Deciphering metastatic signaling cascades in small cell lung cancer

Down to the present-day small cell lung cancer (SCLC) remains one of the deadliest cancer entities. Due to its early metastasis formation and wide therapy resistance, SCLC occupies a special place within cancer research. Given the limited treatment options, particularly in advanced stages, it is vital to examine how certain signaling cascades contribute to the aggressive phenotype, immune evasion and high migration potential of SCLC. My dissertation mainly focuses on the CD29-dependent signaling pathway and MHC-I dependent regulation of the tumor micro-milieu and discusses their involvement in processes of metastasis formation and immune evasion mechanisms of SCLC. In our initial screening, we observed elevated CD29 expression in liver metastases in both human and murine samples. Therefore, CD29 Knockout (KO) clones were generated using CRISPR to test the characteristics on functional and cellular level in comparison to the original liver metastasis-derived cell line. To evaluate migration behavior, the different clones and the wild type were subjected to a series of cell migration and cell–cell interaction tests (Scratch Assay, Boyden Chamber). To elucidate the functional role of CD29 in metastasis formation, the CD29 KO clones were also tested in an in vivo setting. In vivo, tumors were induced via orthotopic and intravenous injection of the respective tumor cells into immunocompetent and immune deficient mouse models. A fluorescence-activated cell sorting (FACS) readout of tumor and metastasis tissue provided insights into immune cell involvement and tumor expression markers. Additionally, immunohistochemistry (IHC) was applied to assess tumor cells and desmoplastic reactions ex vivo. In vitro migration assays demonstrated that CD29 KO abrogates fibronectin- and angiopoietin-2 (ANG-2) stimulated migration. In vivo experiments further revealed that, in contrast to the wild type SCLC cells, CD29 KO clones did not form liver metastasis, which was confirmed by flow cytometry and IHC. Altogether, CD29 is a potent driver of metastasis in SCLC, which can be abrogated by knockout. Furthermore, my dissertation explores MHC-I and its role in SCLC immune evasion and metastasis formation. In a patient sample screen, we found a downregulation of MHC-I in SCLC liver metastases. To investigate the impact of MHC-I loss on tumor-immune interactions, MHC-I-deficient clones (MHC-I KO) were generated by targeted knockout of the β2 microglobulin (B2M) gene. The MHC-I KO was tested in vivo by orthotopic and intravenous injection and presented a more aggressive, fast-growing phenotype. In MHC-I KO tumors, we found an enhanced reaction of the innate immune system in the liver metastases and a decreased T cell answer in the pleural effusion. A comparison between primary tumors and metastases revealed increased phosphorylation of ERBB2 in SCLC metastases. Initial in vitro experiments with an ERBB2 inhibitor demonstrated suppression of key downstream signaling pathways and induction of immune activating signals via TBK1. Notably, in our autochthonous SCLC mouse model, combined blockade of PD-1 and ERBB2 resulted in a significant extension of both overall and progression-free survival. Overall, this study states the importance of the CD29-ANG-2 signaling axis in SCLC metastasis formation and portrays the immune evading effects caused by MHC-I loss. Based on these findings, potential therapeutic approaches are discussed and tested in preclinical setting.