Introduction. Mutational activation of the KRAS proto-oncogene is an initiating event during the development of 40% of human colorectal carcinomas (CRC) and is preserved during tumor progression to metastatic lesions, commonly found in the liver. The aim of this study was to assess the contribution of endogenous mutant Krasto epithelial de-differentiation of colorectal cancer cells and to their capacity to form metastases in the liver. We previously showed that RNA interference-mediated KrasD12 knockdown in C26 mouse CRC cells elicits a potent anti-tumor immune response that prevents subcutaneous tumor growth in the majority of immunocompetent mice. Therefore, we also assessed the contribution of Kras to preventing immune-mediated tumor cell clearance from the liver sinusoids. experimental procedures. We used murine C26 CRC cells transduced by a control lentivirus (C26-pLL) or by a lentivirus targeting the endogenous mutant KrasD12 mRNA by RNA interference (C26-KrasKD). The invasive properties of C26-pLL and C26-KrasKD cells were analyzed in vitro in 24-well matrigel invasion chambers. The expression of epithelial and mesenchymal markers was assessed by Western blotting. Liver metastases were induced by intrasplenic or direct intrahepatic injection of tumor cells in immunocompetent and athymic immunodeficient mice. The formation of liver metastases was analyzed by bioluminescence imaging and intravital microscopy (IVM). Results. C26-KrasKD cells showed strongly reduced invasive capacity in vitro. This was not associated with an epithelial-to-mesenchymal (EMT) transition. Following intrasplenic injection into immunocompetent mice, C26-pLL cells developed massive liver metastases within 12 days. KrasD12 suppressed cells however, completely failed to form metastases. The metastatic potential of C26-KrasKD cells was not restored in immunodeficient mice. The abrogation of liver metastasis formation following Kras suppression can therefore not solely be due to increased immune-mediated clearance of tumor cells. Intravital microscopy analysis showed that C26-pLL and C26-KrasKD cells displayed equal seeding efficiency in the liver sinusoids following intrasplenic injection. However, C26-KrasKD cells lacked the ability to extravasate from the liver sinusoids and were rapidly cleared from the liver. When the need for extravasation was bypassed by directly injecting tumor cell into the liver parenchyma, the tumor-forming potential of KrasD12-deleted cells was restored in immunodeficient but not in immunocompetent mice. Conclusion. The suppression of KrasD12 results in reduced invasive capacity and reduced extravasation, and hence, to prolonged retention in the hostile environment of the liver sinusoids. In addition, suppression of KrasD12 allows tumor cells to be efficiently cleared by the host immune system in the liver, which prevents the formation of liver metastases.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]