Design of Aptamer-Functionalized Substrates: Towards Breast Cancer Stem Cell Isolation and Detection

Abstract

Cancer relapse and metastasis remain one of the main problems in treatment of breast cancer (BC). A small subset from bulk tumor cells, called breast cancer stem cells (BCSCs), is found to be responsible for cancer initiation, recurrence, metastasis and resistance to therapy. Therefore, specifically detecting these cells is an important task in BC diagnosis and management. The main goal of this thesis was to develop aptamer-functionalized substrates which in the future could be used for BCSC isolation and detection. To achieve this objective, the project has been divided into three tasks as will be discussed below. Given small number of available specific ligands against BCSC and their importance in BC, one of the tasks of this thesis was to select and characterize new single stranded DNA aptamers against BCSC. Fluorescently activated cell sorting was utilized to enrich oligonucleotides bound to cells while imaging flow cytometry was used to study their binding. Two of the selected aptamers showed increased binding to target cells than to control cells; however, their binding affinity was not fully studied. They are one of the few ligands reported to date to bind BCSC and were selected against well characterized BCSC derived from a triple-negative breast cancer. Another task of this work was to functionalize stainless steel (SS) wire with aptamers specific to BCSC in order to alleviate the problem of “fishing out” such rare events as BCSC. For this, the wire electropolishing conditions were determined. In order to attach ligand, silanization by electrodeposition was optimized thus determining the most suitable applied potential (–0.8 V), pH of the solution (pH 5 and 5.5) and heat treatment temperature after electrodeposition (130°C). The silanized surface was then immobilized with commercially available CD44 aptamers (marker of BCSC) after being activated by a crosslinker to build a functionalized surface. This wire was able to capture the target cells in an in vitro test. The wires were analyzed by such surface characterization methods as atomic force microscopy (AFM), cyclic voltammetry (CV), scanning electron microscopy (SEM) and fluorescence microscopy. In addition, using the same surface chemistry as in functionalized SS wire, another platform – fiber Bragg grating (FBG) sensor has been explored with a well-studied ligand-analyte pair (thrombin and thrombin-binding aptamer). For this, FBG was made sensitive to the surrounding refractive index (RI) by chemical etching and calibrated in solutions with known RI before being functionalized with aptamers. Then the sensor demonstrated increased Bragg wavelength shift when tested in different thrombin concentrations. In conclusion, the main goal of this thesis – developing aptamer-functionalized substrates with a perspective application in BCSC isolation and detection – was achieved, although each task of the project was completed with different level of success. Binding of aptamers selected against BCSC could not be fully studied. However, they are one of the few reported aptamers against an important subtype of BC. Besides, only a small fraction of aptamer candidates were characterized and better binders could still be revealed. Wires functionalized with CD44 aptamers, after further study, have a potential to be used for in vivo capture of target cells in the blood flow, since their small size allows the insertion as a standard guidewire in biomedical devices. For fabricated EGBF biosensor, selective detection of clinically relevant concentration of thrombin has been demonstrated. The used functionalization method allows a facile fabrication of the sensor not requiring thin film fabrication.