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

Abstract

Colorectal cancers (CRCs) present across a range of differentiation grades, which impact 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 in HG tumours. Mechanistically, multiple genes suppressed in HG tumours harboured promoter methylation, indicative of stable epigenetic silencing. Furthermore, 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.

Fig. 1. Growth of 84 CRC cell lines as xenografts.

A Doubling time of 74 (of 84) CRC cell lines grown as xenografts in Balb/c nu/nu mice. Balb/c nu/nu mice were injected with 2 × 10⁶ cells for each cell line in the right and left flanks, and tumour growth was monitored every 2 or 3 days by caliper measurement. Data shown are the mean doubling time (Dt) and standard error (SEM) of n = 2–4 tumours per cell line for which Dt could be computed (n = 74). B Doubling times of cell lines separated according to MSS (n = 45) or MSI status (n = 29). Lines represent the mean of each group, and groups were compared using a non-parametric Mann–Whitney t-test, **P < 0.005.

Fig. 2. Differentiation grade of CRC cell line xenografts.

A, B Representative H&E images of A low-grade (LG) and B high-grade (HG) CRC cell line xenografts. C, D Association of differentiation grade with C MSI status and B BRAF mutation status. **P < 0.005, Fisher’s exact test.

Fig. 3. Differential gene expression between low-grade and high-grade CRC cell lines.

A Heatmap of 1763 differentially expressed genes between high-grade (n = 20) (orange bar) and low-grade (n = 10) CRC cell lines (blue bar), highlighting differential expression of general markers of colonic epithelial cells (orange text); transcription factors involved in intestinal development and differentiation (green); and transcription factors involved in epithelial-to-mesenchymal transition (purple). B Immunohistochemical staining for CDX2 in n = 4 representative low-grade and high-grade CRC cell line xenografts. C, D Volcano plots showing higher expression of C enterocyte and D goblet cell markers in LG CRC cell lines. E, F Cell type signature GSEA showing significant enrichment of enterocyte and goblet cell markers previously identified by Gao et al. in LG tumours.

Fig. 4. HG CRC cell lines lose expression of markers of colonic epithelial cells, enterocyte and goblet cells.

A, B Immunohistochemical staining of the A colonic epithelial marker Villin (VIL1); B the enterocyte marker KRT20; and C, D the goblet cell markers C MUC2 and D PAS/Alcian Blue (AB) in n = 4 representative low-grade and n = 4 representative high-grade CRC cell line xenografts.

Fig. 5. HG CRC cell lines lose expression of markers of LGR5+ colon stem cells and gain expression of markers of fetal-like intestinal stem cells.

A Downregulation of LGR5+ colon stem cell signature in HG CRC cell lines. B Expression (mRNA) of LGR5+ colonic stem cell markers in a subset of HG and LG CRC cell lines determined by qPCR. Values shown are mean + SEM of a representative experiment performed in technical triplicate. C Expression of corresponding LGR5+ colonic stem cell markers in HG (n = 58) vs LG (n = 202) primary CRCs in the TCGA cohort (RNAseq data). D Enrichment of fetal-like intestinal stem cell signature in HG CRC cell lines. E mRNA expression of fetal-like stem cell markers in HG and LG CRC cell lines determined by qPCR. Values shown are mean + SEM of a representative experiment performed in technical triplicate. F Corresponding mRNA expression of fetal-like intestinal stem cell markers in HG (n = 58) vs LG (n = 202) primary CRCs in the TCGA cohort (RNAseq data). Values shown in (C and F) are mean + SEM, and groups were compared using unpaired t-tests. **P < 0.01, ***P < 0.005, ****P < 0.001.

Fig. 6. HG CRC cell lines upregulate cell proliferation genes and EMT markers.

A Cell type signature GSEA showing significant enrichment of the “mesenchymal” gene signature previously identified by Gao et al. in HG CRC cell lines. B, C Images of cell migration over 24 h of representative B high and C low-grade cell lines. D Quantitation of the cell migration rates of LG (n = 13) and HG (n = 22) CRC cell lines, assessed over 24 h in vitro. Data shown are mean ± SEM. E Representative images of metastasis of n = 2 low-grade and n = 2 high-grade CRC cell lines in a zebrafish model in vivo. Cells were injected into the perivitelline space of zebrafish embryos and seeding of cells in the avascular fin tip (F) quantified after 3 days. Data shown in (F) are mean ± SEM of n = 21–37 zebrafish embryos per cell line. In all cases, groups were compared using unpaired t-tests. **P < 0.01, ***P < 0.005, ****P < 0.001.

Fig. 7. Loss of differentiation markers in HG CRC cell lines is associated with promoter methylation.

A Heatmap of corresponding methylation changes in promoters of 519 of the 1763 genes differentially expressed between HG and LG CRC cell lines. B, C Correlation of differential promoter methylation and differential gene expression for genes with B lower expression in HG cell lines (n = 346) and C lower expression in LG cell lines (n = 173).

Fig. 8. Transcriptional differences between HG and LG CRC cell lines grown in vitro are retained in xenografts, PDTOs and primary CRCs.

A–C Pearson correlations of the magnitude of differential expression of genes between HG and LG cell lines (logFC) with the corresponding change in expression between HG versus LG A xenografts, B PDTOs and C TCGA cases. D, E Canberra clustering of D n = 95 LG and n = 9 HG transcriptionally profiled patient-derived tumour organoids, and E n = 202 LG and n = 58 HG primary CRCs profiled by the TCGA, based on a 90 gene signature derived from the 1763 DEGs in cell lines using an elastic net approach.