PO-185 Towards dynamic targeting of TGF-β in metastatic melanoma using intravital microscopy

Abstract

IntroductionMelanoma patients diagnosed with liver metastasis have a poor prognosis. TGF-β inhibitors give mixed results in clinical trials with metastatic melanoma patients. To more specifically target TGF-β in liver metastasis melanoma patients, it is necessary to unravel the spatio-temporal function of the TGF-β pathway in hepatic colonisation of melanoma cells. TGF-β is a multifunctional cytokine that signals via TGF-β receptors and downstream SMAD effector proteins. SMAD proteins regulate transcription by binding, among others, to CAGA elements in the DNA. It can exert both pro-and anti-tumorigenic functions, depending on cellular context. It acts on tumour cells, as well as the tumour micro-environment and the immune system. In metastatic melanoma cells, TGF-β can stimulate invasion and metastasis. To unravel its exact role during the different processes of metastasis, we will investigate the spatio-temporal patterns of TGF-β signalling during metastatic colonisation using intravital microscopy (IVM).Material and methodsSpatio-temporal patterns of TGF-β signalling will be studied in vitro and in vivo. For our in vivo studies, we are using an experimentally induced liver metastasis model, injecting highly aggressive B16F10 melanoma cells in immune competent C57BL/6 mice. By injecting these tumour cells in the mesenteric vein, cells will be transported directly to the liver, the first capillary network the cells will encounter. An abdominal imaging window will be placed after cell injection to visualise the different steps of metastasis in real-time using IVM. We developed a rapid CAGA12-GFP-based transcriptional reporter, which expresses a fluorophore upon TGF-β receptor activation and SMAD binding. Upon the expression of this reporter in B16F10 cells, activation of the TGF-β pathway can be monitored over time. By genetic manipulation of B16F10 cells to express dominant negative or constitutively active TGF-β receptors, the role of TGF-β can be assessed for the different steps of metastasis.Results and discussionsWe confirmed earlier reports showing that B16F10 cells show a transcriptional CAGA response upon TGF-β3 stimulation. Using our liver metastasis model, the injected B16F10 cells are able to perform the steps of metastasis and form liver metastasis within a short time frame. The use of the rapid TGF-β reporter during intravital imaging will show the involvement of the TGF-β pathway in the different phases of metastasis.ConclusionB16F10 cells have a functional TGF-β pathway and are able to colonise the liver.