The tumor microenvironment (TM), consisting of the extracellular matrix (ECM), fibroblasts, endothelial cells, and immune cells, might affect tumor invasiveness and the outcome of standard chemotherapy. This study investigated the cross talk between germ cell tumors (GCT) and surrounding TM cells (macrophages, T‐lymphocytes, endothelial cells, and fibroblasts) at the transcriptome and secretome level. Using high‐throughput approaches of three‐dimensional (3D) co‐cultured cellular aggregates, this study offers newly identified pathways to be studied with regard to sensitivity toward cisplatin‐based chemotherapy or tumor invasiveness as a consequence of the cross talk between tumor cells and TM components. Mass‐spectrometry‐based secretome analyses revealed that TM cells secreted factors involved in ECM organization, cell adhesion, angiogenesis, and regulation of insulin‐like growth factor (IGF) transport. To evaluate direct cell–cell contacts, green fluorescent protein (GFP)‐expressing GCT cells and mCherry‐expressing TM cells were co‐cultured in 3D. Afterward, cell populations were separated by flow cytometry and analyzed by RNA sequencing. Correlating the secretome with transcriptome data indicated molecular processes such as cell adhesion and components of the ECM being enriched in most cell populations. Re‐analyses of secretome data with regard to lysine‐ and proline‐hydroxylated peptides revealed a gain in proteins, such as collagens and fibronectin. Cultivation of GCT cells on collagen I/IV‐ or fibronectin‐coated plates significantly elevated adhesive and migratory capacity, while decreasing cisplatin sensitivity of GCT cells. Correspondingly, cisplatin sensitivity was significantly reduced in GCT cells under the influence of conditioned medium from fibroblasts and endothelial cells. This study sheds light on the cross talk between GCT cells and their circumjacent TM, which results in deposition of the ECM and eventually promotes a pro‐tumorigenic environment through enhanced migratory and adhesive capacity, as well as decreased cisplatin sensitivity. Hence, our observations indicate that targeting the ECM and its cellular components might be a novel therapeutic option in combination with cisplatin‐based chemotherapy for GCT patients.

Beauvericin (BEA), a mycotoxin of the enniatin family produced by various toxigenic fungi, has been attributed multiple biological activities such as anti-cancer, anti-inflammatory, and anti-microbial functions. However, effects of BEA on dendritic cells remain unknown so far. Here, we identified effects of BEA on murine granulocyte–macrophage colony-stimulating factor (GM-CSF)-cultured bone marrow derived dendritic cells (BMDCs) and the underlying molecular mechanisms. BEA potently activates BMDCs as signified by elevated IL-12 and CD86 expression. Multiplex immunoassays performed on myeloid differentiation primary response 88 (MyD88) and toll/interleukin-1 receptor (TIR) domain containing adaptor inducing interferon beta (TRIF) single or double deficient BMDCs indicate that BEA induces inflammatory cytokine and chemokine production in a MyD88/TRIF dependent manner. Furthermore, we found that BEA was not able to induce IL-12 or IFNβ production in Toll-like receptor 4 (Tlr4)-deficient BMDCs, whereas induction of these cytokines was not compromised in Tlr3/7/9 deficient BMDCs. This suggests that TLR4 might be the functional target of BEA on BMDCs. Consistently, in luciferase reporter assays BEA stimulation significantly promotes NF-κB activation in mTLR4/CD14/MD2 overexpressing but not control HEK-293 cells. RNA-sequencing analyses further confirmed that BEA induces transcriptional changes associated with the TLR4 signaling pathway. Together, these results identify TLR4 as a cellular BEA sensor and define BEA as a potent activator of BMDCs, implying that this compound can be exploited as a promising candidate structure for vaccine adjuvants or cancer immunotherapies.