|dc.contributor.author||Tucker, Louise Helen||
|dc.description.abstract||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.||en
|dc.contributor.sponsor||Engineering and Physical Sciences Research Council (EPSRC)||en
|dc.publisher||The University of Edinburgh||en
|dc.relation.hasversion||Tucker, L. H.; Conde-González, A.; Cobice, D.; Hamm, G. R.; Goodwin, R. J. A.; Campbell, C. J.; Clarke, D. J.; Mackay, C. L. Anal. Chem. 2018, 90 (15), 8742– 8749.||en
|dc.subject||three-dimensional cell cultures||en
|dc.subject||3D cell culture||en
|dc.subject||multicellular tumour spheroids||en
|dc.subject||mass spectrometry imaging||en
|dc.subject||hexosamine biosynthetic pathway||en
|dc.title||High spectral resolution mass spectrometry imaging of three-dimensional cell culture||en
|dc.type||Thesis or Dissertation||en
|dc.type.qualificationname||PhD Doctor of Philosophy||en