Tumorstammzell-gerichtete Therapie mit Rapamycin und Cyclopamin sensitiviert chemotherapieresistente Pankreaskarzinome gegenüber 5-Fluorouracil

Abstract

Background: The contribution of cancer stem cells (CSC) to pancreatic cancer has not been established yet. Furthermore, the presence of cancer stem cells in chemotherapy-resistant pancreatic cancer has not been demonstrated. In the present study, we aimed to identify and to characterize putative CSC within different human pancreatic cancer cell lines as well as within 5-Fluorouracil-resistant pancreatic cancer cell lines established in our laboratory. Methods: Pancreatic CSC were identified and characterized by flow cytometry, using the following antibodies: anti-CD133/1 (clone AC133), anti-CD133/2 (clone 293C3), anti-CD44, anti-CD34, all individually or in combination. The relative expression of stem cell markers was confirmed by Western blotting. CSC from 5-FU-resistant cell lines were isolated by magnetic bead sorting using the MACS system and the CSC self-renewal pathways (hedgehog, Notch, Wnt/beta-catenin) were further analyzed by RT-PCR. Isolated CSC within 5-FU-resistant cell lines were exposed to the cancer stem cell targeted therapeutics (Rapamycin, Cyclopamine) and stained for Propidium iodide and BrdU. Furthermore, isolated CSC as well as non-tumorigenic cancer cells within 5-FU-resistant cell lines were orthotopically xenografted in nude mice and the efficacy of stem-cell-targeted therapy in combination with 5-FU was investigated. Results: All human pancreatic cancer cell lines as well as their 5-FU-resistant sublines were different in the CSC content. Interestingly, flow cytometry and Western blotting analyses revealed a significantly high amount of CSC in all chemotherapy-resistant cell lines tested. The stem cell phenotype of CD133/2+ cells isolated from 5-FU-resistant cell lines was contributed by the expression of pancreatic progenitor cell transcriptional factor Sox9 and identification of Bmi-1, Notch1, WNT1/beta-catenin and MYC signaling. The combined use of 5-FU with cancer stem cell targeted therapeutics Rapamycin or Cyclopamine selectively and efficiently killed chemotherapy-resistant pancreatic cancer cell lines as detected by Propidium iodide and BrdU staining. In vivo the combination of 5-FU with cancer stem cell targeted therapeutics Rapamycin or Cyclopamine significantly decreased the tumorigenic potential of CD133/2+ cells derived from 5-FU-resistant cell lines and dramatically reduced primary pancreatic tumor volume and weight. Conclusions: Our results demonstrate for the first time that human pancreatic cancer cells contain a persistent percentage of CSC. Furthermore, the chemotherapy-resistant cancer cells contain the increased CSC population (as compared to their parental sensitive cells) that is highly resistant to standard chemotherapy but not towards CSC-targeted therapy in vitro and in vivo. The further characterization of such cells might therefore lead to the development of new molecular and pharmaceutical therapeutics and better anti-cancer strategies.