Fighting Cancer with Sugars: Towards the Synthesis of Mass Tag Labelled CMP-Sialic Acid Probes to Detect Cancerous Tissue via Mass Spectrometry Imaging
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Authors
Vogt, Pascal M.
Date
Type
thesis
Language
eng
Keyword
sialic acid , mass spectrometry imaging , cancer , carbohydrates , sugars , hematoxylin , eosin , staining
Alternative Title
Abstract
Cancer is one of the biggest global health problems causing several million deaths annually. Efforts to improve early detection of cancer at stage 0 or I may save more human lives than treatments for late-stage cancer (stage II to IV). A standard technique for cancer detection is hematoxylin and eosin (H&E) staining of tissue biopsies. While the technique is relatively fast to determine cancerous tumours in tissue specimens, it is also unspecific, requires pathologists to conduct assays and analyze results, and thus is subject to human error. Here, we propose a more specific and robust approach using mass spectrometry imaging (MSI) to detect cancerous tumours in tissue specimens. MSI offers improved spatial resolution of tissue specimens and requires fewer steps in sample preparation for analysis. Our approach uses cytidine-5’-monophosphate sialic acid (CMP-Sia) probes bearing mass tags for mass spectrometric detection (MS-tags) and sialyltransferases (STs) that selectively add the CMP-Sia probes onto cancer-associated carbohydrate antigens. Once the antigens are labelled, the MS-tags will be photocleaved by 355 nm laser irradiation and detected by mass spectrometry (MS). The MS-tags provide simple MS spectra, high sensitivities, and are the keystone in our MS-supported approach to image tissue specimens and reveal tumour margins. Six MS-tag derivatives based on 6,11-dihydrothio-chromeno[4,3-b]indole bearing an alkyl group of one to four carbon atoms were synthesized chemically. Four spacers with either a terminal alkyne or azide group were installed on the ethyl MS-tag. The CMP-Sia probes bearing linkers with either a terminal azide or alkyne were chemoenzymatically synthesized using a one-pot three-enzyme synthesis and will be conjugated to the alkyne- or azide-modified MS-tags through copper-catalyzed azide-alkyne cycloaddition (CuAAC). The STs ST3Gal2 and ST6GalNAc1 were expressed in mammalian Expi293 cells and will be used to introduce the MS-tag labelled CMP-Sia probes to cancer-associated carbohydrate antigens. Our approach will be generalized to a platform methodology using several STs to label different cancer-associated antigens with MS-tags for MSI. We intend to introduce our method in cancer screening of tissue specimens and make H&E staining obsolete. Additionally, our method may support surgeons to locate and remove cancerous tumours more exactly during surgery.
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ProQuest PhD and Master's Theses International Dissemination Agreement
Intellectual Property Guidelines at Queen's University
Copying and Preserving Your Thesis
This publication is made available by the authority of the copyright owner solely for the purpose of private study and research and may not be copied or reproduced except as permitted by the copyright laws without written authority from the copyright owner.
Attribution-NonCommercial 4.0 International