Project Summary
In contrast to their expected cytotoxic function, macrophages can actively promote tumor cell invasion. We have previously demonstrated that non-canonical signaling via Wnt 5a is critical for this process. We now asked whether Wnt-signaling is also important for for the last step of tumor progression, the metastatic dissemination into distant tissues.
During the first funding period we could confirm that microglia, the resident macrophages of the brain, in fact enhance invasion and colonization of brain tissue by breast cancer cells. Apart from classical invasion via epithelial-mesenchymal transition, we characterized a different type of “assisted invasion” in a newly established organotypic slice coculture model, where microglia served both as active transporters and guiding rails for tumor cells with preserved epithelial features. Correspondent histological findings in human brain metastases underlined the significance of these results. Microglia-induced invasion was dependent on active Wnt signaling as it was antagonized by the Wnt-inhibitor Dickkopf-2. It was also dependent on activation of the JNK pathway. Pro-invasive microglia demonstrated altered morphology, but neither regulation of the constitutively expressed ligands Wnt 5a and b nor a detectable switch into a typical M2-phenotype as in peripheral blood-derived macrophages. However, stimulation of the TLR4 pathway by bacterial lipopolysacharide (LPS) shifted tumor-educated microglia into a classical M1-phenotype, reduced their pro-invasive function and unmasked inflammatory and Wnt signaling in gene expression arrays as the most important cascades. Under these conditions, the most strongly regulated gene was the chemokine receptor CXCR4, well-known for its role in tumor cell invasion. In summary this demonstrates that resident macrophages are essential for colonization of distant tissues and that this depends on active Wnt signaling with cross-links to inflammatory pathways.
In the second funding period, we plan to further dissect the involved Wnt cascades mediating the pro-invasive effect of different types of macrophages by use of Wnt inhibitors, modulators of the different sub-pathways as well as various reporter assays. In vitro findings will be confirmed by immunohistochemistry of human brain metastasis, coculture assays and organotypic slice cocultures using macrophages and brain tissues from different mouse models (Wnt 5a +/-, Wnt 5a -/-, Wnt 6 -/- ). The in vivo relevance of these results will be investigated in a syngeneic mouse model of brain metastasis by intracerebral inoculation of breast cancer cells and modulation of metastatic progression via local injection of Wnt-antagonists as well as coinjection of ex vivo manipulated migroglia. Last, we will characterize the links between the Wnt and the TLR pathway to further clarify the antiinvasive function of inflammatory signaling via LPS, which may provide a target for antimetastatic strategies.