Identifikation und Charakterisierung von hochmetastatischen und therapieresistenten Tumorstammzellen beim Pankreaskarzinom

Abstract

Despite our advancing knowledge of tumor cell biology, the treatment efficacy in most cancers has not significantly improved over the past decades. Accumulating evidence suggests that cancer may actually be a disease of stem cells. It has been shown that a small fraction of cancer cells bear stem cells properties and one possible explanation would be that these cancer stem cells (CSC) are derived from normal tissue stem cells [1, 2]. So far, however, in solid tumors these CSC have only been described for brain and breast cancer, and colon and their biological properties with respect to tumor progression remain enigmatic to date [3]. Therefore, the purpose of the present study was to identify CSC in pancreatic cancer and define their role in tumor metastasis. Material and Methods: Putative cancer stem cells were identified by flow cytometry using the stem cell marker prominin-1 (CD133) [4] and isolated by means of magnetic microbeads cell sorting. We analyzed pancreatic tissue as well as pancreatic tumors from patients with pancreatic cancer. Moreover, we evaluated non-metastatic human FG pancreatic cancer cells and highly-metastatic human L3.6pl pancreatic cancer cells with respect to their CSC content in vitro and in vivo. The functional activity of the cells was assessed in vitro using a modified Boyden chamber assay (membrane coated with Matrigel? and in vivo using a murine model of orthotopic pancreatic cancer (female 8 week old NMRI nu/nu mice). Results: Human pancreatic cancer tissue contained a variable, but distinct CD133 positive CSC population ranging from 1 to 3 % of the total cell count (n = 3). SELDI-TOF mass spectrometry analysis indicated a marked overlap in the protein expression pattern between pancreatic stem cells and pancreatic cancer stem cells supporting the hypothesis that CSC are indeed derived from tissue resident stem cells. Cultured non-metastatic human FG pancreatic cancer cells contained only very few CD133 positive cells (0.02 %) whereas highly-metastatic human L3.6pl pancreatic cancer cells were characterized by a distinct CD133 positive stem cells population ranging from 1 to 2 % (n = 5 each). The migratory capacity of isolated CSC from L3.6pl tumors was significantly higher (+ 242 %) as compared to CD133 negative tumor cells (P < 0.01) and primarily mediated by the CXCR4 receptor. A significantly lower migratory activity was observed for cancer cells isolated from FG tumors with no difference between CD133 positive and CD133 negative cells. After orthotopic implantation in NMRI nu/nu mice, the developed tumors derived from FG cells contained a very small population of CD133 positive cells (0.2 to 0.4 %; n = 8) whereas tumors derived from L3.6pl cells include a robust CD133 positive CSC population representing about 2 % of the tumor cell population (n=10). Intriguingly, as few as 103 CD133 positive CSC isolated from L3.6pl tumors were capable of inducing new tumors (5/8 animals) whereas no tumor developed in mice receiving 106 CD133 negative tumors (0/8 animals). In 4 out of 5 animals with developing tumors after injection of CD133 positive CSC, metastatic lesions were observed. Although the anti-metabolite gemcitabine was capable of inducing apoptosis of CD133 negative tumor cells, it completely failed to induced apoptosis or cell death in CD133 positive CSC. Long-term in vitro treatment produced tumor cells that were markedly enriched for tumor stem cells. In vivo, gemcitabine significantly reduced tumor burden but also did not affect cancer stem cells resulting in a 7.6-fold increase in the relative stem cell content as compared to untreated animals. Conclusions: Here we demonstrate that human pancreatic cancers contain a low, but persistent percentage of CD133 positive cancer stem cells with highly invasive characteristics. Consistently, the highly-invasive human L3.6pl pancreatic cancer cells also contained a distinct cancer stem cell population whereas in non-metastatic FG pancreatic cancer cells this population was hardly detectable. The isolated CSC demonstrated a strong tumor repopulating capacity as well as a marked invasive activity which may explain at least in part the different metastatic activity of these two cell types. Most importantly, cancer stem cells are resistant to standard treatment. Thus, cancer stem cells represent a challenging novel target for the development of advanced molecular and pharmaceutical therapeutics.