Establishment of a tissue-engineered colon cancer model for comparative analysis of cancer cell lines

To overcome the limitations of in vitro two-dimensional (2D) cancer models in mimicking the complexities of the native tumor milieu, three-dimensional (3D) engineered cancer models using biomimetic materials have been introduced to more closely recapitulate the key attributes of the tumor microenvironment. Specifically, for colorectal cancer (CRC), a few studies have developed 3D engineered tumor models to investigate cell-cell interactions or efficacy of anti-cancer drugs. However, recapitulation of CRC cell line phenotypic differences within a 3D engineered matrix has not been systematically investigated. Here, we developed an in vitro 3D engineered CRC (3D-eCRC) tissue model using the natural-synthetic hybrid biomaterial PEG-fibrinogen and three CRC cell lines, HCT 116, HT-29, and SW480. To better recapitulate native tumor conditions, our 3D-eCRC model supported higher cell density encapsulation (20 × 10⁶  cells/mL) and enabled longer term maintenance (29 days) as compared to previously reported in vitro CRC models. The 3D-eCRCs formed using each cell line demonstrated line-dependent differences in cellular and tissue properties, including cellular growth and morphology, cell subpopulations, cell size, cell granularity, migration patterns, tissue growth, gene expression, and tissue stiffness. Importantly, these differences were found to be most prominent from Day 22 to Day 29, thereby indicating the importance of long-term culture of engineered CRC tissues for recapitulation and investigation of mechanistic differences and drug response. Our 3D-eCRC tissue model showed high potential for supporting future in vitro comparative studies of disease progression, metastatic mechanisms, and anti-cancer drug candidate response in a CRC cell line-dependent manner.