Precise regulation of signaling pathways in single cells underlies tissue development, maintenance and repair in multicellular organisms, but our ability to monitor signaling dynamics in living vertebrates is currently limited. We implemented kinase translocation reporter (KTR) technology to create DREKA (“dynamic reporter of Erk activity”) zebrafish, which allow one to observe Erk activity in vivo at single cell level with high temporal resolution. DREKA zebrafish faithfully reported Erk activity after muscle cell wounding and revealed the kinetics of small compound uptake. Our results promise that kinase translocation reporters can be adapted for further applications in developmental biology, disease modeling, and in vivo pharmacology in zebrafish.

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Di-peptides derived from the human host defense peptide lactoferricin were previously described to specifically interact with the negatively charged lipid phosphatidylserine exposed by cancer cells. In this study one further derivative, namely R-DIM-P-LF11-334 is shown to exhibit even increased cancer toxicity in vitro and in vivo while non-neoplastic cells are not harmed.In liposomal model systems composed of phosphatidylserine mimicking cancerous and phosphatidylcholine mimicking non-cancerous membranes the specific interaction with the cancer marker PS was confirmed by specific induction of membrane perturbation and permeabilization in presence of the peptide. In vitro studies with cell lines of human malignant melanoma, such as A375, or primary cells of human melanoma metastases to the brain, as MUG Mel1, and non-neoplastic human dermal fibroblasts NHDF revealed high cytotoxic effect of R-DIM-P-LF11-334 on melanoma cells of A375 and MUG Mel1, whereas only minor effect on the dermal fibroblasts NHDF was observed, yielding an about 20-fold killing-specificity for A375 and MUG-Mel1. The LC50 values for melanoma A375 and MUG Mel1 were about 10 μM. Analysis of secondary structure of the peptide revealed an increase in the proportion of β-sheets exclusively in presence of the cancer mimic. Stability studies further indicated a potential adequate stability in blood or under stringent conditions. Importantly the cytotoxic effect on cancer cells was also proven in vivo in mouse xenografts of human melanoma, where peptide treatment induced strong tumor regression and in average a tumor area reduction of 85% compared to tumors of control mice without peptide treatment.

Glioblastoma (GBM) is characterized by a particularly invasive phenotype, supported by oncogenic signals from the fibroblast growth factor (FGF)/ FGF receptor (FGFR) network. However, a possible role of FGFR4 remained elusive so far. Several transcriptomic glioma datasets were analyzed. An extended panel of primary surgical specimen-derived and immortalized GBM (stem)cell models and original tumor tissues were screened for FGFR4 expression. GBM models engineered for wild-type and dominant-negative FGFR4 overexpression were investigated regarding aggressiveness and xenograft formation. Gene set enrichment analyses of FGFR4-modulated GBM models were compared to patient-derived datasets. Despite widely absent in adult brain, FGFR4 mRNA was distinctly expressed in embryonic neural stem cells and significantly upregulated in glioblastoma. Pronounced FGFR4 overexpression defined a distinct GBM patient subgroup with dismal prognosis. Expression levels of FGFR4 and its specific ligands FGF19/FGF23 correlated both in vitro and in vivo and were progressively upregulated in the vast majority of recurrent tumors. Based on overexpression/blockade experiments in respective GBM models, a central pro-oncogenic function of FGFR4 concerning viability, adhesion, migration, and clonogenicity was identified. Expression of dominant-negative FGFR4 resulted in diminished (subcutaneous) or blocked (orthotopic) GBM xenograft formation in the mouse and reduced invasiveness in zebrafish xenotransplantation models. In vitro and in vivo data consistently revealed distinct FGFR4 and integrin/extracellular matrix interactions. Accordingly, FGFR4 blockade profoundly sensitized FGFR4-overexpressing GBM models towards integrin/focal adhesion kinase inhibitors. Collectively, FGFR4 overexpression contributes to the malignant phenotype of a highly aggressive GBM subgroup and is associated with integrin-related therapeutic vulnerabilities.

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Ewing sarcoma is a pediatric bone and soft tissue cancer for which new therapies to improve disease outcome and to reduce adverse effects of current standard treatments are urgently needed. To identify new and effective drugs, phenotypic drug screening has proven to be a powerful method and a cancer model ideally suited for this approach is the larval zebrafish xenograft system. Complementing mouse xenografts, zebrafish offer high-througput screening possibilities in an intact complex vertebrate organism. Here, we generated Ewing sarcoma xenografts in zebrafish larvae and established a workflow for automated imaging of xenografts, tumor cell recognition within transplanted zebrafish and quantitative tumor size analysis over consecutive days by high-content imaging. The increased throughput of our in vivo screening setup allowed us to identify combination therapies effective against Ewing sarcoma cells. Especially, combined inhibition of MCL-1 and BCL-XL, two anti-apoptotic proteins, was highly efficient at eradicating tumor cells in our zebrafish xenograft assays with two Ewing sarcoma cell lines and with patient-derived cells. Transcriptional analysis across Ewing sarcoma cell lines and tumors revealed that MCL-1 and BCL2L1, coding for BCL-XL, are the most abundantly expressed anti-apoptotic genes, suggesting that combined MCL-1/BCL-XL inhibition might be a broadly applicable strategy for Ewing sarcoma treatment.

Models that more accurately reflect Ewing sarcoma (ES) will enable the prioritization of novel targeted agents from bench to clinic. To date, development of an ES mouse model have been unsuccessful1 and engraftment of human ES in mice is variable2. Coupled with a moral obligation to minimize the use of mammals, we have established and characterized models using patient-derived ES cultures in vitro and in larval zebrafish. The transcriptomes of ES cell lines and patient-derived ES cultures3 were analyzed and compared using total RNAseq followed by DESeq2 and Gene Set Enrichment analysis. Cells in 2D, as 3D spheroids, in combination with cells of the tumor microenvironment and in larval zebrafish (Danio rerio) were characterized. Six cell lines clustered independently from patient-derived ES cultures (n=20), reflecting differential expression of 15,709 RNAs (p<0.01). Interrogation of the differentially expressed genes using Reactome, KEGG and Gene Ontology identified up regulation of genes associated with mitotic spindle and G2/M G1/S DNA damage spindle checkpoints, homologous recombination and the cell cycle in the cell lines compared to patient-derived cultures (p<0.01). These data are consistent with the quicker doubling time (DT) and increased sensitivity of ES cell lines to chemotherapy (actinomycin D, doxorubicin, vincristine, etoposide; DT range=19-33h, EC50 range=4-34nM) compared to patient-derived cultures (DT range=55-197h, EC50>10μM, p<0.05). All patient-derived ES cultures formed spheroids of similar diameter (range 105-258µm) at 96h (p>0.05), although there was heterogeneity in spheroid weight (range 205-3639ng, p<0.05) and migration index (MI; range 30-170, p<0.05). Treatment with actinomycin D (1-10 µM) significantly increased spheroid diameter (p<0.05), whereas 4-hyperoxyifosfamide (active metabolite of ifosfamide, 7-30µM) decreased spheroid diameter (p<0.05). Doxorubicin did not significantly change the diameter of spheroids. When propagated in combination with mesenchymal stem cells there was a 2-fold increase in viable cell number (p<0.0001) compared to ES cultures alone. Patient-derived ES cultures in zebrafish were observed at the injection site (perivitelline space). Some cells were detected in circulation 3 days post injection. This is consistent with heterogeneity and the high MI of patient-derived ES cultures. The transcriptome of ES cell lines is significantly different from that of ES patient-derived cultures and is associated with increased doubling times and response to chemotherapies. This highlights the importance of developing preclinical models using patient-derived ES cultures. Patient-derived ES cultures in 2D and 3D models in vitro and in zebrafish may provide a reliable cost-effective preclinical pipeline 1Minas et al. 2017.Oncotarget,8(21):34141-34163.2Nanni et al. 2019.Sci Rep,9(1):12174 3Roundhill et al. 2021.Cell Oncol,44(5):1065-1085. Citation Format: Elizabeth A. Roundhill, Elton J. Vasconcelos, David R. Westhead, Sarah Grissenberger, Martin Distel, Susan A. Burchill. Developing human Ewing sarcoma in vitro models to prioritise new treatments. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4683.