|dc.description.abstracteng||Cancer development is frequently linked to signaling pathways that are required for normal embryonic patterning. One of the pathways important in patterning and growth of the embryo is the Hedgehog (Hh) signaling cascade. Activation or inappropriate maintenance of this pathway in the adult organism is frequently caused by mutations in the Hh receptor Patched (Ptch) or overexpression of Hh and can result in the development of a variety of tumors. Whereas the pathway’s function in tissues such as skin, brain, lung, muscle, bladder, breast and prostate is well studied, the knowledge about its role and tumorigenic potential in cells composing the immune system is very limited. The original goal of this thesis was to elucidate the function of Ptch in macrophages. For this purpose Ptchflox/flox mice were crossed with LysMcre mice. The latter mice express a cre recombinase specifically in lysozyme M (LysM) positive cells that are mainly macrophages, granulocytes and dendritic cells (DC), which are derived from the myeloid lineage.
Surprisingly, the Ptch mutation in LysM positive cells resulted in multiple tumors arising from the wall of the stomach or the intestine. In the tumors Hh signaling was activated. Furthermore the histology, localization, responsiveness to imatinib, and molecular analysis were suggestive of Gastrointestinal Stromal Tumors (GIST). Because human GIST are considered to arise from KIT- or PDGFRA-expressing cells of the smooth muscle layer of the GI tract, this observation was inconsistent with the Ptch mutation in the myeloid lineage. To resolve this discrepancy, a lineage tracing experiment was performed. The data showed that the tumors indeed arose from LysM-expressing cells. In addition, these cells were Kit-negative and sometimes expressed Pdgfrα. Similar relationships of Kit and Pdgfrα expression were found in the GIST-like tumors, which were all negative for Kit, but highly expressed Pdgfrα. Since HH- and PDGFRA, but not KIT-signaling pathways cooperated in oncogenic transformation, the data suggest that the GIST-like tumors in Ptch mutant mice developed due to the cooperativity between Hh and Pdgfrα pathways from Kit-negative cells in the intestine.
In addition, the function of Ptch-deficient macrophages was analyzed. First, the response to inflammatory stimuli was investigated. For this purpose, bone-marrow derived macrophages (BMDM) were isolated from Ptchflox/floxLysMcre+/- animals and were challenged with lipopolysaccharide (LPS) or bacterial lipoprotein (BLP). The preliminary analyses showed that Ptch was important for IL-6 expression and macrophage proliferation after BLP stimulation.
Second, in order to study the role of Ptch-deficient macrophages (and other myeloid cells) in tumor immune surveillance, tumors (other than GIST) were induced in Ptchflox/floxLysMcre+/- mice. In a first approach, syngeneic melanoma cells were injected i.p. or i.v. into Ptchflox/floxLysMcre+/- and respective control mice. The analysis revealed that the Ptch-deficiency had no impact on primary or metastatic tumor growth. Similar observations were made after i.p. injection of the slow-growing ovarian carcinoma cell line ID8-LUC and monitoring of tumor growth by chemoluminescence. Finally, Ptchflox/floxLysMcre+/- and control mice were subjected to the two-stage chemical carcinogenesis DMBA/TPA protocol, which results in the development of papilloma and melanoma-like nevi. Whereas papilloma growth was not affected by the Ptch mutation, the nevi growth was significantly enhanced in Ptchflox/floxLysMcre+/- animals which were associated with a stronger infiltration with skin macrophages. Taken together, these results suggest that Ptch-deficiency in LysM-expressing cells such as macrophages modulates inflammatory responses and may promote growth of melanoma.||de