Pilocytic astrocytomas (PAs) as well as other pediatric low-grade gliomas (pLGGs) exhibit genetic events leading to aberrant activation of the MAPK pathway. The most common alterations are KIAA1549:BRAF fusions, BRAF V600E and NF1 mutations. Novel drugs targeting the MAPK pathway (MAPKi) are prime candidates for the treatment of these singlepathway diseases. We aimed to develop an assay suitable for pre-clinical testing of MAPKi in pLGGs with the goal to identify novel MAPK pathway suppressing synergistic drug combinations.A reporter plasmid (pDIPZ) with a MAPK-responsive ELK-1-binding element driving the expression of destabilized firefly luciferase was generated and packaged using a lentiviral vector system. Pediatric glioma cell lines with a BRAF fusion (DKFZ-BT66) and a BRAF V600E mutation (BT-40) background, respectively, were stably transfected. Modulation of the MAPK pathway activity by MAPKi was measured using the luciferase reporter and validated by detection of phosphorylated protein levels. A screen of a MAPKi library was performed and synergy of selected combinations was calculated.Screening of a MAPKi library revealed MEK inhibitors as the class inhibiting the pathway with the lowest IC50s, followed by ERK and next-generation RAF inhibitors. Combination treatments with different MAPKi classes showed synergistic effects in BRAF fusion as well as BRAF V600E mutation backgrounds.We here report a novel reporter assay for medium-to high-throughput pre-clinical drug testing in pLGG cell lines. The assay confirmed MEK, ERK and next-generation RAF inhibitors as potential treatment approaches for KIAA1549:BRAF and BRAF V600E mutated pLGGs. In addition, the assay revealed that combination treatments synergistically suppressed MAPK pathway activity.
(1) Background: We describe a 4D cell culture platform with which we tried to detect and to characterize migration dynamics of single hematopoietic stem cells in polymer film microcavity arrays integrated into a microtiter plate. (2) Methods: The system was set up with CD34-expressing KG-1a cells as a surrogate for hematopoietic stem cells. We then evaluated the system as an artificial hematopoietic stem cell niche model comprised of a co-culture of human hematopoietic stem cells from cord blood (cord blood CD34+ cells, hHSCs) and human mesenchymal stromal cells (hMSCs) from bone marrow over a period of 21 days. We used a software-based cell detection method to count single hematopoietic stem cells (HSCs) in microcavities. (3) Results: It was possible to detect single HSCs and their migration behavior within single microcavities. The HSCs displayed a pronounced migration behavior with one population of CD34-expressing cells located at the bottom of the microcavities and one population located in the middle of the microcavities at day 14. However, at day 21 the two populations seemed to unite again so that no clear distinction between the two was possible anymore. (4) Conclusions: Single cell migration detection was possible but microscopy and flow cytometry delivered non-uniform data sets. Further optimization is currently being developed.
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