WNT5A induces release of exosomes containing pro-angiogenic and immunosuppressive factors from malignant melanoma cells.

BACKGROUND: Wnt proteins are important for developmental processes and certain diseases. WNT5A is a non-canonical Wnt protein that previously has been shown to play a role in the progression of malignant melanoma. High expression of WNT5A in melanoma tumors correlates to formation of distant metastasis and poor prognosis. This has partly been described by the findings that WNT5A expression in melanoma cell lines increases migration and invasion. METHODS: Malignant melanoma cell lines were treated with rWNT5A or WNT5A siRNA, and mRNA versus protein levels of soluble mediators were measured using RT-PCR, cytokine bead array and ELISA. The induced signaling pathways were analyzed using inhibitors, Rho-GTPase pull down assays and western blot. Ultracentrifugation and electron microscopy was used to analyze microvesicles. Gene expression microarray data obtained from primary malignant melanomas was used to verify our data. RESULTS: We show that WNT5A signaling induces a Ca2+-dependent release of exosomes containing the immunomodulatory and pro-angiogenic proteins IL-6, VEGF and MMP2 in melanoma cells. The process was independent of the transcriptional machinery and depletion of WNT5A reduced the levels of the exosome-derived proteins. The WNT5A induced exosomal secretion was neither affected by Tetanus toxin nor Brefeldin A, but was blocked by the calcium chelator Bapta, inhibited by a dominant negative version of the small Rho-GTPase Cdc42 and was accompanied by cytoskeletal reorganization. Co-cultures of melanoma/endothelial cells showed that depletion of WNT5A in melanoma cells decreased endothelial cell branching, while stimulation of endothelial cells with isolated rWNT5A-induced melanoma exosomes increased endothelial cell branching in vitro. Finally, gene expression data analysis of primary malignant melanomas revealed a correlation between WNT5A expression and the angiogenesis marker ESAM. CONCLUSIONS: These data indicate that WNT5A has a broader function on tumor progression and metastatic spread than previously known; by inducing exosome-release of immunomodulatory and pro-angiogenic factors that enhance the immunosuppressive and angiogenic capacity of the tumors thus rendering them more aggressive and more prone to metastasize.

Longitudinal clearance of seasonal influenza A viral RNA measured by real-time polymerase chain reaction in patients identified at a hospital emergency department.

To study influenza virus shedding during acute infection, viral load was longitudinally measured by quantitative PCR in nasal flocked swabs from patients with seasonal H3N2 influenza at a Swedish emergency department, including both hospitalized patients and outpatients. Influenza A was detected in 65/184 patients. Sampling was repeated every 3-4 days in 45 patients, with the aim of continuing sampling until day 12 after disease onset. Home visits were offered. Antibodies were measured on paired sera in 95/184 patients. Fifty percent of the patients remained polymerase chain reaction (PCR)-positive 8 days after disease onset in a Kaplan-Meier survival curve. The longest observed duration of viral shedding was 12 days. The average viral load was initially low, peaked on days 2-3 of disease and then declined. Viral decline results remained similar when all 15 (25%) oseltamivir-treated patients were excluded. Significant antibody titre changes were seen in all the 35 PCR verified cases with available paired sera and in 8 of the 58 patients with negative PCR tests on acute phase nasal samples. In conclusion, quantitative PCR testing indicated the presence of influenza virus for up to 12 days, which could have implications for disease transmission and infection control.