cells Article Inhibition of Vasculogenic Mimicry and Angiogenesis by an Anti-EGFR IgG1-Human Endostatin-P125A Fusion Protein Reduces Triple Negative Breast Cancer Metastases Seung-Uon Shin 1,† , Hyun-Mi Cho 1,† , Rathin Das 2 , Hava Gil-Henn 3 , Sundaram Ramakrishnan 4 , Ahmed Al Bayati 1,5 , Stephen F. Carroll 2 , Yu Zhang 1 , Ankita P. Sankar 6 , Christian Elledge 6 , Augustin Pimentel 7 , Marzenna Blonska 1 and Joseph D. Rosenblatt 1, *   Citation: Shin, S.-U.; Cho, H.-M.; Das, R.; Gil-Henn, H.; Ramakrishnan, S.; Al Bayati, A.; Carroll, S.F.; Zhang, Y.; Sankar, A.P.; Elledge, C.; et al. Inhibition of Vasculogenic Mimicry and Angiogenesis by an Anti-EGFR IgG1-Human Endostatin-P125A Fusion Protein Reduces Triple Negative Breast Cancer Metastases. Cells 2021, 10, 2904. https://doi.org/ 10.3390/cells10112904 Academic Editor: Sima Lev Received: 11 August 2021 Accepted: 19 October 2021 Published: 27 October 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1 Sylvester Comprehensive Cancer Center, Department of Medicine, Division of Hematology, University of Miami Miller School of Medicine (UMMSOM), Miami, FL 33136, USA; sshin@med.miami.edu (S.-U.S.); hcho@med.miami.edu (H.-M.C.); ahmed.albayati@uky.edu (A.A.B.); y.zhang4@med.miami.edu (Y.Z.); MBlonska@med.miami.edu (M.B.) 2 Synergys Biotherapeutics Inc., Alamo, CA 94507, USA; rathindas@synergysbio.com (R.D.); sfcarroll@earthlink.net (S.F.C.) 3 The Azrieli Faculty of Medicine, Bar-Ilan University, Safed 1311502, Israel; Hava.Henn@biu.ac.il 4 Department of Surgery, University of Miami Miller School of Medicine (UMMSOM), Miami, FL 33136, USA; sramakrishnan@miami.edu 5 Kentucky Clinic, University of Kentucky, Lexington, KY 40536, USA 6 Sheila and David Fuente Graduate Program in Cancer Biology, Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine (UMMSOM), Miami, FL 33136, USA; aps195@miami.edu (A.P.S.); Mce58@med.miami.edu (C.E.) 7 Sylvester Comprehensive Cancer Center, Department of Medicine, Division of Medical Oncology, University of Miami Miller School of Medicine (UMMSOM), Miami, FL 33136, USA; apimentel@med.miami.edu * Correspondence: jrosenblatt@med.miami.edu; Tel.: +1-305-243-4618; Fax: +1-305-243-9161 † Shin and Cho both recognized as co-first authors. Abstract: Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. Metastasis is the major cause of TNBC mortality. Angiogenesis facilitates TNBC metastases. Many TNBCs also form vascular channels lined by tumor cells rather than endothelial cells, known as ‘vasculogenic mimicry’ (VM). VM has been linked to metastatic TNBC behavior and resistance to anti-angiogenic agents. Epidermal growth factor receptor (EGFR) is frequently expressed on TNBC, but anti-EGFR antibodies have limited efficacy. We synthesized an anti-EGFR antibody– endostatin fusion protein, αEGFR IgG1-huEndo-P125A (αEGFR-E-P125A), designed to deliver a mutant endostatin, huEndo-P125A (E-P125A), to EGFR expressing tumors, and tested its effects on angiogenesis, TNBC VM, and motility in vitro, and on the growth and metastasis of two independent human TNBC xenograft models in vivo. αEGFR-E-P125A completely inhibited the ability of human umbilical vein endothelial cells to form capillary-like structures (CLS) and of TNBC cells to engage in VM and form tubes in vitro. αEGFR-E-P125A treatment reduced endothelial and TNBC motility in vitro more effectively than E-P125A or cetuximab, delivered alone or in combination. Treatment of TNBC with αEGFR-E-P125A was associated with a reduction in cytoplasmic and nuclear β-catenin and reduced phosphorylation of vimentin. αEGFR-E-P125A treatment of TNBC xenografts in vivo inhibited angiogenesis and VM, reduced primary tumor growth and lung metastasis of orthotopically implanted MDA-MB-468 TNBC cells, and markedly decreased lung metastases following intravenous injection of MDA-MB-231-4175 lung-tropic TNBC cells. Combined inhibition of angiogenesis, VM, and TNBC motility mediated by αEGFR-E-P125A is a promising strategy for the prevention of TNBC metastases. Keywords: EGFR; endostatin; vasculogenic mimicry; triple negative breast cancer Cells 2021, 10, 2904. https://doi.org/10.3390/cells10112904 https://www.mdpi.com/journal/cells