Human Lymphatic Endothelial Cells

ESCC Cell-Derived Exosomes Promoted the Migration, Invasion, and Lymphangiogenesis of HLECs

Esophageal squamous carcinoma (ESCC) is a common malignancy with late-stage diagnosis and frequent recurrence. Lymph node metastasis (LNM) is a typical sign of ESCC treatment failure. Lymphangiogenesis is associated with LNM in ESCC, but the underlying mechanisms remain unclear. In this study, Yao’s team analyzed the expression of circ_0026611 in ESCC cells and exosomes by RT-qPCR, and explored the effect and mechanism of circ_0026611 on lymphangiogenesis.

Exosome labeling and tracking assay showed that ESCC exosomes could be absorbed by human lymphatic endothelial cells (HLECs). After 6 h of culture, PKH67-labeled exosomes from ESCC cells were internalized by HLECs (Fig. 1A). The expression of circ_0026611 in HLECs was significantly upregulated after absorbing exosomes from KYSE30. In addition, after HLECs absorbed exosomes from EC109, circ_0026611 was more significantly upregulated (Fig. 1B). Migration and invasion of HLECs after KYSE30-Exos treatment were significantly enhanced, while the effects were more significant after EC109-Exos treatment (Fig. 1C, D). Tube formation assay showed that KYSE30-Exos treatment promoted the tube formation ability of HLECs, and EC109-Exos had a stronger effect (Fig. 1E). The expression of lymphangiogenesis-related markers (VEGF-C, VEGF-D, VEGFR3, LYVE-1, podoplanin, PROX1) was significantly upregulated in HLECs after KYSE30-Exos treatment, while the effects were more significant after EC109-Exos treatment (Fig. 1F, G). Therefore, exosomes secreted by ESCC cells promote lymphangiogenesis.

BoTA Influences Lymphangiogenesis by Significantly Decreasing the Maximum Tube Length of Cultured LECs

Botulinum toxin A (BoTA) is a neurotoxin with a wide range of medical applications, primarily affecting muscles. Its effects on other systems, including blood vessels, have been studied, but its impact on the lymphatic vascular system remains unknown. Vasella et al. evaluated the effects of BoTA on human lymphatic endothelial cells (LECs) using in vitro culture systems and various analytical techniques.

To determine the direct influence of BoTA on LECs, they compared LEC proliferation after applying different BoTA dosages, and different controls (starvation medium, full medium, VEGF-A medium in starvation medium, and PBS). LEC proliferation was significantly higher in full medium than in any other medium, while PBS was the worst. However, BoTA dosages, independent of its strength, did not show significant effect on LEC proliferation and hence showed no direct effect on LECs (Fig. 2). The cord/tube formation assay serves as a model to understand the re-organization phase during lymphangiogenesis. Maximum tube length was significantly increased in negative (starvation medium) and positive (VEGF-A) control. In all other conditions, FM and both BoTA groups, tube length was significantly shorter. The average tube length was significantly increased in the VEGF-A medium. Non-significant increase was also observed in FM and BoTA medium. (Fig. 3a and b).