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Gur, Hatice Deniz

Cell and Molecular Biology Program

2008

M.S.

In the United States, breast cancer is the second leading cause of cancer-related deaths in women. The overall mortality for breast cancer has been declining recently due to advances in early detection and prevention. However the survival of patients with metastatic disease has not changed significantly over the past several decades despite the extensive effort to develop targeted therapies with enhanced specificity and decreased toxicity. One possible explanation came from recent research applying biological information coming from stem cell studies to cancer. These studies support the presence of a small population of cancer cells within tumors, sharing the signature characteristics of adult stem cells: the ability to self renew and to differentiate. In breast cancer, researchers have isolated 'breast cancer stem cells' which were capable of recreating the tumor in vivo. Generated new tumors contained both additional numbers of cancer stem cells and diverse mixed populations of cells present in the initial tumor, supporting the intriguing self renewal and differentiation characteristics. Moreover, as few as 100 cells with the phenotype of 'breast cancer stem cells' were able to form tumors in mice, whereas tens of thousands of cells with alternate phenotypes failed to form tumors. In addition to their self renewal and differentiation ability, cancer stem cells are thought to be quiescent, infrequently dividing cells with a natural resistance to chemotherapeutic agents. The implication of these studies is that therapies which effectively treat the majority of tumor cells but 'miss' the stem cell population will fail while therapies directed at cancer stem cells can potentially eradicate tumors. This dissertation was focused on identifying breast cancer stem cell specific ligands that can distinguish these cells from the remaining cell population of the tumor using phage display technology. A antibody phage library was used to search for phage displayed-single chain antibodies (scFv) with selective affinity to specific targets on breast cancer stem cells. A set of ligands binding preferentially to the cancer stem cell population isolated from SUM159PT cancer cell line was identified. Two of the identified clones bound selectively to cancer stem cells compared to non-stem cancer cells. Importantly these clones could be used to identify cancer stem cells in paraffin embedded sections. Their applicability to paraffin blocks made them good candidates to be used in archived breast cancer specimens. They can be used to determine the cancer stem cell status of tumors which has both diagnostic and prognostic values. Screening the antibody library on live breast cancer stem cells revealed another phage clone which preferentially bound to cancer stem cells compared to non-stem cancer cells. Another focus of the study was to identify clones that were selectively endocytosed by cancer stem cells. Recovering phage clones from within cancer stem cells could increase not only selection efficiency, but could also generate clones that have more desirable biological properties. Such clones could possibly mediate intracellular drug delivery, induction of apoptosis or inhibition of proliferation. With this panning approach, 11 clones were recovered and identified as clones that were internalized by cancer stem cells. The set of clones identified as selective binders to breast cancer stem cell specific targets will be useful in accelerating steps involved in development of cancer stem cell targeted therapy.