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.

Highlights d IGF-1 and FGF-2 improve human intestinal organoid plating and genome editing efficiencies d Organoids maintain self-renewal and multi-differentiation capacity in refined condition d Refined condition enables long-term culture of healthy and diseased intestinal organoids d Human small intestinal crypts and organoids compared with droplet-based scRNA-seq

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.

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Genetic lineage tracing has revealed that Lgr5 murine colon stem cells (CoSCs) rapidly proliferate at the crypt bottom. However, the spatiotemporal dynamics of human CoSCs in vivo have remained experimentally intractable. Here we established an orthotopic xenograft system for normal human colon organoids, enabling stable reconstruction of the human colon epithelium in vivo. Xenografted organoids were prone to displacement by the remaining murine crypts, and this could be overcome by complete removal of the mouse epithelium. Xenografted organoids formed crypt structures distinctively different from surrounding mouse crypts, reflecting their human origin. Lineage tracing using CRISPR-Cas9 to engineer an LGR5-CreER knockin allele demonstrated self-renewal and multipotency of LGR5 CoSCs. In contrast to the rapidly cycling properties of mouse Lgr5 CoSCs, human LGR5 CoSCs were slow-cycling in vivo. This organoid-based orthotopic xenograft model enables investigation of the functional behaviors of human CoSCs in vivo, with potential therapeutic applications in regenerative medicine.

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Arginine is a versatile additive to prevent protein aggregation. This paper shows that arginine ethylester (ArgEE) prevents heat-induced inactivation and aggregation of hen egg lysozyme more effectively than arginine or guanidine. The addition of ArgEE decreased the melting temperature of lysozyme. This data could be interpreted in terms of ArgEE binding to unfolded lysozyme, possibly through the ethylated carboxyl group, which leads to effective prevention of intermolecular interaction among aggregation-prone molecules. The data suggest that ArgEE could be used as an additive to prevent inactivation and aggregation of heatlabile proteins.Keywords: arginine; arginine ethylester; lysozyme; protein aggregation; thermal inactivation.Protein aggregation is a serious problem for both biotechnology and cell biology. Diseases such as prion misfolding, Alzheimer's, and other amyloidoses are phenomena for which protein aggregation in our living cells is of considerable relevance [1][2][3][4]. In the field of biotechnology, aggregation, resulting in the formation of inclusion bodies, is a major problem in bacterial recombinant systems [5][6][7].Under unfolding conditions, irreversible aggregation competes with correct folding. The classical model by Lumry-Eyring has been used to describe protein aggregation [8][9][10]:where N, A, and Agg represent a native state, a non-native state, and aggregates. Equation (1) involves a first-order reversible folding/unfolding reaction and subsequent intermolecular association with a higher-order irreversible process. The kinetics and equiliblium of Eqn (1) are dependent on solution conditions, such as temperature, pH, and the presence of additives. The additives may influence both the solubility and the stability of proteins in the N and A states. They also may change the folding rate to prevent or accelerate the nonspecific aggregation from A to Agg. Guanidine and urea are well established as aggregation suppressors that weaken the hydrophobic intermolecular interaction of proteins [11,12]. In particular, these denaturants increase the solubility of aggregation-prone unfolded molecules, but decrease the stability of the native state. Among nondenaturing reagents, arginine is the most widely used additive for increasing refolding yields by decreasing aggregation, for example when it is used in experiments with a single chain antibody [11,13]. Arginine does not facilitate refolding, but suppresses aggregation, with only a minor effect on protein stability [14], while it enhances the solubility of aggregates-prone molecules, leading to an increase in refolding yields [15][16][17]. Although other additives, such as proline, glycerol, glycine, and ethylene glycol, have been used [12], these are not enough to solve the problems of protein aggregation and misfolding. Recently we reported that polyamines, typically spermine and spermidine, prevent heat-induced inactivation and aggregation of lysozyme [18,19]. As part of a series of studies to develop additives, this paper shows a new candidat...

We have found that the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 produces an extracellular chitinase. The gene encoding the chitinase (chiA) was cloned and sequenced. ThechiA gene was found to be composed of 3,645 nucleotides, encoding a protein (1,215 amino acids) with a molecular mass of 134,259 Da, which is the largest among known chitinases. Sequence analysis indicates that ChiA is divided into two distinct regions with respective active sites. The N-terminal and C-terminal regions show sequence similarity with chitinase A1 from Bacillus circulans WL-12 and chitinase from Streptomyces erythraeus (ATCC 11635), respectively. Furthermore, ChiA possesses unique chitin binding domains (CBDs) (CBD1, CBD2, and CBD3) which show sequence similarity with cellulose binding domains of various cellulases. CBD1 was classified into the group of family V type cellulose binding domains. In contrast, CBD2 and CBD3 were classified into that of the family II type. chiA was expressed inEscherichia coli cells, and the recombinant protein was purified to homogeneity. The optimal temperature and pH for chitinase activity were found to be 85°C and 5.0, respectively. Results of thin-layer chromatography analysis and activity measurements with fluorescent substrates suggest that the enzyme is an endo-type enzyme which produces a chitobiose as a major end product. Various deletion mutants were constructed, and analyses of their enzyme characteristics revealed that both the N-terminal and C-terminal halves are independently functional as chitinases and that CBDs play an important role in insoluble chitin binding and hydrolysis. Deletion mutants which contain the C-terminal half showed higher thermostability than did N-terminal-half mutants and wild-type ChiA.