Abstract
Early detection and predicting treatment response in colorectal cancer (CRC) are two key strategies to improve CRC survival. Our increasing understanding of the underlying molecular biology of cancer initiation and progression has led to the development of novel molecular biomarkers in clinical care. However, their clinical translation has also been limited by a lack of sensitivity for early-stage CRC and the biological heterogeneity intrinsic to tumours. A new class of molecular biomarkers, circulating small non-coding RNAs (sncRNAs), show great promise as non-invasive biomarkers given their role in CRC initiation and their extracellular release into blood. Additionally, new technologies including single-cell sequencing, can improve our understanding of intra- and inter-tumour heterogeneity to further propel biomarker and therapeutic discovery in this biologically complex disease.
In this thesis we first profiled circulating sncRNAs in two small CRC cohorts: stage I CRC and locally advanced rectal cancer (LARC) undergoing neoadjuvant long course chemoradiation (LCCR). We utilised small RNA sequencing in baseline (pre-treatment) plasma samples to discover diagnostic and predictive biomarkers. This was followed by RT-qPCR for technical validation of biomarker candidates. We validated a four-microRNA (miRNA) score which had high diagnostic power on receiver operating characteristic analysis for distinguishing stage I CRC from healthy participants. The score also had high diagnostic power for detecting a broader stage I-III CRC cohort, demonstrating its potential utility as a diagnostic test. Meanwhile, when comparing LARC responders and non-responders to LCCR, we did not detect a single circulating sncRNA statistically associated with response. We concluded sncRNAs may not fully encompass the complexity and heterogeneity of this disease state.
Having identified a panel of CRC-associated circulating miRNAs, we next explored their function in CRC as potential therapeutic targets. miRNA expression, extracellular release and behaviour were characterised in CRC tumours and cell lines. All candidate miRNAs were dysregulated in tumour tissue compared to normal mucosa, although substantial inter-individual variation was observed for some miRNAs. We found a strong negative correlation between plasma and tumour expression of miR-25, suggestive of a tumour sequestration process. However, we also observed active secretion of miR-25 from CRC cells. In accordance with complex tumour expression and release, miR-25 function in transfected CRC cells was cell line-specific, demonstrating both oncogenic and tumour suppressive activity. These findings highlight the complexity of miRNA expression and function, complicating their potential as therapeutic targets.
Finally, given the paucity in biomarker translation for predicting response to LCCR in LARC, we explored the utility of massively parallel single-cell sequencing to improve our understanding of tumour heterogeneity and cellular diversity. We specifically used single-nuclei assay for transposase-accessible chromatin sequencing (snATAC-seq) in three LARC tumours. At low clustering resolution of data in aggregate samples, we observed substantial tumour heterogeneity across and within samples. Major cell types including epithelial, immune and stromal cell clusters were identified and several were patient-specific. Epithelial cells also clustered by differentiation status and we observed an expanded population of stem and EMT-like cells in a treatment-resistant tumour, suggestive of a treatment-resistant epigenetic phenotype. These findings demonstrate the utility of snATAC-seq in deconvoluting tumour heterogeneity to identify new biomarker and therapeutic opportunities.
Together, findings from this thesis demonstrate the potential for circulating miRNAs to detect early-stage CRC as a non-invasive blood test. We also provide more insight into CRC molecular heterogeneity, having observed complex dynamics in miRNA biology and epigenetic phenotypic diversity within and across tumours. These observations can inform future larger studies to exploit this tumour heterogeneity for molecular biomarker and therapeutic development.