The biguanides metformin and phenformin inhibit angiogenesis, local and metastatic growth of breast cancer by targeting both neoplastic and microenvironment cells Stefania Orecchioni 1 *, Francesca Reggiani 1 *, Giovanna Talarico 1 *, Patrizia Mancuso 1 , Angelica Calleri 1 , Giuliana Gregato 1 , Valentina Labanca 1 , Douglas M. Noonan 2,3 , Katiuscia Dallaglio 4 , Adriana Albini 4 and Francesco Bertolini 1 1 Laboratory of Hematology-Oncology, European Institute of Oncology, Milan, Italy 2 Scientific and Technologic Park, IRCCS MultiMedica, Italy 3 Department of Biotechnology and Life Sciences, University of Insubria, Varese, Italy 4 Research and Statistics Department, IRCCS "Tecnologie Avanzate e Modelli Assistenziali in Oncologia" Arcispedale S. Maria Nuova, Reggio Emilia, Italy The human white adipose tissue (WAT) contains progenitors with cooperative roles in breast cancer (BC) angiogenesis, local and metastatic progression. The biguanide Metformin (Met), commonly used for Type 2 diabetes, might have activity against BC and was found to inhibit angiogenesis in vivo. We studied Met and another biguanide, phenformin (Phe), in vitro and in vivo in BC models. In vitro, biguanides activated AMPK, inhibited Complex 1 of the respiratory chain and induced apoptosis of BC and WAT endothelial cells. In coculture, biguanides inhibited the production of several angiogenic proteins. In vivo, bigua- nides inhibited local and metastatic growth of triple negative and HER21 BC in immune-competent and immune-deficient mice orthotopically injected with BC. Biguanides inhibited local and metastatic BC growth in a genetically engineered murine model model of HER21 BC. In vivo, biguanides increased pimonidazole binding (but not HIF-1 expression) of WAT progenitors, reduced tumor microvessel density and altered the vascular pericyte/endothelial cell ratio, so that cancer vessels displayed a dysplastic phenotype. Phe was significantly more active than Met both in vitro and in vivo. Considering their safety profile, biguanides deserve to be further investigated for BC prevention in high-risk subjects, in combination with chemo and/or tar- geted therapy and/or as post-therapy consolidation or maintenance therapy for the prevention of BC recurrence. There is increasing preclinical evidence that the biguanide Metformin (Met), commonly used for the therapy of Type 2 diabetes, might have activity against several types of neoplas- tic diseases 1–25 including breast cancer (BC). Another bigua- nide, phenformin (Phe), which was dismissed from the arsenal of antidiabetes drug because of some side effects, has shown preclinical activity in a model of BC. 26 We and others have recently shown that two populations of human white adipose tissue (WAT) CD45-CD341 pro- genitors have cooperative roles in BC angiogenesis, local and metastatic progression. 27–35 In orthotopic murine models we found that (i) purified human WAT CD341CD131 mesen- chymal adipose stromal cell progenitors (ASCs) were not able to migrate but promoted local tumor growth in the mam- mary fat pad and (ii) purified human WAT CD341CD311 endothelial progenitor cells (EPCs) were able to migrate toward lymph nodes and blood and promoted BC cell EMT, migration, invasion and metastatic growth. 34 In another recent study we have found that Met inhibited the formation of capillary-like networks by human umbilical vein endothelial cells (HUVEC), and decreased microvessel density (MVD) in tumor-free mice. 36 Here we report that Met and Phe (with even more efficacy) targeted in vitro and in vivo both BC cells and WAT EPCs, resulting in profound effects on BC angiogenesis, local and metastatic growth that are likely due to additive effects on both tumor and microenvironment cells. These effects were observed both in triple negative and in HER21 models of BC. Material and Methods In vitro studies Human WAT was obtained as previously described 28,34 from women undergoing breast reconstruction after signature of an informed consent. In brief, samples were centrifuged at 1,200g to remove erythrocytes and leukocytes and subsequently digested in Hanks balanced salt solution (HBSS, Gibco, UK) containing 2 mg/mL of collagenase Type I (Sigma-Aldrich, St. Louis, MO) and 3.5% bovine serum albumin (BSA; Sigma) at 37  C with constant shaking for 120 min. The digestion was blocked with RPMI 1640 supplemented by 20% FBS Key words: metformin, phenformin, breast cancer, angiogenesis Additional Supporting Information may be found in the online version of this article. *S.O., F.R. and G.T. contributed equally to this work DOI: 10.1002/ijc.29193 History: Received 2 May 2014; Accepted 27 Aug 2014; Online 6 Sep 2014 Correspondence to: Francesco Bertolini, Laboratory of Hematology-Oncology, European Institute of Oncology, via Ripa- monti 435, 20141 Milan Italy, Tel.: 139-02-57489535, Fax: 139-02- 57489537, E-mail: francesco.bertolini@ieo.it Cancer Cell Biology Int. J. Cancer: 00, 00–00 (2014) V C 2014 UICC International Journal of Cancer IJC