The mechanisms underlying the enrichment and action of glypican-1-positive exosomes in colorectal cancer cells

Abstract

Background: Glypican-1 (GPC1) is overexpressed in several tumors, and GPC1+ exosomes have shown the potential to predict early colorectal cancer (CRC). However, the mechanisms underlying the enrichment and action of GPC1+ exosomes in CRC remain unknown. Methods: The expression of slit guidance ligand 2 (SLIT2), hypoxia-inducible factor (HIF)-1α/2α, and GPC1 in clinical CRC tissues was detected using immunohistochemistry and western blot. Exosomes were isolated from the supernatants of CRC cell cultures. The effects of SLIT2, hypoxia, heparin, and phospholipase C (PLC) on exosomal GPC1 expression and GPC1+ exosome enrichment in CRC cells were analyzed with western blot and flow cytometry. CRC cell proliferation was assessed with MTT and colony formation assays. Co-immunoprecipitation was used to detect the binding of GPC1 and SLIT2 in SW480 cells. Nude mice were subcutaneously inoculated with SW480 cells with different treatments. The Wnt signaling was detected. Results: SLIT2 was poorly expressed and GPC1, HIF-1α, and HIF-2α were highly expressed in human CRC tissues. SLIT2 in CRC cells inhibited GPC1+ exosome enrichment and exosomal GPC1 expression. PLC and heparin increased GPC1+ exosome enrichment in CRC cells in a concentration-dependent manner. Hypoxia increased the enrichment of GPC1+ exosomes in CRC cells depending on HIF-2α expression. GPC1+ exosomes stimulated CRC cell proliferation and xenograft tumor growth through activation of Wnt signaling. Conclusions: GPC1+ exosome enrichment is related to PLC and heparin. Hypoxia increases the enrichment of GPC1+ exosomes in CRC cells by activating HIF-2α and downregulating SLIT2. GPC1+ exosomes further drive CRC progression by activating Wnt signaling.

Keywords

• Colorectal cancer
• Exosome
• Glypican-1
• Slit guidance ligand 2
• Hypoxia