In-depth characterisation of circulating tumour cells in metastatic colorectal cancer via single cell MRNA sequencing

Abstract

Colorectal cancer (CRC) is the third most diagnosed cancer and ranked second in cancer mortality worldwide. Metastasis, recurrence, and drug resistance make surgery and chemotherapy ineffective. Circulating tumour cells (CTCs) could reveal key information about CRC metastasis and tumour heterogeneity. Advancements in nextgeneration sequencing could unravel the interplay between intrinsic cellular processes and gene expression profiles at the single-cell resolution. Thus, investigating CTCs via scRNA-seq is believed to reveal the importance of tumour heterogeneity in CRC metastasis. This study aims to perform an in-depth characterisation of CTCs in metastatic CRC (mCRC) via scRNA-seq. The MILLIPLEX® MAP kit-human circulating cancer biomarker magnetic bead panel 4 was used to profile the abundance of CTC serum markers in CRC patients. It identified three markers (L1CAM, CA9, and HPN) that were significantly higher in mCRC patients compared to primary tumours. EasySep human CD45 depletion kit II was used to isolate pure CTC suspension from peripheral blood mononuclear cell samples via negative selection. The BD Rhapsody single-cell analysis system captured 507 single cells with a 0.9% cell multiplet rate, followed by single-cell sorting, barcoding, whole transcriptomics amplification and library preparation. The libraries were sequenced on Illumina NextSeq and analysed using Seven Bridges and ICARUS. Bioinformatic analysis revealed six distinct CTC clusters in mCRC. The gene MALAT1 was significantly overexpressed in multiple cluster comparisons. A comparison between clusters 4 and 1 revealed four significant differentially expressed genes including SLC4A1 and BCL2L1, which were involved in CRC proliferation, cell cycle and apoptosis. Based on gene ontology and pathway analyses, the interference of PABPC1 in nonsense-mediated decay pathway was predicted to inhibit the degradation of MALAT1 by UPF1 via NF-κB and TGF-β signalling pathways. Co-expression of PABPC1 and MALAT1 could potentially be used as a ‘biomarker signature’ for mCRC CTC. Furthermore, CD74 could increase the survival of tumour cells from immune attacks via immunoediting surface antigen presentation. In conclusion, scRNA-seq identified unique genes associated with CRC metastasis, suggesting the existence of tumour heterogeneity in CTCs from mCRC. This research will build a novel liquid biopsy-based method for the discovery of mCRC biomarkers, resulting in the ultimate development of personalised treatments.