Nanoparticle delivery of miR-122 inhibits colorectal cancer liver metastasis

Abstract

Liver metastasis is a leading cause of cancer morbidity and mortality. Thus, there has been strong interest in the development of therapeutics that can effectively prevent liver metastasis. One potential strategy is to utilize molecules that have broad effects on the liver microenvironment, such as microRNA-122 (miR-122), a liver-specific microRNA (miRNA) that is a key regulator of diverse hepatic functions. Here we report the development of a nanoformulation miR-122 as a therapeutic agent for preventing liver metastasis. We engineered a galactose-targeted lipid calcium phosphate (Gal-LCP) nanoformulation of miR-122. This nanotherapeutic elicited no significant toxicity and delivered miR-122 into hepatocytes with specificity and high efficiency. Across multiple colorectal cancer (CRC) liver metastasis models, treatment with Gal-LCP miR-122 treatment effectively prevented CRC liver metastasis and prolonged survival. Mechanistic studies revealed that delivery of miR-122 was associated with downregulation of key genes in involved in metastatic and cancer inflammation pathways, including several pro-inflammatory factors, matrix metalloproteinases, and other extracellular matrix degradation enzymes. Moreover, Gal-LCP miR-122 treatment was associated with an increased CD8+/CD4+ T-cell ratio and decreased immunosuppressive cell infiltration, which makes the liver more conducive to anti-tumor immune response. Collectively, this work presents a strategy to improve cancer prevention and treatment with nanomedicine-based delivery of miRNA.

Introduction

Cancer metastasis to the liver is one of the most common causes of cancer related morbidity and mortality (1). Therefore, there has been strong interest in the development of therapeutic agents that can effectively prevent and treat liver metastasis. However, the biology of liver metastasis is highly complex, involving many interactions between tumor cells and the liver microenvironment (2). Because of such complex interactions, treatment for liver metastasis has been highly challenging. Recent studies have shown that microRNA (miRNA), either oncogenic miRNAs or tumor suppressor miRNAs, play an important regulatory role in all aspects of liver metastasis (3, 4). One potential strategy to prevent or treat liver metastasis is to utilize miRNAs that have broad effects on the liver microenvironment. An example is miR-122, a liver-specific miRNA and a key regulator in the liver, which accounts for 70% of the total miRNA population within the liver. MiR-122 plays critical roles in liver homeostasis by regulating the expression of a large number of target mRNAs involved in diverse hepatic functions and also by suppressing non-hepatic genes (5). MiR-122 is originally known to play an important role in molecular pathogenesis of viral hepatitis (6). Mir-122 can bind to the 5’ UTR of HCV RNA and increase hepatitis c virus (HCV) replication (7), hence Miraversin (an antagomir of miR-122) is in phase 2 clinical trials in patients with HCV infection. Interestingly, miR-122 is also known as a tumor suppressor miRNA and decreased miR-122 expression is associated with the development of hepatocellular carcinoma (HCC) (8). Mir-122 is also found to be specifically repressed in a subset of HCC with poor prognosis (9). Moreover, it has been shown that restoration of miR-122 to physiologic levels can prevent the development of hepatocellular carcinoma and restricts intrahepatic metastasis of HCC by suppression of angiogenesis (8, 10). In a recent study, we also showed that miR-122 inhibition, in a liver organoid model, can lead to increase in several inflammatory factors as well as a variety of matrix metalloproteinases (MMPs), all of which are important in colonization and expansion of metastasis within liver microenvironment (11). Thus, we hypothesized that miR-122 can be utilized as a therapeutic agent to prevent liver metastasis (Fig 1).

Fig. 1. Nanoparticle delivery of miR-122 to hepatocytes can prevent liver metastasis.

A Schematic of Gal-LCP miR-122 delivery to the liver to prevent liver metastasis. It shows delivery of miR-122 to hepatocytes (A), and suppressive effects of miR-122 which can prevent the growth of tumor cells within liver microenvironment (B).

There has been a variety of viral and non-viral delivery systems developed to maximize miRNA therapeutic efficacy in vivo (12). Nanoparticles-lipid nanoparticles (LNPs) in particular, are known as one of the most common vehicles for delivery of nucleic acids including miRNAs (13). Bio-distribution studies have shown that NPs majorly cleared by the liver, however targeting of liver parenchyma has only recently been achieved through mimicking apolipoproteins for binding to low density lipoprotein receptor (LDLR) (14), and using galactose and its derivatives (i.e. GalNAc) as ligand for binding to asialoglycoprotein receptor (ASGPR) on the surface of hepatocytes (15). Herein, we report on the development of a miR-122-containing galactose-targeted nanoparticle for preventing and treatment of liver metastasis using colorectal cancer (CRC) liver metastasis mouse models.

Materials and Methods

Chemicals

DSPE-PEG–N-hydroxysuccinimide (NHS) was purchased from Nanosoft Polymers (Winston Salem, NC). 4-aminophenyl β-d-galactopyranoside was purchased from Sigma Aldrich (St. Louis, MO). DSPE-PEG-galactose was synthesized through the conjugation of 10 eq. of 4-aminophenyl β-d-galactopyranoside and 1 eq. of DSPE-PEG-NHS in PBS buffer, followed by chloroform extraction and dialysis against water using a MWCO 1000 dialysis tube. All other lipids were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL) miR-122 and miRNC mimics were purchased from Horizon Discovery (Lafayette, CO).

Preparation and characterization of LCP loaded with miRNA.

LCP was prepared using a modified protocol (16). Two separate microemulsions (60 mL each) were prepared of Igepal 520 and cyclohexane (3:7 v/v) and placed under stirring. 80μg of miRNA was added into 1800 μL of 2.5M CaCl2 solution. An (NH4)2HPO4 solution (1800 μL, 50 mM) was also prepared and added to the other microemulsion. Each microemulsion was allowed to stir for 20 min. The microemulsion containing (NH4)2HPO4 was added to the microemulsion containing the CaCl2/miRNA. This solution was allowed to stir for 5 min before addition of 1400 μL of DOPA in CHCl3. After addition of DOPA, the microemulsion was left to stir an additional 30 min. An equal volume of 100% EtOH (120 mL) was added to disrupt the emulsion. The mixture was centrifuged at 10,000 × g for 20 min. After removing the supernatant, the precipitate was washed twice thereafter with 100% EtOH to remove traces of Igepal and/or cyclohexane. The precipitate was then dried under N2, and resuspended in CH2Cl2. This solution was centrifuged at 13,000 × g for 10 min for the removal of large aggregates, and the supernatant containing the LCP cores (miRNA entrapped within a calcium phosphate nanoprecipitate, supporting and surrounded by a lipid monolayer of DOPA) was recovered. Final Gal-LCP miR-122 was produced through desiccation of a mixture of free lipids and cores and rehydration via 5% aqueous sucrose solution. The ratio of cores to outer leaflet lipids for optimal final particle formulation was found to be 15 mg core: 420 μL DOTAP, (40 mM):420 μL Cholesterol (40 mM):666 μL DSPE-PEG2000 (20 mM). Therein, 35 mol% DOTAP, 35 mol% cholesterol, 25 mol% DSPE-PEG and 5 mol% DSPE-PEG-Gal) were utilized as outer leaflet lipids. Zeta potential and particle size of LCP were measured using a Malvern ZetaSizer Nano Series (Westborough, MA). TEM images of LCP were acquired using a JEOL 100CX II TEM (JEOL, Japan)

Encapsulation efficiency of Gal-LCP

To characterize miRNA entrapment efficiency of Gal-LCP, Cy5 labeled miR-122 was purchased from Horizon Discovery (Lafayette, CO). Cy5-miR122 was then formulated into Gal-LCP. The final nanoparticles were dissolved in the same amount of lysis buffer (2mM EDTA and 0.05% Triton X-100 in pH 7.8 Tris buffer) at 65 °C for 10 min to release the entrapped miRNA. The standard Cy5-miRNA solutions were prepared by diluting the original Cy5-MiR122 in the same lysis buffer. Then, 100 μl of the standard and Gal-LCP solution was taken to the 96-well plate and the fluorescence intensity was measured on a plate reader with a 620 nm excitation filter and a 670 nm emission filter.

Bio-distribution of Gal-LCP containing miRNA

To assess bio-distribution of Gal-LCP containing miRNA, fluorescently labeled miRNA (Dy-547-miR-122) was used at a dose of 80 ug of miRNA per mouse. Levels of fluorescence in liver, heart, lungs, spleen, and kidneys were characterized at 24 hr post IV injection.

Blood chemistry and histopathology analysis.

To assess the toxicity profile of nanoparticle, C57BL/6 mice were given I.V. Gal-LCP miR122 at a dose of 80 ug of miRNA per mouse every other day for 3 weeks. Three mice in each group (miRNC vs miR-122) were subjected to chronic toxicity assay and serum samples were collected. Serum biomarkers, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), serum albumin, triglyceride, cholesterol and blood urea nitrogen (BUN) were analyzed. Organs including the heart, liver, lungs, spleen, and kidneys were collected and fixed for hematoxylin and eosin (H&E) as well as trichrome staining at UNC histology facility to evaluate the organ-specific toxicity.

Cell lines

HT-29-luc2, was purchased from Caliper Life Sciences (Hopkinton, MA) and murine CRC CT26-Luc cells were obtained from Tissue Culture Facility—UNC Lineberger Comprehensive Cancer Center. Cell lines used were authenticated by the vendors and tested for mycoplasma contamination by source. HT-29-luc2 cells were cultured in DMEM/F12 (Gibco, Gaithersburg, MD) supplemented with 10% fetal bovine serum (Gibco, MD) and penicillin/streptomycin (Mediatech, Manassas, VA). CT26-Luc cells were cultured by DMEM (Gibco, MD), supplemented with 10% fetal bovine serum (Gibco, MD), penicillin/streptomycin (Mediatech, VA) and 0.4 mg/mL G418 (Gibco, MD).

Western Blotting

Protein solution was diluted by sample buffer (4×) containing reducing reagent and heated at 95°C for 5 minutes. Proteins were separated on 4%-12% gradient sodium dodecyl sulphate-polyacrylamide gel and then transferred to a PVDF membrane (Invitrogen, CA). The membranes were blocked for 1 h in PBS containing 5% nonfat dry milk (v/v), and then incubated for 1 h with the primary antibody at room temperature. After 4 washes with 0.1% (v/v) Tween 20 in PBS (PBS-T), the membranes were incubated for 1 h with the appropriate secondary antibody. Finally, the membranes were washed 4 times with PBS-T, and the bound antibodies were visualized with the Pierce ECL Western Blotting Substrate (ThermoFisher Scientific, IL). Beta actin was used as control (Sigma A3854). ADAM17 (24620-1-AP) and ALDOA (11217-1-AP) were both purchased from proteintech (Rosemont, IL).

Perfusion-based decellularization of liver and lung

Male Sprague-Dawley rats were used to produce liver biomatrix (BMS) through cannulating the portal vein for perfusion of decellularizing reagents. The vasculature was perfused with basal medium until blood was eliminated and then perfused with 250 mL of 1% sodium deoxycholate (SDC) containing 36 units/L phospholipase. Next, organs were perfused with 3.5 M NaCl until the perfusate was negative for proteins. Finally, tissues were rinsed with basal medium, snap frozen and then pulverized into a fine powder using a freezer mill (Spex SamplePrep 6770, Metuchen, NJ).

Preparation of BMS coated tissue culture plates

To determine protein concentrations of BMS materials, BMS were dissolved in a solution composed of 4 M guanidine HCl, 50 mM sodium acetate (pH 5.8), and 25 mM EDTA 300 containing proteinase and phosphatase inhibitor cocktails. BCA assays were then performed to determine total protein concentrations. To prepare BMS coated surfaces, BMSs were suspended in Medium (DMEM/F12), added to tissue culture plates and allowed to dry overnight. Plates were sterilized using 100 Gy of 304 external beam irradiation (Precision X-Ray). Then, HT29-Luc2 cells were grown on coated tissue culture plates until 7–10 when they acquire metastatic features.

Mouse model establishment

Eight-week-old male C57BL/6, BALB/c, and Nu/Nu, mice were obtained from the Jackson Laboratory. All animal testing and research were conducted in compliance with ethical regulations approved by the Institutional Animal Care and Use Committee of UNC Chapel Hill. CT26-Luc liver metastasis model was established in male BALB/c mice. CT26-Luc cells were harvested and washed in phosphate buffered saline (PBS) just prior to spleen implantation. Mice were anesthetized by 2.5% isoflurane and placed in supine position. For splenic inoculation, an incision located below the left rib cage was made to expose the spleen. The spleen was tied and cut into two parts each containing intact vascular pedicle for each half of the spleen. The distal section of the spleen was inoculated with 2 × 10⁵ CT26-Luc cells in PBS. The hemi-spleen containing inoculated cells was resected 5 min after inoculation allowing the cancer cells to enter the portal vein. The hemi-half containing inoculated cells was resected to model primary tumor resection. The other half of the spleen was returned to the cavity and abdominal wall and skin were closed with 4–0 polyglycolic acid suture. In vivo imaging was done the day after surgery and weekly afterward. HT29 hepatic injection for inducing liver metastasis was performed on Nu/Nu mice. HT-29-luc2 cells grown on plastic or liver BMS were trypsinized, harvested and processed into single cell suspensions. Cells were suspended in PBS and administered into Nu/Nu mice through portal vein. Bioluminescence was measured every week using an In Vivo Imaging Systems (IVIS) imaging system.

Luciferase imaging

The in vivo metastatic progression was monitored by intraperitoneal injection of 100 μL of D-luciferin (Perkin Elmer, 20 mg/mL) followed by bioluminescent analysis using an IVIS® Kinetics Optical System (Perkin Elmer, CA).

Real time PCR

Non-tumor liver tissue was extracted at week 4 after completion of their treatment. Total RNA was isolated with miRNAeasy kit (Qiagen, USA). The cDNA was made using RT2 First Strand kit (Qiagen, MD) and mixed with RT2 SYBR Green/ROX qPCR Master Mix (Qiagen, MD) and the mixture was added into a 384-well RT2 PCR Arrays of mouse tumor metastasis (Qiagen, PAMM-028Z) and cancer inflammation and immunity cross talk (Qiagen, PMAM-181Z) that contained primers for 84 key genes in fibrosis and 5 housekeeping genes according to manufacturer’s instruction. Thermal cycling was performed using QuantStudio 6 Pro RT PCT System (Applied Biosystems, USA) with an initial denaturation at 95°C for 10 minutes, 40 cycles at 95°C for 15 seconds, and 60°C for 1 minute. Values of cycle threshold (Ct) obtained in quantification were used for calculations of fold changes in mRNA abundance using 2-ΔΔCt method. Data was analyzed using RT2 profiler PCR array data analysis at GeneGlobe Data Analysis Center online (GeneGlobe, Qiagen, MD).

Flow cytometry

Fluorochrome labeled monoclonal antibodies used in flow cytometry against murine anti-CD8 (Pacific blue BD 558106), CD4 (BV605 BD740336), TCRb (PE-Cy7 BD 560729), Ly-6G (APC-Cy7 BD 560600), CD11b (PerpCP-Cy5.5 BD 550993), and CD11c (BV 711BD 563048) were purchased from BD Biosciences (Franklin Lakes, NJ). Intracellular antibody FoxP3 (FJK-16s 12-5773-82), MHC Class II (I-A/I-E PE-eFluor610#61-5321-82), and Ly6c (APC, # 17-5932-82) were purchased from eBioscience Inc. (San Diego, CA). For cell viability, Alexa 488 Fixable Green Dead Cell Stain kit (L34970) was used from Invitrogen. Single-cell suspensions of murine tumors were obtained by both mechanical and enzymatical digestion. First, resected metastatic tumors were divided to small pieces by razor. Next, digestion was performed using the collagenase, DNase I, hyaluronidase (Sigma-Aldrich, St. Louis, MO), and Liberase (Roche, USA). Cells were washed and staining buffer (PBS with 2% NCS). Lympholyte-M (Cedar lane, NC) gradient and 44% Percoll (Sigma- Aldrich, St. Louis, MO) in complete RPMI media were used for enrichment of lymphocytes. Cells were washed by PBS and were re-suspended in live/dead staining dye diluted in PBS and incubated for 35 min on ice. Next, cells were washed and re-suspended in Fc Block (clone 2.4G2; Bio X Cell, West Lebanon, NH) and incubated further for 20 min on ice. Cells were surface stained by master mix of antibodies for 40 min on ice. Cells were fixed, and permeabilized by Foxp3/transcription factor staining buffer set (eBioscience). Intracellular staining was completed the following day with anti-Foxp3 antibody, and flow cytometry was performed on a LSR II (BD Biosciences). The analysis of acquired data was performed using FlowJo software (TreeStar Inc., Ashland, OR).

Statistical analysis

Statistical analysis were conducted mainly using Prism 8 (Graph- Pad Software) in particular for survival studies which a log rank test was used for comparison. A two-tailed Student’s t-test was used to determine statistical significance when there was only two groups were compared. Ordinary one-way or two-way ANOVA with Turkey’s multiple comparisons was used for comparison between multiple groups. Mann-Whitney and KrusKal-Wallis tests were used when a non-parametric test was needed. (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). The mean and SEM are presented in the Figures.

Data Availability Statement

Data that support the findings of RT PCR in this study have been deposited in NCBI in GEO with accession numbers of GSE158365, GSE158366, and GSE158368 and are available in following links:https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE158365, https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE158366, https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE158368

Results

Characterization and bio-distribution of lipid calcium phosphate containing miR-122

To enable the targeted delivery of miR-122 to hepatocytes and to protect miR-122 from degradation, we utilized a lipid calcium phosphate (LCP) nanoparticle to encapsulate miR-122. The core of the nanoparticle (NP) contains calcium phosphate which complexes with miR-122 to form a stable core ⁽¹⁷⁾. The NP outer layer is a double lipid membrane, comprised of 1,2-dioleoyl-sn-glycero-3-phosphate (DOPA), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), and cholesterol. The cationic surface facilitates endosomal escape. To enable targeting to hepatocytes, the lipid monolayer also contains 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine-N-succinyl (polyethylene glycol) [DSPE-PEG] and galactose (Gal) conjugated to the DSPE-PEG. Galactose enables asialoglycoprotein receptor (ASGPR)-mediated endocytosis by the hepatocytes. The resulting Gal-LCP miR-122 were about 30 nm in diameter (Supp. Fig 1a), with a surface charge of +1.75 (Supp. Fig 1b) according to dynamic light scattering analysis. Transmission electron microscopy (TEM) image also showed the spherical shape and homogenous distribution for the nanoparticle core (Supp. Fig. 1c). Using the Cy5 labeled miR-122, miRNA encapsulation efficiency was determined to be 52 ± 5% (n = 3) as described in method section.

We examined the bio-distribution of Gal-targeted LCP to confirm their preferential distribution to the liver. Using fluorescently labeled miR-122 (80 ug of RNA per mouse), we characterized the level of fluorescence in liver, heart, lungs, spleen, and kidneys at 24 hr post IV injection. We found that Gal-LCP miR-122’s distribution is mainly to the liver (80%) as we expected. This is followed by lungs (19%) and kidneys (1%) (Fig 2a).

Fig 2. Bio-distribution and targeted delivery of Gal-LCP miR-122 to hepatocytes.

a Bio-distribution of Gal-LCP miR-122. Gal-LCP containing fluorescently labeled miR-122 was injected systemically. Mice were sacrificed after 24 hours and fluorescence was quantified in each organ and relative distribution of Gal-LCP miR-122 within liver, heart, lungs, spleen, and kidneys were calculated based on the florescence measurement in each organ. b. Gal-LCP siRNA albumin can reduce albumin production. C57BL/6 mice were injected with Gal-LCP containing siRNA against albumin. Serum albumin was measured at baseline and 48 hours after injection. c. ADAM17 and ALDOA (two known targets of miR-122) protein level change (western blot) with Gal-LCP miR-122 with b-actin and scrambled nucleic acid as controls (*P < 0.05, ** P < 0.01).

To validate that the LCP can indeed effectively deliver nucleic acid therapeutics to hepatocytes, we studied LCP delivery of a siRNA against albumin. C57BL/6 mice were injected (i.v.) once with Gal-LCP containing siRNA against albumin at a dose of 80 ug of siRNA per mouse. Peripheral blood was obtained from mice at baseline as well as 48 hours post treatment. Gal-LCP siAlb treated mice had significantly lower serum albumin level when compared to their baseline albumin levels (3.5 vs 3.9 g/dL, p<0.01) (Fig 2b). This indicates that the Gal-LCP can deliver nucleic acid therapeutic agents in an effective manner.

We then confirmed that Gal-LCP can effectively deliver miR-122 into hepatocytes. To assess this, we characterized the change in expression of two known targets of miR-122: ADAM17 and ALDOA. C57BL/6 mice were treated with Gal-LCP miR-122 at a dose of 80 ug of miRNA per mouse. 48 hours post treatment, hepatocytes were harvested and the expression level of ADAM17 and ALDOA were quantified. These levels were compared to mice that were treated with Gal-LCP formulations of scrambled miRNA (miRNC) sequence. The expression levels of ADAM17 and ALDOA are significantly lower in the Gal-LCP miR-122 treated mice (ADAM17 50±6% decrease, p<0.05; ALDOA 84±16% decrease, p<0.01) (Fig 2c). This data confirms Gal-LCP delivery of miR-122 to hepatocytes and shows that once delivered, miR-122 is biologically active in hepatocytes.

Next, we assessed the toxicity profile of Gal-LCP miR122. Based on the bio-distribution, the key concerns were hepatotoxicity and nephrotoxicity. To characterize both, serum biomarkers, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), serum albumin, triglyceride, cholesterol and blood urea nitrogen (BUN) were analyzed. These values were compared to those in mice given Gal-LCP miRNC. There were no significant differences in these serum biomarkers between the treatment arms, indicating a low toxicity profile of miR-122 (Table S1). Moreover, we harvested heart, liver, lungs, spleen and kidneys and used Hematoxylin & Eosinophil and trichrome-stained histology sections of any organ compared with mice treated with Gal-LCP miRNC (Supp. Fig 2). We did not observe any tissue toxicity in these histologic studies.

Gal-LCP miR-122 inhibits HT29-induced liver metastasis in nude mice

To examine the effectiveness of Gal-LCP miR-122 in preventing liver metastasis, we employed colorectal cancer as a disease model. Colorectal cancer is the third most common cancer and second leading cause of cancer death in the US (18). Liver is the primary site of metastasis and main cause of morbidity and mortality from colorectal cancer (19). We studied Gal-LCP miR-122 in two liver metastasis models. The first model is based on our previous work on engineering colorectal cancer liver metastasis (20). For induction of hepatic metastasis, HT29-Luc2 cells were first grown on decellularized liver for 7–10 days to acquire metastatic features. The cells were then harvested and injected into portal veins of nu/nu mice. Similar to our previous report, 80% of the mice given conditioned HT29-Luc2 developed liver metastasis as compared to 0% in mice given unconditioned HT29-Luc2 (Supp. Fig 3a–b). We assessed the efficacy of Gal-LCP miR-122 treatment in preventing liver metastasis in this tumor model. Mice (n=10 each group) were treated with Gal-LCP miR-122 three times per week for a total of three weeks. The first injection of Gal-LCP miR-122 was given after 24 hours of HT29-Luc2 injection. Control groups included mice given Gal-LCP formulation of a scrambled miRNC sequence as well with no treatment at all. The formation of hepatic metastases was monitored weekly for 4 weeks in vivo using bioluminescent imaging (Fig 3a). Metastases from HT29 developed slowly and only became evident on IVIS 1–2 weeks post injection (Fig 3a, Supp. Fig 3b). Our study showed mice treated with with Gal-LCP miR-122 had significantly lower metastatic (liver) burden (Fig 3a, Supp. Fig 4a). 70% of mice treated with Gal-LCP miR-122 were alive at 100 days while none of control mice treated with Gal-LCP miRNC or untreated control mice survived (p<0.0001, Fig3b). To validate that Gal-LCP miR-122’s mechanism of action is through prevention of metastasis, we sacrificed the surviving mice from Gal-LCP miR-122 treatment group and evaluated resected livers for the presence of tumor. We found that 6 of the 7 mice had no evidence of metastatic cancer in their liver and one mouse had a focal metastasis (Fig 3c).

Fig. 3. miR-122 targeted delivery inhibits liver metastasis in vivo and increases survival in HT29 induced liver metastasis in nude mice.

a Gal-LCP miR-122 treatment prevented liver metastasis in HT29 liver metastasis model. Nude mice (n=10 each group) were treated with Gal-LCP miR-122 or Gal-LCP miRNC three times per week for a total of three weeks. Control group represent untreated mice. Hepatic metastases formation was monitored weekly for 4 weeks in vivo using bioluminescent imaging. b Survival curves of the same 3 groups of mice (n=10 each group) shown in Fig 3a (****P < 0.0001). c Macroscopical examination of surviving mice from Gal-LCP miR-122 treatment group after 100 days. HT29 liver metastatic nude mice with miR-122 delivery which still survived after 100 days were sacrificed and their livers were extracted to examine any potential liver metastasis. Only one of the livers had a focal metastasis (blue arrow).

Gal-LCP miR-122 inhibits CT26-induced liver metastasis in syngeneic mice

We further validated our finding using the CT26 syngeneic and highly aggressive colorectal cancer model. Liver metastases were established through a hemi-splenic injection protocol (21). Similar to previous methods, mice were treated with 9 doses of Gal-LCP miR-122 for 3 weeks. Control arms again included Gal-LCP miRNC, and no treatment. Hepatic metastases formation was monitored weekly for 4 weeks in vivo using bioluminescent imaging (Fig 4a). Thereafter, mice were sacrificed at day 29 to evaluate the liver metastasis growth (Fig 4b). The results of IVIS showed a significant decrease in metastasis burden upon treatment with Gal-LCP miR-122 (Fig 4a, Supp. Fig 4b) In this study, mice in all 3 experimental groups developed liver metastasis by day 8 (Fig 4a). However, there was significant difference in the size of the liver metastases at the end of study. While 80–100% of mice in both control groups showed multiple liver metastasis visible on IVIS imaging by day 29, only 40% of mice treated with Gal-LCP miR122 showed such macroscopic metastasis (Fig 4b). It is noteworthy to mention that mice treated with Gal-LCP miRNC had the least luciferase activity at day 1 among three different groups, however, the level reached that of the other groups after a week and Gal-LCP miRNC group actually turned out to be the group with highest metastatic burden based on the luminescence intensity and macroscopically liver examination at day 28 (Fig 4a–b). Furthermore, a survival study was performed on 5 groups of mice including mice received Gal-LCP miR-122, LCP-miR122 (no Galactose), free miR-122, Gal-LCP miRNC and untreated control mice (n=8 each group). In this survival study, 25% of mice treated with Gal-LCP miR-122 were alive at 85 days and in general they had significantly longer survival (median 79) when compared to the untreated control group (median 21), mice received Gal-LCP miRNC (median 27), mice received LCP-miR122 (median 29) and mice with free miR-122 (median 37) (p<0.0001, Fig 4c).

Fig. 4. miR-122 targeted delivery inhibits liver metastasis in vivo and increases survival in CT26 induced liver metastasis in syngeneic mice.

a Gal-LCP miR-122 treatment prevented liver metastasis in CT26 syngeneic metastatic model (n=5 each group) as well. Hepatic metastases formation was monitored weekly for 4 weeks in vivo using bioluminescent imaging and mice sacrificed at day 29. b livers from mice were examined at day29 for macroscopic tumor evaluation. c Survival curves of 5 groups (n=8 each group) of CT26 syngeneic liver metastasis model (****P < 0.0001). These groups included mice received Gal-LCP miR-122, LCP-miR122 (no Galactose), free miR-122, Gal-LCP miRNC and untreated control mice.

Gal-LCP miR-122 changes gene expression profiles of liver and tumor microenvironment

To understand the mechanisms of action of Gal-LCP miR-122 on the molecular level, we compared hepatic gene expression profiling on 2 different platforms each consisting of 84 Key genes: metastasis pathways and cancer inflammation pathways. These RT PCR profiling tests were performed on non-tumor liver tissues from Gal-LCP miR122 as well as Gal-LCP miRNC treated groups in CT26-induced liver metastasis syngenic mice. Non-tumor liver tissues were extracted in week 4 after completion of their ninth IV injection and total RNA was isolated. Non-tumor liver tissues were extracted from furthest location to tumor in tumor-bearing mice. This reduces the risk of effect of tumor on gene expression profile of healthy liver tissue within the same extracted liver. Interestingly, majority of genes found to have lower expression (blue dots under parallel lines, Fig 5a–b) in mice liver treated with Gal-LCP miR-122 compared with those treated with Gal-LCP miRNC. A list of those metastatic genes with statistically significant lower expression (at least 10-fold) are presented in Fig 5c. In metastatic platform, several matrix metaloproteinases (MMP3, MMP9, MMP10), ECM degradation enzymes (Elane, Plaur) as well as some chemokines which promotes hepatic metastasis (22), were significantly down-regulated upon miR-122 delivery compared with miRNC. Also, treated with Gal-LCP miR-122, their expression kept down close to their baseline levels, opposite to Gal-LCP miRNC group (Supp. Fig 5). Also in cancer inflammation platform, several chemokines and chemokine receptors (CCL2, CXCL1, CCR1) were significantly down-regulated.

Fig. 5. Gene expression changes from miR-122 delivery.

a Scatter plot of expression changes in genes that are known to be involved in cancer metastasis. Mice were treated with Gal-LCP miR-122 with Gal-LCP miRNC as control. The genes which are down-regulated more than 10-fold upon miR-122 delivery are labeled. b Scatter plot of gene expression in genes known to be involved in cancer inflammation and immunity. Mice were treated with Gal-LCP miR-122 compared with mice treated with Gal-LCP miRNC as control. The genes which are down-regulated more than 10-fold upon miR-122 delivery are labeled. c A list of down-regulated genes resulting from Gal-LCP miR-122.

Gal-LCP miR-122 treatment is associated with a less immunosuppressive liver microenvironment

We also characterized the effects of Gal-LCP miR-122 on the liver microenvironment, especially with respect to immune cells (Fig 6, Supp. Fig 6). Flow cytometry analysis of livers from CT26-induced liver metastasis syngeneic mice treated with Gal-LCP miR-122 showed these livers had higher CD8+T cells in the metastases compared to that in mice in the control arms (29% vs 21%, p<0.01; Fig 6). In addition, Gal-LCP miR-122 treated mice had higher CD8+/CD4+ T-cell ratio vs control group (0.49 vs 0.36, p<0.05; Fig 6). MiR-122 treated mice also had a slightly decreased Tregs (CD4+Foxp3+ T-cells) population (3.4% vs 2.8%), which was not statistically significant. Interestingly, miR-122 treated mice had a decrease in population of Myeloid-Derived Suppressor Cells (MDSCs) compared to control (60% vs 75%, p<0.001; Fig 6). This decrease was evident in both monocytic as well as granulocytic subsets of MDSCs. MiR-122 treated mice also had an increase in percentage of Ly6c^low macrophages (37% vs 20%, p<0.001) and lymphoid DCs in liver (40% vs 13%, p<0.05, Fig 6) compared to control group.

Fig 6. miR-122 targeted delivery remodels liver immune microenvironment.

CD8+ T cells, CD4+ T cells, CD8+/CD4+ ratio, activated DCs, Treg cells, MDSCs and macrophages in the liver of mice bearing CT26 syngeneic liver metastasis. Treatment represents mice treated with Gal-LCP miR-122, and control represents mice treated with Gal-LCP miRNC. (*P < 0.05, **P < 0.01, ***P < 0.001).

Discussion

Our Gal-LCP miR-122 treatment dramatically increased survival in both nude mice injected with HT29 cells grown on biomatrix as well as in syngeneic balb/c mice with hemi-splenectomy injection of CT26 cells. 70% of nude mice survived after 100 days compared to none in control groups (p<0.0001) while in syngeneic mice, median survival increased from 21 to 79 (p<0.0001). Delivery of miR-122 was found to be associated with down-regulation of some key genes in metastatic pathways as well as cancer inflammation. We also found miR-122 delivery was associated with increase in CD8+/CD4+ T-cell ratio (0.49 vs 0.36, p<0.05) as well as decrease in MDSCs (60% vs 75%, p<0.001) and increase in Ly6c^low macrophages (37% vs 20%, p<0.001).

MiR-122 has been shown to inhibit hepatocellular carcinoma, but its mechanism of action is through direct action on the oncogenes. Our work has demonstrated that modifying the organ microenvironment through microRNA can prevent the development of hepatic metastases. Interestingly, our findings also reveal some insight into the complex mechanism of metastasis development. Gal-LCP miR-122 suppressed the production of a number of proteins that can directly contribute to metastasis colonization, ECM degradation, and expansion of metastasis, including several MMPs, other ECM degradation enzymes and some pro-inflammatory factors. This is consistent with previous reports (11, 23). The increase in CD8+/CD4+ T-cell ratio and decrease in MDSCs could also have played a critical role in suppressing liver metastasis. An increased percentage of Ly6c^low macrophages also plays a key role in resolution of hepatic inflammation and metastasis (24). However, it is possible that changes of immune cell profile might not be directly caused by miR-122 expression. Nevertheless, the indirect effect of miR-122 in regression of tumors and consequent change in immune profile cannot be overlooked. Further details of suppressive activity of miR-122 on metastasis expansion and its effect on liver immune microenvironment yet to be investigated.

Herein, we report a biologically targeted agent to prevent organ-specific metastasis to the liver. We have shown this approach is feasible and effective. The concept can also be translated clinically. Many patients present with liver-only metastasis. These patients’ existing liver metastases will be treated definitively and their long term outcome depends on disease control in the liver. Based on our work, agents such as Gal-LCP miR-122 can be used in these patients to prevent further metastasis development. This approach can be applied to a number of malignancies with propensity of developing liver metastases, such as breast cancer, pancreatic cancer and gastric cancer.

Significance:

Highly specific and efficient delivery of miRNA to hepatocytes using nanomedicine has therapeutic potential for the prevention and treatment of colorectal cancer liver metastasis.