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Abstract

In pancreatic cancer and other solid tumor entities subpopulations of cancer stem cells (CSCs) or tumor initiating cells (TIC) have recently been identified. These cells were described to be tumorigenic in immunodeficient mice and give rise to the whole heterogeneity of the patient`s tumor. Besides those phenotypic markers previously associated with TIC function, little is known about pancreatic TIC. This thesis project unravels the clonal dynamics of long-term tumor growth in pancreatic cancer, and explores the phenotypic diversity within the pancreatic TIC population. Primary patient tumor samples were xenografted in immunodeficient NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice to remove benign human cells and receive sufficient amounts of tissue. Adherent cultures were established from xenograft tumor tissue in serum-free medium simulated with growth factors. These culture conditions allowed the enrichment of pancreatic TIC without restriction to a certain phenotype. These cultures grew as three-dimensional epithelial colonies with tight cell-cell contacts, and reliably initiated tumors in NSG mice. In order to induce TIC differentiation, culture conditions were changed to 10% FBS containing medium and withdrawal of cytokines. Subsequently, the cells lost three-dimensional growth, formed monolayers and showed irregular morphology. This was accompanied by a down-regulation of markers previously described for pancreatic TIC, stem cells of various entities or normal pancreatic progenitors. However, despite this differentiation-like phenotype, tumor initiation in serial transplantation was not substantially affected. Moreover, sorted CD133- cells formed equally efficient tumors as the CD133+ cell fraction and contained a similar proportion of CD133+ cells in vivo. In sum, these data indicate that pancreatic TIC are diverse with respect to marker expression, and exhibit an previously unknown phenotypic plasticity. To determine the clonal kinetics of individual TIC in vivo early passage serum-free cultures from 3 patients were lentivirally marked and serially transplanted over 3 generations in NSG mice. In primary mice, 0.003-0.113% of all transduced cells were detected to contribute significantly to tumor formation. However, the second and third generation tumor formation was predominantly driven by distinct TIC clones that were not detected in earlier generations, but recruited later to participate in tumor formation. Mathematical modeling indicated profound changes in the proliferation of individual TIC that produced mainly non-tumorigenic progeny with limited capacity of self-renewal. These data indicate that in pancreatic cancer long-term tumor growth is driven by the succession of transiently active TIC generating tumor tissue in temporally restricted bursts. The recruitment of inactive TIC clones to tumor formation after serial transplantation indicates a context-dependent switch between a quiescent and an active status. A clonal relation between tumorassociated fibroblast-like cells and neoplastic cells has been described in breast cancer. To investigate whether pancreatic TIC also give rise to fibroblast-like cell types xenograft tumors were analyzed in detail for their stromal compartment. Xenograft tumors contained no human stroma and attracted murine fibroblast-like cells instead. Human stroma cells were only found in xenograft tumors when these were co-transplanted with tumor cells, but engrafted with very low efficacy. Thus, due to the instability of human stroma cells in xenograft tumors grown in NSG mice, a possible clonal relation between fibroblast-like cells and neoplastic cells could not be investigated conclusively. In total, this study describes a previously unknown phenotypic and functional plasticity of pancreatic TIC. Its data show that TIC in pancreatic cancer have to be defined functionally level in vivo and that this cannot be replaced by the examination of phenotypic markers. Understanding the molecular mechanisms that regulate the activation and quiescence of pancreatic TIC might be important for future therapy approaches against pancreatic cancer.