Colorectal tumor 3D<i>in vitro</i>models: advantages of biofabrication for the recapitulation of early stages of tumour development

Abstract

The majority of cancer-related<i>in vitro</i>studies 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 throughput<i>in vitro</i>system 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<i>α</i>. 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 3D<i>in vitro</i>models 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 other<i>in vitro</i>models. The use of biofabricated<i>in vitro</i>models will improve the translatability of results, in particular when testing strategies for therapeutic intervention.