Organoid cultures of early-onset colorectal cancers reveal distinct and rare genetic profiles

Abstract

ObjectiveSporadic early-onset colorectal cancer (EOCRC) has bad prognosis, yet is poorly represented by cell line models. We examine the key mutational and transcriptomic alterations in an organoid biobank enriched in EOCRCs.DesignWe established paired cancer (n=32) and normal organoids (n=18) from 20 patients enriched in microsatellite-stable EOCRC. Exome and transcriptome analysis was performed.ResultsWe observed a striking diversity of molecular phenotypes, including PTPRK-RSPO3 fusions. Transcriptionally, RSPO fusion organoids resembled normal colon organoids and were distinct from APC mutant organoids, with high BMP2 and low PTK7 expression. Single cell transcriptome analysis confirmed the similarity between RSPO fusion organoids and normal organoids, with a propensity for maturation on Wnt withdrawal, whereas the APC mutant organoids were locked in progenitor stages. CRISPR/Cas9 engineered mutation of APC in normal human colon organoids led to upregulation of PTK7 protein and suppression of BMP2, but less so with an engineered RNF43 mutation. The frequent co-occurrence of RSPO fusions with SMAD4 or BMPR1A mutation was confirmed in TCGA database searches. RNF43 mutation was found in organoid from a leukaemia survivor with a novel mutational signature; and organoids with POLE proofreading mutation displayed ultramutation. The cancer organoid genomes were stable over long culture periods, while normal human colon organoids tended to be subject to clonal dominance over time.ConclusionsThese organoid models enriched in EOCRCs with linked genomic data fill a gap in existing CRC models and reveal distinct genetic profiles and novel pathway cooperativity.

Organoids and Colorectal Cancer

“…for the increased rates in sporadic EOCRC rates are unknown. A first organoid biobank enriched in EOCRC has been recently reported [ 135 ]. Exome and transcriptomic analyses have revealed a diversity of molecular phenotypes, including some unusual alterations such as PTPRK-RSPO3 fusions whose relevance must wait for new and larger studies.…”

Not All Wnt Activation Is Equal: Ligand-Dependent versus Ligand-Independent Wnt Activation in Colorectal Cancer

“…We also demonstrated similar results with high-throughput AXIN2 profiling by quantitative real-time polymerase chain reaction (qRT-PCR). These findings were recently supported by the use of an organoid biobank derived from patients with colorectal cancer, in which organoids with RSPO fusions or RNF43 mutations exhibited lower AXIN2 expression than APC-mutant organoids [ 58 ]. Our analysis of paired qRT-PCR and immunohistochemistry for AXIN2 showed that there was only weak correlation between AXIN2 mRNA and scored AXIN2 protein expression, suggesting that AXIN2 may undergo significant translational regulation, as has been described previously [ 66 ].…”

“…Why polyp subtypes acquire apparent obligatory Wnt disruption through these different mechanisms is unknown, but may be influenced by the variable cell-of-origin in different lesion subtypes ( Figure 2 ). Indeed, APC mutations induce tumorigenesis in vivo if introduced into the LGR5+ intestinal stem cell but not transit-amplifying cells [ 16 ], while RSPO fusions significantly co-occur with loss-of-function mutations in the Bone morphogenic protein (BMP) signaling pathway that are known to induce ectopic crypt formation [ 58 , 59 ]. These data would also suggest that RSPO-mutant colorectal tumors are wholly derived from TSAs.…”

Extracellular Matrix Mechanical Properties and Regulation of the Intestinal Stem Cells: When Mechanics Control Fate

“…Moreover, contrary to spheroids, cells growing into intestinal organoids are able to develop as single-layered epithelium surrounding a central lumen. Colorectal organoids can be obtained from primary intestinal stem cell explants from mice [2] or humans [17] and are now widely used to better understand the intestinal physiology and pathology [18][19][20][21]. Indeed, intestinal and colonic cell lines are mainly derived from tumor samples, thus not allowing studying physiological mechanisms or elucidating those implicated in the switch from physiological towards pathological cell phenotypes.…”