GelMA-based hydrogels for 3D modeling of metastatic Colorectal Cancer patient-derived organoids

Gelatine methacryloyl (GelMA)-based hydrogels are emerging as innovative and promising 3D platforms, increasingly employed in many fields of biomedical research due to their high versatility, biocompatibility and tunability of mechanical properties. Derived from gelatine and functionalized with methacrylic anhydride, GelMA can be precisely tailored by adjusting set up synthesis, enabling the formation of highly customizable 3D matrices via photo-polymerization. This study aimed to assess the potential of GelMA-based hydrogel as a support for mimicking the extracellular matrix (ECM) of metastatic colorectal cancer (mCRC), the third most frequently diagnosed malignant tumour worldwide. Nowadays 3D in-vitro models are widely adopted to investigate the biological processes underlying pathologies as mCRC, and as platforms for the evaluation of new therapeutic agents, due to their ability to faithfully reproduce the 3D native tissue architecture. The first part of this thesis focused on establishing 3D cultures of mCRC in two different types of GelMA (Type A and B) and comparing the performance of these matrices with Matrigel, the current gold standard for 3D models due to its ECM-like properties. However, Matrigel presents several major drawbacks: high cost, batch-to-batch variability, limited tunability, and ethical concerns. GelMA Types A and B differ based on the source of the native gelatine used in their synthesis: porcine and bovine-derived respectively. In-vitro assays show that GelMA A-based matrix is more suitable for culturing metastatic tumour cell line (T84), exhibiting higher viability, morphological features and structural organizations comparable to those observed in Matrigel. To further assess the performance of GelMA A-based hydrogels, we had the valuable opportunity to work with primary biological material: five patient-derived mCRC organoids from the XENTURION Biobank platform, kindly provided by Prof. L. Trusolino. These organoids were generated from liver mCRC patients, KRAS/NRAS-mutated and resistant to conventional treatments. Moreover, patient-derived mCRC organoids show the same mutational profiles and drug sensitivity of the original tumours. The experiments were conducted with GelMA A-based hydrogels synthetized with different Degrees of Functionalization (DoF) and weight on volume (w/v) concentrations in the final solution (High DoF at 12,5% w/v, Medium DoF at 12,5%, 10%, and 7,5% w/v), while maintaining the other experimental parameters (light intensity of 365 nm, and time exposure of 45’) constant. All the experiments were performed with Matrigel as reference. Morphological analyses, based on confocal spinning disk microscopy, and 3D proliferation assays over two weeks cultures, confirmed GelMA A-based hydrogels’ effectiveness in preserving essential cellular functions and interactions. Subsequently, the composition of the matrices has been optimized to achieve mechanical properties and biocompatibility levels adequate to support patient-derived mCRC organoids proliferation and morphological organization. Medium DoF at 7,5% w/v concentration of GelMA A results in the best performing formulation, highlighting it as a promising alternative to conventional materials for drug testing, personalized mCRC therapy and for the study of tumour dynamics. Potential future directions involve long-term cultures to investigate the transcriptome profile and drug response consistency between patient-derived mCRC organoids cultured in GelMA and original tumours.