Untargeted Metabolite Mapping in 3D Cell Culture Models Using High Spectral Resolution FT-ICR Mass Spectrometry Imaging

Abstract

Multicellular tumor spheroids (MTS) are a well-established model system for drug development and are a valuable in vitro research tool for use prior to employing animal models. These 3D-cell cultures are thought to display chemical gradients of oxygen and nutrients throughout their structure, giving rise to distinct microenvironments in radial layers, thus mimicking the pathophysiological environment of a tumor. Little is known about the localized distributions of metabolites within these microenvironments. To address this, here we utilize high spectral resolution Fourier-transform ion cyclotron resonance (FT-ICR), MALDI mass spectrometry imaging (MSI) to image the distribution of endogenous metabolites in breast cancer MCF-7 spheroids. We show that known specific metabolite markers (adenosine phosphates and glutathione) indicate that the central region of these cell culture models experiences increased hypoxic and oxidative stress. By using discriminatory analysis, we have identified which m/z values localize towards the outer proliferative or central hypoxic regions of an MTS and have assigned elemental formula with sub-ppm error. Using this information, we have mapped these metabolites back to distinct pathways to improve our understanding of the molecular environment and biochemistry of these tumor models.

Characterization of an Aggregated Three-Dimensional Cell Culture Model by Multimodal Mass Spectrometry Imaging

“…Proteins including Histone H4 and cytochrome c were localized across the spheroid, and a specific, unidentified peak ( m / z 12 828) was localized within the necrotic core. More recently, Tucker et al 21 employed high spectral resolution Fourier transform-ion cyclotron resonance (FT-ICR) MALDI-MSI to determine metabolite distributions in MCF-7 breast cancer spheroids. The group mapped metabolites associated with hypoxia through biochemical processes including glycolysis and the hexosamine biosynthetic pathway.…”

A Critical and Concise Review of Mass Spectrometry Applied to Imaging in Drug Discovery

“…61 MSI can be used to understand advanced drug delivery, such as tracking the distribution and release from an encapsulation form of the drug from delivery particles that help target specific tissues or protect it from early degradation or metabolism. 62 Further uses include blood-brain barrier penetration, 18,63 imaging microphysiological systems and 3D cell cultures [64][65][66][67] (Fig. 2), assessing tumor hypoxia, 56 and clinical translation and application.…”

“…Chem., 2019. 67 alongside the presence of drug and metabolite. The data were acquired at either 50 µm for the imaging MS and tandem MS (MS/MS) repeat analysis of the single-dosed tissues or 100 µm for the imaging of all dosing conditions in positive-and negative-ion modes.…”

MS imaging of multicellular tumor spheroids and organoids as an emerging tool for personalized medicine and drug discovery

“…16 In the succeeding decade, there have been numerous reports of MSI applied to MCTS, both to explore the underlying biology and to evaluate the penetration and metabolism of new drug compounds. [17][18][19][20][21] This review will focus primarily on the application of MSI to the MCTS model. We will also briefly introduce a novel model system, the patient-derived organoid, as the next generation platform for these types of studies.…”