Clinical Pathology - Theses

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    Characterising tumour and immunological heterogeneity in colorecal cancer
    Shembrey, Carolyn Elizabeth ( 2021)
    Despite improvements in surgical oncology and precision medicine, 5-year survival rates for those with late-stage colorectal cancer (CRC) remain extremely poor and innovative treatment strategies are needed. Although T-cell directed immunotherapies are strikingly effective in many solid cancer types, durable responses are limited to approximately 5% of all CRC patients. Accordingly, there is an urgent need to explore alternate immunotherapeutic strategies which harness other cytotoxic cell types, most notably NK cells. However, the inter-patient variability of NK cell involvement in CRC, and particularly in colorectal liver metastases (CRLMs), is poorly characterised. Moreover, very little is known regarding the influence of tumour heterogeneity on immunotherapy response, mandating the development of novel methodologies which can dissect divergent responses to immuno-, chemo- and targeted therapies at the level of individual tumour cell subpopulations. In this thesis, the scope of tumour and immunological heterogeneity in primary CRC and CRLMs was assessed from transcriptomic, spatial and functional perspectives. A CRC-specific NK cell gene signature which infers the NK cell load of individual tumours from bulk RNAseq data was designed and validated. Differential expression analysis revealed that tumours with high evidence of this NK cell signature were characterised by the upregulation of chemotactic and cytolytic transcriptional programs. Furthermore, amongst patients with primary CRC, those with high NK scores were shown to have better survival outcomes in two independent cohorts. Focussing on metastatic disease, Cell type Identification by Estimating the Relative Subsets of RNA Transcripts (CIBERSORT) analysis revealed significant variance in terms of immune cell composition between CRLMs and adjacent normal (AdjN) liver tissue. Using a novel mIHC panel to quantify the scope of NK cell infiltration and NK cell ligand expression, it was determined that neoadjuvant chemotherapy increased NK cell penetrance of CRLM tissue, restoring NK cell load to levels comparable with the AdjN liver. Furthermore, NK cell densities were comparable in the AdjN liver and CRLM tissue of patients who partially responded to chemotherapy whereas nonresponders showed preferential accumulation in the AdjN liver. Preliminary data is also presented regarding the establishment of a NK cell versus patient-derived organoid (PDO) co-culture killing assay which can be used to assess the functional activity of NK cells against CRLMs. Lastly, a lineage tracing technique termed optical barcoding (OBC) was employed to study the relationship between intra-tumour heterogeneity and treatment response. Stably marking heterogenous populations of CRC cells with unique fluorescent signatures enabled the real-time identification and tracking of different cellular subpopulations under pharmacological selective pressures. Moreover, optimising the fluorescent protein panel allowed the maximum theoretical subpopulation resolution for the OBC technique to be achieved, greatly improving throughput as compared with previously published fluorescent barcoding techniques. Collectively, the results provided herein have deepened our understanding of tumour and immunological heterogeneity in primary CRC and CRLMs, and, by introducing several novel technologies, have laid strong foundations for future studies.