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 miR-122, a liver-specific 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 liver metastasis models, treatment with Gal-LCP miR-122 treatment effectively prevented colorectal cancer liver metastasis and prolonged survival. Mechanistic studies revealed that delivery of miR-122 was associated with downregulation of key genes involved in metastatic and cancer inflammation pathways, including several proinflammatory 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 antitumor immune response. Collectively, this work presents a strategy to improve cancer prevention and treatment with nanomedicine-based delivery of miRNA. 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.

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).

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).

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).

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.

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.

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).