Spheroid on-demand printing and drug screening of endothelialized hepatocellular carcinoma model at different stages

Abstract Hepatocellular carcinoma (HCC) poses a significant threat to human health and medical care. Its dynamic microenvironment and stages of development will influence the treatment strategies in clinics. Reconstructing tumor–microvascular interactions in different stages of the microenvironment is an urgent need for in vitro tumor pathology research and drug screening. However, the absence of tumor aggregates with paracancerous microvascular and staged tumor–endothelium interactions leads to bias in the antitumor drug responses. Herein, a spheroid-on-demand manipulation strategy was developed to construct staged endothelialized HCC models for drug screening. Pre-assembled HepG2 spheroids were directly printed by alternating viscous and inertial force jetting with high cell viability and integrity. A semi-open microfluidic chip was also designed to form a microvascular connections with high density, narrow diameter, and curved morphologies. According to the single or multiple lesions in stages Ⅰ or Ⅰ HCC, endothelialized HCC models from micrometer to millimeter scale with dense tumor cell aggregation and paracancerous endothelial distribution were successively constructed. A migrating stage Ⅰ HCC model was further constructed under TGF-β treatment, where the spheroids exhibited a more mesenchymal phenotype with a loose cell connection and spheroid dispersion. Finally, the stage ⅠHCC model showed stronger drug resistance compared to the stage Ⅰ model, while the stage III showed a more rapid response. The corresponding work provides a widely applicable method for the reproduction of tumor–microvascular interactions at different stages and holds great promise for the study of tumor migration, tumor–stromal cell interactions, and the development of anti-tumor therapeutic strategies.