Cell-matrix interface regulates dormancy in human colon cancer stem cells.

Cancer relapse after chemotherapy remains a main cause of cancer-related death. Although the relapse is thought to result from the propagation of resident cancer stem cells1, a lack of experimental platforms that enable the prospective analysis of cancer stem cell dynamics with sufficient spatiotemporal resolution has hindered the testing of this hypothesis. Here we develop a live genetic lineage-tracing system that allows the longitudinal tracking of individual cells in xenotransplanted human colorectal cancer organoids, and identify LGR5+ cancer stem cells that exhibit a dormant behaviour in a chemo-naive state. Dormant LGR5+ cells are marked by the expression of p27, and intravital imaging provides direct evidence of the persistence of LGR5+p27+ cells during chemotherapy, followed by clonal expansion. Transcriptome analysis reveals that COL17A1-a cell-adhesion molecule that strengthens hemidesmosomes-is upregulated in dormant LGR5+p27+ cells. Organoids in which COL17A1 is knocked out lose the dormant LGR5+p27+ subpopulation and become sensitive to chemotherapy, which suggests that the cell-matrix interface has a role in the maintenance of dormancy. Chemotherapy disrupts COL17A1 and breaks the dormancy in LGR5+p27+ cells through FAK-YAP activation. Abrogation of YAP signalling prevents chemoresistant cells from exiting dormancy and delays the regrowth of tumours, highlighting the therapeutic potential of YAP inhibition in preventing cancer relapse. These results offer a viable therapeutic approach to overcome the refractoriness of human colorectal cancer to conventional chemotherapy.

Organoid screening reveals epigenetic vulnerabilities in human colorectal cancer.

Precision oncology presumes an accurate prediction of drug response on the basis of the molecular profile of tumors. However, the extent to which patient-derived tumor organoids recapitulate the response of in vivo tumors to a given drug remains obscure. To gain insights into the pharmacobiology of human colorectal cancer (CRC), we here created a robust drug screening platform for patient-derived colorectal organoids. Application of suspension culture increased organoid scalability, and a refinement of the culture condition enabled incorporation of normal and precursor organoids to high-throughput drug screening. Drug screening identified bromodomain and extra-terminal (BET) bromodomain protein inhibitor as a cancer-selective growth suppressor that targets genes aberrantly activated in CRC. A multi-omics analysis identified an association between checkpoint with forkhead and ring finger domaines (CHFR) silencing and paclitaxel sensitivity, which was further validated by gene engineering of organoids and in xenografts. Our findings highlight the utility of multiparametric validation in enhancing the biological and clinical fidelity of a drug screening system.

Phylogenies from mitochondrial genomes of 120 species of ticks: Insights into the evolution of the families of ticks and of the genus Amblyomma.

The evolution and phylogenetic relationships of the ticks at both the family and genus levels are contested. The genus Amblyomma and its subgenera are in a state of flux; moreover, the relationships among the three tick families are controversial due to conflicting phylogenetic support for different arrangements of the three families of living ticks. With 18 newly sequenced mitochondrial (mt) genomes of ticks included, we executed the largest mt genome phylogenetic study of ticks so far. Phylogenetic trees were inferred from one sea spider mt genome, one horseshoe crab, five mite mt genomes and 146 tick mt genomes from 120 species: 153 mt genomes in total. Sixteen phylogenetic trees were inferred from 10 datasets using both maximum likelihood and Bayesian inference methods. We describe the first novel mt gene-arrangement for the metastriate Ixodidae in Amblyomma (Africaniella) transversale. Also, three unusual partial 16S rRNA gene inserts were found in the mt genome of Haemaphysalis (Alloceraea) kitaokai: we consider the possible role of past genome translocation events in the formation of these inserts. Our phylogenies revealed evidence that: (i) the genus Amblyomma is polyphyletic with respect to Amblyomma (Africaniella) transversale; (ii) the subgenus Aponomma is apparently embedded in the genus Amblyomma; (iii) Haemaphysalis (Segalia) parva and Haemaphysalis (Alloceraea) kitaokai form a clade to the exclusion of other Haemaphysalis species; and (iv) the phylogenetic position of the family Nuttalliellidae is unstable among phylogenies from different datasets.

An Organoid Biobank of Neuroendocrine Neoplasms Enables Genotype-Phenotype Mapping.

Gastroenteropancreatic (GEP) neuroendocrine neoplasm (NEN) that consists of neuroendocrine tumor and neuroendocrine carcinoma (NEC) is a lethal but under-investigated disease owing to its rarity. To fill the scarcity of clinically relevant models of GEP-NEN, we here established 25 lines of NEN organoids and performed their comprehensive molecular characterization. GEP-NEN organoids recapitulated pathohistological and functional phenotypes of the original tumors. Whole-genome sequencing revealed frequent genetic alterations in TP53 and RB1 in GEP-NECs, and characteristic chromosome-wide loss of heterozygosity in GEP-NENs. Transcriptome analysis identified molecular subtypes that are distinguished by the expression of distinct transcription factors. GEP-NEN organoids gained independence from the stem cell niche irrespective of genetic mutations. Compound knockout of TP53 and RB1, together with overexpression of key transcription factors, conferred on the normal colonic epithelium phenotypes that are compatible with GEP-NEN biology. Altogether, our study not only provides genetic understanding of GEP-NEN, but also connects its genetics and biological phenotypes.

Somatic inflammatory gene mutations in human ulcerative colitis epithelium.

With ageing, normal human tissues experience an expansion of somatic clones that carry cancer mutations1-7. However, whether such clonal expansion exists in the non-neoplastic intestine remains unknown. Here, using whole-exome sequencing data from 76 clonal human colon organoids, we identify a unique pattern of somatic mutagenesis in the inflamed epithelium of patients with ulcerative colitis. The affected epithelium accumulates somatic mutations in multiple genes that are related to IL-17 signalling-including NFKBIZ, ZC3H12A and PIGR, which are genes that are rarely affected in colon cancer. Targeted sequencing validates the pervasive spread of mutations that are related to IL-17 signalling. Unbiased CRISPR-based knockout screening in colon organoids reveals that the mutations confer resistance to the pro-apoptotic response that is induced by IL-17A. Some of these genetic mutations are known to exacerbate experimental colitis in mice8-11, and somatic mutagenesis in human colon epithelium may be causally linked to the inflammatory process. Our findings highlight a genetic landscape that adapts to a hostile microenvironment, and demonstrate its potential contribution to the pathogenesis of ulcerative colitis.

Divergent Routes toward Wnt and R-spondin Niche Independency during Human Gastric Carcinogenesis.

Recent sequencing analyses have shed light on heterogeneous patterns of genomic aberrations in human gastric cancers (GCs). To explore how individual genetic events translate into cancer phenotypes, we established a biological library consisting of genetically engineered gastric organoids carrying various GC mutations and 37 patient-derived organoid lines, including rare genomically stable GCs. Phenotype analyses of GC organoids revealed divergent genetic and epigenetic routes to gain Wnt and R-spondin niche independency. An unbiased phenotype-based genetic screening identified a significant association between CDH1/TP53 compound mutations and the R-spondin independency that was functionally validated by CRISPR-based knockout. Xenografting of GC organoids further established the feasibility of Wnt-targeting therapy for Wnt-dependent GCs. Our results collectively demonstrate that multifaceted genetic abnormalities render human GCs independent of the stem cell niche and highlight the validity of the genotype-phenotype screening strategy in gaining deeper understanding of human cancers.

Visualization and targeting of LGR5+ human colon cancer stem cells.

The cancer stem cell (CSC) theory highlights a self-renewing subpopulation of cancer cells that fuels tumour growth. The existence of human CSCs is mainly supported by xenotransplantation of prospectively isolated cells, but their clonal dynamics and plasticity remain unclear. Here, we show that human LGR5+ colorectal cancer cells serve as CSCs in growing cancer tissues. Lineage-tracing experiments with a tamoxifen-inducible Cre knock-in allele of LGR5 reveal the self-renewal and differentiation capacity of LGR5+ tumour cells. Selective ablation of LGR5+ CSCs in LGR5-iCaspase9 knock-in organoids leads to tumour regression, followed by tumour regrowth driven by re-emerging LGR5+ CSCs. KRT20 knock-in reporter marks differentiated cancer cells that constantly diminish in tumour tissues, while reverting to LGR5+ CSCs and contributing to tumour regrowth after LGR5+ CSC ablation. We also show that combined chemotherapy potentiates targeting of LGR5+ CSCs. These data provide insights into the plasticity of CSCs and their potential as a therapeutic target in human colorectal cancer.

A Colorectal Tumor Organoid Library Demonstrates Progressive Loss of Niche Factor Requirements during Tumorigenesis.

Colorectal tumor is a heterogeneous disease, with varying clinical presentation and prognosis in patients. To establish a platform encompassing this diversity, we generated 55 colorectal tumor organoid lines from a range of histological subtypes and clinical stages, including rare subtypes. Each line was defined by gene expression signatures and optimized for organoid culture according to niche factor requirements. In vitro and in xenografts, the organoids reproduced the histopathological grade and differentiation capacity of their parental tumors. Notably, we found that niche-independent growth is predominantly associated with the adenoma-carcinoma transition reflecting accumulation of multiple mutations. For matched pairs of primary and metastatic organoids, which had similar genetic profiles and niche factor requirements, the metastasis-derived organoids exhibited higher metastatic capacity. These observations underscore the importance of genotype-phenotype analyses at a single-patient level and the value of our resource to provide insights into colorectal tumorigenesis and patient-centered therapeutic development.

Modeling colorectal cancer using CRISPR-Cas9-mediated engineering of human intestinal organoids.

Human colorectal tumors bear recurrent mutations in genes encoding proteins operative in the WNT, MAPK, TGF-ß, TP53 and PI3K pathways. Although these pathways influence intestinal stem cell niche signaling, the extent to which mutations in these pathways contribute to human colorectal carcinogenesis remains unclear. Here we use the CRISPR-Cas9 genome-editing system to introduce multiple such mutations into organoids derived from normal human intestinal epithelium. By modulating the culture conditions to mimic that of the intestinal niche, we selected isogenic organoids harboring mutations in the tumor suppressor genes APC, SMAD4 and TP53, and in the oncogenes KRAS and/or PIK3CA. Organoids engineered to express all five mutations grew independently of niche factors in vitro, and they formed tumors after implantation under the kidney subcapsule in mice. Although they formed micrometastases containing dormant tumor-initiating cells after injection into the spleen of mice, they failed to colonize in the liver. In contrast, engineered organoids derived from chromosome-instable human adenomas formed macrometastatic colonies. These results suggest that 'driver' pathway mutations enable stem cell maintenance in the hostile tumor microenvironment, but that additional molecular lesions are required for invasive behavior.

Function of strawberry notch family genes in the zebrafish brain development.

We previously reported embryonic expression pattern of strawberry notch (sbno) family genes, suggesting involvement in brain development. However function of sbno genes in the vertebrate development has not been known yet. Utilizing zebrafish embryos, we experimentally examined function of sbno genes during brain development in this report. Knockdown experiments of sbno1 and sbno2a disrupted brain morphology, and delayed developmental alteration of gene expression. The earliest effect of loss of function of sbno genes on the zebrafish embryogenesis that we found here was downregulation of otx2 expression. Knockdown of sbno1 specifically affects regionalization along the anterior-posterior axis of the brain. These results suggest essential roles of sbno genes in vertebrate brain development.