Investigating intratumoural heterogeneity and plasticity with multiplexed single-cell assays

Progress has been made in the development of small molecule inhibitors to target KRAS, among the most frequently mutated oncogenes, but patient responses to KRAS inhibitors are limited and variable across cancer types. Treatment resistance remains a universal feature contributing to poor cancer patient outcomes in the clinic and KRAS inhibitors have limited efficacy in patients with KRAS-mutant colorectal cancer (CRC). Genetic mutations alone do not fully explain tumour biology and treatment resistance given the vast genomic and phenotypic heterogeneity in tumours. There is a recent appreciation of the inherent transcriptomic heterogeneity and plastic nature of cancer cells through single-cell RNA-sequencing (scRNA-seq) of patient tumours. Drug combinations are a promising strategy to overcome treatment resistance but little is known about how CRC cells invoke cellular plasticity by shifting their cell states following treatment with KRAS inhibitors and drug combinations. To address questions about tumour plasticity in the context response towards KRAS inhibitors, I applied massively parallel and multiplexed scRNA-seq to profile KRAS-mutant CRC organoids after KRAS inhibition and with drug combinations over time. I reveal biological insight across tumour plasticity, the intratumoural heterogeneity of KRAS-mutant CRC, and mechanisms of treatment adaptation. In this Thesis, I begin by introducing tumour heterogeneity, its contribution to resistance and plasticity, and how this can be measured at single-cell resolution. I initially designed and performed experiments in KRAS-mutant CRC organoids to select different treatment conditions for profiling with scRNA-seq and to investigate resistance towards KRAS inhibition. I compare methods for scRNA-seq profiling, and find combinatorial indexing suitable to interrogate, at scale, questions surrounding tumour plasticity in CRC organoids. I also provide evidence to suggest that CRC organoids recapitulate a wider range of cellular states compared to CRC cell lines, making organoids suitable to address questions surrounding tumour plasticity in the context of treatment response. Following this, I developed an approach that exploits the potential of combinatorial indexing to perform massive parallel and multiplexed scRNA-seq readouts in KRAS-mutant CRC organoids that have been treated with different conditions over time. I find cellular variation in response to KRAS inhibitors and drug combinations. In addition, I identified variation in cell states in the CRC organoids and their differential response to KRAS inhibition. Cells have a slow-cycling and hybrid phenotype after KRAS inhibition which are reminiscent of developmental programs found in the healthy colon. I propose a preliminary model of how CRC cells adapt to KRAS inhibition over time. Finally, I summarize my Thesis findings, demonstrate how they advance our understanding in the context of existing knowledge, and propose further analyses and experiments to further validate them. I propose future directions and discuss maturing technologies to investigate tumour plasticity and heterogeneity. In summary, I developed a scalable platform based on massively parallel scRNA-seq to interrogate intratumoural heterogeneity and plasticity in CRC following different treatment conditions over time. I discover different CRC cell states linked to variation in response to KRAS inhibitors and nominate candidate pathways that explain the behaviour of these cell states after KRAS inhibition. My Thesis forms the basis for a mechanistic understanding of how CRC cells take advantage of cellular plasticity to adapt to KRAS inhibition, with potential longer term implications for developing more effective and durable treatments for patients with KRAS-mutant CRC.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2025

Author dc:creator

• Toh, Tzen Szen

Advisors dc:contributor.advisor

• Garnett, Mathew
• Behjati, Sam

• scRNA-seq
• Tumour plasticity
• KRAS
• Colorectal cancer

Licence

• https://www.repository.cam.ac.uk/bitstreams/1714c874-f5b7-4c2e-8b35-a2332ed69882/download
• https://creativecommons.org/licenses/by/4.0/

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.123926

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/393714