Stammzell-basierte Tie2/Tk Suizidgentherapie des fortgeschrittenen Pankreaskarzinoms

Abstract

Introduction: Stem cells and endothelial progenitor cells play an important role in modulating tumour growth. After recruitment of stem cells from different niches, endothelial transdifferentiation within the tumour neovascularisation has been identified as a major factor [1, 2]. It was our intent to evaluate the role of mesenchymal stem cells (MSCs) in this process and to use them as a vehicle for a suicide gene therapy in a pancreatic tumour model. MSCs have been transfected using non-retroviral gene transfer of herpes-simplex-virus-thymidinkinase (tk) [3] and red fluorescent protein (RFP). Tk and RFP were put under the control of the Tie2 promoter for tissue specific gene expression. Tk leads to phosphorylation of ganciclovir (GCV), which subsequently leads to cell death in the transfected cells and the nearby cells via a bystander effect [4, 5]. Methods: MSCs were obtained from the bone marrow of C57/Bl6 p53 knock out mice. They were characterized and grew adherent and continuously in the cell culture tube. They were transfected with thymidinkinase (tk) and red fluorescent protein (RFP) linked to the Tie2 promoter for tissue specific expression of the construct. Migration assay was performed using a modified Boyden chamber. Stem cells migrated against descending concentrations of supernatant of Panc02 syngenic pancreatic carcinoma cells. For in vivo experiments we used an orthotopic pancreatic carcinoma model in C57Bl/6 mice. RFP and tk transfected stem cells were given systemically via i.v. injections. After 28 days tumour weight was measured and frozen sections were obtained after the animals had been sacrificed. Results: We were able to demonstrate that there is active migration of MSCs towards supernatant of murine pancreatic carcinoma cells using a modified Boyden chamber, which is suggestive of an active homing mechanism. Moreover using fluorescent microscopy we were able to find tissue specific expression of RFP in MSCs within the pancreas carcinoma, but not secondary lymphatic. Using the orthotopic pancreatic tumour model we demonstrated that non-therapeutic stem cells promote tumour growth, whereas therapeutic stem cells together with GCV lead to reduced tumour size (50 %) as well as reduced peritoneal carcinosis in comparison to the untreated group. Discussion: The data shows that MSCs play an important role in pancreatic carcinoma. They home actively in the tumour, where they promote growth. This has been proven using migration assay as well as systemic injections of Tie2/RFP MSC, which have been found in the tumour exclusively. However, the active homing can be used to transport a suicide construct in the tumour: After the tissue specific differentiation signal Ang-1 binds to the Tie2 receptor, activation of the promoter leads to thymidinkinase expression. The enzyme phosphorylates GCV, which leads to cells death of the integrated stem cells as well as cell death of the tumour itself via the so-called bystander effect. The reduction in tumour size and the reduced peritoneal carcinosis are promising for the clinical application of a combined stem cell/suicide gene therapy. The selective homing of stem cells in the tumour and the potential use of the patients’ own stem cells indicates little side effects during future clinical application.