Spatially Programmed 3D‐Bioprinted Tumor–Stromal Assembloids Reveal Progressive Microenvironmental Remodeling and Phase‐Dependent Drug Responses in Hepatocellular Carcinoma

ABSTRACT Hepatocellular csarcinoma (HCC) progression is driven by dynamic interactions between cancer cells and stromal fibroblasts that evolve with spatial proximity and physical association. However, conventional co‐culture models either combine these populations from the outset or maintain them in permanent separation, limiting compartment‐specific analysis of their progressive interaction. Here, we develop a distance‐programmed tumor–stromal assembloid platform in which tumor and stromal microtissues are bioprinted at defined surface‐to‐surface separations. We engineer a collagen–alginate hybrid bioink that restrains spreading, promotes cell–cell cohesion, and preserves discrete tumor and stromal compartments throughout assembly, while interface angle and neck length quantify structural integration, which TGF‐β1 accelerates. Compartment‐specific tracking resolves a graded E‐cadherin‐to‐N‐cadherin transition within the tumor compartment together with progressive activation and spatial expansion of the stromal compartment. Paracrine signaling, fibroblast activation, matrix remodeling, and drug‐resistance programs predominate at distinct phases, producing phase‐dependent responses to pathway‐specific inhibition combined with doxorubicin. Modular repetition of these units extends assembly across a large spatial field, yielding an interconnected centimeter‐scale array with high viability. This platform couples compartment‐resolved analysis of tumor–stromal remodeling with a scalable route to macroscale tumor tissues.