title: In vitro and in vivo antiproliferative activity of metformin on stem-like cells isolated from spontaneous canine mammary carcinomas: translational implications for human tumors.
abstract: BACKGROUND
  Cancer stem cells (CSCs) are considered the cell subpopulation responsible for breast cancer (BC) initiation, growth, and relapse. CSCs are identified as self-renewing and tumor-initiating cells, conferring resistance to chemo- and radio-therapy to several neoplasias. Nowadays, th (about 10mM)e pharmacological targeting of CSCs is considered an ineludible therapeutic goal. The antidiabetic drug metformin was reported to suppress in vitro and in vivo CSC survival in different tumors and, in particular, in BC preclinical models. However, few studies are available on primary CSC cultures derived from human postsurgical BC samples, likely because of the limited amount of tissue available after surgery. In this context, comparative oncology is acquiring a relevant role in cancer research, allowing the analysis of larger samples from spontaneous pet tumors that represent optimal models for human cancer.
  
  
  METHODS
  Isolation of primary canine mammary carcinoma (CMC) cells and enrichment in stem-like cell was carried out from fresh tumor specimens by culturing cells in stem-permissive conditions. Phenotypic and functional characterization of CMC-derived stem cells was performed in vitro, by assessment of self-renewal, long-lasting proliferation, marker expression, and drug sensitivity, and in vivo, by tumorigenicity experiments. Corresponding cultures of differentiated CMC cells were used as internal reference. Metformin efficacy on CMC stem cell viability was analyzed both in vitro and in vivo.
  
  
  RESULTS
  We identified a subpopulation of CMC cells showing human breast CSC features, including expression of specific markers (i.e. CD44, CXCR4), growth as mammospheres, and tumor-initiation in mice. These cells show resistance to doxorubicin but were highly sensitive to metformin in vitro. Finally, in vivo metformin administration significantly impaired CMC growth in NOD-SCID mice, associated with a significant depletion of CSCs.
  
  
  CONCLUSIONS
  Similarly to the human counterpart, CMCs contain stem-like subpopulations representing, in a comparative oncology context, a valuable translational model for human BC, and, in particular, to predict the efficacy of antitumor drugs. Moreover, metformin represents a potential CSC-selective drug for BC, as effective (neo-)adjuvant therapy to eradicate CSC in mammary carcinomas of humans and animals.
authors.affiliation: Dipartimento di Medicina Interna, Sezione di Farmacologia, University of Genova, Genoa, Italy. federica.barbieri@unige.it.|Centro di Eccellenza per la Ricerca Biomedica (CEBR), University of Genova, Genoa, Italy. federica.barbieri@unige.it.
authors.forename: Federica
authors.initials: F
authors.lastname: Barbieri
authors.affiliation: Dipartimento di Medicina Interna, Sezione di Farmacologia, University of Genova, Genoa, Italy. stefano.thellung@unige.it.|Centro di Eccellenza per la Ricerca Biomedica (CEBR), University of Genova, Genoa, Italy. stefano.thellung@unige.it.
authors.forename: Stefano
authors.initials: S
authors.lastname: Thellung
authors.affiliation: Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle D'Aosta, and National Reference Center of Veterinary and Comparative Oncology (CEROVEC), Genoa, Italy. alessandra.ratto@izsto.it.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Ratto
authors.affiliation: Dipartimento di Medicina Sperimentale, University of Genova, Genoa, Italy. elisacarra@hotmail.it.
authors.forename: Elisa
authors.initials: E
authors.lastname: Carra
authors.affiliation: Dipartimento di Medicina Interna, Sezione di Farmacologia, University of Genova, Genoa, Italy. valeria.marini@unige.it.
authors.forename: Valeria
authors.initials: V
authors.lastname: Marini
authors.affiliation: Dipartimento di Medicina Interna, Sezione di Farmacologia, University of Genova, Genoa, Italy. antidpx@gmail.com.
authors.forename: Carmen
authors.initials: C
authors.lastname: Fucile
authors.affiliation: Dipartimento di Medicina Interna, Sezione di Farmacologia, University of Genova, Genoa, Italy. adriana.bajetto@unige.it.
authors.forename: Adriana
authors.initials: A
authors.lastname: Bajetto
authors.affiliation: Dipartimento di Medicina Interna, Sezione di Farmacologia, University of Genova, Genoa, Italy. alpat25@hotmail.com.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Pattarozzi
authors.affiliation: Dipartimento di Medicina Interna, Sezione di Farmacologia, University of Genova, Genoa, Italy. roberto.wurth@unige.it.
authors.forename: Roberto
authors.initials: R
authors.lastname: Würth
authors.affiliation: Dipartimento di Medicina Interna, Sezione di Farmacologia, University of Genova, Genoa, Italy. monica.gatti@unige.it.
authors.forename: Monica
authors.initials: M
authors.lastname: Gatti
authors.affiliation: Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle D'Aosta, and National Reference Center of Veterinary and Comparative Oncology (CEROVEC), Genoa, Italy. chiara.campanella@izsto.it.
authors.forename: Chiara
authors.initials: C
authors.lastname: Campanella
authors.affiliation: Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle D'Aosta, and National Reference Center of Veterinary and Comparative Oncology (CEROVEC), Genoa, Italy. guendalina.vito@izsto.it.
authors.forename: Guendalina
authors.initials: G
authors.lastname: Vito
authors.affiliation: Dipartimento di Medicina Interna, Sezione di Farmacologia, University of Genova, Genoa, Italy. fmattiol@unige.it.
authors.forename: Francesca
authors.initials: F
authors.lastname: Mattioli
authors.affiliation: Dipartimento di Medicina Sperimentale, University of Genova, Genoa, Italy. aldo.pagano@unige.it.|IRCCS AOU San Martino - IST, Genoa, Italy. aldo.pagano@unige.it.
authors.forename: Aldo
authors.initials: A
authors.lastname: Pagano
authors.affiliation: IRCCS AOU San Martino - IST, Genoa, Italy. antonio.daga@hsanmartino.it.
authors.forename: Antonio
authors.initials: A
authors.lastname: Daga
authors.affiliation: Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle D'Aosta, and National Reference Center of Veterinary and Comparative Oncology (CEROVEC), Genoa, Italy. angelo.ferrari@izsto.it.
authors.forename: Angelo
authors.initials: A
authors.lastname: Ferrari
authors.affiliation: Dipartimento di Medicina Interna, Sezione di Farmacologia, University of Genova, Genoa, Italy. tullio.florio@unige.it.|Centro di Eccellenza per la Ricerca Biomedica (CEBR), University of Genova, Genoa, Italy. tullio.florio@unige.it.
authors.forename: Tullio
authors.initials: T
authors.lastname: Florio
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000493:pharmacokinetics / Q000494:pharmacology*; D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D004195:Disease Models, Animal; D004285:Dogs; D019008:Drug Resistance, Neoplasm; D066246:ErbB Receptors / Q000378:metabolism; D047628:Estrogen Receptor alpha / Q000378:metabolism; D005260:Female; D006801:Humans; D018960:Hyaluronan Receptors / Q000378:metabolism; D019394:Ki-67 Antigen / Q000378:metabolism; D015674:Mammary Neoplasms, Animal; D008687:Metformin / Q000493:pharmacokinetics / Q000494:pharmacology*; D051379:Mice; D014411:Neoplastic Stem Cells / Q000187:drug effects* / Q000378:metabolism*; D010641:Phenotype; D023041:Xenograft Model Antitumor Assays
chemical_list: D000970:Antineoplastic Agents; D047628:Estrogen Receptor alpha; D018960:Hyaluronan Receptors; D019394:Ki-67 Antigen; D008687:Metformin; D066246:ErbB Receptors