Meanwhile, collagen turnover in the tumor microenvironment was associated with tumor progression and metastasis. In previous studies, we have developed an injectable gelatin-based transglutaminase-crosslinked gel system for cell culture and drug delivery. Here we focus on the development and validation of novel 3D culture system that simulate the tumor stromal environment by manipulating the Col-Tgel. We demonstrated that biocompatibility and 3D architecture of Col-Tgel were suitable for reproducing the solid tumor microenvironment and it may offer a toolbox to study key events associated with tumor formation, progression, and metastasis and have potential to serve as an antitumor drug testing platform. Col-Tgel is a tailorable collagen-based remodelable hydrogel system able to induce spheroid formation without the use of time and labor intensive protocols such as the hanging drop and linkerengineered method, sophisticated equipment like rotating wall vessel bioreactors, or special handling temperature to prevent selfassembly. In vitro 3D culture systems to induce spheroid tumor formation using synthetic, natural or hybrid materials have been extensively attempted. Biocompatible materials such as agarose, methylcellulose, PMMA, PEG are structurally suitable to provide support for tumor spheroid formation, however, they lack cell adhesion and enzyme cleavage sites correlating in vivo tissue. Previous studies reported that MDA-MB-231 cells failed to form spheroids and lacked E-cadherin expression when grown on semi-solid methylcellulose, or in round bottom culture well coated with a poly-HEMA.These findings suggest that extracellular matrix, such as collagen fragments in Col-Tgel, are important for tumor spheroid formation. Invascu’s work demonstrated that type I collagen, but not fibronectin or type IV collagen, not only enhances, but also participates in MDA-MB231 spheroid formation. Therefore, a biologically functional 3D scaffold is very crucial to simulate the tumor microenvironment. 3D scaffolds fabricated from ECM substrates such as Matrigel, type I collagen, laminin, and fibronectin are cell attachable and remodelable and are ideal materials to construct a tumor tissue scaffold. However, when used in their native purified form, they are unable to provide the wide spectrum of rigidity necessary to mimic normal and pathological conditions. By using collagen peptides with an enzymatic crosslinking technique, Col-Tgel overcomes these limitations and appears suitable for in vitro and in vivo tumor engineering. Importantly, Col-Tgel offers handling flexibility through controlling the 3D construct size, shape, concentration, and crosslinking rate to achieve structural heterogeneity resemble in vivo tumor environment on many aspects, including nutrition diffusion and pH gradients, hypoxia environment, and mechanical restriction. By modulating these parameters according to the tumor progression state, it is possible to bioengineer 3D tumor in vitro to closely resemble cancer cells growing in the in vivo environment. Due to the transparent property of the Col-Tgel, we established imaging based assays to monitor cluster formation, cell morphology, delivery of Navitoclax chemotherapies.