3-layer lung cancer invasion model for evaluating MMP-targeted anti-metastatic therapeutics

Abstract Metastatic lung cancer is a leading cause of cancer mortality with invasion through the extracellular matrix (ECM) as a pivotal early step. This process is largely driven by metalloproteinases (MMPs), making them prime targets for antimetastatic therapy. However, conventional 2D or simplified 3D assays fail to capture the complex tumor-ECM interactions that are essential for evaluating anti-metastatic drugs. In this study, we present a 3D in vitro lung cancer model that faithfully mimics the native three-layer architecture—endothelium, ECM, tumor cells—within a physiologically relevant tumor microenvironment. By sequentially dispensing each layer, the model enables direct visualization and quantitative analysis of invasion dynamics, separating the effects of intrinsic tumor cell motility from platform for high invasiveness and revealing a strong dependence of invasion on ECM density. Furthermore, we analyzed correlation between invasion and MMP9, a critical protein involved in cancer invasion, using an MMP9 knockdown model that has clinical relevance to lung cancer metastasis. This analysis confirmed that cancer cell invasion was effectively suppressed in the MMP9 knockdown model. Finally, we demonstrated the translational application of the platform by quantifying the dose-dependent inhibition of invasion by a selective MMP9 inhibitor and the broad-spectrum MMP inhibitor marimastat. This layered in vitro lung cancer invasion model provides a robust, organ-specific platform for cancer invasion studies and for preclinical screening of MMP-targeted therapeutics, and this approach can be adapted to model other metastatic cancers, enabling versatile evaluation of invasion mechanisms and targeted interventions.