Nodal is highly expressed in various human malignancies, thus supporting the<br>rationale for exploring Nodal as a therapeutic target. Here, we describe the effects<br>of a novel monoclonal antibody (mAb), 3D1, raised against human Nodal. In vitro<br>treatment of C8161 human melanoma cells with 3D1 mAb shows reductions in<br>anchorage-independent growth and vasculogenic network formation. 3D1 treated<br>cells also show decreases of Nodal and downstream signaling molecules, P-Smad2<br>and P-ERK and of P-H3 and CyclinB1, with an increase in p27. Similar effects were<br>previously reported in human breast cancer cells where Nodal expression was<br>generally down-regulated; following 3D1 mAb treatment, both Nodal and P-H3 levels<br>are reduced. Noteworthy is the reduced growth of human melanoma xenografts in<br>Nude mice treated with 3D1 mAb, where immunostaining of representative tumor<br>sections show diminished P-Smad2 expression. Similar effects both in vitro and in<br>vivo were observed in 3D1 treated A375SM melanoma cells harboring the active<br>BRAF(V600E) mutation compared to treatments with IgG control or a BRAF inhibitor,<br>dabrafenib. Finally, we describe a 3D1-based ELISA for the detection of Nodal in<br>serum samples from cancer patients. These data suggest the potential of 3D1 mAb<br>for selecting and targeting Nodal expressing cancers.<br>