Exosome-delivered EGFR regulates liver microenvironment to promote gastric cancer liver metastasis

Abstract

The metastatic organotropism has been one of the cancer's greatest mysteries since the 'seed and soil' hypothesis. Although the role of EGFR in cancer cells is well studied, the effects of secreted EGFR transported by exosomes are less understood. Here we show that EGFR in exosomes secreted from gastric cancer cells can be delivered into the liver and is integrated on the plasma membrane of liver stromal cells. The translocated EGFR is proved to effectively activate hepatocyte growth factor (HGF) by suppressing miR-26a/b expression. Moreover, the upregulated paracrine HGF, which binds the c-MET receptor on the migrated cancer cells, provides fertile 'soil' for the 'seed', facilitating the landing and proliferation of metastatic cancer cells. Thus, we propose that EGFR-containing exosomes derived from cancer cells could favour the development of a liver-like microenvironment promoting liver-specific metastasis.

Figure 1. Clinical analysis of sr-exosome EGFR and HGF-cMET in GC.

(a) Electron microscope scanning of exosomes isolated from human serum. (b) Sr-exosome EGFR is related to the progression of GC. Exosomes were isolated from serum of healthy donors (NC), stage II/III GC patients and stage IV GC patients, respectively (n=20); Alix, TSG101 and CD63 were used as the internal control of exosomes. (c) The expression of HGF, c-MET and p-c-MET in para-carcinoma tissue (liver) and GC liver metastases (n=5). (d) Immunohistochemistry (IHC) analysis of HGF in GC liver metastasis. (e) Positive ratio of HGF and its receptor c-MET in GC liver metastases (n=30). The data represent the mean±s.e.m. *P< 0.05, **P<0.01 (Student's t-test).

Figure 2. SGC-exosomes transport EGFR into liver cells.

(a) Electron microscope scanning of exosomes isolated from the medium of SGC7901 cells. (b) EGFR expression in both SGC exosomes and SGC7901 cells treated with EGFR siRNA. (c) Schematic description of the experimental design. The SGC exosomes were isolated and 50 μg exosomes were used to culture with 5 × 10⁵ primary liver cells. (d) Confocal microscopy image of the internalization of fluorescently labelled exosomes in mixed liver cells. Scale bars, 50 μm. (e) SGC-exosome-mediated EGFR is located in the membrane of mixed primary liver cells. Scale bars, 50 μm. (f) Nanoparticle Tracking Analysis (NTA) of isolated exosomes. The data represent the mean±s.e.m. **P<0.01 (Student's t-test).

Figure 3. Characterization of cells types in primary liver cells.

(a) Western blotting analysis of desmin, α-SMA and F4/80 in mixed primary liver cells (n=3). (b) Quantification analysis of a. (c) Immunofluorescence analysis of exosome-delivered EGFR and stromal cell markers. Scale bars, 50 μm. The data represent the mean±s.e.m. **P<0.01 (Student's t-test).

Figure 4. SGC-exosomes-mediated EGFR activates liver HGF by suppressing miR-26a/b expression.

Forty micrograms of exosomes were used to culture with 1 × 10⁶ primary liver cells seeded in a six-well plate. (a,b) Effects of SGC exosomes delivered EGFR on HGF protein levels (a) and mRNA levels (b) in mixed primary liver cells (n=3). (c,d) Effects of EGFR on the expression of HGF protein (c) and mRNA (d) (n=3). (e) The binding sites of miR-26a/b in the 3′-UTR of HGF mRNA. (f) SGC-exosomes decrease liver miR-26a/b levels (n=3). (g) EGFR is negatively related with miR-26a/b in liver cells (n=3). (h) Absolute quantification of serum miR-26a/b in the progression of GC (n=150). The data represent the mean±s.e.m. **P<0.01, ***P<0.001 (Student's t-test).

Figure 5. Identification of HGF as a direct target of miR-26a/b.

(a) Predicted binding sites of miR-26a/b within the 3′-UTR of HGF mRNA. (b) Direct recognition of HGF 3′-UTR by miR-26a and miR-26b. Primary liver cells were co-transfected with firefly luciferase reporters containing either wild-type or mutant (mut) HGF 3′-UTR with miR-26a/b mimics, inhibitors and the corresponding normal control. The relative luciferase levels were detected using a luciferase kit at 24–36 h after transfection (n=3). (c) Western blot analysis of HGF expression in primary liver cells with the overexpression or suppression of miR-26a or miR-26b (n=3). (d) Relative levels of HGF mRNA in liver cells transfected with miR-26a/b mimics or inhibitors (n=3). The data represent the mean±s.e.m. **P<0.01 (Student's t-test).

Figure 6. Upregulated liver paracrine HGF promotes proliferation and invasion of SGC7901 cells.

Pretreated mixed liver cells were co-cultured with SGC7901 cells using a 0.4 μm membrane and the biology behaviour of SGC7901 cells were measured subsequently. (a) Schematic representation of the in vitro model for cell co-culture. (b) Western blot analysis of HGF expression in primary liver cells treated with HGF shRNA and HGF-overexpressing lentivirus (OE.HGF) (n=3). (c) Effects of SGC exosomes and control exosomes on the HGF secretion from mixed liver cells (n=3). (d) ELISA analysis of HGF secretion from primary liver cells treated with HGF siRNA or OE.HGF lentivirus (n=3). (e–g). Upregulated liver HGF promotes growth (e) and invasion (g) of SGC7901 cells (n=3). (h) c-MET and p-c-MET expression in mixed liver cells treated as above (n=3). The data represent the mean±s.e.m. *P<0.05, **P<0.01 (Student's t-test).

Figure 7. In vivo verification for exosome-EGFR activates liver HGF by inhibiting miR-26a/b.

The livers of mice were pre-treated with HGF shRNA or HGF overexpressing lentivirus by multi-point injection, followed by orthotopic tumour implantation on the 6th day; finally, mice were killed and data were analysed on the 66th day. (a) A flow chart depicting the in vivo experimental design. (b) Immunohistochemistry (IHC) analysis of EGFR expression in primary gastric tumour. (c) Western blotting analysis of exosome EGFR in the serum of tumour-implanted mice (n=30). (d) Levels of HGF in liver tissues of GC tumour-implanted mice (n=30). (e) Electron-microscope scanning of exosomes isolated from mouse serum. (f) Relative levels of miR-26a/b in mouse liver tissues (n=30). The data represent the mean±s.e.m. **P<0.01 (Student's t-test).

Figure 8. Effects of upregulated liver HGF on hepatotropic metastasis.

(a) Ratio of liver metastasis in GC tumor-implanted mice (n=30). (b) Number of metastases in the liver of each mouse (the number in each group was marked). (c) Images of liver metastases in mice (n=30). (d) The weight of tumours in c. (e) Immunohistochemistry (IHC) analysis of HGF and c-MET expression in GC liver metastases. (f) IHC analysis of p-c-MET in the liver metastases of each group. (g) Western blot analysis of HGF, p-c-MET and c-MET in GC liver metastases. The data represent the mean±s.e.m. *P<0.05, **P<0.01 (Student's t-test).