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In Vitro Selections of Mammaglobin B and Mammaglobin A Aptamers for detecting Metastatic Breast Cancer Cells Using Terahertz Chemical Microscopy.

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Hassan, Eman (2017). In Vitro Selections of Mammaglobin B and Mammaglobin A Aptamers for detecting Metastatic Breast Cancer Cells Using Terahertz Chemical Microscopy. Thèse. Québec, Université du Québec, Institut national de la recherche scientifique, Doctorat en sciences de l'énergie et des matériaux, 244 p.

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Résumé

Breast cancer is the most common cancer diagnosed in Canadian women, accounting for 26% of all newly diagnosed cancers in women. Metastatic breast cancer (MBC) is the stage of cancer where the disease has spread to distant parts of the body, further than the axillary lymph nodes. Breast cancer cells that leave the primary tumor to the blood are called circulating tumor cells (CTCs). Studying CTCs allows for better understanding of the metastases process. The detection of CTCs is a challenging task due to the heterogeneous nature of CTCs and lack of biomarkers. There is an urgent need to identify more biomarkers to help in the detection of CTCs from breast cancer. Mammaglobin B (MGB2) and mammaglobin A (MGB1) proteins are small secretory proteins that are believed to have roles in cancer development, immune system regulation, and androgen binding. MGB2 is found at high levels in many secretions, including those from uterine, prostatic, pulmonary, and lacrimal and salivary glands, whereas MGB1 expression is exclusive to the breast tissue. Since both proteins are overexpressed in most cases of metastatic breast cancer,, MGB2 and MGB1 are very attractive biomarkers that could be used in the detection of breast cancer and breast CTCs. Aptamers are powerful recognition elements. They are single-stranded ssDNA or RNA oligonucleotides that bind to their targets with high affinity and selectivity. Aptamers are chemically produced via a process called Systematic Evolution of Ligands by EXponential enrichment (SELEX). SELEX is an iterative in vitro processwhich enriches target-binding sequencesfrom randomized large ssDNA or RNA libraries. In each round of SELEX, target–binding sequences are eluted from target molecules, amplified, and used as the library for subsequent rounds. This process results in an aptamer with high affinity and specificity for the target. In this work, a group of aptamers against MGB2 and MGB1 proteins were developed for the first time using a unique type of SELEX called hybrid SELEX, in which the ssDNA library was alternated between two targets that are the recombinant form of both MGB2, and MGB1 proteins and breast cancer cells MCF7 and MDA-MB-415 respectively. All selected aptamers were then tested for their affinity by determining the dissociation constant (Kd) using flow cytometry. Aptamers were tested for their selectivity by testing their affinities to other normal and cancer cell lines. Aptamers for both targets that had the highest affinity and selectivity were further tested for their binding affinity to their target cancer cell lines in plasma and whole blood lysate environments using flow cytometry. Aptamers for both targets were tested for their binding affinity in blood containing normal blood cells and spiked with different percentages of target breast cancer cells. The binding affinity of the selected aptamers was tested against their target proteins using Electrophoretic Mobility Shift Assay (EMSA). Finally, and for the first time, the selected aptamers against the two targets were tested to detect breast cancer cells using Terahertz Chemical Microscopy (TCM). Results obtained by this study revealed the successful selection of a group of aptamers against MGB2 and MGB1 proteins. Among all the selected aptamers, mammaglobin B1 (MAMB1) against MGB2 (MCF7 cell line), and mammaglobin A2 (MAMA2) against MGB1 (MDA-MB-415 cell line) showed the highest affinities to their targets (as indicated by their Kd values). Moreover, these aptamers showed minimal binding to normal and other cancer cell lines, indicating the high selectivity to their target breast cancer cells. Plasma and whole blood lysate results showed that MAMB1 and MAMA2 aptamers bound specifically to MCF7 and MDA-MB-415 cells respectively. MAMB1 showed higher affinity to MCF7 than MAMA2 showed to MDA-MB-415 cells. Moreover, the study with spiked breast cancer cells showed an increase in the fluorescence intensity with increasing percentages of spiked cells for both aptamers, indicating the great potential to use both aptamers in the detection of breast CTCs. TCM results showed that terahertz (THz) amplitude was higher when MAMB1 was interacting with MCF7 cells and MAMA2 with MDA-MB-415, compared to the control on the surface of the sensing plate. Moreover, TCM could detect the interaction of a low number of cells (10 cells) binding to their aptamers. MAMB1 aptamer was more selective than MAMA2 aptamer, as shown by TCM. Overall results obtained by flow cytometry and TCM were very promising and indicate the possibility of using MAMB1 and MAMA2 aptamers in the detection of breast cancer and breast CTCs.

Type de document: Thèse Thèse
Directeur de mémoire/thèse: Osaki, Tsuneyuki
Co-directeurs de mémoire/thèse: DeRosa, Maria; Willmore, Bill
Mots-clés libres: énergie matériaux
Centre: Centre Énergie Matériaux Télécommunications
Date de dépôt: 09 avr. 2019 21:12
Dernière modification: 09 avr. 2019 21:12
URI: http://espace.inrs.ca/id/eprint/8023

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