Development of a state-of-the-art  multicellular model of the three dimensional (3D) colorectal tumour  microenvironment

Colorectal cancer (CRC) is the second leading cause of cancer-related deaths worldwide. CRC has been classified into four molecular subtypes: CMS1-4. CMS4 accounts for 23% of all CRC cases and is associated with worse prognosis for patients. One of the main characteristics of CMS4 CRC is high stromal infiltration. High levels of stromal cells create an immunosuppressive tumour microenvironment (TME), promoting tumour growth and progression. Further understanding the role stromal cells have in immunosuppression in the TME of CRC could identify novel therapeutic targets for CRC. Three-dimensional (3D) models offer a promising tool for studying interactions in the TME of CRC. This thesis describes the development and optimisation of multicellular 3D spheroids using two different hydrogels. It outlines the relevance of the 3D model in recapitulating the extracellular matrix environment following the addition of stromal cells. I have used a variety of methodologies including flow cytometry, AlamarBlue™, real-time PCR, imaging and confocal microscopy to assess the impact of chemotherapeutics and immunotherapeutics on multicellular 3D models in vitro. I have shown that mesenchymal stromal cells (MSCs) promote viability and outgrowth of cells from the co-culture spheroids. I have also demonstrated that MSCs increase the production of fibronectin which is present in stromal-rich areas in vivo. Using this 3D model, I have demonstrated the effects of Auranofin, a therapeutic previously used in the treatment of rheumatoid arthritis, in killing CRC cells in 2D and 3D models. By incorporating immune cells, isolated from PBMCs, into the 3D model, I have shown that secretome from HCT116 + hMSC spheroids can induce an anti-inflammatory, pro-tumorigenic macrophage phenotype in a 3D CRC model through upregulation of PDL1 and CD163 in the TME. I also optimised the activation and assessed the impact on primary T cell phenotype and function following culture with 3D multicellular spheroids. I have demonstrated that hMSCs induce the production of exhaustion markers LAG3 and PD1 on T cells in co-cultures with HCT116 + hMSC + PBMC spheroids. Finally, I demonstrated that Sialidase and PD1-Sialidase can potentially reverse immunosuppression induced by hMSCs through decreasing the expression of PD1 on both CD4 and CD8+ cells. Overall, the research presented in this thesis demonstrates the development of a multi-cellular collagen-embedded spheroid, that can be used to interrogate stromal-mediated immunosuppression in the TME of CRC. This model can also be used in a drug screening capacity allowing us to analyse the effects hMSCs have on the response to chemotherapies and Sialidase treatment in a 3D CRC model. Overall, the model represents an effective screening platform to identify novel therapeutic targets and assess the impact of combination chemo -and immunotherapies in vitro.

Publisher

University of Galway

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Attribution-NonCommercial-NoDerivatives 4.0 International

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University of Galway Theses (PhD Theses)