CologEM: Hydrogel derivato da pazienti per il miglioramento della medicina personalizzata attraverso la replicazione del microambiente del cancro del colon retto

Introduction: Colorectal cancer (CRC) is among the most prevalent cancers globally and is associated with a high mortality rate, particularly in advanced stages. In the realm of drug discovery, the use of innovative and highly translational preclinical CRC models is essential. Currently, the most relevant in vitro tumor approaches are three dimensional (3D) models. However, most 3D models of solid tumors are based either on synthetic materials or animal-derived commercial hydrogels, which fail to accurately mimic the biology of native tissues and originate from non-human sources. In contrast, hydrogels derived from human decellularized extracellular matrix (ECM) retain signaling cues from native tissue and represent a bioactive mechanical structure that can foster tumor cell growth in a tissue-specific 3D in vitro environment. Aim of the study: The objective of this thesis is to develop and characterize a patient-derived decellularized ECM hydrogel, identified as CologEM, from human colon biopsies. Furthermore, the formulated CologEM will be used as culture platoform CRC patient-derived organoid (PDOs) with the final goal of producing an integrated and reliable 3D in vitro preclinical model that accurately replicates CRC biology. Methods: Healthy colon biopsies, obtained through surgical resection, were decellularized using a detergent-enzymatic treatment (DET). The decellularized tissues were then lyophilized and ground into powder that was pepsin-digested to formulate CologEM. The physical, chemical, and structural properties of CologEM were evaluated through DNA quantification, immunofluorescence, immunohistochemistry, swelling tests, fluorescence recovery after photobleaching, scanning electron microscopy, and gelation, collagenase, hydroxyproline, and diffusion assays. CRC cell lines, including HCT-116, HCT-15, and HT-29, were then embedded in CologEM. To evaluate the biocompatibility of CologEM, cell viability assays, drug treatments, and gene expression analysis were performed. Additionally, PDOs were also embedded into CologEM and biocompatibility was assessed. Furthermore, the interaction between PDOs and CologEM was also investigated. Results: This study demonstrated that patient-derived decellularized colon ECM can be processed into a hydrogel, producing CologEM, suitable for the culture of CRC cells, PDOs and suitable for drug screening studies. The decellularization protocol preserved key ECM proteins, such as collagens and glycosaminoglycans, while leaving only insignificant levels of residual DNA. CologEM displayed a fibrous ultrastructure with interconnected pores, with notable differences observed between 1% and 3% (w/V) CologEM. Both 1% and 3% CologEM showed good biocompatibility, with 3% CologEM demonstrating a higher propensity to induce a mesenchymal phenotype and resistance to antitumor drugs. CRC PDOs from CRC biopsies was isolated and characterized. The biocompatibility of CologEM for PDOs culture was also successfully demonstrated. CRC PDOs in CologEM reveal not only a different growth pattern, but also a higher differentiation from the same PDOs line cultured in commercial hydrogel. Finally, the ability of PDOs to remodel and interact with the surrounding environment was also demonstrated. Conclusions: CologEM is a suitable scaffold for 3D CRC models as it replicates critical characteristics of the tumor microenvironment. This model holds promise for facilitating the discovery and development of chemotropic drugs for cancer treatment.