Genetic editing of colonic organoids provides a molecularly distinct and orthotopic preclinical model of serrated carcinogenesis

Abstract

Objective: Serrated colorectal cancer (CRC) accounts for approximately 25% of cases and includes tumours that are among the most treatment resistant and with worst outcomes. This CRC subtype is associated with activating mutations in the mitogen-activated kinase pathway gene, BRAF, and epigenetic modifications termed the CpG Island Methylator Phenotype, leading to epigenetic silencing of key tumour suppressor genes. It is still not clear which (epi-)genetic changes are most important in neoplastic progression and we begin to address this knowledge gap herein.

Design: We use organoid culture combined with CRISPR/Cas9 genome engineering to sequentially introduce genetic alterations associated with serrated CRC and which regulate the stem cell niche, senescence and DNA mismatch repair.

Results: Targeted biallelic gene alterations were verified by DNA sequencing. Organoid growth in the absence of niche factors was assessed, as well as analysis of downstream molecular pathway activity. Orthotopic engraftment of complex organoid lines, but not Braf^V600E alone, quickly generated adenocarcinoma in vivo with serrated features consistent with human disease. Loss of the essential DNA mismatch repair enzyme, Mlh1, led to microsatellite instability. Sphingolipid metabolism genes are differentially regulated in both our mouse models of serrated CRC and human CRC, with key members of this pathway having prognostic significance in the human setting.

Conclusion: We generate rapid, complex models of serrated CRC to determine the contribution of specific genetic alterations to carcinogenesis. Analysis of our models alongside patient data has led to the identification of a potential susceptibility for this tumour type.

Keywords: cancer genetics; colorectal cancer; gene mutation; methylation; oncogenes.

Figure 1. Co-occurring molecular events in stem-cell niche, microsatellite instability and senescence pathways in BRAF^V600E mutant serrated CRC

Of the 50 patients with BRAF^V600E CRC from the TCGA CRC cohort (n=527 patients total), we depict the co-alteration (non-synonymous mutation and/or hyper-methylation) of selected genes in these pathways. Number of patients with each alterations is indicated by bar graph on right, % of BRAF^V600E cases containing the alteration is indicated by numbers on left. Coloured blocks indicate gene is altered in the sample, grey is unaltered.

Figure 2. Introduction of genetic alterations associated with serrated CRC promotes independence from niche factor requirements

A, activation of MAPK pathway in Braf^V600E organoids visualized by phosphorylation of the ERK1/2 effector protein, 4-hydroxytamoxifen (4-OHT). B, Generation of a ‘serratoid’ series from normal mouse colonic organoids through sequential CRISPR/Cas9 targeting and in vitro selection. Braf^V600E, Braf^V600ETgfbr2^Δ/Δ (Braf^V600EΔT), Braf^V600E Tgfbr2^Δ/Δ Rnf43^Δ/Δ/Znrf3^Δ/Δ p16Ink4a^Δ/Δ (Braf^V600E ΔTRZI), Braf^V600E Tgfbr2^Δ/ΔRnf43^Δ/Δ/Znrf3^Δ/Δ p16Ink4a^Δ/ΔMlh1^Δ/Δ (Braf^V600E ΔTRZIM). Normal media components required as stem cell niche factors Wnt-3a (W), Rspo-2 (R), Noggin (N), additional selection with TGFβ1 (T) and chemotherapeutic agent, 5-Fluorouracil (5FU). C, DNA sequence verification of biallelic insertion/deletion (indel) mutations that result in prematurely truncated proteins.

Figure 3. Braf^V600E alone is not sufficient for colon tumour formation, but with increasing serrated pathway genetic alterations tumour penetrance and growth rate increases

A, colonoscopic images showing injection and rapid growth of serratoid Braf^V600E ΔTRZI line. B, Tumour penetrance as a percentage of the number of organoid injections that gave rise to a tumour/mouse in 3 months for each line, with 1–3 injections/mouse. C, Colonoscopic scoring of largest tumour in each mouse (n=5 mice per group, Becker scale). D, Kaplan-Meier plot showing overall survival post-injection with organoid lines Braf^V600E n=4 mice, Braf^V600E ΔT n=12 mice, Braf^V600E ΔTRZI n=11 mice, Braf^V600E ΔTRZIM n=8 mice, compared to Braf^V600E using a Bonferroni adjustment for multiple comparisons. ns=not significant, *=p≤0.05, **=p≤0.01, ***=p≤0.001.

Figure 4. Multi-hit ‘serratoids’ generate invasive adenocarcinoma with features of human serrated CRC

Colonoscopy (A) and histology (B–H) images of mouse colon orthotopically injected with mutant organoid lines Braf^V600E, Braf^V600E ΔT, Braf^V600E ΔTRZI, Braf^V600E ΔTRZIM. B, H&E stained sections of whole colon. C, higher magnification H&E, arrows denote position of muscularis mucosae, * denotes remnant ink from injection. D, immunohistochemical staining for mutant Braf^V600E protein clearly delineates serratoid derived tumour cells. E, representative images of desmoplastic stromal response and F, tumour budding (circled). G, tumour stromal response stains positive for alpha-smooth muscle actin (aSMA). H, mucin lakes present in mucinous adenocarcinoma visualised using Alcian Blue stain. Scale bars are (B) 500um, (C–H) 100um.

Figure 5. Serrated pathway tumours are molecularly distinct from conventional pathway tumours

A, multi-dimensional scaling plot of RNA expression data from normal mouse colon (black), serrated pathway Braf^V600E ΔT (blue), Braf^V600E ΔTRZI (red) and Braf^V600E ΔTRZIM (yellow) and conventional pathway tumours Kras^G12D;Apc^Δ/Δ (grey), n=4 samples per group. B, gene set enrichment analysis (GSEA) for Sphingolipid de novo biosynthesis Reactome between Braf^V600EΔTRZI serrated tumour and normal mouse colon. Enrichment score (ES), normalised enrichment score (NES), false discovery rate (FDR). C, Expression of Sphk1 is increased and Sgpp1 is decreased in mouse Braf^V600E serrated CRC compared to normal mouse colon. Fold induction of mRNA expression is normalized to Gapdh, with transcript level in normal colon set to 1. Results from at least four animals with triplicate technical replicates are shown, error bars denote standard deviation. Two-tailed t-test used for pair-wise statistical analysis. D, Violin plots depicting z-score values for normalized expression of SPHK1 (top) and SGPP1 (bottom) transcripts in 622 human TCGA CRC and normal colon samples separated into wild-type BRAF/KRAS, KRAS mutant and BRAF^V600E CRC. E, Kaplan-Meier plots showing TCGA patient survival probability based on expression level of SPHK1 (top, SPHK1 high group n=139 in red, low group n=483 in green) or SGPP1 (bottom, SGPP1 low group n=145 in green, high group n=477 in red). *=p≤0.05, **=p≤0.01, ***=p≤0.001, NS= not significant.