Chemically induced neurite-like outgrowth reveals a multicellular network function in patient-derived glioblastoma cells

Silva, Bárbara da; Irving, Bronwyn K.; Polson, Euan S.; Droop, Alastair P.; Griffiths, Hollie B. S.; Mathew, Ryan; Marrison, Joanne; Williams, Courtney; Williams, Jennifer M.; Short, Susan; Scarcia, Margherita; O’Toole, Peter; Allison, Simon J.; Mavria, Georgia; Wurdak, Heiko

doi:10.1242/jcs.228452

Cited by 6 publications

(7 citation statements)

References 51 publications

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“…As expected, Calcium exchange increased upon TGF-β1 stimulation and was inhibited by LY2157299. Network formation and increase in Calcium exchange in GBM cells in vitro was also observed by da Silva et al using ROCK inhibition [9].…”

Section: Discussionsupporting

confidence: 56%

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TGF-β promotes microtube formation in glioblastoma through Thrombospondin 1

Joseph

Magaut

Storevik

et al. 2021

Preprint

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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.

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Section: Discussionsupporting

confidence: 56%

“…Calcium signal spread across cells was carried out as previously described [9]. Fluo-3-AM (Thermo Fisher Scientific, F23915) was added to the cells and incubated for 30 min in the dark at room temperature.…”

Section: Methodsmentioning

confidence: 99%

TGF-β promotes microtube formation in glioblastoma through Thrombospondin 1

Joseph

Magaut

Storevik

et al. 2021

Preprint

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“…Network formation and increase in calcium exchange in GBM cells in vitro was also observed by da Silva et al using ROCK inhibition. 19 The majority of our cell lines responded to TGF-β1 with increase in MT formation. Interestingly, we identified one cell line (GG6), which did not respond and also showed very limited invasive capacity upon TGF-β stimulation using ex vivo brain organoid co-cultures and in vivo xenografts.…”

Section: Discussionmentioning

confidence: 87%

“…Calcium signal spread across cells was carried out as previously described. 19 Fluo-3-AM intensity was determined using the ImageJ z-axis profile function. Mean fluorescence intensity was determined from 20 time lapses per condition.…”

Section: In Vitro Calcium Imagingmentioning

confidence: 99%

TGF-β promotes microtube formation in glioblastoma through thrombospondin 1

et al. 2021

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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.

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“…In some cases, ROCKi significantly decreased invasion and provided significant survival benefits in animal models of high-grade glioma [36,37]. Other times, ROCKi significantly increased invasion [38], stabilized pro-invasive tumor microtube networks, and decreased sensitivity to standard of care radiation and chemotherapy [39]. We previously demonstrated that both global activation and inhibition of mDia are effective anti-invasive and anti-tumor microtube strategies in GBM, with activation representing the superior approach [12,40].…”

Section: Introductionmentioning

confidence: 99%

The Cytoskeleton Effectors Rho-Kinase (ROCK) and Mammalian Diaphanous-Related (mDia) Formin Have Dynamic Roles in Tumor Microtube Formation in Invasive Glioblastoma Cells

Becker

Pettee

Sugrue

et al. 2022

Cells

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Glioblastoma (GBM) is a progressive and lethal brain cancer. Malignant control of actin and microtubule cytoskeletal mechanics facilitates two major GBM therapeutic resistance strategies—diffuse invasion and tumor microtube network formation. Actin and microtubule reorganization is controlled by Rho-GTPases, which exert their effects through downstream effector protein activation, including Rho-associated kinases (ROCK) 1 and 2 and mammalian diaphanous-related (mDia) formins (mDia1, 2, and 3). Precise spatial and temporal balancing of the activity between these effectors dictates cell shape, adhesion turnover, and motility. Using small molecules targeting mDia, we demonstrated that global agonism (IMM02) was superior to antagonism (SMIFH2) as anti-invasion strategies in GBM spheroids. Here, we use IDH-wild-type GBM patient-derived cell models and a novel semi-adherent in vitro system to investigate the relationship between ROCK and mDia in invasion and tumor microtube networks. IMM02-mediated mDia agonism disrupts invasion in GBM patient-derived spheroid models, in part by inducing mDia expression loss and tumor microtube network collapse. Pharmacological disruption of ROCK prevented invasive cell-body movement away from GBM spheres, yet induced ultralong, phenotypically abnormal tumor microtube formation. Simultaneously targeting mDia and ROCK did not enhance the anti-invasive/-tumor microtube effects of IMM02. Our data reveal that targeting mDia is a viable GBM anti-invasion/-tumor microtube networking strategy, while ROCK inhibition is contraindicated.

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Chemically induced neurite-like outgrowth reveals a multicellular network function in patient-derived glioblastoma cells

Cited by 6 publications

References 51 publications

TGF-β promotes microtube formation in glioblastoma through Thrombospondin 1

TGF-β promotes microtube formation in glioblastoma through Thrombospondin 1

TGF-β promotes microtube formation in glioblastoma through thrombospondin 1

The Cytoskeleton Effectors Rho-Kinase (ROCK) and Mammalian Diaphanous-Related (mDia) Formin Have Dynamic Roles in Tumor Microtube Formation in Invasive Glioblastoma Cells

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