Project Summary Cancer is one of the major causes of morbidity and mortality. The cancer microenvironment is highly complex, and is highly dynamic with distinctive key features present at each of the different stage of the disease. In particular, the large-scale growth of a tumor ultimately requires a blood supply. In comparison to healthy tissues, the tumor vessels are structurally and functionally abnormal. Moreover, the tumor vascularization is stage-dependent and significantly contributes to the phenotype determination as well as behaviors of the tumors such as their oxygenation levels. To fully grasp the complexity of the tumor microenvironment, as well as screening of various anti-cancer drugs, it has been increasingly realized that in vitro engineered human cancer models are strongly desired, as the conventional animal-based xenograft cancer models do not necessarily recapitulate the human physiology and drug responses. However, major challenges associated with current 3D in vitro tumor models include their oversimplified structures and limited vascularization potential. To overcome these limitations, recent advances in rapid prototyping methods in bioprinting together with novel bioinks now potentially enable us with precise architectural control to fabricate biomimetic tumor microenvironment that may potentially better reproduce their phenotypes and functions, which have been rarely demonstrated so far. We propose that, by optimizing a stereolithographic bioprinter that allows for high-definition manipulation over microarchitectures, it would be possible to, for the first time, precisely reproduce the structure of a tumor and its associated microenvironmental cues. By combining image analysis to allow for bioprinting of tumor models with stage-matching vascularization patterns (thus local oxygenation levels), we hypothesize that their phenotypes can be more accurately preserved in vitro. In Aim 1, we will optimize our custom-designed multi-material stereolithographic bioprinting system and tumor matrix materials for the bioprinting of breast cancer microenvironment including its associated vasculature. In Aim 2, we will integrate image analysis with the bioprinting system to generate breast cancer models with stage- matched vascularization patterns, analyze their oxygenation, and conduct preliminary validation on the phenotypic maintenance of these in vitro models with their in vivo counterparts in mice.