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.

Dynamic Nuclear Polarization for Sensitivity Enhancement in Biomolecular Solid-State NMR

“…For example, in a collaborative effort by van Bergen en Henegouwen and Baldus, an approach to combine 3D tumor model systems (spheroids) with DNP was presented. Their findings showed that the three-dimensional cellular environment is intact after rotation at low MAS frequencies of up to 5 kHz and the introduction of EGFR-specific nanobodies could be followed by DNP within the spheroids . Very recently, a proof-of-concept study utilized fluorescence-activated cell sorting (FACS) by flow cytometry to enhance signal intensity further by deselecting nonfluorescent and thus inactive cells without GFP production .…”

Biomolecular and Biological Applications of Solid-State NMR with Dynamic Nuclear Polarization Enhancement

“…A 13 C, 15 N-labeled nanobody (single domain antibody) known to bind with high affinity to EGFR, was incorporated to the unlabeled spheroids. Despite the low amount of bound nanobody in the spheroid (only about 50% of the EGFR amount), DNP-enhanced 2D 13 C– 13 C DQ-SQ correlation experiments were obtained showing specific resonances from the nanobody, illustrating the feasibility and potential of the approach …”

A review of manufacturing capabilities of cell spheroid generation technologies and future development

“…Overall, hanging drop is an easy to use method and can be used to generate cell spheroids with good control over spheroid size in the laboratories (Damman et al, 2020). It is scalable as users can easily transfer their process from manual pipetting to liquid handling stations.…”