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.
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IJS, KISLJ, NUK, SBMB, UL, UM, UPUK
Cellular diversity that shapes tissue architecture and function is governed by multiple niche signals. Nonetheless, maintaining cellular diversity in human intestinal organoids has been challenging. ...Based on niche ligands present in the natural stem cell milieu, we establish a refined organoid culture condition for intestinal epithelia that allows human intestinal organoids to concurrently undergo multi-differentiation and self-renewal. High-throughput screening reveals that the combination of insulin-like growth factor 1 (IGF-1) and fibroblast growth factor 2 (FGF-2) enhances the clonogenic capacity and CRISPR-genome engineering efficiency of human intestinal stem cells. The combination equally enables long-term culture of a range of intestinal organoids, including rat small intestinal organoids. Droplet-based single-cell RNA sequencing further illustrates the conservation of the native cellular diversity in human small intestinal organoids cultured with the refined condition. The modified culture protocol outperforms the conventional method and offers a viable strategy for modeling human intestinal tissues and diseases in an in vivo relevant context.
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•IGF-1 and FGF-2 improve human intestinal organoid plating and genome editing efficiencies•Organoids maintain self-renewal and multi-differentiation capacity in refined condition•Refined condition enables long-term culture of healthy and diseased intestinal organoids•Human small intestinal crypts and organoids compared with droplet-based scRNA-seq
Sato and colleagues develop a modified culture condition for human intestinal organoids that improves the culture efficiency and maintains their long-term multi-differentiation capacity. scRNA-seq of human small intestinal crypts and organoids demonstrates that in vivo cellular diversity can be preserved in organoids cultured with the refined condition.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Human colorectal tumors bear recurrent mutations in genes encoding proteins operative in the WNT, MAPK, TGF-β, 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.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SBMB, UILJ, UKNU, UL, UM, UPUK
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.
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•An organoid library with diverse colorectal tumor grades and subtypes was established•A refined culture method improves colorectal tumor organoid establishment efficiency•Tumor organoids faithfully recapitulate clinical phenotypes of patient tumors•Niche factor dependency decreases along with adenoma-carcinoma transition
Fujii et al. generated a comprehensive organoid library from colorectal cancer patients. Each organoid line was characterized by gene expression and as xenografts recapitulating the original clinical phenotype. By optimizing niche factor requirements and derivation efficiency, they were able to encompass a range of clinical stages and rare subtypes and reveal that niche-independent growth is progressively associated with the adenoma-carcinoma transition.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
With ageing, normal human tissues experience an expansion of somatic clones that carry cancer mutations
. 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 mice
, 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.
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FZAB, GEOZS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Bats (order, Chiroptera) account for more than one-fifth of all mammalian species in the world and are infected by various intra-erythrocytic parasites of the family Plasmodiidae (Apicomplexa: ...Haemosporida), including
Polychromophilus
Dionisi, 1899. Recent advance in the molecular characterization of haemosporidian isolates has enabled their accurate identification, particularly in the last decade. Studies are actively conducted in tropical regions, Europe, and Australia; however, data on haemosporidian infection in bats in Asian temperate areas, including Japan, remain limited. In this study, 75 bats of 4 species (
Miniopterus fuliginosus
,
Myotis macrodactylus
,
Rhinolophus nippon
, and
Rhinolophus cornutus
) were captured at three sites in western Japan (Yamaguchi Prefecture), and haemosporidian parasites were screened microscopically and molecularly via nested polymerase chain reaction (PCR) targeting the cytochrome
b
(
cytb
), cytochrome
c
oxidase subunit I (
cox-1
), apicoplast caseinolytic protease C (
clpc
), and nuclear elongation factor 2 (EF2) genes. The survey detected
Polychromophilus melanipherus
in 15 (40.5%) miniopterid bats (
M. fuliginosus
) and
Polychromophilus murinus
in 6 (46.2%) vespertilionid bats (
M. macrodactylus
), whereas none of the 25 rhinolophid bats (
R. nippon
and
R. cornutus
) was infected, indicating the robust host specificity for miniopterid (
P. melanipherus
) and vespertilionid (
P. murinus
) bats regardless of orthotopic nesting. The 15
Polychromophilus cytb
sequences obtained from 11 miniopterid and 4 vespertilionid bats were classified into six
cytb
haplotypes (three for each species), showing no region-specific variation in a phylogenetic tree of
Polychromophilus
isolates in the Old World. Similarly, multiple haplotypes (seven for
cox-1
and nine for
clpc
) and genotypes (three for EF2) were characterized for the Japanese isolates of
Polychromophilus
, and the results were consistent with those based on a haemosporidian
cytb
analysis. Bat flies (
Nycteribia allotopa
and another undetermined
Nycteribia
sp.) collected from the body surface of bats harbored
Polychromophilus
oocysts on the external surface of the midgut. This is the first study to report the isolation and molecular characterization of
Polychromophilus
spp. in miniopterid and vespertilionid bats in the temperate area of Asia (western Japan). Future studies should evaluate the global prevalence of haemosporidian infections in bats.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
We confirmed infection of 2 patients with Borrelia miyamotoi in Japan by retrospective surveillance of Lyme disease patients and detection of B. miyamotoi DNA in serum samples. One patient also ...showed seroconversion for antibody against recombinant glycerophosphodiester phosphodiesterase of B. miyamotoi. Indigenous relapsing fever should be considered a health concern in Japan.
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DOBA, IZUM, KILJ, NUK, ODKLJ, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Borrelia miyamotoi, a member of the tick-borne relapsing fever spirochetes, shows a serum-resistant phenotype in vitro. This ability of B. miyamotoi may contribute to bacterial evasion of the host ...innate immune system. To investigate the molecular mechanism of serum-resistance, we constructed a membrane protein-encoding gene library of B. miyamotoi using Borrelia garinii strain HT59G, which shows a transformable and serum-susceptible phenotype. By screening the library, we found that bom1093 and bom1515 of B. miyamotoi provided a serum-resistant phenotype to the recipient B. garinii. These B. miyamotoi genes are predicted to encode P35-like antigen genes and are conserved among relapsing fever borreliae. Functional analysis revealed that BOM1093 bound to serum vitronectin and that the C-terminal region of BOM1093 was involved in the vitronectin-binding property. Importantly, the B. garinii transformant was not serum-resistant when the C terminus-truncated BOM1093 was expressed. We also observed that the depletion of vitronectin from human serum enhances the bactericidal activity of BOM1093 expressing B. garinii, and the survival rate of BOM1093 expressing B. garinii in vitronectin-depleted serum is enhanced by the addition of purified vitronectin. Our data suggests that B. miyamotoi utilize BOM1093-mediated binding to vitronectin as a mechanism of serum resistance.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Many Rickettsia species of the spotted fever group (SFG) cause tick‐borne diseases known as “spotted fever.” One of the candidate SFG Rickettsia species is “Candidatus Rickettsia kotlanii,” which was ...first detected in Haemaphysalis concinna in Hungary in 2006. However, its precise phylogenetic position in the SFG is not clear because only single‐gene sequence–based phylogenetic analyses were performed using very limited genes. Here, we present the complete genome sequences of two Japanese “Ca. R. kotlanii” isolates, which differed only by a 135 bp insertion/deletion (InDel). Using these genomes and publicly available whole genome sequences of other Rickettsia species, the precise phylogenetic position of “Ca. R. kotlanii” in Rickettsia was determined to be in a clade of the SFG. The phylogenetic relationships and average nucleotide identity of “Ca. R. kotlanii” relative to the other species indicated that “Ca. R. kotlanii” is an independent taxon in the SFG. Notably, although the genomes of the two isolates were almost identical, the isolates were obtained from different tick species in different regions and years, suggesting extremely low genomic diversity in “Ca. R. kotlanii.” While the genome of “Ca. R. kotlanii” is the smallest in the transitional group and SFG Rickettsia sequenced to date, we identified genes uniquely present or absent in “Ca. R. kotlanii,” but most were apparently degraded. Therefore, analyses of differences at the sequence (single nucleotide polymorphisms and small InDels) or gene expression level will be required to understand the functional or physiological features unique to “Ca. R. kotlanii.”
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
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