Despite advances in treatment for metastatic melanoma patients, patients with liver metastasis have an unfavorable prognosis. A better understanding of the development of liver metastasis is... Show moreDespite advances in treatment for metastatic melanoma patients, patients with liver metastasis have an unfavorable prognosis. A better understanding of the development of liver metastasis is needed. The multifunctional cytokine Transforming Growth Factor β (TGF-β) plays various roles in melanoma tumors and metastasis, affecting both tumor cells and cells from the surrounding tumor microenvironment. To study the role of TGF-β in melanoma liver metastasis, we created a model to activate or repress the TGF-β receptor pathway in vitro and in vivo in an inducible manner. For this, we engineered B16F10 melanoma cells to have inducible ectopic expression of a constitutively active (ca) or kinase-inactive (ki) TGF-β receptor I, also termed activin receptor-like kinase (ALK5). In vitro, stimulation with TGF-β signaling and ectopic caALK5 expression reduced B16F10 cell proliferation and migration. Contrasting results were found in vivo; sustained caALK5 expression in B16F10 cells in vivo increased the metastatic outgrowth in liver. Blocking microenvironmental TGF-β did not affect metastatic liver outgrowth of both control and caALK5 expressing B16F10 cells. Upon characterizing the tumor microenvironment of control and caALk5 expressing B16F10 tumors, we observed reduced (cytotoxic) T cell presence and infiltration, as well as an increase in bone marrow-derived macrophages in caALK5 expressing B16F10 tumors. This suggests that caALK5 expression in B16F10 cells induces changes in the tumor microenvironment. A comparison of newly synthesized secreted proteins upon caALK5 expression by B16F10 cells revealed increased secretion of matrix remodeling proteins. Our results show that TGF-β receptor activation in B16F10 melanoma cells can increase metastatic outgrowth in liver in vivo, possibly through remodeling of the tumor microenvironment leading to altered infiltration of immune cells. These results provide insights in the role of TGF-β signaling in B16F10 liver metastasis and could have implications regarding the use of TGF-β inhibitors for the treatment of melanoma patients with liver metastasis. Show less
Marvin, D.L.; You, L.; Bornes, L.; Dinther, M. van; Peters, N.; Dang, H.; ... ; Ritsma, L. 2022
Transforming growth factor-beta (TGF-beta) signaling is tightly controlled in duration and intensity during embryonic development and in the adult to maintain tissue homeostasis. To visualize the... Show moreTransforming growth factor-beta (TGF-beta) signaling is tightly controlled in duration and intensity during embryonic development and in the adult to maintain tissue homeostasis. To visualize the TGF-beta/SMAD3 signaling kinetics, we developed a dynamic TGF-beta/SMAD3 transcriptional fluorescent reporter using multimerized SMAD3/4 binding elements driving the expression of a quickly folded and highly unstable GFP protein. We demonstrate the specificity and sensitivity of this reporter and its wide application to monitor dynamic TGF-beta/SMAD3 transcriptional responses in both 2D and 3D systems in vitro, as well as in vivo, using live-cell and intravital imaging. Using this reporter in B16F10 cells, we observed single cell heterogeneity in response to TGF-beta challenge, which can be categorized into early, late, and non-responders. Because of its broad application potential, this reporter allows for new discoveries into how TGF-beta/SMAD3-dependent transcriptional dynamics are affected during multistep and reversible biological processes. Show less
Pardali, E.; Sanchez Duffhues, G.; Gomez-Puerto, M.C.; Dijke, P. ten 2017
