DLL4/Notch3/WNT5B axis mediates bidirectional prometastatic crosstalk between melanoma and lymphatic endothelial cells
Sanni Alve, … , Kari Vaahtomeri, Päivi M. Ojala
Sanni Alve, … , Kari Vaahtomeri, Päivi M. Ojala
Published November 16, 2023
Citation Information: JCI Insight. 2024;9(1):e171821. https://doi.org/10.1172/jci.insight.171821.
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Research Article Cell biology Vascular biology

DLL4/Notch3/WNT5B axis mediates bidirectional prometastatic crosstalk between melanoma and lymphatic endothelial cells

Abstract

Despite strong indications that interactions between melanoma and lymphatic vessels actively promote melanoma progression, the molecular mechanisms are not yet completely understood. To characterize molecular factors of this crosstalk, we established human primary lymphatic endothelial cell (LEC) cocultures with human melanoma cell lines. Here, we show that coculture with melanoma cells induced transcriptomic changes in LECs and led to multiple changes in their function. WNT5B, a paracrine signaling molecule upregulated in melanoma cells upon LEC interaction, was found to contribute to the functional changes in LECs. Moreover, WNT5B transcription was regulated by Notch3 in melanoma cells following the coculture with LECs, and Notch3 and WNT5B were coexpressed in melanoma patient primary tumor and metastasis samples. Moreover, melanoma cells derived from LEC coculture escaped efficiently from the primary site to the proximal tumor-draining lymph nodes, which was impaired upon WNT5B depletion. This supported the role of WNT5B in promoting the metastatic potential of melanoma cells through its effects on LECs. Finally, DLL4, a Notch ligand expressed in LECs, was identified as an upstream inducer of the Notch3/WNT5B axis in melanoma. This study elucidated WNT5B as a key molecular factor mediating bidirectional crosstalk between melanoma cells and lymphatic endothelium and promoting melanoma metastasis.

Authors

Sanni Alve, Silvia Gramolelli, Joonas Jukonen, Susanna Juteau, Anne Pink, Atte A. Manninen, Satu Hänninen, Elisa Monto, Madeleine H. Lackman, Olli Carpén, Pipsa Saharinen, Sinem Karaman, Kari Vaahtomeri, Päivi M. Ojala

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Figure 1

Melanoma cells induce functional changes in LECs.

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Melanoma cells induce functional changes in LECs. (A) Schematic of the w...
(A) Schematic of the workflow for the monotypic and coculture cell models. Representative immunofluorescence images are shown, with green representing the GFP-expressing melanoma cells and red the LECs labeled with anti-CD31. Nuclei were counterstained with Hoechst 33342 (blue). Figure generated by BioRender.com. (B) Spheroid-sprouting assay of LECs cultured as a monotypic control culture (LEC) or as a melanoma cell coculture (LEC* followed by name of the utilized melanoma cell line) for 2 days before cell separation by FACS. Representative images of spheroids after 4 days in fibrin are shown. Quantification of sprouting area from 3 independent experiments of at least 3 spheroids per condition is shown in the right panel. (C) Tube formation assay of LECs and LECs* cultured and sorted as in A and seeded on Cultrex for 16 hours. Representative images from 3 independent experiments are shown on the left panel and quantification of branch length on the right panel. (D) Monotypic control LECs and LECs* originating from a coculture with WM852 melanoma cell line were analyzed by an electrical cell impedance assay after 2 days of culture. A representative assay of 2 independent experiments is shown. Thicker lines indicate mean values and thinner lines ±SD. (E) Immunofluorescence images of the indicated proteins of monotypic LECs or LECs* originating from a coculture with the WM852 melanoma cell line. Nuclei were counterstained with Hoechst 33342. Representative images from 3 independent experiments are shown. Scale bar: 50 μm. Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ****P < 0.0001 by 1-way ANOVA followed by Tukey’s multiple-comparison test (B, C, and E) or AUC analysis followed by unpaired, 2-tailed t test (D).