Development of a syngeneic, orthotopic microsatellite instability mouse model of colorectal cancer
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(Library staff access only.)
Date
2023
Authors
Thomas, Elaine May
Editors
Advisors
Woods, Susan
Wright, Josephine (SAHMRI)
Page, Amanda
Wright, Josephine (SAHMRI)
Page, Amanda
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Thesis
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Abstract
Colorectal cancer (CRC) is a common and deadly disease. CRC can be broadly divided into microsatellite stable (MSS) CRC (85% of cases) and microsatellite instability (MSI) CRC (15% of cases). MSI is strongly associated with inactivation of MutL homolog 1 (MLH1), a key DNA mismatch repair (MMR) gene. This leads to an accumulation of errors in the microsatellite tracts, which in turn induces high neoantigen expression and an immune cellinfiltrated phenotype. Immunotherapy has seen success in a proportion of MSI cases, but remains ineffective for MSS CRC, despite preclinical success. This points to a lack of predictive value in our current models, highlighting a need for a more sophisticated approach to preclinical immunotherapy research. To this end, this thesis focuses on creating an immune-competent preclinical model of MSI CRC to 1) investigate mechanisms of immune escape and 2) to drive a translationally focused investigation of immunotherapy options for CRC. This was achieved by knocking out Mlh1 in an established MSS CRC murine organoid model using CRISPR/Cas9 technology. After selection and validation of Mlh1 knockout organoids, they were cultured in vitro for up to 3 months and orthotopically injected into the colons of immunocompetent and immunocompromised mice, alongside MSS organoids. In vivo, rate of engraftment of MSI tumours was significantly reduced in immunocompetent mice compared to the MSS tumours, while remaining similar in NSG mice. To create an MSI model with a higher rate of establishment, tumours from the established MSI tumours were harvested, processed into organoids, and then reinjected back into syngeneic hosts. These mouse-passaged organoids produced established tumours at a higher rate than the MSI organoids initially tested. Whole exome sequencing revealed that the mutational signatures of MSI organoids correlated with MMR deficient profiles, while that of the MSS organoids more closely aligned with a clocklike mutational signature. Bulk RNA sequencing revealed some differential gene expression between both the MSS and MSI organoid lines. However, there was no obvious differential pathway regulation between the lines. Developing bacterial cancer therapies (BCTs) for solid tumours such as CRC has sparked interest in recent years due to the lack of effective, tolerable treatment strategies for late-stage tumours. In collaboration with the Danino laboratory, I aimed to test tumour colonisation of an immunotherapeutic BCT (SLIC3) in orthotopic organoid CRC models of MSS and MSI CRC, and investigate avenues to visualise SLIC3 in vivo. The results revealed that oral dosing of SLIC3 enabled specific tumour colonisation in these models, with no significant off-target colonisation of healthy organs. As SLIC3 colonisation had only previously been tested in less sophisticated, subcutaneous models, this was an important finding. However, there was significant variation in colonisation success in the MSS model, and highlighted difficulty in identifying colonised and uncolonised tumours in vivo via IVIS imaging. Overall, this thesis represents an important step towards improving our ability to accurately model CRC and test novel preclinical treatments using sophisticated organoid-based systems.
School/Discipline
Adelaide Medical School
Dissertation Note
Thesis (MPhil) -- University of Adelaide, Adelaide Medical School, 2024
Provenance
This thesis is currently under embargo and not available.