Construction of 3D tumor <i>in vitro</i> models with an immune microenvironment exhibiting similar tumor properties and biomimetic physiological functionality

Tumors pose a serious threat to people's lives and health, and the complex tumor microenvironment is the biggest obstacle to their treatment. In contrast to the basic protein matrices typically employed in 2D or 3D cell culture systems, decellularized extracellular matrix (dECM) can create complex microenvironments. In this study, a combination of physicochemical methods was established to obtain liver decellularized extracellular matrix scaffolds (dLECMs) to provide mechanical support and cell adhesion sites. By co-culturing tumor cells, tumor-associated stromal cells and immune cells, an <i>in vitro</i> 3D tumor model with a biomimetic immune microenvironment was constructed. By utilizing microenvironment data obtained from human liver tumor tissues and refining the double seeding modeling process, 3D <i>in vitro</i> liver tumor-like tissues with a tumor immune microenvironment (TIME) were obtained and designated as reconstructed human liver cancer (RHLC). These tissues demonstrated similar tumor characteristics and exhibited satisfactory physiological functionality. The results of metabolic characterisation and mouse tumorigenicity testing verified that the constructed RHLC significantly increased <i>in vitro</i> drug resistance while also closely mimicking <i>in vivo</i> tissue metabolism. This opens up new possibilities for creating effective <i>in vitro</i> models for screening chemotherapy drugs.