Development and characterization of patient derived ECM hydrogel as support for colorectal cancer 3D modelling

Introduction: Colorectal cancer (CRC) is one of the most common cancers worldwide, characterized by a high mortality rate in advanced-stage disease. In the field of drug discovery, the use of innovative and highly translational preclinical CRC models is of key importance. Currently the most relevant in vitro tumour approaches are 3D models. At present, the majority of 3D models of solid tumors rely either on synthetic materials or on animal tissue derived commercial hydrogels. However, these systems fail to mimic the biology of real tissues and have a non-human origin. In contrast, hydrogels derived from human decellularized extracellular matrix (ECM) may retain signalling cues from the native tissue, representing an important mechanical and bioactive structure that can promote tumor cell growth in a tissue-specific in vitro 3D environment. Aim of the study: The objective of this thesis is to develop, and characterize a patient derived decellularized ECM hydrogel (pdCH) from human colon biopsies, to produce a reliable 3D in vitro model that can accurately recapitulate CRC biology. Methods: Surgically resected healthy colon biopsies were decellularized by detergent-enzymatic treatment (DET). The decellularized samples were then lyophilized and reduced to powder. Then, the powder was pepsin-digested to produce the pdCH. Physical, chemical, and structural properties of pdCHs were evaluated by DNA quantification, immunofluorescence, immunohistochemistry, swelling test, fluorescence recovery after photobleaching, scanning electron microscopy and gelation, collagenase, hydroxyproline and diffusion assays. HCT-116, HCT-15 and HT-29 cell lines were then embedded into the pdCH. To assess the biocompatibility of the developed pdCHs, vitality assay, drug treatments and evaluation of gene expressions were conducted. Lastly, human intestinal organoids were cultured in pdCH to evaluate its biocompatibility with freshly isolated human-derived cells. Results: In this study we demonstrated that patient decellularized colon ECM can be processed to form hydrogels suitable for CRC cells culture and drug screening studies. Decellularization protocol allowed the conservation of main ECM proteins as collagens and glycosaminoglycans while unsignificant DNA levels were detected. We demonstrated that hydrogels have a fibrous ultrastructure with interconnected pores with some differences between 1% and 3% (w/V) pdCH. The biocompatibility of 1% and 3% pdCHs was also verified as well as the greater tendency of 3% hydrogel in inducing a mesenchymal phenotype and resistance to antitumoral drug. We obtained also promising data about the possibility of grow human intestinal organoids in pdCH. Conclusions: The developed pdCH is a suitable scaffold for 3D CRC models since it recapitulates important tumour microenvironment characteristics and could potentially promote discovery and development of chemotropic drug for cancer treatment.