Model systems and unique biological features of high and low-grade colorectal cancer (CRC) revealed by xenografting 84 human CRC cell lines

Colorectal cancers (CRCs) present across a range of differentiation grades which impacts patient outcome and management, however the molecular features and drivers of differentiation status are not fully understood. To address this, 84 commonly used human CRC cell lines were grown as xenografts in mice, revealing models of low-grade (LG) and high-grade (HG) CRC. Transcriptional profiling revealed coordinate downregulation of multiple transcription factors involved in intestinal development and differentiation, markers of colonic lineage-specific differentiation, and effectors of normal functions of the colonic epithelium. Mechanistically, multiple genes suppressed in HG tumours harbored promoter methylation, indicative of stable epigenetic silencing. Strikingly, markers of LGR5+ colon stem cells were suppressed in HG tumours, while markers of cell proliferation, fetal-like intestinal stem cells, and non-canonical cell types including mesenchymal cells were increased. These changes manifested in HG cell line displaying increased proliferation, migration and metastatic capacity. Importantly, CRC cell line-derived transcriptional profiles of differentiation grade were reflected in LG and HG patient-derived tumour organoids and primary CRCs, revealing cell lines accurately model differentiation grade. The models and tumour differentiation-related properties identified herein may inform new approaches for tailored CRC treatments based on tumour grade. Overall design: 2 million cells from 8 colorectal cancer cell lines were injected subcutaneously into the flank of eight-week old male Balb/c nu/nu mice in a 150 µL suspension consisting of a 1:1 mixture of cells resuspended in DMEM-F12 (Invitrogen) and BD Matrigel Basement Matrix (BD Biosciences). When tumours reached 1 cm3 xenografts were harvested and snap frozen. RNA was isolated from each xenograft and quantified using a Qubit 4.0 Fluorometer. Library preparation for RNAseq and next generation sequencing was outsourced to the Australian Genome Research Facility (AGRF).