Background Microtubes (MTs), cytoplasmic extensions of glioma cells, are important cell communication structures promoting invasion and treatment resistance through network formation. MTs are abundant in chemoresistant gliomas, in particular glioblastomas (GBMs), while they are uncommon in chemosensitive IDH-mutant and 1p/19q co-deleted oligodendrogliomas. The aim of this study was to identify potential signaling pathways involved in MT formation. Methods Bioinformatics analysis of TCGA was performed to analyze differences between GBM and oligodendroglioma. Patient-derived GBM stem cell lines were used to investigate microtube formation under TGF-βstimulation and inhibition in vitro and in vivo in an orthotopic xenograft model. RNA sequencing and proteomics were performed to detect commonalities and differences between GBM cell lines stimulated with TGF-β Results Analysis of TCGA data showed that the TGF-β pathway is highly activated in GBMs compared to oligodendroglial tumors. We demonstrated that TGF-β1 stimulation of GBM cell lines promotes enhanced MT formation and communication via Calcium signaling. Inhibition of the TGF-β pathway significantly reduced MT formation and its associated invasion in vitro and in vivo. Downstream of TGF-β, we identified thrombospondin 1 (TSP1) as a potential mediator of MT formation in GBM through SMAD activation. TSP1 was upregulated upon TGF- β stimulation and enhanced MT formation, which was inhibited by TSP1 shRNAs in vitro and in vivo. Conclusion TGF-β and its downstream mediator TSP1 are important mediators of the MT network in GBM and blocking this pathway could potentially help to break the complex MT driven invasion/ resistance network.
Microtubes (MTs), cytoplasmic extensions of glioma cells, are important cell communication structures promoting invasion and treatment resistance through network formation. MTs are abundant in chemoresistant gliomas, in particular glioblastomas (GBMs), while they are uncommon in chemosensitive IDH-mutant and 1p/19q co-deleted oligodendrogliomas. To identify potential signaling pathways involved in microtube formation we performed a bioinformatics analysis of TCGA data showing that the TGF-beta pathway is highly activated in GBMs compared to oligodendroglial tumors. In particular we observed that signaling pathways involved in extracellular matrix organization are differentially expressed between these tumor entities. Using patient-derived GBM stem cell lines, we demonstrated that TGF-beta 1 stimulation promotes enhanced MT formation and communication via Calcium signaling. Inhibition of the TGF-beta pathway significantly reduced MT formation and its associated invasion in vitro and in vivo. Downstream of TGF-beta, we identified thrombospondin 1 (TSP1) as a potential mediator of MT formation in GBM through SMAD activation. TSP1 was upregulated upon TGF-beta stimulation and enhanced microtube formation, which was inhibited by TSP1 shRNAs in vitro and in vivo. In conclusion, TGF-beta and its downstream mediator TSP1 are important mediators of the microtube network in GBM and blocking this pathway could potentially help to break the complex MT driven invasion/ resistance network.
Microtubes (MTs) are cytoplasmic extensions of glioma cells serving as important cell communication structures while also promoting invasion and treatment resistance through network formation. MTs are abundant in chemoresistant gliomas, in particular glioblastomas, while they are uncommon in chemosensitive IDH mutated and 1p/19q co-deleted oligodendrogliomas. By performing a bioinformatics analysis on data from The Cancer Genome Atlas (TCGA) we identified the TGF-b pathway as being distinctly upregulated in glioblastomas compared to oligodendrogliomas, making this a signaling pathway potentially involved in MT formation. Based on patient-derived GBM stem cell line models we demonstrated that stimulation of TGF-b increased MT formation, while inhibition of TGF-b reduced MT formation. MT formation was verified by expression of GAP43 and nestin, which have previously been shown to be important structural proteins of MTs. Interestingly, we also observed a responder/non-responder relationship between GBM cell lines P3 and GG16/ GG6 regarding MT formation upon TGF-b stimulation. To determine downstream signaling mediators of the TGF-b pathway crucial for MT formation, we subsequently performed RNA sequencing of these cell lines. From the 34 initial candidates common to responders, but absent in non-responders, only 3 genes were left after filtering through TCGA data and in vivo RNA sequencing data of a GBM xenograft model derived from P3. Thrombospondin 1 (TSP1) emerged as the most interesting candidate as we have previously shown that transcription of this gene is activated by TGF-b/SMAD signaling and TSP1 also promotes invasiveness of GBM. TSP1 was upregulated by TGFB1 stimulation in responder cells and promoted MT formation. Transcriptional activation of TSP1 was absent in the non-responder cell line GG6 and could be reversed in the responder cell line P3 by TSP1 shRNAs in vitro and in vivo. Thus, TSP1 was experimentally verified as an important mediator of microtube formation downstream of TGF-b signaling.
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