Molecular analysis of pancreatic cancer metastasis in a genetically engineered mouse model

Pancreatic ductal adenocarcinoma (PDAC) is the 4th most common cause for cancer associated death and has a poor 5-year survival rate below 5%. One major problem in treating the disease is its early metastatic dissemination. However, the genes involved in metastasis of PDAC remain mostly unknown. To identify metastasis genes and to analyze the molecular mechanism of the metastatic process, we investigated gene expression profiles of primary tumours and their corresponding metastases in genetically engineered oncogenic KrasG12D based mouse models of pancreatic ductal adenocarcinoma, which accurately recapitulate the human disease. We compared gene expression profiles of non-metastasized and metastasized primary tumours and their metastases in liver, lung and lymph nodes. Furthermore, to enhance understanding of the metastatic process I isolated tumour cells from primary tumours, disseminated tumour cells and metastases and tested their metastatic capacity via an orthotopic transplantation model in vivo. These isolated tumour cells were also analyzed in regard to their gene expression profiles using microarray analysis. Intriguingly, we identified a high number of genes and gene sets significantly up- or down-regulated in metastases compared to primary tumours. Thus metastases display a distinct transcription profile in comparison to their corresponding primary tumours. In contrast, the expression profile of non-metastasized primary tumours did not differ from metastasized primary tumours indicating that gene-expression profiling of primary pancreatic tumours cannot predict metastasis risk. Cells isolated from primary tumours did not reflect the metastatic capacity of their original primary tumour whereas cells isolated from metastases did so. Analysis of organ-speficity revealed that even though metastasis was not organ-specific there might be a signature for metastasis via the lymphatic system opposed to metastasis via the bloodstream. In conclusion, I suggest that metastasis does not occur as progression from a premetastatic to a metastatic state of the primary tumour. Instead, my results support the model that metastatic cells arise from a small subset of rare, variant tumour cells that disseminate early during tumorigenesis. To verify the relevance of these data and to functionally characterize the candidate genes in an endogenous genetically defined mouse model a knock-in mouse line termed LSL-R26Tva-lacZ with conditional expression of TVA has been generated in our laboratory. TVA is a receptor for the avian sarcoma-leucosis retrovirus RCAS which transduces exclusively mammalian cells genetically engineered to express the avian retroviral receptor. In my thesis, I set out to characterize and establish this novel mouse model as a tool to functionally validate the identified genes relevant for metastasis in pancreatic cancer. TVA mediated infection of genetically engineered mice with endogenous expression of KrasG12D in pancreatic cancer cells using RCAS virus carrying the reporter gene EGFP proved that conditional expression of TVA enables spatio-temporal gene expression and knock-down in a small subset of somatic cells in vivo. This enables selective manipulation and investigation of candidate genes and the relevant signal transduction pathways in pancreatic cancer metastasis. Combined with the growing number of Cre expression models, RCAS-TVA based gene expression and knock-down systems open up promising perspectives for analysis of gene function in a time-controlled and tissue-specific fashion in vivo.