Immune cells and Patient derived extracellular matrix hydrogel integration as 3D platform for the in vitro study of colorectal cancer microenvironment

Colorectal cancer (CRC) is a widespread malignancy worldwide, associated with high mortality rates in its advanced stages. Innovative and translational preclinical CRC models are essential for drug discovery, with three-dimensional (3D) in vitro cell culture models being the most relevant for both basic research and translational applications. However, most existing 3D tissue culture models utilize synthetic materials or animal tissue-derived hydrogels, which have limited fidelity to actual tissue biology due to their non-human origin. In contrast, hydrogels derived from human decellularized extracellular matrix (ECM) preserve native tissue signalling cues, providing a crucial mechanical and bioactive framework that supports tumor cell growth in a tissue-specific 3D environment. The objective of the work was to establish a reliable 3D in vitro model able to accurately recapitulate CRC microenvironment. My thesis aimed to study the interaction between patient-derived ECM ColoGEM and a monocytic stabilized cell line, further integrated with CRC patient derived organoids (PDOs) to develop a co-culture model, and investigation of the polarization of monocytes toward macrophage state induced by the ECM. This has been investigated by gene expression changes and phenotypic changes in monocytes. Specifically, THP1 stabilized cells were cultured in 3D into ColoGEM or in the commercial scaffold Geltrex™ as control. Biocompatibility was evaluated through cell viability assays and immunofluorescence. Polarization toward macrophages was assessed through cytofluorimetry, immunofluorescence, proteomic analysis and gene expression analysis for macrophage-specific genes. Functional assays such as migration, viability and ECM remodelling were also assessed. PDOs were also embedded into ColoGEM or Geltrex™ and biocompatibility was assessed. For the co-culture model, cell viability assays were performed, together with immunofluorescence and response to 5-fluorouracil (5-FU) treatment. In conclusion, this thesis established a 3D in vitro model aimed at faithfully recapitulating the microenvironment of CRC. By integrating patient-derived ECM ColoGEM with a monocytic stabilized cell line and CRC PDOs, this study investigated the polarization of monocytes towards a macrophage state induced by the ECM, trying to replicate crucial aspects of the tumor microenvironment and enhancing the comprehension of cancer biology.