Colorectal tumor 3Dmodels: advantages of biofabrication for the recapitulation of early stages of tumour development.

The majority of cancer-relatedstudies are conducted on cell monolayers or spheroids. Although this approach has led to key discoveries, it still has a poor outcome in recapitulating the different stages of tumor development. The advent of novel three-dimensional (3D) systems and technological methods for their fabrication is set to improve the field, offering a more physiologically relevant and high throughputsystem for the study of tumor development and treatment. Here we describe the fabrication of alginate-based 3D models that recapitulate the early stages of colorectal cancer, tracking two of the main biomarkers for tumor development: CD44 and HIF-1. We optimized the fabrication process to obtain alginate micro-beads with controlled size and stiffness, mimicking the early stages of colorectal cancer. Human colorectal HCT-116 cancer cells were encapsulated with controlled initial number, and cell viability and protein expression of said 3Dmodels was compared to that of current gold standards (cell monolayers and spheroids). Our results evidenced that encapsulated HCT-116 demonstrated a high viability, increase in stem-like cell populations (increased expression of CD44) and reduced hypoxic regions (lower HIF-1a expression) compared to spheroid cultures. In conclusion we show that our biofabricated system is a highly reproducible and easily accessible alternative to study cell behavior, allowing to better mimic the early stages of colorectal cancer in comparison to othermodels. The use of biofabricatedmodels will improve the translatability of results, in particular when testing strategies for therapeutic intervention.