Decoding the basis of histological variation in human cancer.

Molecular abnormalities that shape human neoplasms dissociate their phenotypic landscape from that of the healthy counterpart. Through the lens of a microscope, tumour pathology optically captures such aberrations projected onto a tissue slide and has categorized human epithelial neoplasms into distinct histological subtypes based on the diverse morphogenetic and molecular programmes that they manifest. Tumour histology often reflects tumour aggressiveness, patient prognosis and therapeutic vulnerability, and thus has been used as a de facto diagnostic tool and for making clinical decisions. However, it remains elusive how the diverse histological subtypes arise and translate into pleiotropic biological phenotypes. Molecular analysis of clinical tumour tissues and their culture, including patient-derived organoids, and add-back genetic reconstruction of tumorigenic pathways using gene engineering in culture models and rodents further elucidated molecular mechanisms that underlie morphological variations. Such mechanisms include genetic mutations and epigenetic alterations in cellular identity codes that erode hard-wired morphological programmes and histologically digress tumours from the native tissues. Interestingly, tumours acquire the ability to grow independently of the niche-driven stem cell ecosystem along with these morphological alterations, providing a biological rationale for histological diversification during tumorigenesis. This Review comprehensively summarizes our current understanding of such plasticity in the histological and lineage commitment fostered cooperatively by molecular alterations and the tumour environment, and describes basic and clinical implications for future cancer therapy.

Genotype-phenotype mapping of a patient-derived lung cancer organoid biobank identifies NKX2-1-defined Wnt dependency in lung adenocarcinoma.

Human lung cancer is a constellation of tumors with various histological and molecular properties. To build a preclinical platform that covers this broad disease spectrum, we obtained lung cancer specimens from multiple sources, including sputum and circulating tumor cells, and generated a living biobank consisting of 43 lines of patient-derived lung cancer organoids. The organoids recapitulated the histological and molecular hallmarks of the original tumors. Phenotypic screening of niche factor dependency revealed that EGFR mutations in lung adenocarcinoma are associated with the independence from Wnt ligands. Gene engineering of alveolar organoids reveals that constitutive activation of EGFR-RAS signaling provides Wnt independence. Loss of the alveolar identity gene NKX2-1 confers Wnt dependency, regardless of EGFR signal mutation. Sensitivity to Wnt-targeting therapy can be stratified by the expression status of NKX2-1. Our results highlight the potential of phenotype-driven organoid screening and engineering for the fabrication of therapeutic strategies to combat cancer.

Ionoacoustic application of an optical hydrophone to detect proton beam range in water.

BACKGROUND: Proton range uncertainty has been the main factor limiting the ability of proton therapy to concentrate doses to tumors to their full potential. Ionoacoustic (IA) range verification is an approach to reducing this uncertainty by detecting thermoacoustic waves emitted from an irradiated volume immediately following a pulsed proton beam delivery; however, the signal weakness has been an obstacle to its clinical application. To increase the signal-to-noise ratio (SNR) with the conventional piezoelectric hydrophone (PH), the detector-sensitive volume needs to be large, but it could narrow the range of available beam angles and disturb real-time images obtained during beam delivery. PURPOSE: To prevent this issue, we investigated a millimeter-sized optical hydrophone (OH) that exploits the laser interferometric principle. For two types of IA waves [γ-wave emitted from the Bragg peak (BP) and a spherical IA wave with resonant frequency (SPIRE) emitted from the gold fiducial marker (GM)], comparisons were made with PH in terms of waveforms, SNR, range detection accuracy, and signal intensity robustness against the small detector misalignment, particularly for SPIRE. METHODS: A 100-MeV proton beam with a 27 ns pulse width and 4 mm beam size was produced using a fixed-field alternating gradient accelerator and was irradiated to the water phantom. The GM was set on the beam's central axis. Acrylic plates of various thicknesses, up to 12 mm, were set in front of the phantoms to shift the proton range. OH was set distal and lateral to the beam, and the range was estimated using the time-of-flight method for γ-wave and by comparing with the calibration data (SPIRE intensity versus the distance between the GM and BP) derived from an IA wave transport simulation for SPIRE. The BP dose per pulse was 0.5-0.6 Gy. To measure the variation in SPIRE amplitude against the hydrophone misalignment, the hydrophone was shifted by ± 2 mm at a maximum in lateral directions. RESULTS: Despite its small size, OH could detect γ-wave with a higher SNR than the conventional PH (diameter, 29 mm), and a single measurement was sufficient to detect the beam range with a submillimeter accuracy in water. In the SPIRE measurement, OH was far more robust against the detector misalignment than the focused PH (FPH) used in our previous study [5%/mm (OH) versus 80%/mm (FPH)], and the correlation between the measured SPIRE intensity and the distance between the GM and BP agreed well with the simulation results. However, the OH sensitivity was lower than the FPH sensitivity, and about 5.6-Gy dose was required to decrease the intensity variation among measurements to less than 10%. CONCLUSION: The miniature OH was found to detect weak IA signals produced by proton beams with a BP dose used in hypofractionated regimens. The OH sensitivity improvement at the MHz regime is worth exploring as the next step.

Identification of Quiescent LGR5+ Stem Cells in the Human Colon.

BACKGROUND & AIMS: In the mouse intestinal epithelium, Lgr5+ stem cells are vulnerable to injury, owing to their predominantly cycling nature, and their progenies de-differentiate to replenish the stem cell pool. However, how human colonic stem cells behave in homeostasis and during regeneration remains unknown. METHODS: Transcriptional heterogeneity among colonic epithelial cells was analyzed by means of single-cell RNA sequencing analysis of human and mouse colonic epithelial cells. To trace the fate of human colonic stem or differentiated cells, we generated LGR5-tdTomato, LGR5-iCasase9-tdTomato, LGR5-split-Cre, and KRT20-ERCreER knock-in human colon organoids via genome engineering. p27+ dormant cells were further visualized with the p27-mVenus reporter. To analyze the dynamics of human colonic stem cells in vivo, we orthotopically xenotransplanted fluorescence-labeled human colon organoids into immune-deficient mice. The cell cycle dynamics in xenograft cells were evaluated using 5-ethynyl-2'-deoxyuridine pulse-chase analysis. The clonogenic capacity of slow-cycling human stem cells or differentiated cells was analyzed in the context of homeostasis, LGR5 ablation, and 5-fluorouracil-induced mucosal injury. RESULTS: Single-cell RNA sequencing analysis illuminated the presence of nondividing LGR5+ stem cells in the human colon. Visualization and lineage tracing of slow-cycling LGR5+p27+ cells and orthotopic xenotransplantation validated their homeostatic lineage-forming capability in vivo, which was augmented by 5-FU-induced mucosal damage. Transforming growth factor-ß signaling regulated the quiescent state of LGR5+ cells. Despite the plasticity of differentiated KRT20+ cells, they did not display clonal growth after 5-FU-induced injury, suggesting that occupation of the niche environment by LGR5+p27+ cells prevented neighboring differentiated cells from de-differentiating. CONCLUSIONS: Our results highlight the quiescent nature of human LGR5+ colonic stem cells and their contribution to post-injury regeneration.

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.

Elimination of Off-Target Effect by Chemical Modification of 5'-End of siRNA.

In this study, the efficiency of RNA interference of small interfering RNAs (siRNAs) bearing 5'-O-methyl-2'-deoxythymidine (X) and 5'-amino-2', 5'-dideoxythymidine (Z) at the 5'-end of the sense strand and the antisense strand of siRNA was investigated in HeLa cells stably expressing enhanced green fluorescent protein. The results indicated that when one strand of siRNA was modified with X or Z and the other was unmodified, the X or Z modification was predominant in the process of strand selection and the unmodified strand was selected as a guide strand. When both strands are modified with X or Z, the modified antisense strand with X or Z will be selected as a guide strand with a certain probability. The resulting mature RNA-induced silencing complex exerted reduced, but still moderate silencing activity remained. These results suggest that the modification of the sense strand with X or Z eliminates the off-target effects caused by the sense strand without affecting the silencing efficiency of the siRNA.

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.

Protein disulfide isomerase A1­associated pathways in the development of stratified breast cancer therapies.

The oxidoreductase protein disulfide isomerase A1 (PDIA1) functions as a cofactor for many transcription factors including estrogen receptor α (ERα), nuclear factor (NF)­κB, nuclear factor erythroid 2­like 2 (NRF2) and regulates the protein stability of the tumor suppressor p53. Taking this into account we hypothesized that PDIA1, by differentially modulating the gene expression of a diverse subset of genes in the ERα­positive vs. the ERα­negative breast cancer cells, might modify dissimilar pathways in the two types of breast cancer. This hypothesis was investigated using RNA­seq data from PDIA1­silenced MCF­7 (ERα­positive) and MDA­MB­231 (ERα­negative) breast cancer cells treated with either interferon Î³ (IFN­Î³) or etoposide (ETO), and the obtained data were further analyzed using a variety of bioinformatic tools alongside clinical relevance assessment via Kaplan­Meier patient survival curves. The results highlighted the dual role of PDIA1 in suppressing carcinogenesis in the ERα(+) breast cancer patients by negatively regulating the response to reactive oxygen species (ROS) and promoting carcinogenesis by inducing cell cycle progression. In the ERα(­) breast cancer patients, PDIA1 prevented tumor development by modulating NF­κΒ and p53 activity and cell migration and induced breast cancer progression through control of cytokine signaling and the immune response. The findings reported in this study shed light on the differential pathways regulating carcinogenesis in ERα(+) and ERα(­) breast cancer patients and could help identify therapeutic targets selectively effective in ERα(+) vs. ERα(­) patients.

Direct derivation of human alveolospheres for SARS-CoV-2 infection modeling and drug screening.

Although the main cellular target of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is thought to be alveolar cells, the absence of their tractable culture system precludes the development of a clinically relevant SARS-CoV-2 infection model. Here, we establish an efficient human alveolosphere culture method and sphere-based drug testing platform for SARS-CoV-2. Alveolospheres exhibit indolent growth in a Wnt- and R-spondin-dependent manner. Gene expression, immunofluorescence, and electron microscopy analyses reveal the presence of alveolar cells in alveolospheres. Alveolospheres express ACE2 and allow SARS-CoV-2 to propagate nearly 100,000-fold in 3 days of infection. Whereas lopinavir and nelfinavir, protease inhibitors used for the treatment of human immunodeficiency virus (HIV) infection, have a modest anti-viral effect on SARS-CoV-2, remdesivir, a nucleotide prodrug, shows an anti-viral effect at the concentration comparable with the circulating drug level. These results demonstrate the validity of the alveolosphere culture system for the development of therapeutic agents to combat SARS-CoV-2.

An organoid-based organ-repurposing approach to treat short bowel syndrome.

The small intestine is the main organ for nutrient absorption, and its extensive resection leads to malabsorption and wasting conditions referred to as short bowel syndrome (SBS). Organoid technology enables an efficient expansion of intestinal epithelium tissue in vitro1, but reconstruction of the whole small intestine, including the complex lymphovascular system, has remained challenging2. Here we generate a functional small intestinalized colon (SIC) by replacing the native colonic epithelium with ileum-derived organoids. We first find that xenotransplanted human ileum organoids maintain their regional identity and form nascent villus structures in the mouse colon. In vitro culture of an organoid monolayer further reveals an essential role for luminal mechanistic flow in the formation of villi. We then develop a rat SIC model by repositioning the SIC at the ileocaecal junction, where the epithelium is exposed to a constant luminal stream of intestinal juice. This anatomical relocation provides the SIC with organ structures of the small intestine, including intact vasculature and innervation, villous structures, and the lacteal (a fat-absorbing lymphatic structure specific to the small intestine). The SIC has absorptive functions and markedly ameliorates intestinal failure in a rat model of SBS, whereas transplantation of colon organoids instead of ileum organoids invariably leads to mortality. These data provide a proof of principle for the use of intestinal organoids for regenerative purposes, and offer a feasible strategy for SBS treatment.

Wnt Signaling Shapes the Histologic Variation in Diffuse Gastric Cancer.

BACKGROUND AND AIMS: Diffuse-type gastric cancer (GC) is currently subdivided into signet-ring cell carcinoma (SRCC) and non-SRCC, referred to as poorly cohesive carcinoma not otherwise specified (PCC-NOS). Although these subtypes are considered to be independent, they often coexist in the same tumors, raising a question of whether they clonally differ or not. To tackle this question, we established an experimental platform for human diffuse GC that enables accurate modeling of histologic subtypes. METHODS: Seven patient-derived diffuse GC organoid lines were established, characterized by histopathologic analysis, in situ hybridization, and gene expression analysis. For genetic modeling of diffuse GC, we knocked out CDH1 and/or TP53 in human normal gastric organoids. Green fluorescent protein-labeled GC organoids were xenotransplanted into immune-deficient mice for in vivo assessment. RESULTS: PCC-NOS organoids transformed into SRCC-like structures on removal of Wnt and R-spondin from the culture medium. This morphologic change paralleled downregulation of Wnt-target and gastric stem cell genes, including LGR5, and elevation of differentiation markers, such as KRT20 and MUCs. The association between Wnt target gene expression and histologic subtypes was confirmed in 3 patient-derived GC tissues. In vivo, single clone-derived organoids formed tumors that comprised 2 distinct histologic compartments, each corresponding to SRCC and PCC-NOS. The transition from PCC-NOS to SRCC histology reflected the abundance of surrounding R-spondin-expressing fibroblasts. CONCLUSIONS: SRCC and PCC-NOS were clonally identical, and their morphology was regulated by extracellular Wnt and R-spondin expression. Our results decoded how genetic mutations and the tumor environment shape pathohistologic and biologic phenotypes in human diffuse GCs.

Somatic cell-derived organoids as prototypes of human epithelial tissues and diseases.

Recent progress in our understanding of the regulation of epithelial tissue stem cells has allowed us to exploit their abilities and instruct them to self-organize into tissue-mimicking structures, so-called organoids. Organoids preserve the molecular, structural and functional characteristics of their tissues of origin, thus providing an attractive opportunity to study the biology of human tissues in health and disease. In parallel to deriving organoids from yet-uncultured epithelial tissues, the field is devoting a growing amount of effort to model human diseases using organoids. This Review describes multidisciplinary approaches for creating organoid models of human genetic, neoplastic, immunological and infectious diseases, and details how they have contributed to our understanding of disease biology. We further highlight the potential role as well as limitations of organoids in clinical practice and showcase the latest achievements and approaches for tuning the organoid culture system to position organoids in biologically defined settings and to grant organoids with better representation of human tissues.

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.

Organoid Derivation and Orthotopic Xenotransplantation for Studying Human Intestinal Stem Cell Dynamics.

Intestinal stem cells continuously self-renew throughout life to maintain gut homeostasis. With the advent of the organoid culture system, we are now able to indefinitely expand healthy and diseased tissue-derived human intestinal stem cells in vitro and use them for various applications. Nonetheless, investigating the behavior of human intestinal stem cells in vivo still remains challenging. We recently developed an orthotopic xenotransplantation system that realizes in vivo reconstruction of human intestinal epithelial tissue with preserved stem cell hierarchy by engrafting human normal colon organoids onto the mouse colon surface. We also introduced new growth factors, namely, insulin-like growth factor-1 (IGF-1) and fibroblast growth factor-2 (FGF-2), into the culture condition for human intestinal organoids that significantly increase scalability and transfectability of the organoids. By integrating these recent advances, we organized a tissue-oriented platform encompassing derivation of patient-derived intestinal organoids and their orthotopic xenotransplantation. The research platform based on orthotopic xenotransplantation of human intestinal organoids provides a powerful tool for studying human intestinal stem cell biology in native tissue-relevant contexts as well as for establishing novel disease modeling systems.

Regulated IFN signalling preserves the stemness of intestinal stem cells by restricting differentiation into secretory-cell lineages.

Intestinal stem cells (ISCs) are located at the crypt base and fine-tune the balance of their self-renewal and differentiation1,2, but the physiological mechanism involved in regulating that balance remains unknown. Here we describe a transcriptional regulator that preserves the stemness of ISCs by restricting their differentiation into secretory-cell lineages. Interferon regulatory factor 2 (IRF2) negatively regulates interferon signalling3, and mice completely lacking Irf24 or with a selective Irf2 deletion in their intestinal epithelial cells have significantly fewer crypt Lgr5hi ISCs than control mice. Although the integrity of intestinal epithelial cells was unimpaired at steady state in Irf2-deficient mice, regeneration of their intestinal epithelia after 5-fluorouracil-induced damage was severely impaired. Similarly, extended treatment with low-dose poly(I:C) or chronic infection of lymphocytic choriomeningitis virus clone 13 (LCMV C13)5 caused a functional decline of ISCs in wild-type mice. In contrast, massive accumulations of immature Paneth cells were found at the crypt base of Irf2-/- as well as LCMV C13-infected wild-type mice, indicating that excess interferon signalling directs ISCs towards a secretory-cell fate. Collectively, our findings indicate that regulated interferon signalling preserves ISC stemness by restricting secretory-cell differentiation.

Telomerase inhibition, telomere attrition and proliferation arrest of cancer cells induced by phosphorothioate ASO-NLS conjugates targeting hTERC and siRNAs targeting hTERT.

Telomerase activity has been regarded as a critical step in cellular immortalization and carcinogenesis and because of this, regulation of telomerase represents an attractive target for anti-tumor specific therapeutics. Recently, one avenue of cancer research focuses on antisense strategy to target the oncogenes or cancer driver genes, in a sequence specific fashion to down-regulate the expression of the target gene. The protein catalytic subunit, human telomerase reverse transcriptase (hTERT) and the template RNA component (hTERC) are essential for telomerase function, thus theoretically, inhibition of telomerase activity can be achieved by interfering with either the gene expression of hTERT or the hTERC of the telomerase enzymatic complex. The present study showed that phosphorothioate antisense oligonucleotide (sASO)-nuclear localization signal (NLS) peptide conjugates targeting hTERC could inhibit telomerase activity very efficiently at 5 µM concentration but less efficiently at 1 µM concentration. On the other hand, siRNA targeting hTERT mRNA could strongly suppress hTERT expression at 200 nM concentration. It was also revealed that siRNA targeting hTERT could induce telomere attrition and then irreversible arrest of proliferation of cancer cells.

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.

Chromosome Engineering of Human Colon-Derived Organoids to Develop a Model of Traditional Serrated Adenoma.

BACKGROUND & AIMS: Traditional serrated adenomas (TSAs) are rare colorectal polyps with unique histologic features. Fusions in R-spondin genes have been found in TSAs, but it is not clear whether these are sufficient for TSA development, due to the lack of a chromosome engineering platform for human tissues. We studied the effects of fusions in R-spondin genes and other genetic alterations found in TSA using CRISPR-Cas9-mediated chromosome and genetic modification of human colonic organoids. METHODS: We introduced chromosome rearrangements that involve R-spondin genes into human colonic organoids, with or without disruption of TP53, using CRISPR-Cas9 (chromosome-engineered organoids). We then knocked a mutation into BRAF encoding the V600E substitution and overexpressed the GREM1 transgene; the organoids were transplanted into colons of NOG mice and growth of xenograft tumors was measured. Colon tissues were collected and analyzed by immunohistochemistry or in situ hybridization. We also established 2 patient-derived TSA organoid lines and characterized their genetic features and phenotypes. We inserted a bicistronic cassette expressing a dimerizer-inducible suicide gene and fluorescent marker downstream of the LGR5 gene in the chromosome-engineered organoids; addition of the dimerizer eradicates LGR5+ cells. Some tumor-bearing mice were given intraperitoneal injections of the dimerizer to remove LGR5-expressing cells. RESULTS: Chromosome engineering of organoids required disruption of TP53 or culture in medium containing IGF1 and FGF2. In colons of mice, organoids that expressed BRAFV600E and fusions in R-spondin genes formed flat serrated lesions. Patient-derived TSA organoids grew independent of exogenous R-spondin, and 1 line grew independent of Noggin. Organoids that overexpressed GREM1, in addition to BRAFV600E and fusions in R-spondin genes, formed polypoid tumors in mice that had histologic features similar to TSAs. Xenograft tumors persisted after loss of LGR5-expressing cells. CONCLUSIONS: We demonstrated efficient chromosomal engineering of human normal colon organoids. We introduced genetic and chromosome alterations into human colon organoids found in human TSAs; tumors grown from these organoids in mice had histopathology features of TSAs. This model might be used to study progression of human colorectal tumors with RSPO fusion gene and GREM1 overexpression.

Neutral and Negatively Charged Phosphate Modifications Altering Thermal Stability, Kinetics of Formation and Monovalent Ion Dependence of DNA G-Quadruplexes.

The effect of phosphate group modifications on formation and properties of G-quadruplexes (G4s) has not been investigated in detail. Here, we evaluated the structural, thermodynamic and kinetic properties of the parallel G-quadruplexes formed by oligodeoxynucleotides d(G4 T), d(TG4 T) and d(TG5 T), in which all phosphates were replaced with N-methanesulfonyl (mesyl) phosphoramidate or phosphoryl guanidine groups resulting in either negatively charged or neutral DNA sequences, respectively. We established that all modified sequences were able to form G-quadruplexes of parallel topology; however, the presence of modifications led to a decrease in thermal stability relative to unmodified G4s. In contrast to negatively charged G4s, assembly of neutral G4 DNA species was faster in the presence of sodium ions than potassium ions, and was independent of the salt concentration used. Formation of mixed G4s composed of both native and neutral G-rich strands has been detected using native gel electrophoresis, size-exclusion chromatography and ESI-MS. In summary, our results indicate that the phosphate modifications studied are compatible with G-quadruplex formation, which could be used for the design of biologically active compounds.