Exosomes from cervical cancer cells facilitate pro-angiogenic endothelial reconditioning through transfer of Hedgehog-GLI signaling components

Background: Angiogenic switch is a hallmark feature of transition from low-grade to high-grade cervical intraepithelial neoplasia (CIN) in cervical cancer progression. Therefore, early events leading to locally-advanced cervical metastatic lesions demand a greater understanding of the underlying mechanisms. Recent leads indicate the role of tumor-derived exosomes in altering the functions of endothelial cells in cervical cancer, which needs further investigation. Methods: Exosomes isolated from cervical cancer cell lines were assessed for their angiogenic effect on the human umbilical vein endothelial cells (HUVEC) using tube formation and wound healing assay. The exosomal uptake by HUVEC cells was monitored using PKH-67 labelling followed by fluorescence microscopy. Alterations in Hh-GLI signaling components, PTCH1 and GLI1, in HUVEC were measured by immunoblotting. Changes in angiogenesis-related transcripts of vascular endothelial growth factor VEGF-A, VEGF-B, VEGFR2 and angiopoietin-1, angiopoietin-2, osteopontin were measured in exosome-treated HUVEC and in the exosomal RNA by RT-PCR. Results: Enhanced tube formation, with an increased number of nodes and branching was observed in HUVEC's treated with exosomes derived from different cervical cancer cell lines. HPV-positive (SiHa and HeLa) cells' exosomes were more angiogenic. Exosome-treated HUVEC showed increased migration rate. PKH-67 labelled exosomes were found internalized in HUVEC. A high level of PTCH1 protein was detected in the exosome-treated endothelial cells. Subsequent RT-PCR analysis showed increased transcripts of Hh-GLI downstream target genes VEGF-A, VEGFR2, angiopoietin-2, and decreased expression of VEGF-B, and angiopoietin-1, suggestive of active Hh-GLI signaling. These effects were more pronounced in HUVEC's treated with exosomes of HPV-positive cells. However, these effects were independent of tumor-derived VEGF-A as exosomal cargo lacked VEGF-A transcripts or proteins. Conclusion: Overall, the data showed cervical cancer exosomes promote pro-angiogenic response in endothelial cells via upregulation of Hh-GLI signaling and modulate downstream angiogenesis-related target genes. The study provides a novel exosome-mediated mechanism potentially favoring cervical angiogenesis and thus identifies the exosomes as potential pharmacological targets against locally-advanced metastatic cervical lesions.