This thesis is focused on the metabolomic study of human cancer tissues by ex vivo High
Resolution-Magic Angle Spinning (HR-MAS) nuclear magnetic resonance (NMR) spectroscopy.
This new technique allows for the acquisition of spectra directly on intact tissues (biopsy or
surgery), and it has become very important for integrated metabonomics studies. The objective is to
identify metabolites that can be used as markers for the discrimination of the different types of
cancer, for the grading, and for the assessment of the evolution of the tumour. Furthermore, an
attempt to recognize metabolites, that although involved in the metabolism of tumoral tissues in low
concentration, can be important modulators of neoplastic proliferation, was performed. In addition,
NMR data was integrated with statistical techniques in order to obtain semi-quantitative information
about the metabolite markers. In the case of gliomas, the NMR study was correlated with gene
expression of neoplastic tissues.
Chapter 1 begins with a general description of a new “omics” study, the metabolomics. The
study of metabolism can contribute significantly to biomedical research and, ultimately, to clinical
medical practice. This rapidly developing discipline involves the study of the metabolome: the total
repertoire of small molecules present in cells, tissues, organs, and biological fluids. Metabolomic
approaches are becoming increasingly popular in disease diagnosis and will play an important role
on improving our understanding of cancer mechanism. Chapter 2 addresses in more detail the basis
of NMR Spectroscopy, presenting the new HR-MAS NMR tool, that is gaining importance in the
examination of tumour tissues, and in the assessment of tumour grade. Some advanced
chemometric methods were used in an attempt to enhance the interpretation and quantitative
information of the HR-MAS NMR data are and presented in chapter 3. Chemometric methods
seem to have a high potential in the study of human diseases, as it permits the extraction of new and
relevant information from spectroscopic data, allowing a better interpretation of the results.
Chapter 4 reports results obtained from HR-MAS NMR analyses performed on different
brain tumours: medulloblastoma, meningioms and gliomas. The medulloblastoma study is a case
report of primitive neuroectodermal tumor (PNET) localised in the cerebellar region by Magnetic
Resonance Imaging (MRI) in a 3-year-old child. In vivo single voxel 1H MRS shows high
specificity in detecting the main metabolic alterations in the primitive cerebellar lesion; which
consist of very high amounts of the choline-containing compounds and of very low levels of
creatine derivatives and N-acetylaspartate. Ex vivo HR-MAS NMR, performed at 9.4 Tesla on the
neoplastic specimen collected during surgery, allows the unambiguous identification of several
metabolites giving a more in-depth evaluation of the metabolic pattern of the lesion. The ex vivo
HR-MAS NMR spectra show higher detail than that obtained in vivo. In addition, the spectroscopic
data appear to correlate with some morphological features of the medulloblastoma. The present
study shows that ex vivo HR-MAS 1H NMR is able to strongly improve the clinical possibility of in
vivo MRS and can be used in conjunction with in vivo spectroscopy for clinical purposes.
Three histological subtypes of meningiomas (meningothelial, fibrous and oncocytic) were
analysed both by in vivo and ex vivo MRS experiments. The ex vivo HR-MAS investigations are
very helpful for the assignment of the in vivo resonances of human meningiomas and for the
validation of the quantification procedure of in vivo MR spectra. By using one- and two
dimensional experiments, several metabolites in different histological subtypes of meningiomas,
were identified. The spectroscopic data confirmed the presence of the typical metabolites of these
benign neoplasms and, at the same time, that meningomas with different morphological
characteristics have different metabolic profiles, particularly regarding macromolecules and lipids.
The profile of total choline metabolites (tCho) and the expression of the Kennedy pathway
genes in biopsies of human gliomas were also investigated using HR-MAS NMR, and microfluidic
genomic cards. 1H HR-MAS spectra, allowed the resolution and relative quantification by LCModel
of the resonances from choline (Cho), phosphorylcholine (PC) and glycerolphorylcholine (GPC),
the three main components of the combined tCho peak observed in gliomas by in vivo 1H MRS
spectroscopy. All glioma biopsies depicted an increase in tCho as calculated from the addition of
Cho, PC and GPC HR-MAS resonances. However, the increase was constantly derived from
augmented GPC in low grade NMR gliomas or increased PC content in the high grade gliomas,
respectively. This circumstance allowed the unambiguous discrimination of high and low grade
gliomas by 1H HR-MAS, which could not be achieved by calculating the tCho/Cr ratio commonly
used by in vivo 1H MR spectroscopy. The expression of the genes involved in choline metabolism
was investigated in the same biopsies. The present findings offer a convenient procedure to classify
accurately glioma grade using 1H HR-MAS, providing in addition the genetic background for the
alterations of choline metabolism observed in high and low gliomas grade.
Chapter 5 reports the study on human gastrointestinal tract (stomach and colon) neoplasms.
The human healthy gastric mucosa, and the characteristics of the biochemical profile of human
gastric adenocarcinoma in comparison with that of healthy gastric mucosa were analyzed using ex
vivo HR-MAS NMR. Healthy human mucosa is mainly characterized by the presence of small
metabolites (more than 50 identified) and macromolecules. The adenocarcinoma spectra were
dominated by the presence of signals due to triglycerides, that are usually very low in healthy
gastric mucosa. The use of spin-echo experiments enable us to detect some metabolites in the
unhealthy tissues and to determine their variation with respect to the healthy ones. Then, the ex vivo
HR-MAS NMR analysis was applied to human gastric tissue, to obtain information on the
molecular steps involved in the gastric carcinogenesis. A microscopic investigation was also carried
out in order to identify and locate the lipids in the cellular and extra-cellular environments.
Correlation of the morphological changes detected by transmission (TEM) and scanning (SEM)
electron microscopy, with the metabolic profile of gastric mucosa in healthy, gastric atrophy
autoimmune diseases (AAG), Helicobacter pylori-related gastritis and adenocarcinoma subjects,
were obtained. These ultrastructural studies of AAG and gastric adenocarcinoma revealed lipid
intra- and extra-cellularly accumulation associated with a severe prenecrotic hypoxia and
mitochondrial degeneration.
A deep insight into the metabolic profile of human healthy and neoplastic colon tissues was
gained using ex vivo HR-MAS NMR spectroscopy in combination with multivariate methods:
Principal Component Analysis (PCA) and Partial Least Squares Discriminant Analysis (PLS-DA).
The NMR spectra of healthy tissues highlight different metabolic profiles with respect to those of
neoplastic and microscopically normal colon specimens (these last obtained at least 15 cm far from
the adenocarcinoma). Furthermore, metabolic variations are detected not only for neoplastic tissues
with different histological diagnosis, but also for those classified identical by histological analysis.
These findings suggest that the same subclass of colon carcinoma is characterized, at a certain
degree, by metabolic heterogeneity. The statistical multivariate approach applied to the NMR data
is crucial in order to find metabolic markers of the neoplastic state of colon tissues, and to correctly
classify the samples. Significant different levels of choline containing compounds, taurine and myoinositol,
were observed.
Chapter 6 deals with the metabolic profile of normal and tumoral renal human tissues
obtained by ex vivo HR-MAS NMR. The spectra of human normal cortex and medulla show the
presence of differently distributed osmolytes as markers of physiological renal condition. The
marked decrease or disappearance of these metabolites and the high lipid content (triglycerides and
cholesteryl esters) is typical of clear cell renal carcinoma (RCC), while papillary RCC is
characterized by the absence of lipids and very high amounts of taurine. This research is a
contribution to the biochemical classification of renal neoplastic pathologies, especially for RCCs,
which can be evaluated by in vivo MRS for clinical purposes. Moreover, these data help to gain a
better knowledge of the molecular processes envolved in the onset of renal carcinogenesis.
| Identifer | oai:union.ndltd.org:unibo.it/oai:amsdottorato.cib.unibo.it:647 |
| Date | 09 June 2008 |
| Creators | Righi, Valeria <1979> |
| Contributors | Tugnoli, Vitaliano, Mucci, Adele |
| Publisher | Alma Mater Studiorum - Università di Bologna |
| Source Sets | Università di Bologna |
| Language | English |
| Detected Language | English |
| Type | Doctoral Thesis, PeerReviewed |
| Format | application/pdf |
| Rights | info:eu-repo/semantics/openAccess |
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