TGF-β promotes microtube formation in glioblastoma through thrombospondin 1

Abstract

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.

Refining the Role of Pyruvate Dehydrogenase Kinases in Glioblastoma Development

“…Cell morphology analysis showed that PDHK2 KO and DCA-treated cells increased in size. DCA-treated cells were also found to be more elongated, as shown by an increase in aspect ratio and form factor parameters ( Figure 2 F–H), correlating with a higher number of microtubes ( Figure 2 F, lower panels), membrane structures that have been described previously [ 24 ]. Finally, the mitochondrial network was analyzed based on anti-Tom20 staining and no difference was observed in the mitochondrial mass between conditions, but the mitochondrial networks appeared more fragmented in PDHK2 KO and DCA-treated cells ( Figure 2 I), indicating mitochondrial dysfunction.…”

“…To determine the role of PDHK1, PDHK2 and PDH phosphorylation in GB development ( Figure 2 A), we used a stem-like cell model (P3 cells), as previously characterized [ 16 , 24 ]. P3 cells were transduced with CRISPR-Cas9 constructs against either PDHK1 or PDHK2 gene, and protein depletion was confirmed by Western blot analysis at 21% and 0.1% O 2 ( Figure 2 B).…”

Tumor Cell Infiltration into the Brain in Glioblastoma: From Mechanisms to Clinical Perspectives

“…Overactivation of Ephrin pathway elements, especially EphA2 and EphA3, has been associated with invasiveness and poor patient outcomes [ 34 , 68 ]. It was recently shown that TGFβ is associated with microtube formation and invasion in glioma cells through SMAD and Tsp1 signaling, as microtube formation is one of the drivers of invasion [ 108 ]. The Rho family of GTPases from the Ras superfamily, Rho-A, Rac1, and Cdc42, are responsible for the spatial regulation of glioblastoma invasion.…”

Glioblastoma Microenvironment: From an Inviolable Defense to a Therapeutic Chance

“…Of note, it has been demonstrated that GBM cells can manipulate the microenvironment surrounding themselves developing a niche sustaining tumor growth and development (14). This process involves immune cells, astrocytes, glial cells, neurons, extra-cellular matrix (ECM), vascular cells, and other cell types (15)(16)(17)(18)(19)(20). There are several ways by which GBM can communicate with the surrounding tissue.…”

“…GBM cells can also promote the creation of gap junction and cytoplasmatic connections (15)(16)(17)(18)(19)(20). Finally, the secretion of microvesicles, extracellular vesicles, and exosomes can promote communication with also very distant cells or tissues (15)(16)(17)(18)(19)(20).…”