Harnessing High Density Lipoproteins to Block Transforming Growth Factor Beta and to Inhibit the Growth of Liver Tumor Metastases Jose ´ Medina-Echeverz 1 , Jessica Fioravanti 1 , Nancy Dı´az-Valde ´s 1,2 , Kathrin Frank 2 , Fernando Aranda 1 , Celia Gomar 1 , Nuria Ardaiz 1 , Javier Dotor 3 , Viktor Umansky 2 , Jesu ´ s Prieto 1,4 *, Pedro Berraondo 1 * 1 Division of Hepatology and Gene Therapy, Center for Applied Medical Research, University of Navarra, Pamplona, Spain, 2 Skin Cancer Unit, German Cancer Research Center, Heidelberg and Department of Dermatology, Venereology and Allergology, University Medical Center Mannheim, Ruprecht-Karl University of Heidelberg, Mannheim, Heidelberg, Germany, 3 DIGNA Biotech, Madrid, Spain, 4 Liver Unit, University of Navarra Clinic, Networked Biomedical Research Center of Hepatic and Digestive Diseases, Pamplona, Spain Abstract Transforming growth factor b (TGF-b) is a powerful promoter of cancer progression and a key target for antitumor therapy. As cancer cells exhibit active cholesterol metabolism, high density lipoproteins (HDLs) appear as an attractive delivery system for anticancer TGFb-inhibitory molecules. We constructed a plasmid encoding a potent TGF-b-blocking peptide (P144) linked to apolipoprotein A-I (ApoA-I) through a flexible linker (pApoLinkerP144). The ApoLinkerP144 sequence was then incorporated into a hepatotropic adeno-associated vector (AAVApoLinkerP144). The aim was to induce hepatocytes to produce HDLs containing a modified ApoA-I capable of blocking TGF-b. We observed that transduction of the murine liver with pApoLinkerP144 led to the appearance of a fraction of circulating HDL containing the fusion protein. These HDLs were able to attenuate TGF-b signaling in the liver and to enhance IL-12 -mediated IFN-c production. Treatment of liver metastasis of MC38 colorectal cancer with AAVApoLinkerP144 resulted in a significant reduction of tumor growth and enhanced expression of IFN-c and GM-CSF in cancerous tissue. ApoLinkerP144 also delayed MC38 liver metastasis in Rag2 2/ 2 IL2rc 2/2 immunodeficient mice. This effect was associated with downregulation of TGF-b target genes essential for metastatic niche conditioning. Finally, in a subset of ret transgenic mice, a model of aggressive spontaneous metastatic melanoma, AAVApoLinkerP144 delayed tumor growth in association with increased CD8 + T cell numbers in regional lymph nodes. In conclusion, modification of HDLs to transport TGF-b-blocking molecules is a novel and promising approach to inhibit the growth of liver metastases by immunological and non-immunological mechanisms. Citation: Medina-Echeverz J, Fioravanti J, Dı ´az-Valde ´s N, Frank K, Aranda F, et al. (2014) Harnessing High Density Lipoproteins to Block Transforming Growth Factor Beta and to Inhibit the Growth of Liver Tumor Metastases. PLoS ONE 9(5): e96799. doi:10.1371/journal.pone.0096799 Editor: Lu-Zhe Sun, University of Texas Health Science Center, United States of America Received December 27, 2013; Accepted April 11, 2014; Published May 5, 2014 Copyright: ß 2014 Medina-Echeverz et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the agreement between FIMA and the ‘‘UTE project CIMA’’, Red de Inmunoterapia IMMUNONET-SOE1/P1/E014, Instituto de Salud Carlos III, Fondo de Investigacio ´ n Sanitaria (FIS PI10/00264), Fundacio ´ n Mutua Madrilen ˜ a, Dr. Mildred Scheel Foundation for Cancer Research (grant 108992, to V.U.) and by the Initiative and Networking Fund of the Helmholtz Association within the Helmholtz Alliance on Immunotherapy of Cancer (to V.U.). J.F. and J.M-E. were supported by a fellowship of Spanish Fondo de Investigacio ´ n Sanitaria. N.D-V. was supported by a Martin Escudero fellowship. P.B. was supported by a Miguel Servet contract from Spanish Fondo de Investigacio ´ n Sanitaria. J.D. was employee of Digna Biotech until June 2013. He has neither rights nor benefits from the data presented in this manuscript. This does not alter the authors’ adherence to PLOS ONE policies on sharing data and materials. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: pberraondol@unav.es (PB); jprieto@unav.es (JP) Introduction Transforming growth factor b (TGF-b) has long been recog- nized as a key promoter of tumor growth and a critical initiator of the metastatic process [1]. Most established tumors develop mutations in the TGF-b-signaling pathway. These mutations confer the tumor cells the possibility of bypassing TGF-b– mediated growth inhibition while making use of the ability of this cytokine to enhance tumor growth, invasion, and metastasis [2]. TGF-b is a potent inducer of epithelial-mesenchymal transition (EMT) [3], a process by which tumor cells lose epithelial characteristics and acquire enhanced migratory and invasive capabilities. Moreover, TGF-b directly promotes the expression of key angiogenic factors such as VEGF [4] and induces metallo- proteases like MMP9 [5], facilitating tumor vascularization and matrix remodeling which are critical events involved in condi- tioning the metastatic niche [6]. TGF-b also stimulates tumor invasion and metastasis by inhibiting anti-tumor immune surveil- lance and promoting local immune suppression. Along this line, TGF-b directly inhibits NK and T cell proliferation and suppresses CD8 + T cell cytotoxicity by transcriptional repression of genes encoding perforin, granzymes and cytotoxins [7]. In addition, TGF-b induces FoxP3 thereby generating inducible regulatory T lymphocytes and exerts additional immunosuppres- sive functions by acting on myeloid-derived suppressor cells, tumor-associated neutrophils and tumor-associated macrophages [1,7]. Thus, TGF-b is a polyvalent cytokine that favors tumor growth and spread by a diversity of mechanisms. Accordingly, antitumor strategies directed to effectively block TGF-b have long been sought. However, attempts to antagonize TGF-b in malignant processes were not followed by clear clinical benefit PLOS ONE | www.plosone.org 1 May 2014 | Volume 9 | Issue 5 | e96799