Ricerc@Sapienza

In this study we aimed to develop a new in vivo bioluminescence-based tool to monitor and to quantify colon cancer (CC) liver metastasis development. HCT 116 cells were transducted with pLenti6/V5-DEST- fLuc for constitutive expression of firefly luciferase. Infection was monitored analyzing endogenous bioluminescence using the IVIS Lumina II in vivo Imaging System and a positive clone constitutively expressing luciferase (HCT 116-fLuc) was isolated. HCT 116-fLuc cells were left untreated or treated with 1 M GDC-0449, a Hedgehog pharmacological inhibitor. Moreover, 1 × 106 HCT 116-fLuc cells were implanted via intra-splenic injection in nude mice. Bioluminescence was analyzed in these mice every 7 days for 5 weeks. After that, mice were sacrificed and bioluminescence was analyzed on explanted livers. We found that in vitro bioluminescence signal was significantly reduced when HCT 116-fLuc cells were treated with GDC-0449. Regarding in vivo data, bioluminescence sources consistent with hepatic anatomical localization were detected after 21 days from HCT 116-fLuc intrasplenic injection and pro- gressively increased until the sacrifice. The presence of liver metastasis was further confirmed by ex-vivo bioluminescence analysis of explanted livers. Our in vitro results suggest that inhibition of Hedgehog pathway may hamper CC cell proliferation and impel for further studies. Regarding in vivo data, we set- up a strategy for liver metastasis visualization, that may allow follow-up and quantification of the entire metastatic process. This cost-effective technique would reduce experimental variability, as well as the number of sacrificed animals.