Organoid models to characterise colonic stem cell plasticity

Cellular plasticity is the capability of cells to alter their phenotype in response to cues in the surrounding milieu, a vital property in several biological processes. The colon is a prime example of the importance of cellular plasticity in homeostasis as its epithelial lining is extremely dynamic, with cells constantly selecting between proliferation, differentiation, migration and apoptosis, depending on the needs of the tissue. While plasticity plays an important role in healthy tissues, genetic alterations and microenvironmental influences can also generate aberrant cellular plasticity which can mediate numerous diseases, including colorectal cancer. In colorectal cancer, the dysregulation of cellular plasticity causes phenotypic heterogeneity within the tumour, which is known to drive resistance to therapy. Consequently, despite progress in drug development in the past decades, curative treatment for this highly heterogeneous and metastatic disease remains difficult, with a significant proportion of cancer-related mortality worldwide attributable to colorectal cancer.
Understanding of the cellular plasticity mediating treatment resistance in colorectal cancer is a key step towards improving patient outcomes. In this study, in order to elucidate how genetic alterations and microenvironmental influences affected the cellular plasticity exhibited by colonic stem cells, a library of colon organoids (colonoids) harbouring driver gene mutations common in colorectal cancer were generated. Study of cellular plasticity was achieved by analysing this colonoid library with a novel, high-throughput, and unbiased organoid phenotyping platform, termed Shape, Appearance and Motion (SAM), which identified instantaneous mutant colonoid states and generated phenotype trajectories to compare mutant trait development over time. SAM analyses revealed that colorectal cancer-associated driver mutations significantly alter the cellular plasticity of colonoids and generate distinct genotype-phenotype profiles unique to each driver mutation. Notably, Apc mutant colonoids demonstrated a phenotypic dynamic that was particularly distinct from colonoids carrying all other driver mutations assessed, adopting a unique two-dimensional eccentric appearance.
To understand the molecular basis for the differences seen in cellular plasticity between the various mutant colonoids, phenotypic findings were integrated with single-cell RNA-sequencing. The distinct spatiotemporal dynamics exhibited by Apc mutant colonoids were found to be associated with a unique gene expression profile characterised by elevated Yap signalling and the acquisition of a regenerative stem cell and foetal gene signature. Furthermore, Apc mutant colonoids had decreased expression of tight junction proteins and increased expression of Yap-dependent extracellular matrix remodelling proteins. Together these findings suggest that Apc mutant colonoids are engaged in a Yap-mediated regenerative response, a process normally observed in the repairing intestinal epithelium. By combining these data with microscopic observation of developing Apc mutant colonoids, a mechanistic model for the unique Apc mutant colonoid phenotype is developed, whereby the two-dimensional eccentric appearance activates Yap-mediated mechanotransduction pathways and pushes the colonoids towards the regenerative stem cell molecular phenotype. Accordingly, the usual basal stem cell molecular phenotype imposed by the Apc mutation is overwritten by microenvironmentally influenced plasticity, which is able to induce a phenotypic shift towards a regenerative stem cell state regardless of the normal basal stem cell molecular phenotype associated with this mutation.
To extend this phenotypic study of mutant colonoids to model the subclonal dynamics and intratumoral heterogeneity driving tumour evolution and drug resistance in colorectal cancer, co-culture of mutant colonoids was employed. Findings suggested that mutant colonoids exhibit cooperative interactions in co-culture which significantly alter their individual phenotypes and increase their capacity for growth. scRNA-seq analysis on these co-cultured mutant colonoids showed that, while most mutant genotypes showed considerable transcriptomic changes during co-culture, Apc mutant colonoids were less susceptible to modifications of their transcriptomic profile in response to the presence of other mutant genotypes.
Finally, to advance the co-culture approach towards highly defined structures that more closely represent the clonal architecture of the tumour milieu, a novel organoid co-culture protocol which provides spatial control and patterning of mutant colonoids and facilitates the incorporation of cells usually present in the tumour microenvironment was developed. The mixed colonoid model represents an exciting tool for future research to further understand the direct subclonal interactions, intratumoural heterogeneity, and cellular plasticity present in colorectal cancer tumours and clarify the influence that the tumour microenvironment has on tumour evolution. In summary, the findings and methods developed in this work using a driver mutation colonoid library provide further insight into how driver mutations co-opt cellular plasticity to drive colorectal cancer.

Keywords: yap-mediated mechanotransduction
stem cell plasticity
spatiotemporal dynamics
colorectal cancer
Advisor: Lu, X
Leedham, S