DLL4/Notch3/WNT5B axis mediates bidirectional prometastatic crosstalk between melanoma and lymphatic endothelial cells
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
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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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 3

Melanoma cell–derived WNT5B contributes to the functional changes in LECs.

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Melanoma cell–derived WNT5B contributes to the functional changes in LEC...
(A) Quantification of the relative branch length of a tube formation assay with LECs cultured in conditioned media (CM) from monotypic LECs, WM852 cells, or LEC+WM852 coculture for 24 hours and analyzed by a 16-hour tube formation assay. Experiment was performed 2 independent times. (B) qRT-PCR of WNT5B mRNA levels in the indicated monotypic or LEC-cocultured melanoma cell lines from 3 independent experiments. (C) Immunofluorescence images of monotypic WM852 and WM852+LEC cultures labeled with an antibody against WNT5B. Small inserts identify the GFP-expressing melanoma cells. Nuclei were counterstained with Hoechst 33342. Representative images from 3 independent experiments are shown in the left panel, and quantification of WNT5B relative signal intensity from at least 100 cells/experiment/condition is shown in the right panel. Scale bar: 50 μm. (D) Quantification of the spheroid-sprouting assay of monotypic LECs and LECs cocultured with the indicated melanoma cells (LEC*). Prior to the coculture, melanoma cells were pretreated with control (siCtrl) or WNT5B-targeting siRNAs (siWNT5B) for 24 hours. Graph shows the mean of 3 independent experiments, each with at least 4 spheroids/condition quantified. WM852 and WM165 melanoma cell–LEC cocultures were performed at the same time and therefore the same LEC control spheroids were used for analysis. (E) Quantification of the tube formation assay with LECs cultured and treated as in D (n = 3). WM165 and WM793 melanoma cell–LEC cocultures were performed at the same time and therefore the same LEC control samples were used for analysis. (F) The electrical cell impedance assay with LECs cultured and treated as in D. Data are presented as mean ± SD for each sample. Representative experiment of 2 experiments is shown. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by 1-way ANOVA followed by Tukey’s multiple-comparison test (A, B, and D), unpaired, 2-tailed t test (B and C), or AUC analysis followed by 1-way ANOVA with Tukey’s multiple-comparison test (F).