Abstract 3613: Effect of thrombomodulin and plasminogen interaction on melanoma spheroid invasion

Abstract

Abstract Background: Thrombomodulin (TM), a transmembrane glycoprotein that acts as a plasminogen (Plg) receptor, has been found as a potential molecular and prognostic marker that impacts the spread of various cancers, including malignant melanoma. A previous study showed that TM plays a vital function in angiogenesis activity [1] but its role in the malignant melanoma invasion is unclear. Here, we investigate the role of TM-Plg by using melanoma spheroid (MS) embedded in an extracellular matrix (ECM) modeled to understand how TM-Plg affects the progression of MS invasion. Methods: In this work, A2058 (A2058-GFP and A2058-TMGFP) and MeWo (siC and siTM) MS with different TM expressions were formed, harvested, and embedded in type I collagen gel for invasion assay. TM and Plg expression were determined by western blot. The surface of A2058 MS was observed by SEM imaging. Human Plg, MMPs, and Plg inhibitors, tranexamic acid (TXA) and epsilon-aminocaproic acid (ε-ACA), were utilized to investigate the effect of TM on the plasmin system. Immunofluorescence of TM and F-actin were validated. Results: When the two cell lines expressed with TM, the formed MS invaded to the surrounding matrix more distinctively than that of the TM-silencing ones, with a 2.5-fold difference in the ratio invasive area change. Upon placing the two A2058-TMGFP MS in the vicinity of each other within the ECM, they showed a strong interaction with an observed distance between the two MS from 50 μm to 280 μm. Immunofluorescence of TMGFP assessed the role of TM in MS invasion and interaction in ECM. Microvilli structures were found on the surface of A2058-TMGFP cells that may help the cell have more cellular processes such as adhesion, signal transduction, and metabolic activities. Furthermore, TXA, ε-ACA, and MMP inhibitors inhibited the invasion of A2058-TMGFP MSs in a dose-dependent manner. Human Plg stimulated invasion in TM-expressing MSs while no effect on non-TM MSs. The results suggested that the human Plg only significantly increased the MS invasion with the presence of TM. Conclusion: We concluded that with the presence of TM, the MS invasion and interaction become stronger than MS without TM expression. Moreover, TM enhances MS invasion in the 3D model via interacting Plg. This study provides evidence for the development of TM antagonists in the future to analyze the drug's potential as an anti-metastasis treatment.[1] P. K. Chen et al., “Thrombomodulin functions as a plasminogen receptor to modulate angiogenesis,” FASEB J., vol. 27, no. 11, pp. 4520-4531, 2013, doi: 10.1096/fj.13-227561. Citation Format: Thi Kim Ngan Ngo, Hua-Lin Wu, Cheng-Hsiang Kuo, Ting-Yuan Tu. Effect of thrombomodulin and plasminogen interaction on melanoma spheroid invasion. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3613.