spinning rate. For large quadrupolar coupling (C Q) values, however, static acquisitions are often preferable, leading to challenges in extracting local structural information. This study explores the use of two-dimensional QUadrupolar Isotope Correlation SpectroscopY (QUICSY) experiments as a means to increase the NMR spectral resolution and enrich the characterization of quadrupolar NMR patterns under static conditions. QUICSY seeks to correlate the solid-state NMR powder line shapes for two quadrupolar isotopes belonging to the same element via a 2D experiment. In general, two isotopes of the same element will have different nuclear quadrupole moments, gyromagnetic ratios, and spin numbers but essentially identical chemical environments. The possibility then arises of obtaining sharp "ridges" in these 2D correlations, even in static samples showing large quadrupolar effects, which lead to second-order line shapes that are several kilohertz wide. Moreover, pairs of quadrupolar isotopes are recurrent in the periodic table and include important elements such as 35,37Cl, 69,71Ga, 79,81Br, and 85,87Rb. The potential of this approach is explored theoretically and experimentally on two rubidium-containing salts: RbClO4 and Rb2SO4. We find that each compound gives rise to distinctive 2D QUICSY line shapes, depending on the quadrupolar and chemical shift anisotropy (CSA) parameters of its sites. These experimental line shapes show good agreement with analytically derived 2D spectra relying on literature values of the quadrupolar and CSA tensors of these compounds. The approach underlined here paves the way toward better characterization of wideline NMR spectra of quadrupolar nuclei possessing different nuclear isotopes.

Project description:Structural information about the coordination environment of calcium present in bone is highly valuable in understanding the role of calcium in bone formation, biomineralization, and bone diseases like osteoporosis. While a high-resolution structural study on bone has been considered to be extremely challenging, NMR studies on model compounds and bone minerals have provided valuable insight into the structure of bone. Particularly, the recent demonstration of (43)Ca solid-state NMR experiments on model compounds is an important advance in this field. However, application of (43)Ca NMR is hampered due to the low natural-abundance and poor sensitivity of (43)Ca. In this study, we report the first demonstration of natural-abundance (43)Ca magic angle spinning (MAS) NMR experiments on bone, using powdered bovine cortical bone samples. (43)Ca NMR spectra of bovine cortical bone are analyzed by comparing to the natural-abundance (43)Ca NMR spectra of model compounds including hydroxyapatite and carbonated apatite. While (43)Ca NMR spectra of hydroxyapatite and carbonated apatite are very similar, they significantly differ from those of cortical bone. Raman spectroscopy shows that the calcium environment in bone is more similar to carbonated apatite than hydroxyapatite. A close analysis of (43)Ca NMR spectra reveals that the chemical shift frequencies of cortical bone and 10% carbonated apatite are similar but the quadrupole coupling constant of cortical bone is larger than that measured for model compounds. In addition, our results suggest that an increase in the carbonate concentration decreases the observed (43)Ca chemical shift frequency. A comparison of experimentally obtained (43)Ca MAS spectra with simulations reveal a 3:4 mol ratio of Ca-I/Ca-II sites in carbonated apatite and a 2.3:3 mol ratio for hydroxyapatite. 2D triple-quantum (43)Ca MAS experiments performed on a mixture of carbonated apatite and the bone protein osteocalcin reveal the presence of protein-bound and free calcium sites, which is in agreement with a model developed from X-ray crystal structure of the protein.

Dataset Information

Development of in vitro-grown spheroids as a 3D tumor model system for solid-state NMR spectroscopy.

Publications

Development of in vitro-grown spheroids as a 3D tumor model system for solid-state NMR spectroscopy.

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