Differentiation of tissue- and devlopment of tumor stem cells

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Cells comprising tissues and organs are in a constant state of turnover. Senescent, differentiated cells are removed by cell shedding or apoptosis, and are replaced by cells derived from tissue specific stem or progenitor cells. Similarly, tumors are composed of terminally differentiated as well as stem cell like cells. This latter subpopulation is believed to drive tumor growth and to underly resistance to therapy. Although well established as a concept, tumor stem cells are notoriously difficult to identify. So far no unique marker for cancer stem cells has been described. Furthermore, the cell of origin of tumor stem cells remains unclear. Stem cells are defined as cells that can undergo self renewing symmetric and differentiating asymmetric mitosis. They can differentiate into several specialized cell types. Though stable markers are unavailable, tissue stem cells can be identified by surface markers or by expression of transcription factors such as Oct4, nanog, or Sox2. Stem cells reside in a highly specialized microenvironment, the stem cell niche, which tightly regulates stem cell maintenance, proliferation, and differentiation. Signaling molecules described in the regulation of stem cell behavior include Wnt, Notch, FGF-2, and TGF-beta. TGF-beta is a pleiotropic cytokine that is involved in developmental processes and in tissue maintenance and repair. TGF-beta signals through several intracellular signaling cascades such as the Smad2/3 pathway, and MAPK pathways, and extensively interacts with other signaling cascades such as PI3K/mTOR and BMP signaling, and as such participates in a signaling network that regulates differentiation, survival, proliferation and apoptosis of stem cells and differentiated cells. Additionally, TGF-beta influences cell biology by altering the composition of the extracellular matrix; generally, TGF-beta increases matrix rigidity via increased stromal collagen production. It has further been shown that stem cell differentiation is strongly influenced by the matrix rigidity itself. In addition to these factors, nuclear restricted protein/brain (NRP/B) has been shown to play a critical regulatory role in malignant transformation of various tumors and to be overexpressed in differentiated embryonic stem cells. In order to work with stable culture of tumor stem cells, we need a tool to analyze stem cell characteristics of a given cell population in the absence of a robust marker. In our hands, FACS-sorting cell lines and primary cells for CD15, CD24, CD44, CD133, and aldehyde dehydrogenase activity did not cells with stem cells properties. Our next step will be to use fluorescence protein expressing reporter plasmids for transcription factors that are expressed in tissue stem cells such as Oct4 and nanog to identify stem cell like subpopulation in epithelial cells, stromal cells, and tumor cells. We recently established the isolation and culture of human breast epithelial and stromal cells under serum free conditions. Currently, we identify subpopulations in these cultures by immunofluorescence and FACS analysis, and optimize the culture conditions to extend the culture time during which cells remain in a stable morphological phenotype. To test the influence of substances such as vitamins, amino acids, or lipids on cell proliferation and morphology we employ high throughput imaging (Opera system) that enables us to investigate cell number, cell vitality / death, cell morphology, and expression of proteins from the same cell population. Using a human breast cancer cell line we observed asymmetric activation of TGF-beta in a subset of mitotic tumor cells. These data imply that TGF-beta may determine the fate of daughter cells during stem cell mitosis. However, since since stable markers for tumor stem cells are not yet identified, we currently employ cortical neuronal stem cells (NSC) to investigate the influence of TGF-beta on stem cell mitosis and differentiation in collaboration with Ron McKay. We were able to demonstrate by immunofluorescence that NSCs endogenously express active TGF-beta, and that TGF-beta decreases NSC death in a concentration dependent fashion, while the ALK5 inhibitor SB431542 increased cell death. We will next investigate the role of the TGF-beta signaling network on NSC differentiation using high throughput imaging techniques (Opera system) and time lapse imaging (fluorescence / phase contrast / DIC) which are established and available in the McKay lab. We will use exogenous TGF-beta and small molecule inhibitors to influence signaling cascades. Cell proliferation, differentiation, and activation of signaling cascades will be monitored by using fluorescent vital dyes, fluorescent protein coupled reporter assays, and immunocytohemistry / immunofluorescence. Using these methods we aim to identify not only if TGF-beta affects stem cells differentiation, but also in which time frame signaling is activated. All methods can be readily applied to other types of stem cells, in particular breast tissue/tumor stem cells as soon as those are identified. Since matrix elasticity influences stem cells differentiation on on side, and is involved in tumorigenesis and tumor progression in breast cancer on the other side, we here aim to identify whether matrix elasticity influences the tumor progression by altering the size of the tumor stem cell pool. We established protocols for generating acrylamide and silicone matrices of defined elasticity that can be functionalized with matrix components such as fibronectin. Preliminary experiments using cell lines indicate that with increasing matrix density breast epithelial cells indeed show alterations of protein expression that are typically found in tumor cells. We will now investigate the biological relevance of matrix-related changes of the stem cell, transiently amplifying cell, and progenitor cell populations. Particularly, we will ask whether matrix density increases malignancy in epithelial cells using the already established MCF10A model, and if this is due to the expansion of a stem cell like cell pool. We will furthermore investigate if tissue stem cell like cells can give rise to tumor cell populations if grown on a matrix of inappropriate density for the tissue (e.g. if neuronal stem cell are grown on matrix of the rigidity of bone). We have also begun to establish the following projects: Using the Smad null mouse model, we will identify the specific alteration(s) or modification(s) of heterochromatin structure between normal and breast cancer stem cells. We further plan to identify the involved in the maintenance of stem cell proliferation and differentation by utilizing microarray and Affymetrix analysis of mammary epithelial and hematopoetic stem cells obtained from Smad wild type and knockout mice. Molecular/biochemical mechanisms of the target molecule(s) will be determined and in vivo studies are planned to verify the significance of the identified molecules on the maintenance of stem cell properties. Finally, we will study the molecular mechanism of NRP/B's action on the differentiation of embryonic stem cell. NRP/B inducible knockout mice will be generated and used for the biological function of NRP/B on the embryonic development and stem cell differentiation.