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

Reinier Damman; Alessandra Lucini Paioni; Katerina T. Xenaki; Irati Beltrán Hernández; Paul M.P. van Bergen en Henegouwen; Marc Baldus

doi:10.1007/s10858-020-00328-8

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

Reinier Damman
, Alessandra Lucini Paioni
, Katerina T. Xenaki
, Irati Beltrán Hernández
, Paul M.P. van Bergen en Henegouwen
, Marc Baldus^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › Academic › peer-review

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.

Original language	English
Pages (from-to)	401-412
Number of pages	12
Journal	Journal of Biomolecular NMR
Volume	74
Issue number	8-9
DOIs	https://doi.org/10.1007/s10858-020-00328-8
Publication status	Published - 19 Jun 2020

Funding

We thank Johan van der Zwan and Dr. Klaartje Houben for technical assistance and Dr. Hugo van Ingen for providing access to the solution-state NMR instrument. We are indebted to Professor Paul Tordo and Dr. Olivier Ouari (Aix-Marseille University, Marseille) for providing AMUPol. This work received financial support from the Dutch Science Foundation NWO (Grant numbers 718.015.001 and 718.018.008 to M.B.) and from iNEXT-Discovery, project number 871037, a project funded by the Horizon 2020 program of the European Commission. Experiments at the 950 MHz instrument were supported by uNMR-NL, an NWO-funded Roadmap NMR Facility (No. 184.032.207).

Keywords

DNP
EGFR
In-cell NMR
Nanobodies
Solid-state NMR
Spheroids

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title = "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.",

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author = "Reinier Damman and \{Lucini Paioni\}, Alessandra and Xenaki, \{Katerina T.\} and \{Beltr{\'a}n Hern{\'a}ndez\}, Irati and \{van Bergen en Henegouwen\}, \{Paul M.P.\} and Marc Baldus",

year = "2020",

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doi = "10.1007/s10858-020-00328-8",

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AU - Beltrán Hernández, Irati

AU - van Bergen en Henegouwen, Paul M.P.

AU - Baldus, Marc

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N2 - 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.

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

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