Integration of Immune Cells and Patient-Derived ECM Hydrogel in a Microfluidic 3D Platform to Model the Colorectal Cancer Microenvironment

Colorectal cancer (CRC) is one of the most prevalent malignancies globally and is characterized by high mortality, particularly in its advanced stages. The development of innovative and translational preclinical models is critical for effective drug discovery. Among these, three-dimensional (3D) in vitro culture systems have emerged as particularly valuable for both fundamental research and translational studies. Despite their relevance, many current 3D culture models rely on synthetic scaffolds or hydrogels derived from animal tissues, which often fall short in recapitulating the complexity of human tissue biology. In contrast, hydrogels formulated from decellularized human extracellular matrix (ECM) retain native biochemical and mechanical signals, offering a biologically faithful microenvironment that supports tumor cell proliferation in a tissue-specific 3D context.The objective of the work was to establish a reliable 3D in vitro model able to accurately recapitulate CRC microenvironment.In this context, my thesis aimed to study the interaction between colorectal cancer organoids derived from the patients and a monocytic stabilized cell line, cultivated in a patient-derived ECM ColoGEM seeded inside a microfluidic 3D platform. Biocompatibility was assessed through cell viability assays and measurement of organoids perimetry, using the commercial Geltrex™ as a control. The ability of the model to recapitulate the cancer microenviroment was investigated through the polarization of monocytes toward macrophage state. This was assessed through cytofluorimetry, cytokine array for specific markers and gene expression analysis for macrophage-specific genes. Finally I evaluate how the co-culture and the patient-derived ECM change the response to 5-fluorouracil (5-FU) treatment through cell titer viability assay. In conclusion, this thesis established a 3D in vitro model aimed at faithfully recapitulating the microenvironment of CRC.