Identification, isolation and characterization of human LGR5-positive colon adenoma cells

The intestine is maintained by stem cells located at the base of crypts and distinguished by the expression of LGR5. Genetically engineered mouse models have provided a wealth of information about intestinal stem cells, whereas less is known about human intestinal stem cells owing to difficulty detecting and isolating these cells. We established an organoid repository from patient-derived adenomas, adenocarcinomas and normal colon, which we analyzed for variants in 71 colorectal cancer (CRC)-associated genes. Normal and neoplastic colon tissue organoids were analyzed by immunohistochemistry and fluorescent-activated cell sorting for LGR5. LGR5-positive cells were isolated from four adenoma organoid lines and were subjected to RNA sequencing. We found that LGR5 expression in the epithelium and stroma was associated with tumor stage, and by integrating functional experiments with LGR5-sorted cell RNA sequencing data from adenoma and normal organoids, we found correlations between LGR5 and CRC-specific genes, including dickkopf WNT signaling pathway inhibitor 4 (DKK4) and SPARC-related modular calcium binding 2 (SMOC2). Collectively, this work provides resources, methods and new markers to isolate and study stem cells in human tissue homeostasis and carcinogenesis. Summary: Immunohistochemical and transcriptomic analyses of organoids generated from precancerous adenoma, colon adenocarcinoma and normal human tissue shows that the intestinal stem cell marker LGR5 is a colon cancer prognostic factor.

[1]  Study of the root transcriptome of bread wheat using high-throughput RNA sequencing (RNA-SEQ) , 2020, Bioinformatics of Genome Regulation and Structure/ Systems Biology.

[2]  E. Vakiani,et al.  Logarithmic expansion of LGR5+ cells in human colorectal cancer. , 2018, Cellular signalling.

[3]  A. Regev,et al.  Anatomically and Functionally Distinct Lung Mesenchymal Populations Marked by Lgr5 and Lgr6 , 2017, Cell.

[4]  A. Saadatpour,et al.  Dynamic Reorganization of Chromatin Accessibility Signatures during Dedifferentiation of Secretory Precursors into Lgr5+ Intestinal Stem Cells. , 2017, Cell stem cell.

[5]  Hanlee P. Ji,et al.  Intestinal Enteroendocrine Lineage Cells Possess Homeostatic and Injury-Inducible Stem Cell Activity. , 2017, Cell stem cell.

[6]  Camille Stephan-Otto Attolini,et al.  Mex3a Marks a Slowly Dividing Subpopulation of Lgr5+ Intestinal Stem Cells. , 2017, Cell stem cell.

[7]  Shinya Sugimoto,et al.  Visualization and targeting of LGR5+ human colon cancer stem cells , 2017, Nature.

[8]  M. Caron,et al.  Inhibiting clathrin-mediated endocytosis of the leucine-rich G protein-coupled receptor-5 diminishes cell fitness , 2017, The Journal of Biological Chemistry.

[9]  J. Karp,et al.  Clonal Expansion of Lgr5-Positive Cells from Mammalian Cochlea and High-Purity Generation of Sensory Hair Cells. , 2017, Cell reports.

[10]  下川 真理子.  Visualization and targeting of LGR5⁺ human colon cancer stem cells(審査報告) , 2017 .

[11]  D. Schlessinger,et al.  Molecular dynamics of Dkk4 modulates Wnt action and regulates meibomian gland development , 2016, Development.

[12]  B. Győrffy,et al.  Iron Uptake via DMT1 Integrates Cell Cycle with JAK-STAT3 Signaling to Promote Colorectal Tumorigenesis. , 2016, Cell metabolism.

[13]  T. Ried,et al.  Targeting colorectal cancer (stem-like) cells using LGR5 directed antibody drug conjugates. , 2016, Annals of translational medicine.

[14]  Alexander van Oudenaarden,et al.  Reg4+ deep crypt secretory cells function as epithelial niche for Lgr5+ stem cells in colon , 2016, Proceedings of the National Academy of Sciences.

[15]  Toshio Uraoka,et al.  A Colorectal Tumor Organoid Library Demonstrates Progressive Loss of Niche Factor Requirements during Tumorigenesis. , 2016, Cell stem cell.

[16]  Sukhen C Ghosh,et al.  LGR5-Targeted Antibody–Drug Conjugate Eradicates Gastrointestinal Tumors and Prevents Recurrence , 2016, Molecular Cancer Therapeutics.

[17]  Jason R Spence,et al.  Organoid Models of Human Gastrointestinal Development and Disease. , 2016, Gastroenterology.

[18]  G. Kang,et al.  Distribution of intestinal stem cell markers in colorectal precancerous lesions , 2016, Histopathology.

[19]  J. Takagi,et al.  Active and water-soluble form of lipidated Wnt protein is maintained by a serum glycoprotein afamin/α-albumin , 2016, eLife.

[20]  A. Kobielak,et al.  Lgr5 Marks Neural Crest Derived Multipotent Oral Stromal Stem Cells , 2016, Stem cells.

[21]  Alexander van Oudenaarden,et al.  Replacement of Lost Lgr5-Positive Stem Cells through Plasticity of Their Enterocyte-Lineage Daughters. , 2016, Cell stem cell.

[22]  Dudley Lamming,et al.  High fat diet enhances stemness and tumorigenicity of intestinal progenitors , 2016, Nature.

[23]  Manasvi Shah,et al.  Dormant Intestinal Stem Cells Are Regulated by PTEN and Nutritional Status. , 2015, Cell reports.

[24]  Mallika Singh,et al.  Targeting LGR5+ cells with an antibody-drug conjugate for the treatment of colon cancer , 2015, Science Translational Medicine.

[25]  P. Aloy,et al.  Isolation of Human Colon Stem Cells Using Surface Expression of PTK7 , 2015, Stem cell reports.

[26]  O. Klein,et al.  Transcriptome-wide Analysis Reveals Hallmarks of Human Intestine Development and Maturation In Vitro and In Vivo , 2015, Stem cell reports.

[27]  N. Nagarajan,et al.  Cloning and Variation of Ground State Intestinal Stem Cells , 2015, Nature.

[28]  N. Nishimura,et al.  Chemokine-Targeted Mouse Models of Human Primary and Metastatic Colorectal Cancer , 2015, Nature Biotechnology.

[29]  Hayley E. Francies,et al.  Prospective Derivation of a Living Organoid Biobank of Colorectal Cancer Patients , 2015, Cell.

[30]  J. Eastham‐Anderson,et al.  Castration-Resistant Lgr5+ Cells Are Long-Lived Stem Cells Required for Prostatic Regeneration , 2015, Stem cell reports.

[31]  Hans Clevers,et al.  Sequential cancer mutations in cultured human intestinal stem cells , 2015, Nature.

[32]  T. Graham,et al.  Characterization of LGR5 stem cells in colorectal adenomas and carcinomas , 2015, Scientific Reports.

[33]  Takanori Kanai,et al.  Modeling colorectal cancer using CRISPR-Cas9–mediated engineering of human intestinal organoids , 2015, Nature Medicine.

[34]  Camille Stephan-Otto Attolini,et al.  Stromal gene expression defines poor-prognosis subtypes in colorectal cancer , 2015, Nature Genetics.

[35]  G. von Heijne,et al.  Tissue-based map of the human proteome , 2015, Science.

[36]  Hans Clevers,et al.  In vitro expansion of human gastric epithelial stem cells and their responses to bacterial infection. , 2015, Gastroenterology.

[37]  Mathias Leblanc,et al.  TRPV1 Pain Receptors Regulate Longevity and Metabolism by Neuropeptide Signaling , 2014, Cell.

[38]  Olivier Gevaert,et al.  Oncogenic transformation of diverse gastrointestinal tissues in primary organoid culture , 2014, Nature Medicine.

[39]  S. Buczacki,et al.  Side population sorting separates subfractions of cycling and non-cycling intestinal stem cells. , 2014, Stem cell research.

[40]  J. Varani,et al.  Human colonic crypts in culture: segregation of immunochemical markers in normal versus adenoma-derived , 2014, Laboratory Investigation.

[41]  N. Barker.  Adult intestinal stem cells: critical drivers of epithelial homeostasis and regeneration , 2013, Nature Reviews Molecular Cell Biology.

[42]  J. Karp,et al.  Niche-independent high-purity cultures of Lgr5+ intestinal stem cells and their progeny , 2013, Nature Methods.

[43]  W. Kim,et al.  Distribution of LGR5 + Cells and Associated Implications during the Early Stage of Gastric Tumorigenesis , 2013, PloS one.

[44]  Hiroyuki Miyoshi,et al.  In vitro expansion and genetic modification of gastrointestinal stem cells in spheroid culture , 2013, Nature Protocols.

[45]  H. Clevers,et al.  Retroviral gene expression control in primary organoid cultures. , 2013, Current protocols in stem cell biology.

[46]  E. Boerwinkle,et al.  dbNSFP v2.0: A Database of Human Non‐synonymous SNVs and Their Functional Predictions and Annotations , 2013, Human mutation.

[47]  Matthias Stelzner,et al.  Isolation and characterization of intestinal stem cells based on surface marker combinations and colony-formation assay. , 2013, Gastroenterology.

[48]  T. Imai,et al.  Genetic reconstitution of tumorigenesis in primary intestinal cells , 2013, Proceedings of the National Academy of Sciences.

[49]  A. Bosio,et al.  Abstract 3757: Novel monoclonal antibodies for the analysis and isolation of Lgr5 positive cells. , 2013 .

[50]  J. Spence,et al.  A Gutsy Task: Generating Intestinal Tissue from Human Pluripotent Stem Cells , 2013, Digestive Diseases and Sciences.

[51]  A. Sivachenko,et al.  Sensitive detection of somatic point mutations in impure and heterogeneous cancer samples , 2013, Nature Biotechnology.

[52]  Hans Clevers,et al.  In vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration , 2013, Nature.

[53]  A. McCullough.  Comprehensive molecular characterization of human colon and rectal cancer , 2013 .

[54]  Thomas R. Gingeras,et al.  STAR: ultrafast universal RNA-seq aligner , 2013, Bioinform..

[55]  H. Clevers,et al.  Redundant sources of Wnt regulate intestinal stem cells and promote formation of Paneth cells. , 2012, Gastroenterology.

[56]  A. Levine,et al.  LGR5‐Positive Colon Cancer Stem Cells Interconvert with Drug‐Resistant LGR5‐Negative cells and are Capable of Tumor Reconstitution , 2012, Stem cells.

[57]  W. de Lau,et al.  Monoclonal Antibodies Against Lgr5 Identify Human Colorectal Cancer Stem Cells , 2012, Stem cells.

[58]  H. Clevers,et al.  Developmental stage‐specific contribution of LGR5+ cells to basal and luminal epithelial lineages in the postnatal mammary gland , 2012, The Journal of pathology.

[59]  Kenny Q. Ye,et al.  An integrated map of genetic variation from 1,092 human genomes , 2012, Nature.

[60]  H. Clevers,et al.  Lgr5(+ve) stem/progenitor cells contribute to nephron formation during kidney development. , 2012, Cell reports.

[61]  Hans Clevers,et al.  Lineage Tracing Reveals Lgr5+ Stem Cell Activity in Mouse Intestinal Adenomas , 2012, Science.

[62]  Steven J. M. Jones,et al.  Comprehensive molecular characterization of human colon and rectal cancer , 2012, Nature.

[63]  Alexander van Oudenaarden,et al.  The Lgr5 Intestinal Stem Cell Signature: Robust Expression of Proposed Quiescent ' Þ 4' Cell Markers , 2022 .

[64]  Wendy S. W. Wong,et al.  Strelka: accurate somatic small-variant calling from sequenced tumor-normal sample pairs , 2012, Bioinform..

[65]  C. Röcken,et al.  The Spatial Distribution of LGR5+ Cells Correlates With Gastric Cancer Progression , 2012, PloS one.

[66]  Q. Lin,et al.  LGR5 Interacts and Cointernalizes with Wnt Receptors To Modulate Wnt/β-Catenin Signaling , 2012, Molecular and Cellular Biology.

[67]  Pablo Cingolani,et al.  © 2012 Landes Bioscience. Do not distribute. , 2022 .

[68]  Bruce J. Aronow,et al.  The Pan-ErbB Negative Regulator Lrig1 Is an Intestinal Stem Cell Marker that Functions as a Tumor Suppressor , 2012, Cell.

[69]  Christopher A. Miller,et al.  VarScan 2: somatic mutation and copy number alteration discovery in cancer by exome sequencing. , 2012, Genome research.

[70]  M. Ebert,et al.  TFAP2E-DKK4 and chemoresistance in colorectal cancer. , 2012, The New England journal of medicine.

[71]  M. Capecchi,et al.  The intestinal stem cell markers Bmi1 and Lgr5 identify two functionally distinct populations , 2011, Proceedings of the National Academy of Sciences.

[72]  Hans Clevers,et al.  Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium. , 2011, Gastroenterology.

[73]  Hans Clevers,et al.  Lgr5 homologues associate with Wnt receptors and mediate R-spondin signalling , 2011, Nature.

[74]  E. Boerwinkle,et al.  dbNSFP: A Lightweight Database of Human Nonsynonymous SNPs and Their Functional Predictions , 2011, Human mutation.

[75]  C. Kersten,et al.  Expression of the adult intestinal stem cell marker Lgr5 in the metastatic cascade of colorectal cancer. , 2011, International journal of clinical and experimental pathology.

[76]  M. DePristo,et al.  A framework for variation discovery and genotyping using next-generation DNA sequencing data , 2011, Nature Genetics.

[77]  Hans Clevers,et al.  Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts , 2011, Nature.

[78]  K. Mimori,et al.  Significance of Lgr5+ve Cancer Stem Cells in the Colon and Rectum , 2011, Annals of Surgical Oncology.

[79]  B. Garcia,et al.  Proteomics , 2011, Journal of biomedicine & biotechnology.

[80]  Camilla A. Richmond,et al.  Mouse telomerase reverse transcriptase (mTert) expression marks slowly cycling intestinal stem cells , 2010, Proceedings of the National Academy of Sciences.

[81]  R. Sellers,et al.  Recommendations for Pathology Peer Review , 2010, Toxicologic pathology.

[82]  Hans Clevers,et al.  Intestinal Crypt Homeostasis Results from Neutral Competition between Symmetrically Dividing Lgr5 Stem Cells , 2010, Cell.

[83]  Louis Vermeulen,et al.  Wnt activity defines colon cancer stem cells and is regulated by the microenvironment , 2010, Nature Cell Biology.

[84]  Xiangshan Fan,et al.  Expression of Lgr5 in human colorectal carcinogenesis and its potential correlation with β-catenin , 2010, International Journal of Colorectal Disease.

[85]  Aaron R. Quinlan,et al.  BIOINFORMATICS APPLICATIONS NOTE , 2022 .

[86]  Hans Clevers,et al.  Lgr5(+ve) stem cells drive self-renewal in the stomach and build long-lived gastric units in vitro. , 2010, Cell stem cell.

[87]  Mark D. Robinson,et al.  edgeR: a Bioconductor package for differential expression analysis of digital gene expression data , 2009, Bioinform..

[88]  Shinichi Takano,et al.  DICKKOPF‐4 and ‐2 genes are upregulated in human colorectal cancer , 2009, Cancer science.

[89]  Hans Clevers,et al.  OLFM4 is a robust marker for stem cells in human intestine and marks a subset of colorectal cancer cells. , 2009, Gastroenterology.

[90]  Richard Durbin,et al.  Sequence analysis Fast and accurate short read alignment with Burrows – Wheeler transform , 2009 .

[91]  H. Clevers,et al.  Single Lgr5 stem cells build crypt–villus structures in vitro without a mesenchymal niche , 2009, Nature.

[92]  Helmut Blum,et al.  Dickkopf-4 is frequently down-regulated and inhibits growth of colorectal cancer cells. , 2009, Cancer letters.

[93]  H. Mashimo,et al.  Immunostaining of Lgr5, an Intestinal Stem Cell Marker, in Normal and Premalignant Human Gastrointestinal Tissue , 2008, TheScientificWorldJournal.

[94]  Hans Clevers,et al.  Lgr5 marks cycling, yet long-lived, hair follicle stem cells , 2008, Nature Genetics.

[95]  C. Carlberg,et al.  DICKKOPF-4 is induced by TCF/β-catenin and upregulated in human colon cancer, promotes tumour cell invasion and angiogenesis and is repressed by 1α,25-dihydroxyvitamin D3 , 2008, Oncogene.

[96]  Hans Clevers,et al.  The intestinal stem cell. , 2008, Genes & development.

[97]  H. Clevers,et al.  Identification of stem cells in small intestine and colon by marker gene Lgr5 , 2007, Nature.

[98]  M. Katoh,et al.  Conserved POU-binding site linked to SP1-binding site within FZD5 promoter: Transcriptional mechanisms of FZD5 in undifferentiated human ES cells, fetal liver/spleen, adult colon, pancreatic islet, and diffuse-type gastric cancer. , 2007, International journal of oncology.

[99]  Wenwu Cui,et al.  qPrimerDepot: a primer database for quantitative real time PCR , 2006, Nucleic Acids Res..

[100]  Hiroyuki Ogata,et al.  KEGG: Kyoto Encyclopedia of Genes and Genomes , 1999, Nucleic Acids Res..

[101]  Y. Benjamini,et al.  Controlling the false discovery rate: a practical and powerful approach to multiple testing , 1995 .