title: Humanization of the mouse mammary gland by replacement of the luminal layer with genetically engineered preneoplastic human cells.
abstract: BACKGROUND
  The cell of origin for estrogen receptor α-positive (ERα+) breast cancer is probably a luminal stem cell in the terminal duct lobular units. To model these cells, we have used the murine myoepithelial layer in the mouse mammary ducts as a scaffold upon which to build a human luminal layer. To prevent squamous metaplasia, a common artifact in genetically-engineered breast cancer models, we sought to limit activation of the epidermal growth factor receptor (EGFR) during in vitro cell culture before grafting the cells.
  
  
  METHODS
  Human reduction mammoplasty cells were grown in vitro in WIT medium. Epidermal growth factor in the medium was replaced with amphiregulin and neuregulin to decrease activation of EGFR and increase activation of EGFR homologs 3 and 4 (ERBB3 and ERBB4). Lentiviral vectors were used to express oncogenic transgenes and fluorescent proteins. Human mammary epithelial cells were mixed with irradiated mouse fibroblasts and Matrigel, then injected through the nipple into the mammary ducts of immunodeficient mice. Engrafted cells were visualized by stereomicroscopy for fluorescent proteins and characterized by histology and immunohistochemistry.
  
  
  RESULTS
  Growth of normal mammary epithelial cells in conditions favoring ERBB3/4 signaling prevented squamous metaplasia in vitro. Normal human cells were quickly lost after intraductal injection, but cells infected with lentiviruses expressing CCND1, MYC, TERT, BMI1 and a short-hairpin RNA targeting TP53 were able to engraft and progressively replace the luminal layer in the mouse mammary ducts, resulting in the formation of an extensive network of humanized ducts. Despite expressing multiple oncogenes, the human cells formed a morphologically normal luminal layer. Expression of a single additional oncogene, PIK3CA-H1047R, converted the cells into invasive cancer cells. The resulting tumors were ERα+, Ki67+ luminal B adenocarcinomas that were resistant to treatment with fulvestrant.
  
  
  CONCLUSIONS
  Injection of preneoplastic human mammary epithelial cells into the mammary ducts of immunodeficient mice leads to replacement of the murine luminal layer with morphologically normal human cells. Genetic manipulation of the injected cells makes it possible to study defined steps in the transformation of human mammary epithelial cells in a more physiological environment than has hitherto been possible.
authors.affiliation: INSERM U916, Bergonié Cancer Institute, University of Bordeaux, 229 cours de l'Argonne, Bordeaux, 33076, France. stefverbeke@aol.com.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Verbeke
authors.affiliation: INSERM U916, Bergonié Cancer Institute, University of Bordeaux, 229 cours de l'Argonne, Bordeaux, 33076, France. e.richard@bordeaux.unicancer.fr.
authors.forename: Elodie
authors.initials: E
authors.lastname: Richard
authors.affiliation: INSERM U916, Bergonié Cancer Institute, University of Bordeaux, 229 cours de l'Argonne, Bordeaux, 33076, France. e.monceau@bordeaux.unicancer.fr.
authors.forename: Elodie
authors.initials: E
authors.lastname: Monceau
authors.affiliation: INSERM U916, Bergonié Cancer Institute, University of Bordeaux, 229 cours de l'Argonne, Bordeaux, 33076, France. xenia.schmidt@inserm.fr.|School of Medicine, University of St Andrews, Medical and Biological Sciences Building, North Haugh, St Andrews, KY16 9TF, UK. xenia.schmidt@inserm.fr.
authors.forename: Xenia
authors.initials: X
authors.lastname: Schmidt
authors.affiliation: Animalerie A2, University of Bordeaux, 146 rue Léo Saignat, 33076, Bordeaux, France. benoit.rousseau@u-bordeaux2.fr.
authors.forename: Benoit
authors.initials: B
authors.lastname: Rousseau
authors.affiliation: INSERM U916, Bergonié Cancer Institute, University of Bordeaux, 229 cours de l'Argonne, Bordeaux, 33076, France. v.velasco@bordeaux.unicancer.fr.|Pathology Department, Bergonié Cancer Institute, 229 cours de l'Argonne, 33076, Bordeaux, France. v.velasco@bordeaux.unicancer.fr.
authors.forename: Valerie
authors.initials: V
authors.lastname: Velasco
authors.affiliation: INSERM U1052, Centre Leon Berard, University of Lyon, 28 rue Laennec, 69008, Lyon, France. david.bernard@lyon.unicancer.fr.
authors.forename: David
authors.initials: D
authors.lastname: Bernard
authors.affiliation: INSERM U916, Bergonié Cancer Institute, University of Bordeaux, 229 cours de l'Argonne, Bordeaux, 33076, France. h.bonnefoi@bordeaux.unicancer.fr.
authors.forename: Herve
authors.initials: H
authors.lastname: Bonnefoi
authors.affiliation: INSERM U916, Bergonié Cancer Institute, University of Bordeaux, 229 cours de l'Argonne, Bordeaux, 33076, France. g.macgrogan@bordeaux.unicancer.fr.|Pathology Department, Bergonié Cancer Institute, 229 cours de l'Argonne, 33076, Bordeaux, France. g.macgrogan@bordeaux.unicancer.fr.
authors.forename: Gaetan
authors.initials: G
authors.lastname: MacGrogan
authors.affiliation: INSERM U916, Bergonié Cancer Institute, University of Bordeaux, 229 cours de l'Argonne, Bordeaux, 33076, France. r.iggo@bordeaux.unicancer.fr.|School of Medicine, University of St Andrews, Medical and Biological Sciences Building, North Haugh, St Andrews, KY16 9TF, UK. r.iggo@bordeaux.unicancer.fr.
authors.forename: Richard D
authors.initials: RD
authors.lastname: Iggo
mesh_terms: D000230:Adenocarcinoma / Q000235:genetics*; D000818:Animals; D002471:Cell Transformation, Neoplastic / Q000235:genetics; D017690:Cell Transplantation; D019938:Cyclin D1 / Q000235:genetics; D004195:Disease Models, Animal; D004847:Epithelial Cells / Q000378:metabolism / Q000637:transplantation*; D005260:Female; D005818:Genetic Engineering; D006801:Humans; D008321:Mammary Glands, Animal / Q000378:metabolism*; D042361:Mammary Glands, Human / Q000166:cytology* / Q000378:metabolism; D008325:Mammary Neoplasms, Experimental / Q000235:genetics*; D051379:Mice; D009368:Neoplasm Transplantation; D009857:Oncogenes; D063150:Polycomb Repressive Complex 1 / Q000235:genetics; D011230:Precancerous Conditions / Q000235:genetics*; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D020893:Receptor, ErbB-3 / Q000235:genetics; D066247:Receptor, ErbB-4 / Q000235:genetics; D019098:Telomerase / Q000235:genetics; D019076:Transgenes; D016159:Tumor Suppressor Protein p53 / Q000235:genetics
chemical_list: C069077:BMI1 protein, human; C530104:CCND1 protein, human; C489427:MYC protein, human; D016271:Proto-Oncogene Proteins c-myc; D034741:RNA, Small Interfering; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D019938:Cyclin D1; D063150:Polycomb Repressive Complex 1; C581292:ERBB3 protein, human; C587689:ERBB4 protein, human; D020893:Receptor, ErbB-3; D066247:Receptor, ErbB-4; C509186:TERT protein, human; D019098:Telomerase