Leccia, Felicia (2013) Identification of new markers for the characterization and isolation of breast cancer stem cells. [Tesi di dottorato]

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Abstract

Breast cancer is the first human carcinoma for which a putative cancer stem cell subpopulation (BCSC) has been isolated with the CD44+/CD24-/low phenotype. However, several studies have highlighted that CD44+/CD24-/low cannot be considered the universal marker phenotype of BCSCs. The aim of this thesis is to identify novel BCSCs markers. We focused our work on the basal-like breast cancers that seem to derive from a developmental stage of mammary epithelial cell that is different from the primitive stem cell, namely the luminal progenitor. In the first part of the present thesis we have characterized breast cancer cell lines resembling different tumor molecular subtypes, by using polychromatic flow cytometry. These analyses led us to hypothesize that the basal-A cell line HCC1937 could contain a putative CD24+CD338+ CSC subpopulation. We have next found that CD338 overexpressing cells, namely CD338bright, overlap, almost completely, with the side population in the HCC1937 cell line. Since side population is a property of stem/progenitor cells, this result suggested a key role of CD338 in determining stem-like properties of HCC1937 cell line. To explore this hypothesis, we next isolated the putative BCSCs, on the base of CD24 and CD338 expression, and we further studied them to explore their stem-like and tumorigenic properties. We found that CD24hi cells display a higher mammosphere forming efficiency than CD24- cells and, among CD24hi cells, CD338 overexpressing cells are able to form mammospheres with higher efficiency than CD338- cells. This result confirmed a relevant role of CD338 in stem-like properties of HCC1937 cell line. Furthermore, the soft-agar colony assays revealed that CD338 positive cells are transformed in contrary to the negative ones. We next evaluated the tumorigenic potential of the different CD338 sorted subpopulations and we found that cells expressing CD338 at an intermediate level, namely CD338low, were the most tumorigenic ones. Furthermore, the analysis of CD338bright and CD338low sorted sub-populations, after their growth in culture for several weeks, revealed that CD338bright population is able to divide asymmetrically giving rise to its CD338low progeny which, in contrast, is not able to divide asymmetrically. This result suggested that CD338bright population could constitute the most immature population and its CD338low progeny the more differentiated progenitor cells. Taken together, these results strongly support the presence in the HCC1937 cell line of a putative BCSC subpopulation overexpressing CD338 and bearing the phenotype CD44+CD24+CD338bright, i.e. an antigenic combination different from the classical CD44+/CD24-/low. Concurrently to the study of breast cancer cell lines, a complementary work was carried out to standardize the flow-cytomertic methods to study primary cultures established from human breast cancer tissues. This work will be subsequently used to isolate and study the CD44+CD24+CD338bright CSC population from human breast cancer tissues. In this study, we have also tried to determine whether patient-derived breast cancer cell cultures maintain the cellular heterogeneity of primary tissues and may therefore be used for in vitro modeling of breast cancer subtypes. To this aim, we used a much larger vocabulary of surface markers than those used in previous studies to characterize primary cultures. Most of surface antigens analyzed were heterogeneously expressed. On the other hand, breast cancer cell cultures displayed concomitant high expression of the basal marker CD10/CALLA and low expression of CD326/EpCAM. Furthermore, we found that they express CK5, SMA and vimentin, and are weakly positive for CK19 and CK18-negative. Our results, while confirming that in vitro culturing of breast cancer cells reduces luminal lineage-type of cells, indicate the increased propensity for the selection of myoepithelial/basal breast cancer cells. Furthermore, we have demonstrated that breast cancer cell cultures preserve inter-tumor heterogeneity and express stem/progenitor markers that can be identified, quantified and categorized by flow cytometry.

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