title: In Vivo Selection of Intermediately- and Highly-Malignant Variants of Triple-negative Breast Cancer in Orthotopic Nude Mouse Models.
abstract: Triple-negative breast cancer (TNBC), defined by the absence of receptors for estrogen, progesterone and human epithelial receptor 2 (HER2), is a recalcitrant disease in need of effective therapy. We previously isolated very-highly metastatic variants of the TNBC cell line MDA-MB-231 using serial orthotopic implantation of MDA-MB-231 human breast cancer cells in nude mice.
  
  
  
  MDA-MB-231 cells expressing red fluorescent protein (MDA-MB-231-RFP) (1×107cells/site) were initially injected subcutaneously in the flank of nude mice. After the subcutaneous tumors grew, they were harvested and cut into small pieces for orthotopic implantation into the right lower mammary gland. After the orthotopic tumors grew, they were resected and cut into small pieces and orthotopically re-implanted into the mammary gland of nude mice. The tumors grew and metastasized to lymph nodes. The lymph node metastases were harvested and cut into small pieces and orthotopically re-implanted into the mammary gland of nude mice. After the orthotopic tumors grew, the tumor was removed leaving residual cancer cells, which grew and metastasized to lymph nodes. The lymph node metastases were harvested, cut into pieces and orthotopically re-implanted into the mammary gland of nude mice for three or seven cycles in order to isolate intermediately, or highly-metastatic variants, respectively.
  
  
  
  The degree of malignancy of isolated variants of MDA-MB-231 depends on the extent of orthotopic transplantation. Serial transplantation resulted in MDA-MB-231-RFP which significantly produced larger tumors compared with the parental MDA-MB-231-RFP. Serial orthotopic implantation for three cycles resulted in intermediately-metastatic variants of MDA-MB-231-RFP. MDA-MB-231-RFP serially orthotopically transplanted seven times significantly metastasized more to lymph nodes compared with parental MDA-MB-231-RFP cells and the intermediately-metastatic variant. The highly-metastatic variant MDA-MB-231-RFP cells significantly metastasized to the lung to a greater extent compared with parental MDA-MB-231-RFP and intermediate variants of MDA-MB-231-RFP.
  
  
  
  These results suggest that the number of serial orthotopic transplantations of MDA-MB-231-RFP correlates positively with tumor aggressiveness, and the intermediately- and highly-metastatic variants can serve as models of metastatic progression of TNBC.
authors.affiliation: AntiCancer, Inc., San Diego, CA, U.S.A.|Department of Surgery, University of California San Diego, CA, U.S.A.|Department of Gastroenterological Surgery, Okayama University, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan.
authors.forename: Shuya
authors.initials: S
authors.lastname: Yano
authors.affiliation: AntiCancer, Inc., San Diego, CA, U.S.A.|Department of Surgery, University of California San Diego, CA, U.S.A.|Department of Gastroenterological Surgery, Okayama University, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan.
authors.forename: Kiyoto
authors.initials: K
authors.lastname: Takehara
authors.affiliation: Department of Gastroenterological Surgery, Okayama University, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Kishimoto
authors.affiliation: Center for Innovative Clinical Medicine, Okayama University Hospital, Okayama, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Tazawa
authors.affiliation: Oncolys BioPharm Inc., Tokyo, Japan.
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Urata
authors.affiliation: Department of Gastroenterological Surgery, Okayama University, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan.
authors.forename: Shunsuke
authors.initials: S
authors.lastname: Kagawa
authors.affiliation: Department of Surgery, University of California San Diego, CA, U.S.A.
authors.forename: Michael
authors.initials: M
authors.lastname: Bouvet
authors.affiliation: Department of Gastroenterological Surgery, Okayama University, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan all@anticancer.com toshi_f@md.okayama-u.ac.jp.
authors.forename: Toshiyoshi
authors.initials: T
authors.lastname: Fujiwara
authors.affiliation: AntiCancer, Inc., San Diego, CA, U.S.A. all@anticancer.com toshi_f@md.okayama-u.ac.jp.|Department of Surgery, University of California San Diego, CA, U.S.A.
authors.forename: Robert M
authors.initials: RM
authors.lastname: Hoffman
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D004195:Disease Models, Animal; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D064726:Triple Negative Breast Neoplasms / Q000378:metabolism*
keywords: MDA-MB-231; RFP; TNBC; Triple-negative breast cancer; in vivo selection; nude mice; orthotopic; red fluorescent protein