Grapefruit-derived nanovectors deliver miR-18a for treatment of liver metastasis of colon cancer by induction of M1 macrophages

Liver metastasis accounts for many of the cancer deaths in patients. Effective treatment for metastatic liver tumors is not available. Here, we provide evidence for the role of miR-18a in the induction of liver M1 (F4/80+interferon gamma (IFNγ)+IL-12+) macrophages. We found that miR-18a encapsulated in grapefruit-derived nanovector (GNV) mediated inhibition of liver metastasis that is dependent upon the induction of M1 (F4/80+IFNγ+IL-12+) macrophages; depletion of macrophages eliminated its anti-metastasis effect. Furthermore, the miR-18a mediated induction of macrophage IFNγ by targeting IRF2 is required for subsequent induction of IL-12. IL-12 then activates natural killer (NK) and natural killer T (NKT) cells for inhibition of liver metastasis of colon cancer. This conclusion is supported by the fact that knockout of IFNγ eliminates miR-18a mediated induction of IL-12, miR-18a treatment has an anti-metastatic effects in T cell deficient mice but there is no anti-metastatic effect on NK and NKT deficient mice. Co-delivery of miR-18a and siRNA IL-12 to macrophages did not result in activation of co-cultured NK and NKT cells. Taken together our results indicate that miR-18a can act as an inhibitor for liver metastasis through induction of M1 macrophages.

Oncotarget 5 www.impactjournals.com/oncotarget SUPPLEMENTAL PROCEDURES Mouse model study 8-to 12-week-old female BALB/C mice, Interferon γ(IFNγ) knockout mice, athymic immunodeficient nude mice and Central Institute for Experimental Animals (CIEA) NOG (NOD/Shi-scid,IL-2RYnull) mice. The NOG mice lack mature T cells, B cells, functional NK cells, and are also deficient in cytokine signaling. The athymic immunodeficient nude mice lack T cells but have NK activity. All mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and housed under specific pathogen-free conditions. Animal care was performed following the Institute for Laboratory Animal Research (ILAR) guidelines and all animal experiments were done in accordance with protocols approved by the University of Louisville Institutional Animal Care and Use Committee (Louisville, KY). The mice were acclimated for at least 1 week before any experiments were conducted.
For animal model of colon cancer with liver metastasis, mice were anaesthetized with a mixture of ketamine and xylazine. 1 × 10 6 of CT26 colon cancer cells were administered via intra-splenic injection as previously described (Jiang, Wang et al., 2014). The treatment schedule was arranged as following: thirty 7-week old male BALB/c mice were randomly divided into two groups, with 15 mice per group. The first group of mice (n = 15) was treated as following: 10/15 of mice were intra-spenic injected with CT26 tumor cells at day 0 and 5/15 mice were injected with PBS as a control. The second group of mice (n = 15) were housed in the same environment as first group mice until day 12. At day 12, 10/15 of the second group of mice were intrasplenic injected with CT26 tumor cells and 5/15 mice were injected with PBS as a control. For both groups of mice, day 2 after intra-splenic injection 200 nM OGNVs packed with 2 nM of miR-18 (OGNVs/miR18a) or Scramble miRNA (OGNVs/Ctrl) as a control were administrated to mice via tail veil injection. For the first group of mice, mice were treated three times per week for 2 weeks. For the second group of mice, the mice were treated one time on day 2 after CT26 injection. On day 14 mice were sacrificed and various organs were removed for examination. The percentages of NK, NKT, and T cells isolated from each group of treated mice were also FACS analyzed.

Liver macrophage depletion
Mice were injected with approximately 110 mg/kg of clodronate liposomes (FormuMax Scientific Inc.) i.p. or an equal volume of PBS liposomes. The injection was repeated three days later and experiments were performed 4 days after the first injection.

Antibodies and reagents
The following monoclonal antibodies (eBioscience) were used for flow cytometry:

Flow cytometry
Liver and spleen from mice were removed and gently pressed through nylon cell strainers (70 μm in diameter, Fisher Scientific) to obtain single-cell suspensions in RPMI-1640 containing 5% FBS. Hepatocytes were removed from liver-cell suspensions by colloidal silica particle (Percoll, Invitrogen) gradient centrifugation in phosphate-buffered saline. Erythrocytes in liver and spleencell suspensions were then removed using ammonium-chloride-potassium (ACK) lysing buffer (0.15 M NH4Cl, 10 mM KHCO 3 , 0.1 mM EDTA). Washed cells were stained for 40 min at 4°C with the appropriate fluorochrome-conjugated antibodies in PBS with 2% FBS. To detect intracellular antigens, washed cells were incubated in diluted Fixation/ Permeabilization solution (eBioscience Cat# 00-5123) at 4°C for 30 min. Characterization and phenotyping of the various lymphocytes subsets from liver or spleen were performed by flow cytometry. Data were acquired on BD FACS Canto (BD Biosciences, San Jose, CA) and were analyzed using FlowJo software (Tree Star Inc., Ashland, OR). Numbers above bracketed lines in FACS figures indicate percent of positive stained cells, and the results of cells stained with a isotype-matched control antibody are shown in gray color.

Site-directed mutagenesis within the IRF2 promoter
We utilized two algorithms that predict the mRNA targets of miRNAs, TargetScan (http://www.targetscan. org) and microRNA (http://www.microRNA.org), and Pictar (http://http://pictar.mdc-berlin.de/. IRF2 was selected by both online tools with strong conserved 3′untranslated region (3′UTR) sites. To determine the ability of miR-18a to target the 3′UTR-IRF2 activity, a luciferase reporter containing 1,234 bp of the IRF2 3′UTR in the pEZX-MT01 vector was purchased from GeneCopoeia (Cat# MmiT027452-MT01, Rockville, MD). The mutant of IRF2 3′UTR was generated with the oligonucleotide primer IRF2-Mut, which was designed to specifically disrupt putative IRF2 at its 3′UTR site. Q5 ® Site-Directed Mutagenesis Kit (New England Biolabs, MA, USA) was used in conjunction with specific primers (Supplementary Table 1) to introduce IRF2 3′UTR mutations in the pEZX-MT01 construct according to the manufacturer's instructions. After mutant strand synthesis and ligation, resultant plasmids were introduced into E. coli and transformants were selected using kanamycin resistance. Further DNA sequence of mutant was confirmed by DNA sequencing.

Labeling OGNVs with PKH67
OGNVs were labeled with PKH67 Fluorescent Cell Linker Kits (Sigma) in accordance with the manufacturer's instructions. OGNVs were suspended in 250 μl of Diluent C with 1 μl of PKH67 and mixed with 250 μl of dye solution for subsequent incubated with an equal volume of 1% BSA for 1 min at 22°C. After centrifugation for 5 minutes at 13,000 rpm, 20 μl of resuspended labeled OGNVs were loaded on a slide for assessment of viability using confocal microscopy (Nikon).

Quantitative Real-Time PCR (qPCR) analysis of miRNA and mRNA expression
Total RNA was isolated from lymphocyte cells with a miRNeasy mini kit (Qiagen) and reverse-transcribed using a miRNA reverse transcription kit (Qiagen). Mature miR-18a expression was quantified by quantitative realtime PCR (qPCR) using a miScript II RT kit (Qiagen) and miScript SYBR Green PCR Kit (Qiagen) with Qiagen predesigned primers. All kits were used according to the manufacturer's instructions. U6 transcript was used as an internal control to normalize RNA input. For analysis of IL-12, IFNy, MHCII, TGFp, IRF1,IRF2, Smad2, ESR1, ESR2 mRNA expression, 1 μg of total RNA was reverse transcribed by SuperScript III reverse transcriptase (Invitrogen) and quantitation was performed using primers (Eurofins) with SsoAdvancedTM Universal SYBR Green Supermix (BioRad) and p-actin was used for normalization. The primer sequences are listed in Supplementary table 1. qPCR was run using BioRad CFX96 qPCR System with each reaction run in triplicate. Analysis and fold change were determined using the comparative threshold cycle (Ct) method. The change in miRNA or mRNA expression was calculated as foldchange.

Western blotting
Cells were treated as indicated in individual Figure legends and whole cell extracts (WCE) were prepared in modified RIPA buffer (Sigma) with addition of protease and phosphatase inhibitors (Roche). Western analysis was performed and quantitated as described (Jiang et al., 2014).