Targeted stem cell based RANTES/TK suicide gene-therapy in a murine pancreatic cancer tumour model

Abstract

Introduction: The influence of tumour associated stromal cells on tumour growth and on the potential to metastasize is already issue of current research [1,2,3]. In preclinical studies of a breast-carcinoma model it was shown that mesenchymal stem-cells (MSCs) within the tumour stroma produce increased levels of the cytokine RANTES (CCL5). The secretion of RANTES leads to an higher incidence of lung metastases [4]. It was our intent to evaluate the potential role of MSCs and RANTES in a pancreatic cancer tumour-model and to use MSCs as a vehicle for a suicide gene construct. MSCs were engineered to express the herpes simplex virus (HSV) thymidine kinase (Tk) under the control of the RANTES promoter for tissue specific expression. TK leads to phosphorylation and therefore activation of intraperitoneally administered ganciciclovir (GCV) which then leads to cell death of the integrated stem cells, as well as to cell death of tumour cells by the so called bystander effect [5]. Material and Methods: MSCs were obtained from the bone marrow of C57/Bl6 p53 knock-out mice and characterized. The cells grew adherent in the cell culture tube. The cells were stably transfected with HSV-Tk, red fluorescent protein (RFP) and enhanced green fluorescent protein (eGFP) linked to the RANTES promoter using electroporation. For in vivo experiments we used an orthotopic pancreatic carcinoma model in C57/Bl6 mice with syngenic Panc02 tumour cells. RFP, eGFP and HSV-Tk transfected MSCs were given systemically via i.v. injections once a week for 3 weeks. The HSV-Tk therapy group was administered GCV on day 5, 6, 7 after each stem cell injection via i.p. injections. All mice were sacrificed after 36 days and the tumours were isolated, weighed and the macroscopically detectable metastases were determined. Moreover tissue samples were extracted for frozen sections, immunohistology and RNA-isolation. Results: Using fluorescence microscopy we were able to detect eGPF and RFP signals in the tumours of the corresponding stem cell groups but not in the control group. In the orthotopic pancreatic carcinoma model the HSV-Tk transfected MSCs in combination with GCV lead to a significant reduction of tumour mass (50% compared to the untreated control, p<0,0003, student’s t-test). Furthermore a reduction of metastases into the spleen (30% vs. 100%) was observed whereas all non therapeutic stem cells lead to an increased rate of peritoneal carcinosis compared to the control (0% vs. 67% eGFP, 75% untransfected MSCs, 87,5% RFP). Discussion: We were able to show recruitment to the tumour and activation of RFP and eGFP MSCs after integration into the tumour stroma. The suicide gene-therapy resulted in a reduction of tumour mass and incidence of metastases whereas non therapeutic MSCs promoted tumour growth. Targeting the tumour stroma using the RANTES/Tk suicide gene-therapy offers a promising strategy in the treatment of pancreatic cancer. Nevertheless further experimental studies are necessary to optimize this possible approach.