Investigating the effect of chemotherapy on heterogeneity and plasticity of colorectal cancer liver metastases

Abstract

Chemotherapy is the most common treatment for colorectal cancer liver metastases (CRC LMs), which can significantly prolong patient survival. However, limited chemotherapy response and high rates of disease recurrence are common challenges contributing to poor patient outcome. A better understanding of how chemotherapy impacts specific cell types and states in LMs could aid development of improved therapeutic strategies for metastatic CRC. In this thesis, I aimed to characterise how chemotherapy alters the transcriptional and spatial landscape of cancer and tumour microenvironment cells in CRC LMs. Firstly, I analysed single-cell RNA-sequencing data from 87 CRC LM patients to annotate fine-grained cancer and tumour microenvironment cell subsets, characterising cellular heterogeneity across all major cellular compartments. Comparing transcriptional profiles and abundance of detailed subsets between chemotherapy-treated and untreated LMs revealed significant differences related to treatment. In treated LMs, greater abundance of dendritic cells and inflammatory macrophages, alongside increased expression of TNF-α signalling via NF-κB across multiple cell types, suggested an enhanced inflammatory response. Treated LMs also displayed reduced abundance of exhausted CD8 T cells and pro-tumoural SPP1+ macrophages suggesting that chemotherapy may alleviate immunosuppression. Comparisons based on responsiveness to chemotherapy elucidated that certain cancer cell states in better responding patients upregulated regenerative/fetal, apoptotic and inflammatory pathway signatures, and downregulated proliferation genes, compared to worse responders and untreated LMs. Therefore, the retention of cancer cells in a slower-cycling, regenerative state may relate to favourable chemotherapy outcome. Subsequent spatial mapping of fine-grained cell types using Visium spatial transcriptomics data of treated and untreated LMs supported differences in transcriptional pathways identified in single-cell data. Finally, bulk RNA-sequencing of CRC LM patient-derived organoids revealed that 5-fluorouracil, SN-38 and oxaliplatin chemotherapies induced inflammatory and regenerative signatures as an early response to treatment. Profiling recovery of patient-derived organoids after 5-fluorouracil treatment suggested transitions towards alternative non-canonical states that may relate to worse response to therapy. Overall, our results supported chemotherapy-induced alterations across cancer and tumour microenvironment cells in CRC LMs, which was dependent on patient response to chemotherapy. Our findings provide insights into the wide-ranging effects of chemotherapy at single-cell resolution that could inform identification of therapeutic targets