LGR5 Research Articles

Comparative Analysis of Commercial and Home-Made Media on RSPO1/S6R Axis in Organoids with Different Wnt Backgrounds: A Methodological Guide for the Selection of Intestinal Patient-Derived Organoids Culture Media.

WNT3A is an intestinal ligand triggering the Wnt/β-catenin (Wnt) pathway, which can be enhanced by R-spondin 1 (RSPO1) through the RSPO1-LGR axis or antagonized by the adenomatous polyposis coli (APC) protein supporting β-catenin-degradation. Wnt interplays with several pathways including PI3K/mTOR (mTOR). In this study, we evaluated the influence of WNT3A-commercial and home-made culture media and RSPO1 protein on the Wnt and mTOR interplay in non-APC and APC-mutated intestinal patient-derived organoids (PDOs). Normal mucosa (NM) of sporadic CRC and FAP PDOs were cultured with: WNT3A-lacking/containing commercial (A/A+B) or home-made (BASAL/WNT3A-conditioned medium (CM)±RSPO1) media. In non-APC-mutated-PDOs (CRC-NM), WNT3A-CM, over commercial A+B, strongly activated Wnt-target-genes CCND1 and c-MYC. Most importantly, the addition of RSPO1 to home-made WNT3A-CM or A+B led to the downregulation of the mTOR-downstream-effector phospho-S6 ribosomal protein (p-S6R), highlighting the activation of the RSPO1-pS6R in both non-APC (CRC-NM) and APC-mutated (FAP-NM) PDOs, independently from LGR5 gene expression modulation. Our work demonstrates that home-made WNT3A-CM strongly impacts the crosstalk between Wnt and mTOR over commercial media, and proposes RSPO1 as a key regulator of the RSPO1-p-S6R axis in both non-APC and APC-mutated PDOs. Together, these findings represent an important methodological guide for scientists working in these fields to select the most appropriate intestinal PDO media.

Lactiplantibacillus plantarum JM113 alleviates deoxynivalenol induced intestinal damage by microbial modulation in broiler chickens

Deoxynivalenol (DON) contamination causes the grievous injury in public and animal health, poultry suffer from the greater toxin challenge. Probiotic have been considered as a potential way to mitigate the deleterious effects of DON. In this study, a total of 144 1-day-old Arbor Acres chickens were randomly assigned into 3 groups: control group, DON group (5 mg/kg DON diet), DJ group (1×109 cfu Lactiplantibacillus plantarum JM113/kg DON diet). The results showed that Lactiplantibacillus plantarum JM113 (L. plantarum JM113) increased the growth performance of 21-day-old broilers that challenged by the DON (P < 0.05), and the DON-induced disorder of jejunal morphology was recovered in DJ group (P < 0.05). Compared with the DON group, the mRNA and protein levels of Nrf2 and NQO-1 were upregulated in jejunum of DJ group broilers (P < 0.05). Meanwhile, administration of L. plantarum JM113 effectively increased the expression level of barrier-related genes, and the protein abundance of occludin and claudin1 (P < 0.05). L. plantarum JM113 restored the mRNA and protein abundance of PCNA, and proliferation-linked gene (Lgr5 and Bmi1) expression levels in jejunum of DON-insulted broilers (P < 0.05). Furthermore, administration of L. plantarum JM113 significantly enhanced the relative abundance of s_Limosilactobacillus_reuteri in jejuna of DON-challenged broilers (P < 0.05). Spearman correlation analysis showed that s_Limosilactobacillus_reuteri was positively associated with the jejunal barrier related genes (P < 0.05). In conclusion, L. plantarum JM113 alleviated the toxic effects of DON by regulating the jejunal function through microbial adjustment. Our findings proposed a viable approach to mitigating the adverse effects of deoxynivalenol exposure in broilers.

Abstract LB301: BRAFV600E drives WNT signalling in colorectal cancer via aberrant DNA methylation

Abstract Objective: The BRAF V600E mutation drives the generation of an aggressive subtype of colorectal cancer (CRC). Although activation of WNT signalling is a generic hallmark of CRC, mutations in APC are uncommon in BRAF V600E-mutant CRC. Instead, mutations in RNF43 are the suspected WNT signalling driving event in these tumours. Here, we aimed to uncover how the WNT pathway is activated in BRAF V600E-mutant colon cancer. Design: CRISPR-LbCpf1-corrected BRAF (V600E) and RNF43 (P441fs) organoids were analysed for their growth potential in the presence or absence of growth factors, for their ability to form tumours, and by immunoblotting, RNA sequencing, and DNA methylation assays. Results: BRAF E600V organoids regained dependency on epidermal growth factor (EGF) receptor signalling for ERK pathway activation and proliferation, and completely lost tumorigenic potential. Correction of BRAF V600E, but not RNF43 P441fs, caused suppression of WNT target genes, while genes reflecting epithelial differentiation and negative regulators of WNT signalling were upregulated in BRAF E600V organoids. DNA methylation analysis revealed promoter hypermethylation of WNT antagonist genes in BRAF V600E organoids. Gene body hypermethylation, associated with transcriptional upregulation, was observed in key WNT-driven intestinal stem cell marker genes LGR5 and EPHB2, and in the WNT-effector TCF4. Treatment of BRAF V600E organoids with the DNA demethylating agent 5-aza-2′-deoxycytidine resulted in upregulation of WNT antagonist genes and a marked decrease in WNT target gene expression. Conclusions: BRAFV600E, rather than mutant RNF43, is a prominent force behind WNT pathway activation, and is essential for maintaining tumorigenic capacity. BRAFV600E causes widespread dysregulation of DNA methylation leading to activation of WNT signalling. Citation Format: Layla El Bouazzaoui, Jeroen M. Bugter, Emre Küçükköse, André Verheem, Jasmin B. Post, Nicola Fenderico, Inne H. Borel Rinkes, Hugo J. Snippert, Madelon M. Maurice, Onno Kranenburg. BRAFV600E drives WNT signalling in colorectal cancer via aberrant DNA methylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB301.

Abstract 1566: Cancer metastasis and stemness: The metastasis-inducer MACC1 regulates LGR5 to promote cancer stem cell-like properties in colorectal cancer

Abstract Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths worldwide. The high mortality is directly associated with metastatic disease which is thought to be initiated by colon cancer stem cells according to cancer stem cell (CSC) model. Consequently, early identification of those patients who are at high risk for metastasis is crucial for improved treatment and patient outcome. Metastasis-associated in colon cancer 1 (MACC1) is a novel prognostic biomarker for tumor progression and metastasis formation independent of tumor stage. We previously showed an involvement of MACC1 in cancer stemness in the mouse intestine of our transgenic mouse models. However, the expression of MACC1 in human CSCs and possible implications remain elusive. Here, we explored the molecular mechanisms by which MACC1 regulates stemness and CSC-associated invasive phenotype based on patient-derived 3D cell culture models (PD3D), patient-derived xenografts (PDX) and human CRC cell lines. We showed that CD44-enriched CSCs from PD3D models express significantly higher levels of MACC1 and display higher tumorigenicity in immunocompromised mice. Similarly, RNA sequencing performed on PD3D and PDX models demonstrated significantly increased MACC1 expression in ALDH1(+) CSCs, highlighting its involvement in cancer stemness. We further showed the correlation of MACC1 with CSC markers CD44, NANOG and LGR5 in PD3D models as well as established cell lines. Additionally, MACC1 increased stem cell gene expression, clonogenicity and sphere formation. Strikingly, we showed that MACC1 binds as a transcription factor to the LGR5 gene promoter uncovering the long-known CSC marker LGR5 as novel essential signaling mediator used by MACC1 to induce CSC-like properties in human CRC patients. Our in vitro findings were further substantiated by significant positive correlation of MACC1 and LGR5 in CRC cell lines as well as CRC patient tumors. Taken together, this study indicates that the metastasis-inducer MACC1 act as a cancer stem cell-associated marker. Interventional approaches targeting MACC1 would potentially improve further targeted therapies for colorectal cancer patients to eradicate CSCs, prevent cancer recurrence and distant metastasis formation. Citation Format: Müge Erdem, Kyung Hwan Lee, Markus Hardt, Joseph Regan, Dennis Kobelt, Wolfgang Walther, Christian Regenbrecht, Ulrike S. Stein. Cancer metastasis and stemness: The metastasis-inducer MACC1 regulates LGR5 to promote cancer stem cell-like properties in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1566.

Mechanic evaluation of Wu-Mei-Pill on colitis-associated colorectal cancer: An integrated transcriptomics, metabolomics, and experimental validation study

BackgroundChronic intestinal inflammatory diseases play a crucial role in the onset of colorectal cancer (CRC). Effectively impeding the progression of colitis-associated colorectal cancer (CAC) can be instrumental in hindering CRC development. Wu-Mei-Pill (WMP), a formulation comprising various herbal extracts, is clinically employed for CAC treatment, yet the underlying mechanism of WMP's efficacy in CAC remains unclear. Our study firstly demonstrated the effects and mechanisms of WMP on transcriptional and metabolic levels based on integrated transcriptomics and untargeted metabolomics and relative experimental validations. Materials and methodsA CAC mouse model was established through a single injection of azoxymethane (AOM) followed by intermittent dextran sodium sulfate (DSS) intervention, with subsequent WMP administration. Initially, the therapeutic impact of WMP on the CAC model was assessed by observing survival rate, body weight change, colon length, tumor number, tumor load, and pathological changes in the colon tissue of CAC mice post-WMP intervention. Subsequently, differential genes and metabolites in the colorectal tissue of CAC mice following WMP intervention were identified through transcriptomics and non-targeted metabolomics. Finally, the influence of WMP on the peroxisome proliferator activated receptor (PPAR) pathway, Wnt pathway, and CC motif chemokine ligand 3 (CCL3)/ CC motif chemokine receptor 1 (CCR1) axis in CAC mice was verified through western blot, immunofluorescence, and ELISA based on the results of transcriptomics and non-targeted metabolomics. ResultsWMP intervention enhanced survival, alleviated body weight loss, shortened colon length, tumor occurrence, and pathological changes in the colorectal tissue of CAC mice, such as glandular damage, tumourigenesis, and inflammatory cell infiltration. Transcriptomic and non-targeted metabolomic results revealed that WMP intervention up-regulated the expression of key regulatory mechanisms of fatty acid oxidation PPAR pathway-related genes (Pparg, Ppara, Cpt1a, and Acadm) and metabolites (L-carnitine and L-palmitoylcarnitine). Additionally, it down-regulated Wnt pathway-related genes (Wnt3, Axin2, Tcf7, Mmp7, Lgr5, Wnt5a, Fzd6, Wnt7b, Lef1, and Fzd10 etc.) and pro-inflammatory related genes (Il1b, Il6, Il17a, Ccl3, and Ccr1 etc.). Experimental validation demonstrated that WMP up-regulated PPAR pathway-related proteins [PPARγ, PPARα, carnitine palmitoyltransferase 1A (CPT1A), and acyl-CoA dehydrogenase medium chain (ACADM)] in the colorectal tissue of CAC mice. It also down-regulated Wnt pathway-related proteins [β-catenin, T-cell factor (TCF), lymphoid enhancer-binding factor (LEF), and matrix metallopeptidase 7 (MMP7)], inhibited the nuclear translocation of the key transcription factor β-catenin in the Wnt pathway, and suppressed epithelial-to-mesenchymal transition (EMT) activation induced by the Wnt pathway (up-regulated E-cadherin and down-regulated Vimentin). Furthermore, WMP intervention reduced pro-inflammatory factors [interleukin (IL)-6, IL-1β, and IL-17A] and decreased CCL3/CCR1 axis factors, including CCL3 protein levels and diminished F4/80+CCR1+ positive expressed cells. ConclusionWMP significantly inhibits CAC tumorigenesis by up-regulating PPARα-mediated fatty acid oxidation, inhibiting the Wnt signaling pathway-mediated EMT, and suppressing CCL3/CCR1-mediated inflammatory responses.

Stratification of Colorectal Patients Based on Survival Analysis Shows the Value of Consensus Molecular Subtypes and Reveals the CBLL1 Gene as a Biomarker of CMS2 Tumours.

The consensus molecular subtypes (CMSs) classification of colorectal cancer (CRC) is a system for patient stratification that can be potentially applied to therapeutic decisions. Hakai (CBLL1) is an E3 ubiquitin-ligase that induces the ubiquitination and degradation of E-cadherin, inducing epithelial-to-mesenchymal transition (EMT), tumour progression and metastasis. Using bioinformatic methods, we have analysed CBLL1 expression on a large integrated cohort of primary tumour samples from CRC patients. The cohort included survival data and was divided into consensus molecular subtypes. Colon cancer tumourspheres were used to analyse the expression of stem cancer cells markers via RT-PCR and Western blotting. We show that CBLL1 gene expression is specifically associated with canonical subtype CMS2. WNT target genes LGR5 and c-MYC show a similar association with CMS2 as CBLL1. These mRNA levels are highly upregulated in cancer tumourspheres, while CBLL1 silencing shows a clear reduction in tumoursphere size and in stem cell biomarkers. Importantly, CMS2 patients with high CBLL1 expression displayed worse overall survival (OS), which is similar to that associated with CMS4 tumours. Our findings reveal CBLL1 as a specific biomarker for CMS2 and the potential of using CMS2 with high CBLL1 expression to stratify patients with poor OS.

Epigenetic characterization of sarcopenia-associated genes based on machine learning and network screening

To screen characteristic genes related to sarcopenia by bioinformatics and machine learning, and to verify the accuracy of characteristic genes in the diagnosis of sarcopenia. Download myopia-related data sets from geo public database, find the differential genes through R language limma package after merging, STRING database to build protein interaction network, and do Go analysis and GSEA analysis to understand the functions and molecular signal pathways that may be affected by the differential genes. Further screen the characteristic genes through LASSO and SVM-RFE machine algorithms, make the ROC curve of the characteristic genes, and obtain the AUC value. 10 differential genes were obtained from the data set, including 7 upregulated genes and 3 downregulated genes. Eight characteristic genes were screened by a machine learning algorithm, and the AUC value of characteristic genes exceeded 0.7. In patients with sarcopenia, the expression of TPPP3, C1QA, LGR5, MYH8, and CDKN1A genes are upregulated, and the expression of SLC38A1, SERPINA5, and HOXB2 genes are downregulated. The above genes have high accuracy in the diagnosis of sarcopenia. The research results provide new ideas for the diagnosis and mechanism research of sarcopenia.

The Anti-Müllerian Hormone as Endocrine and Molecular Marker Associated with Reproductive Performance in Holstein Dairy Cows Exposed to Heat Stress.

Anti-Müllerian hormone (AMH) is proposed as a biomarker for fertility in cattle, yet this associative relationship appears to be influenced by heat stress (HS). The objective was to test serum AMH and AMH-related single nucleotide polymorphisms (SNPs) as markers potentially predictive of reproductive traits in dairy cows experiencing HS. The study included 300 Holstein cows that were genotyped using BovineSNP50 (54,000 SNP). A genome-wide association study was then executed. Nine intragenic SNPs within the pathways that influence the AMH gene were found important with multiple comparisons adjustment tests (p < 1.09 × 10-6). A further validation study was performed in an independent Holstein cattle population, which was divided into moderate (MH; n = 152) and severe heat-stressed (SH; n = 128) groups and then subjected to a summer reproductive management program. Serum AMH was confirmed as a predictor of fertility measures (p < 0.05) in MH but not in the SH group. Cows were genotyped, which revealed four SNPs as predictive markers for serum AMH (p < 0.01), reproductive traits (p < 0.01), and additional physiological variables (p < 0.05). These SNPs were in the genes AMH, IGFBP1, LGR5, and TLR4. In conclusion, serum AMH concentrations and AMH polymorphisms are proposed as predictive markers that can be used in conjunction with genomic breeding value approaches to improve reproductive performance in Holstein cows exposed to summer HS conditions.

LGR5, a prognostic stem cell target, promotes endometrial cancer proliferation through autophagy activation

Endometrial cancer (EC) is a common malignant tumor in women worldwide. Although early EC has a good prognosis, advanced endometrial cancer is still associated with the risk of drug resistance and recurrence. Cancer stem cells (CSCs), a category closely related to drug resistance and recurrence, are rarely studied at present. Here, we constructed a risk model containing ten stemness-related prognostic genes. Compared with patients in the low-risk group, patients in the high-risk group had a shorter overall survival time. The accuracy of this model was verified by ROC in the TCGA (AUC = 0.779) and Peking University People's Hospital (PKUPH, AUC = 0.864) cohorts. The risk score and stage were independent risk factors in the multivariate regression analysis, which was subsequently used to construct the nomogram and verified in the TCGA cohort. LGR5 was significantly correlated with overall survival and involvement in the Wnt signaling pathway. In addition, LGR5 was highly expressed in EC tissues and was related to age, stage, histological type, and menopause status in the TCGA database. Overexpression of LGR5 accelerated the proliferation rate of EC cells, which may be related to autophagy activation. Taken together, our study established a prognostic model based on transcription sequencing data from the TCGA database and verified it in the PKUPH cohort, which has prospective clinical implications for the prognostic evaluation of EC. We systematically studied the code gene LGR5 in EC, which may help clinicians make personalized prognostic assessments and effective clinical decisions for EC.

Abstract 18338: TBX4 Loss-of-Function Within the Embryonic Lung Mesenchyme Disrupts Postnatal Lung Development and Increases Susceptibility to Pulmonary Hypertension

Introduction: The T-box 4 (TBX4) transcription factor is recognized as a risk gene in pulmonary arterial hypertension (PAH). TBX4 is expressed within the lung mesenchyme in the developing lung and is required for lung branching morphogenesis. Genetic studies report that PAH cases with TBX4 mutations display an earlier onset of the disease. In addition, studies have reported carriers of TBX4 mutations with diffused alveolar development and pulmonary hypertension (PH). Currently, there is a lack of evidence on the effect of TBX4 loss-of-function (LoF) on later stages of lung development and predisposition to lung disease. Hypothesis: Lung mesenchyme Tbx4 deletion within the developing lung disrupts essential gene regulatory networks leading to abnormal postnatal lung development and susceptibility to pulmonary hypertension. Methods: We have generated Tbx4 conditional knockout ( Tbx4-CKO ) mice in which Cre recombinase removes the exon 5 of Tbx4 within the embryonic lung mesenchyme, creating a null allele. To examine alveolarization, we harvested postnatal day 14 (P14) and 36 (P36) lungs and calculated mean linear intercept (MLI). RNA-seq was performed at both stages to identify disrupted gene networks in Tbx4-CKO lungs. RVSP was measured by right heart catheterization in six-month-old mice to evaluate for PH. Results: Tbx4-CKO lungs show increasing alveolar simplification at P14 and P36, as confirmed by increases in MLI of 24.8% and 34.4%, respectively. Tbx4-CKO mice show a statistically significant increase of 10.4% in RVSP, and preliminary analysis of vessel wall thickness suggests mild vascular remodeling in their lungs. RNA-seq analysis shows enrichment of pathways (canonical WNT, VEGF, and BMP signaling) and genes ( Lgr5 , Tnc , Wnt3a , Areg , Gdf2 , and Bmper ) relevant to lung alveologenesis, angiogenesis, and PH. Conclusions: TBX4 LoF affects lung alveolar development, similar to observations made in human cases with TBX4 mutations. The clinical presentation of such cases is broad, but our mice show features of bronchopulmonary dysplasia, including alveolar simplification and PH. Further analysis of our RNA-seq data and planned single-cell analyses should provide insight into the role of TBX4 in alveolarization and PH risk.

DNA methylation and expression of LGR6 gene in ankylosing spondylitis: A case-control study

ObjectiveThe objectives of the present research were to ascertain the relationship of Leucine-Rich Repeat-Containing G-Protein Coupled Receptors 6 (LGR6) methylation and transcript levels with ankylosing spondylitis (AS). MethodsTargeted bisulfite sequencing was applied to analyze LGR6 DNA methylation in 81 AS cases and 81 controls. Besides, the LGR6 transcription level of peripheral blood mononuclear cells (PBMCs) from 70 AS cases and 64 controls was measured utilizing quantitative real-time transcription-polymerase chain reaction (qRT-PCR). ResultsThe study detected the methylation levels of 43 sites in two CpG (cytosine-guanine dinucleotide) islands of LGR6 and found that LGR6 were significantly hypomethylated in AS patients (LGR6_1: P = 0.002; LGR6_2: P < 0.001). LGR6 transcript level was obviously reduced in AS (P = 0.001) and was positively related to DNA methylation level (CpG-1: P = 0.010; CpG-2: P = 0.007). Besides, the Receiver operating characteristic curve (ROC) exhibited good diagnostic performance of LGR6 methylation level (AUC = 0.676, 95% CI = 0.594–0.758, P < 0.001). Further subgroup analysis revealed that gender may affect the LGR6_1 methylation pattern. ConclusionThe present study revealed that LGR6 DNA methylation dysregulation may be involved in the pathogenesis of AS from an epigenetic perspective for the first time, with the aim of providing new directions for biomarker identification and treatment development for AS patients.

Myocardial Remodeling in Early Chronic Kidney Disease—Mineral and Bone Disorder Model with Low Bone Turnover

Chronic kidney disease—mineral and bone disorder (CKD-MBD) plays a significant role in causing cardiovascular morbidity and mortality related to CKD. CKD-MBD has been studied during advanced stages when changes in inorganic phosphate (Pi) and its hormonal regulation are obvious. The initial phases of myocardial remodeling (MR) in early CKD-MBD remain poorly understood. We induced mild CKD-MBD in spontaneously hypertensive rats using 3/4 nephrectomy. Animals were fed standard chow, containing 0.6% phosphate. In each animal, we analyzed indices of chronic kidney injury, bone turnover and Pi exchange, and assessed the myocardial histology and gene expression profile. Applied CKD-MBD models corresponded to human CKD S1-2 with low bone turnover and without an increase in systemic Pi-regulating factors (parathyroid hormone and fibroblast growth factor 23). In mild CKD-MBD models, we found MR features characterized by cardiomyocyte hypertrophy, interstitial and perivascular fibrosis, intramyocardial artery media thickening, along with alterations in Ppp3ca, Mapk1, Jag1, Hes1, Ptch1, Numb, Lgr4 and Bmp4 genes. Among other genes, the down-regulation of Jag1 was most tightly associated with either myocardial hypertrophy or fibrosis. Myocardial alterations concurrently occurred with mild CKD-MBD and comprised fibrosis preceding cardiomyocyte hypertrophy. The histological features of MR were associated with myocardial P accumulation in settings of low bone turnover, prior to a response of systemic Pi-regulating factors and with alterations in calcineurin, ERK1/2, Notch, BMP and Hedgehog genes.

Breastfeeding lifespan control of growth, maintenance, and metabolism of small intestinal epithelium.

Gastrointestinal epithelial cells respond to milk-born molecules throughout breastfeeding, influencing growth, and development. The rapid renewal of the small intestine depends on the proliferation in the crypt that drives cell fates. We used early weaning model to investigate immediate and late effects of breastfeeding on proliferation, differentiation of jejunal epithelial cells. Wistar rats were either allowed to suckle (S) until 21 postnatal days or submitted to early weaning (EW) at 15 days. By comparing ages (18, 60, and 120 days), we found that EW decreased Ki67 indices and villi height at 18 and 60 days (p < 0.05), and at 120 days they were similar between diets. Proliferative reduction and augmented expression of Cdkn1b (p27 gene) were parallel. In the stem cell niche, EW increased the number and activity (Defa24) of Paneth cells at 18 and 60 days (p < 0.05), and Lgr5 and Ascl2 genes showed inverted responses between ages. Among target cells, EW decreased goblet cell number at 18 and 60 days (p < 0.05) and increased it at 120 days (p < 0.05), whereas enteroendocrine marker genes were differentially altered. EW reduced enterocytes density at 18 days (p < 0.05), and at 120 days this population was decreased (vs. 60 days). Among cell fate crypt-controlling genes, Notch and Atoh1 were the main targets of EW. Metabolically, intraperitoneal glucose tolerance was immediately reduced (18 days), being reverted until 120 days (p < 0.05). Currently, we showed that breastfeeding has a lifespan influence on intestinal mucosa and on its stem cell compartment. We suggest that, although jejunum absorptive function is granted after early weaning, the long lasting changes in gene expression might prime the mucosa with a different sensitivity to gut disorders that still have to be further explored.

Genome-Wide Association Study Identifies 4 Novel Risk Loci for Small Intestinal Neuroendocrine Tumors Including a Missense Mutation in LGR5

Small intestinal neuroendocrine tumor (SI-NET) is a rare disease, but its incidence has increased over the past 4 decades. Understanding the genetic risk factors underlying SI-NETs can help in disease prevention and may provide clinically beneficial markers for diagnosis. Here the results of the largest genome-wide association study of SI-NETs performed to date with 405 cases and 614,666 controls are reported. Samples from 307 patients with SI-NETs and 287,137 controls in the FinnGen study were used for the identification of SI-NET risk-associated genetic variants. The results were also meta-analyzed with summary statistics from the UK Biobank (n= 98 patients with SI-NET and n= 327,529 controls). We identified 6 genome-wide significant (P < 5× 10-8) loci associated with SI-NET risk, of which 4 (near SEMA6A, LGR5, CDKAL1, and FERMT2) are novel and 2 (near LTA4H-ELK and in KIF16B) have been reported previously. Interestingly, the top hit (rs200138614; P= 1.80× 10-19) was a missense variant (p.Cys712Phe) in the LGR5 gene, a bona-fide marker of adult intestinal stem cells and a potentiator of canonical WNT signaling. The association was validated in an independent Finnish collection of 70 patients with SI-NETs, as well as in the UK Biobank exome sequence data (n= 92 cases and n= 392,814 controls). Overexpression of LGR5 p.Cys712Phe in intestinal organoids abolished the ability of R-Spondin1 to support organoid growth, indicating that the mutation perturbed R-Spondin-LGR5 signaling. Our study is the largest genome-wide association study to date on SI-NETs and reported 4 new associated genome-wide association study loci, including a novel missense mutation (rs200138614, p.Cys712Phe) in LGR5, a canonical marker of adult intestinal stem cells.

Abstract 5545: Assessing LGR5 expression levels on colorectal cancer tissue samples for use in LGR5-targeting CAR-T cell therapy clinical trial

Abstract Leucine-rich repeat containing G-protein coupled receptor (LGR5) is stem cell marker that potentiates canonical signaling in the Wnt/β-catenin signaling pathway (Barker et al. 2007). LGR5 is known to be expressed on several tissues; particularly the stem cells within intestinal crypts and the stem cell niche at the base of hair follicles (Barker et al. 2007 and Jaks et al. 2008). The plasticity and stem phenotype of LGR5+ cells has led to their identification as a cancer stem cell marker for some solid tumors including colorectal cancer (CRC) (Barker et al. 2009). This presents LGR5 as a potential target molecule for CAR-T cell directed specific tumor cell killing. For treatment of CRC with LGR5-directed CAR-T cells, it is critical to gain an understanding of the level of LGR5 expression on normal tissue compared with cancerous tissue. The aim of this study was to develop a screening technique for patient selection, and importantly identify potential responders to LGR5-targeting CAR-T cell therapy.Utilizing commercially purchased tissue microarrays a cohort of patient primary tumor biopsies (n=472) were assessed for LGR5 expression; ~60% displayed moderate to high level LGR5 positivity, with a trend toward higher grade, later stage tumors having higher levels of LGR5. LGR5 expression levels on lymph node metastases showed a positive correlation to LGR5 expression on patient-matched primary tumor tissue. Seven out of ten liver biopsies from metastatic colon cancer lesions showed a medium to high level LGR5 staining, indicating this may be used as a screening tool for metastatic CRC patients. We also observed restricted expression of LGR5 across the many normal tissues, suggesting limited off-target effects of LGR5-targeting CAR-T cells.The data obtained in this study provides evidence that LGR5 IHC of biopsies could be used as a diagnostic screening tool of CRC patients for inclusion into a Phase1a/2 clinical trial. Barker N, et al., 2007, Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 449: 1003–1007 Barker N. et al. 2009, Crypt stem cells as the cells-of-origin of intestinal cancer. Nature 457: 608–611Jaks V. et al. 2008, Lgr5 marks cycling, yet long-lived, hair follicle stem cells. Nat Genet. Nov;40(11):1291-9. Citation Format: Emma J. Thompson, Veronika Bandara, Batjargal Gundsambuu, Silvana Napoli, Stuart Mills, Jade Foeng, Dylan McPeake, Timona Tyllis, Caitlin Abbott, Allison Cowin, Timothy Sadlon, Shaun McColl, Carmela Ricciardelli, Simon C. Barry, Claudine S. Bonder. Assessing LGR5 expression levels on colorectal cancer tissue samples for use in LGR5-targeting CAR-T cell therapy clinical trial. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5545.

Loss of LGR5 through Therapy-induced Downregulation or Gene Ablation Is Associated with Resistance and Enhanced MET-STAT3 Signaling in Colorectal Cancer Cells.

Leucine-rich repeat-containing, G protein-coupled receptor 5 (LGR5) is highly expressed in colorectal cancer and cancer stem cells (CSCs) that play important roles in tumor initiation, progression, and metastasis. Loss of LGR5 has been shown to enhance therapy resistance. However, the molecular mechanisms that mediate this resistance remain elusive. In this study, we demonstrate conversion of LGR5+ colorectal cancer cells to an LGR5- state in response to chemotherapy, LGR5- targeted antibody-drug conjugates (ADCs), or LGR5 gene ablation led to activation of STAT3. Further investigation revealed increased STAT3 activation occurred as a result of increased mesenchymal epithelial transition (MET) factor receptor activity. LGR5 overexpression decreased MET-STAT3 activity and sensitized colorectal cancer cells to therapy. STAT3 inhibition suppressed MET phosphorylation, while constitutively active STAT3 reduced LGR5 levels and increased MET activity, suggesting a potential feedback mechanism. Combination treatment of MET-STAT3 inhibitors with irinotecan or antibody-drug conjugates (ADCs) substantiated synergistic effects in colorectal cancer cells and tumor organoids. In colorectal cancer xenografts, STAT3 inhibition combined with irinotecan enhanced tumor growth suppression and prolonged survival. These findings suggest a mechanism by which drug-resistant LGR5- colorectal cancer cells acquire a survival advantage through activation of MET-STAT3 and provide rationale for new treatment strategies to target colorectal cancer.

Murine and Human Gastric Tissue Establishes Organoids after 48 Hours of Cold Ischemia Time during Shipment.

An inadequate supply of fresh tissue is a major limitation of three-dimensional patient-derived gastric organoid research. We propose that tissue procurement for organoid culture could be increased by developing a cold storage shipment protocol for fresh surgical tissues. Sixty stomach specimens from C57BL/6J mice were resected, of which forty-five were stored in Hank's Balanced Salt (HBSS), University of Wisconsin (UW), or Histidine-Tryptophan-Ketoglutarate (HTK) solutions for subsequent organoid culture. Stomachs were dissociated and processed into gastric organoids as fresh tissue or after transport at 4 °C for 24 or 48 h. All gastric organoid cultures were established and maintained for 10 passages. Cold storage for 24 or 48 h did not significantly affect organoid viability. Although cold storage was associated with decreased organoid growth rate, there were no differences in viability, cytotoxic dose response, or LGR5 and TROY stem cell gene expression compared to organoids prepared from fresh tissue. As a proof of concept, six human gastric cancer organoids were established after 24 or 48 h of storage. Patient-derived gastric organoids from mouse and human gastric tissue can be established after 48 h of cold ischemia. Our method, which only requires ice packs, standard shipping containers, and HBSS is feasible and reliable. This method does not affect the reliability of downstream dose-response assays and maintains organoid viability for at least 10 passages. The shipment of fresh tissue for organoid procurement could serve to enhance multicenter collaboration and achieve more elaborate or controlled organoid experimentation.

Chronic GPER activation prompted the proliferation of ileal stem cell in ovariectomized mice depending on Paneth cell-derived Wnt3.

Menopausal women often face long-term estrogen treatment. G protein-coupled estrogen receptor (GPER) expressed in intestinal crypt was activated by estrogen therapy, but it was unclear whether chronic GPER activation during menopause had an effect on intestinal stem cells (ISCs). We tested the effect of chronic GPER activation on ISCs of ovariectomized (OVX) mice by injection of the selective GPER agonist G-1 for 28 days, or G-1 stimulation of organoids derived from crypts of OVX mice. G-1 up-regulated crypt depth, the number of Ki67+, bromodeoxyuridine+ cells and Olfm4+ ISCs, and the expression of ISCs marker genes (Lgr5, Olfm4 and Axin2). G-1 administration promoted organoid growth, increased the number of EdU+ cells per organoid and protein expression of Cyclin D1 and cyclin B1 in organoids. After G-1 treatment in vivo or in vitro, Paneth cell-derived Wnt3, Wnt3 effector β-catenin and Wnt target genes c-Myc and Cyclin D1 increased in ileum or organoids. Once blocking the secretion of Wnt3 from Paneth cells, the effects of G-1 on organoids growth, ISCs marker genes and Wnt/β-catenin signaling were abolished. G-1 did not affect the number of Paneth cells in ex vivo organoids, while activated Mmp7/cryptdin program in Paneth cells, promoted their maturation, and increased the expression of lysozyme protein. G-1 pretreatment in OVX mice inhibited radiation-induced ISCs proliferation injury and enhanced the resistance of mice to intestinal injury. In conclusion, chronic GPER activation prompted the Wnt3 synthesis in Paneth cells, thus increased the proliferation of ISCs via activation of Wnt3/β-catenin signaling in OVX mice.

Age-related glomerular histogenesis in inbred indigenous rabbit (Oryctolagus cuniculus): A morphological, morphometrical, and immunohistochemical study with emphasis on Lgr5-positive cells

Although the regeneration of renal glomeruli and nephrons after injuries especially in adult mammals is not possible, understanding normal glomerular histogenesis is important. Here, we sought to study the morphometrical and histological development of the normal renal glomeruli of rabbits from birth until postnatal day 40. Moreover, we immunohistochemically evaluated the extent and rate of the Lgr5 expression in the immature renal stem/progenitor cells. The untreated, clinically healthy inbred indigenous rabbits (from Duhok city of Iraqi Kurdistan) were sacrificed at postnatal days 1, 10, 15, 30, and 40. After being processed and embedded in paraffin, rabbit anti-human Lgr5 as a primary antibody and rabbit ImmunoCruz LSAB as a staining kit were used for the immunohistochemical detection of Lgr5+ve cells. For normal histology, hematoxylin and eosin were used. The peak generation and regression of renal corpuscles were at postnatal days 10, and 40, respectively, with 50% decrease. The glomeruli diameter significantly increased (1.3-fold, p = 0.001), whereas the Bowman’s space diameter decreased (50%, p < 0.0001) from postnatal day 1–40. The immature nephrons were seen only in one-day postnatal rabbits. While the superficial glomeruli were compact and small, the juxtamedullary glomeruli were larger and segmented. The formation and development of the juxtaglomerular apparatus were documented at postnatal days 30 and 40 only. Our data revealed highly expressed Lgr5 protein at postnatal day one, and the expression level decreased gradually with advancing age. It was moderately expressed on day 10 and mildly expressed on day 15, whereas no expression was recorded on days 30 and 40 postnatally. Our study provides evidence that the Lgr5 gene, within multipotent stem cells and their lineage progeny, was activated within newly formed glomeruli throughout the early postnatal stages of nephrogenesis.

Custom gene expression panel for evaluation of potential molecular markers in hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is the second leading cause of cancer-related mortality worldwide. It is a highly heterogeneous disease with poor prognosis and limited treatment options, which highlights the need for reliable biomarkers. This study aims to explore molecular markers that allow stratification of HCC and may lead to better prognosis and treatment prediction.Materials and methodsWe studied 20 candidate genes (HCC hub genes, potential drug target genes, predominant somatic mutant genes) retrieved from literature and public databases with potential to be used as the molecular markers. We analysed expression of the genes by RT-qPCR in 30 HCC tumour and adjacent non-tumour paired samples from Vietnamese patients. Fold changes in expression were then determined using the 2−∆∆CT method, and unsupervised hierarchical clustering was generated using Cluster v3.0 software.ResultsClustering of expression data revealed two subtypes of tumours (proliferative and normal-like) and four clusters for genes. The expression profiles of the genes TOP2A, CDK1, BIRC5, GPC3, IGF2, and AFP were strongly correlated. Proliferative tumours were characterized by high expression of the c-MET, ARID1A, CTNNB1, RAF1, LGR5, and GLUL1 genes. TOP2A, CDK1, and BIRC5 HCC hub genes were highly expressed (> twofold) in 90% (27/30), 83% (25/30), and 83% (24/30) in the tissue samples, respectively. Among the drug target genes, high expression was observed in the GPC3, IGF2 and c-MET genes in 77% (23/30), 63% (19/30), and 37% (11/30), respectively. The somatic mutant Wnt/ß-catenin genes (CTNNB1, GLUL and LGR5) and TERT were highly expressed in 40% and 33% of HCCs, respectively. Among the HCC marker genes, a higher percentage of tumours showed GPC3 expression compared to AFP expression [73% (23/30) vs. 43% (13/30)].ConclusionThe custom panel and molecular markers from this study may be useful for diagnosis, prognosis, biomarker-guided clinical trial design, and prediction of treatment outcomes.