File S1 - Harnessing High Density Lipoproteins to Block Transforming Growth Factor Beta and to Inhibit the Growth of Liver Tumor Metastases

Contains the following files: Figure S1. In vivo assays to select the best anti-TGF-β inhibitor. (A) A plasmid encoding IL-12 (pIL-12) was administered to C57BL/6 mice by hydrodynamic injection together with pApo, pSpP144, or pApoLinkerP144. Four days later, IFN-γ serum levels were quantified by ELISA. **p5 CT26 tumor cells. Results are displayed as Kaplan-Meier plot of tumor occurrence. Treatment groups were compared using the log-rank test. Data are representative of one of two independent experiments (n = 6 mice/group). *p5 MC38 cells were plated in a 6-well plates. After an overnight incubation, cells were left untreated or treated with 100 pM TGF-β with or without 100 µg/ml rApoLinkerP144. 60 minutes later, cells were harvested and Western blot analysis was performed to determine the phosphorylation status of Smad2, Smad3 and Smad1/5/8. Figure S3. Scavenger receptor class B type I (SRB1) expression on MC38 tumor cell line. 3×105 tumor cells were cultured in 6-well plates for 48 hours. Then, cells were harvested and stained to show SRB1 surface expression by flow cytometry analysis. Expression was compared with isotype control. Data are representative of two independent experiments. Figure S4. Antitumor efficacy of P144. 5×105 MC38 colon carcinoma cells were intrasplenically injected. The same day, we started a daily treatment with vehicle (carbonate buffer pH 9.5) or 200 µg P144 in carbonate buffer (pH 9,5) for 15 days (n = 6/group). Mice were sacrificed at day 15 after tumor cell inoculation and tumor area in the liver was measured quantifying the pixels over a threshold color using Matlab software. Mean±SEM *, P5 MC38 colon carcinoma cells were intrasplenically injected. At the same time, 4×1012 AAVApo or AAVApoLinkerP144 vg/mice were i.p injected (n = 6/group). Mice were sacrificed at day 15 after tumor cell inoculation and liver were fixed with formalin and embedded in paraffin. Immunhistochemical staining of pAKT was performed. A representative picture of pAKT in the metastatic nodules in the liver is shown (Magnification: 100×). Table S1. Primers designed to clone the different peptide fusions and the primers used for quantitative PCR analysis. (DOC)