Decellularised Normal and Tumour Scaffolds for Cancer Organoid Cultures as a Model of Colorectal Peritoneal Metastases

Varinelli, Luca (2022). Decellularised Normal and Tumour Scaffolds for Cancer Organoid Cultures as a Model of Colorectal Peritoneal Metastases. PhD thesis The Open University.



Peritoneal metastasis (PM) is one of the most common routes of dissemination for colorectal cancer and remains a lethal disease. PM development is caused by a cross-talk between invading cancer cells and the rearrangement of the extracellular matrix (ECM). This interplay is governed by biochemical and biomechanical events that allow the development of a specific microenvironment: the so-called metastatic niche. ECM remodeling may be critical for PM spread. In fact, it has been demonstrated that ECMs are not only able to provide structural support to the exfoliated neoplastic cells, but also to trigger specific molecular pathways, paving the path for the seed of cancer cells, directly to their "pre-educated" soil. The mechanisms that determine the interactions within cancer cells and the ECM are still obscure and could be elucidated by an in vitro 3D-culture system that integrates all the elements involved in PM development. Cancer organoids have shown a profound impact in the field of oncology since they better reflect the main characteristics of the native organs compared to the traditional cell culture models. However, they still fail to represent the heterogeneity of the microenvironment. Methodologies have been recently established to remove cells from tissues and obtain matrices in which ECM and tissue architecture are maintained (dECM models), that could be used as the most representative scaffold on which implant 3D cultures.

I aimed to obtain a 3D-model that closely recapitulates the microenvironment where the PM develops and includes d-ECM repopulated with PM-derived organoids (3D-dECM model). I removed the cellular component of ECMs derived from peritoneal cavity obtained from both PM samples and r matched normal peritoneum using detergents and enzymatic methods. dECMs analyses demonstrated that the procedure maintained the specific characteristics of their tissue of origin also in terms of distribution, localization, and architectural organization of ECM-related proteins. The obtained dECMs showed a different spatial rearrangement between normal and PM-derived peritoneum, suggesting that dECM scaffolds closely recapitulate the native PM microenvironment. Moreover, when I repopulated dECMs with PM-derived organoids I found that PM- and normal peritoneum-derived dECMs differentially regulated the localization and organization of the seeded organoids, which was the same as in the original tissue. The two 3D-ECM models presented different ability in supporting cell proliferation, where PM-derived 3D-dECMs showed a higher proliferation index and a major ability to maintain the stemness phenotype. PM- and normal peritoneum-derived 3D-dECMs differently modulated cell homeostasis and proliferation ratio.

A gene expression analysis of organoids, grown on different substrates reflected faithfully the clinical and biological characteristics of the organoids. The impact of the ECM on the response to standard chemotherapy treatment for PM was also observed.

This demonstrated the value of ex vivo 3D models obtained by combining patient-derived extracellular matrices depleted of cellular components and organoids to mimic the metastatic niche, which could provide tools to develop new therapeutic strategies in a biologically relevant context, to personalize treatments and increase their efficacy.

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