Visualization and targeting of LGR5+ human colon cancer stem cells

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.

[1]  Robert A. Weinberg,et al.  Poised Chromatin at the ZEB1 Promoter Enables Breast Cancer Cell Plasticity and Enhances Tumorigenicity , 2013, Cell.

[2]  H. Clevers,et al.  Tracking down the stem cells of the intestine: strategies to identify adult stem cells. , 2007, Gastroenterology.

[3]  Guy S. Salvesen,et al.  SnapShot: Caspases , 2011, Cell.

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

[5]  R. Russell,et al.  Intestinal label-retaining cells are secretory precursors expressing Lgr5 , 2013, Nature.

[6]  A. Krešo,et al.  Evolution of the cancer stem cell model. , 2014, Cell stem cell.

[7]  Michael D. Brooks,et al.  Therapeutic Implications of Cellular Heterogeneity and Plasticity in Breast Cancer. , 2015, Cell stem cell.

[8]  M. Caligiuri,et al.  A cell initiating human acute myeloid leukaemia after transplantation into SCID mice , 1994, Nature.

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

[10]  M. Todaro,et al.  Colorectal cancer stem cells: from the crypt to the clinic. , 2014, Cell stem cell.

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

[12]  J. Kaiser.  The cancer stem cell gamble. , 2015, Science.

[13]  Z. Werb,et al.  The cancer stem cell niche: how essential is the niche in regulating stemness of tumor cells? , 2015, Cell stem cell.

[14]  O. Klein,et al.  A reserve stem cell population in small intestine renders Lgr5-positive cells dispensable , 2012, Nature.

[15]  Targeting colorectal cancer stem cells with inducible caspase-9 , 2012, Apoptosis.

[16]  David A. Scott,et al.  Genome engineering using the CRISPR-Cas9 system , 2013, Nature Protocols.

[17]  Hans Clevers,et al.  The intestinal stem cell signature identifies colorectal cancer stem cells and predicts disease relapse. , 2011, Cell stem cell.

[18]  Michael Kahn,et al.  Targeting Notch, Hedgehog, and Wnt pathways in cancer stem cells: clinical update , 2015, Nature Reviews Clinical Oncology.

[19]  Benjamin D. Simons,et al.  Defining the mode of tumour growth by clonal analysis , 2012, Nature.

[20]  Mamoru Ito,et al.  NOD/SCID/gamma(c)(null) mouse: an excellent recipient mouse model for engraftment of human cells. , 2002, Blood.

[21]  M. Todaro,et al.  IL-4-mediated drug resistance in colon cancer stem cells , 2008, Cell cycle.

[22]  Toshiro Sato,et al.  Efficient genetic engineering of human intestinal organoids using electroporation , 2015, Nature Protocols.

[23]  Peter Dirks,et al.  Cancer stem cells: an evolving concept , 2012, Nature Reviews Cancer.

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

[25]  Alexander van Oudenaarden,et al.  The Lgr 5 intestinal stem cell signature : robust expression of proposed quiescent ‘ þ 4 ’ cell markers , 2012 .

[26]  Mamoru Ito,et al.  NOD / SCID / cnull mouse : an excellent recipient mouse model for engraftment of human cells , 2002 .

[27]  H. Hermeking,et al.  Colorectal Cancers Mimic Structural Organization of Normal Colonic Crypts , 2014, PloS one.

[28]  J. Dick,et al.  A human colon cancer cell capable of initiating tumour growth in immunodeficient mice , 2007, Nature.

[29]  A. Oudenaarden,et al.  Dll1+ secretory progenitor cells revert to stem cells upon crypt damage , 2012, Nature Cell Biology.

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

[31]  A. van Oudenaarden,et al.  Induced Quiescence of Lgr5+ Stem Cells in Intestinal Organoids Enables Differentiation of Hormone-Producing Enteroendocrine Cells. , 2017, Cell stem cell.

[32]  Pradeep S Rajendran,et al.  Single-cell dissection of transcriptional heterogeneity in human colon tumors , 2011, Nature Biotechnology.

[33]  Johannes E. Schindelin,et al.  Fiji: an open-source platform for biological-image analysis , 2012, Nature Methods.

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

[35]  F. D. de Sauvage,et al.  Lgr5+ stem cells are indispensable for radiation-induced intestinal regeneration. , 2014, Cell stem cell.

[36]  L. Ricci-Vitiani,et al.  Identification and expansion of human colon-cancer-initiating cells , 2007, Nature.

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

[38]  Teresa L. Mastracci,et al.  Krt19(+)/Lgr5(-) Cells Are Radioresistant Cancer-Initiating Stem Cells in the Colon and Intestine. , 2015, Cell stem cell.

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

[40]  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.

[41]  F. Cordelières,et al.  A guided tour into subcellular colocalization analysis in light microscopy , 2006, Journal of microscopy.

[42]  C. Blanpain,et al.  Cancer Stem Cells: Basic Concepts and Therapeutic Implications. , 2016, Annual review of pathology.

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