Colorectal Cancer-derived Small Extracellular Vesicles Create an Inflammatory Pre-metastatic Niche through Macrophage Polarization in Liver Metastasis

Colon and liver have an inseparable relationship since embryological development. More than half of colorectal cancer patients developed liver metastases accompanied with poor prognosis. Recent evidence suggests that distant metastatic organs are “educated” by primary tumor-derived extracellular vesicles. Here we found that the liver-specific pro-metastatic effect of colorectal cancer (CRC)-derived small extracellular vesicles (sEVs). In vivo experiments showed that the injected fluorescence-labeled CRC-derived sEVs were accumulated and taken up by macrophages in the mouse liver. MicroRNA sequencing revealed the highest expression of microRNA (miR)-21-5p in CRC-derived sEVs. In vitro co-culture experiments showed that the sEVs polarized macrophages toward an interleukin-6(IL-6)-secreting type of macrophages. Further mouse experiments and microarray studies indicated that the CRC-sEVs increased macrophage invasion and induced an inflammatory microenvironment in the liver, which promoted liver metastasis of orthotropic- transplanted CRC cells. Most importantly, the TCGA data analysis and clinical sample examinations demonstrated that miR-21-5p expressions were sharply raised in the CRC tissues. We apply Tethered Cationic Lipoplex Nanoparticles (tCLN) technology in detecting CRC patients’ sEVs miR-21 in plasma and found strong correlation between sEVs-miR-21 and CRC liver metastasis. The serum IL-6 level was much higher in CRC patients with liver metastasis, and the liver metastasis relevance of MiR-21, macrophages, and IL-6 in CRC patients. Thus, we propose the role of CRC-derived sEVs in the formation of an inflammatory pre-metastatic niche in liver for CRC metastasis through macrophage polarization via a miR-21/TLRs pro-inflammation pathway. Purified SW620-derived sEVs were injected to nude mice via tail vein every other day for 21 days (each injection with 20 μg sEVs, totally 10 injections per mouse). The mice in the control group were injected with sterile PBS. Each group had 5 nude mice, all of which were alive normally before harvested, and there was no obvious weight difference between the experimental and control groups. Three nude mice were randomly picked from the experimental group, and after gas anesthesia, their livers were dissected and immediately stored in liquid nitrogen. Microarray screenings demonstrated that the expression of genes associated with inflammation, cell apoptosis, and excretal cellular matrix (ECM) remodeling was significantly altered.