High spectral resolution mass spectrometry imaging of three-dimensional cell culture
Tucker, Louise Helen
Three-dimensional (3D) cell culture combines the simplicity of two-dimensional (2D) cell culture systems with the complex interplay of factors resembling the multifaceted physiology of tissues in vivo. These microscale spherical cell clusters – known as multicellular tumour spheroids (MTS) – replicate the oxygen, nutrient, and waste gradients observed within tumours, and provide useful model systems to improve our understanding of cancer biology. Matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry imaging (MSI) is an analytical technique that permits broad spectral and label-free analysis to observe the distribution of compounds without requiring any significant prior knowledge. MALDI-MSI can be used as a global untargeted approach to elucidate the various microenvironments within MTS at high spatial resolution. Here, method development for MALDI-MSI of MTS will be reported. Breast cancer (MCF-7) and prostate cancer (PC3) spheroids were grown to diameters of approximately 500 μm using the hanging drop method. For MALDI imaging, the spheroids were embedded in gelatin, cryosectioned, and coated with a matrix. Using the optimised protocol, up to eight spheroids were embedded in a gelatin block, and up to 100 spheroid sections were mounted onto a slide. To discern the ionisable metabolome of an MCF-7 spheroid, MALDI mass spectrometry (MS) analysis was employed to compile a list of tentative metabolite identifications. Using various matrices in both polarities, over 760 tentative formulae were assigned at sub-ppm errors. A targeted MALDI-MSI approach suggested that adenosine triphosphate (ATP), adenosine diphosphate (ADP), and glutathione can be used as metabolite markers to indicate regions of increased oxidative stress and hypoxia. ATP was found to be primarily localised to the outer region, whereas ADP was more uniformly distributed, suggesting there is a decreasing oxygen gradient through the spheroid. Subsequently, an untargeted approach of discriminatory analysis tentatively identified the metabolites that colocalised to these areas. The assignments were used to investigate the regional flux through specific metabolic branch pathways. The hexosamine biosynthetic pathway (HBP) was found to be upregulated in the regions of the spheroid with greater access to oxygen, whereas there was greater glycolytic flux within the regions limited by hypoxia. MALDI-MSI is useful for elucidating the absorption, distribution, metabolism, and excretion (ADME) of drugs within MTS. Therefore, the developed protocol was employed to observe the time-dependent distribution of the hypoxia marker pimonidazole within PC3 spheroids. Due to the low signal-to-noise (S/N) of pimonidazole and its metabolites, continual accumulation of ions (CASI) was used to effectively lower the limit of detection and increase the signal intensities. Over 24 hours, pimonidazole was distributed throughout the spheroid and underwent reduction. Furthermore, its reduction derivatives showed a central localisation throughout the time course, suggestive of a hypoxic core. Finally, a 3D printer and other parts commonly found in an analytical chemistry lab were employed as a low-cost alternative to commercial sprayers for matrix deposition. Using printed rhodamine B microarrays and fluorescence imaging, matrix application conditions were optimised to effectively reduce delocalisation from 403% to 9.4%. Subsequently, MALDI-MSI of MTS was used to compare the optimised conditions of the home-built sprayer to a commercially available matrix application platform. Using this system, robust and reproducible distributions of endogenous metabolite distributions with a high spatial resolution were observed.