A 3D Spheroid Model for Glioblastoma

Guyon, Joris; Andrique, Laëtitia; Pujol, Nadège; Røsland, Gro Vatne; Recher, Gaëlle; Bikfalvi, Andréas; Daubon, Thomas

doi:10.3791/60998

Cited by 16 publications

(14 citation statements)

References 11 publications

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“…Images of CSCs cultured in individual chambers were acquired from Day 0 to Day 4 with an inverted microscope (Nikon). The surface area of each CSC spheroid was measured using the Fiji Macro image analysis program [ 35 ]. For CSCs invasion assay, spheroids were mixed with Type I Collagen (Corning, NY, USA) and incubated separately in each well plate in the presence or absence of the PC inhibitors.…”

Section: Methodsmentioning

confidence: 99%

“…For CSCs invasion assay, spheroids were mixed with Type I Collagen (Corning, NY, USA) and incubated separately in each well plate in the presence or absence of the PC inhibitors. All the images were captured with the same setting parameters and invasion of collagen type I was measured by the deduction of the total area from the central area, using the Fiji Macro analysis program [ 35 ].…”

Section: Methodsmentioning

confidence: 99%

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Role of Furin in Colon Cancer Stem Cells Malignant Phenotype and Expression of LGR5 and NANOG in KRAS and BRAF-Mutated Colon Tumors

Descarpentrie

Araúzo-Bravo

et al. 2022

Cancers

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Proprotein convertases or PCs are known to regulate the malignant phenotype of colon cancer cells by different mechanisms, but their effects on cancer stem cells (CSCs) have been less widely investigated. Here, we report that PCs expression is altered in colon CSCs, and the inhibition of their activity reduced colon CSCs growth, survival, and invasion in three-dimensional spheroid cultures. In vivo, repression of PCs activity by the general PC inhibitors α1-PDX, Spn4A, or decanoyl-RVKR-chloromethylketone (CMK) significantly reduced tumor expression levels of the stem cell markers LGR5 and NANOG that are associated with reduced tumor xenografts. Further analysis revealed that reduced tumor growth mediated by specific silencing of the convertase Furin in KRAS or BRAF mutated-induced colon tumors was associated with reduced expression of LGR5 and NANOG compared to wild-type KRAS and BRAF tumors. Analysis of various calcium regulator molecules revealed that while the calcium-transporting ATPase 4 (ATP2B4) is downregulated in all the Furin-silenced colon cancer cells, the Ca2+-mobilizing P2Y receptors, was specifically repressed in BRAF mutated cells and ORAI1 and CACNA1H in KRAS mutated cells. Taken together, our findings indicate that PCs play an important role in the malignant phenotype of colon CSCs and stem cell markers’ expression and highlight PCs repression, particularly of Furin, to target colon tumors with KRAS or BRAF mutation.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Role of Furin in Colon Cancer Stem Cells Malignant Phenotype and Expression of LGR5 and NANOG in KRAS and BRAF-Mutated Colon Tumors

Descarpentrie

Araúzo-Bravo

et al. 2022

Cancers

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“…All the protocols used for spheroid assays were previously described by Guyon et al [ 23 ]. Briefly, 10 4 cells per well were seeded in cell medium supplemented with 0.4% methylcellulose in a 96-well round-bottomed plate to obtain a single spheroid after 3 days of incubation at 37 °C, 5% CO 2.…”

Section: Methodsmentioning

confidence: 99%

Refining the Role of Pyruvate Dehydrogenase Kinases in Glioblastoma Development

Larrieu

Storevik

Guyon

et al. 2022

Cancers

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Glioblastoma (GB) are the most frequent brain cancers. Aggressive growth and limited treatment options induce a median survival of 12–15 months. In addition to highly proliferative and invasive properties, GB cells show cancer-associated metabolic characteristics such as increased aerobic glycolysis. Pyruvate dehydrogenase (PDH) is a key enzyme complex at the crossroads between lactic fermentation and oxidative pathways, finely regulated by PDH kinases (PDHKs). PDHKs are often overexpressed in cancer cells to facilitate high glycolytic flux. We hypothesized that targeting PDHKs, by disturbing cancer metabolic homeostasis, would alter GB progression and render cells vulnerable to additional cancer treatment. Using patient databases, distinct expression patterns of PDHK1 and PDHK2 in GB tissues were obvious. To disturb protumoral glycolysis, we modulated PDH activity through the genetic or pharmacological inhibition of PDHK in patient-derived stem-like spheroids. Striking effects of PDHKs inhibition using dichloroacetate were observed in vitro on cell morphology and metabolism, resulting in increased intracellular ROS levels and decreased proliferation and invasion. In vivo findings confirmed a reduction in tumor size and better survival of mice implanted with PDHK1 and PDHK2 knockout cells. Adding a radiotherapeutic protocol further resulted in a reduction in tumor size and improved mouse survival in our model.

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“…Cell migration was assessed using spheroid-on-Matrigel assay, as described (Guyon et al 2020). Briefly, cells were counted as described (El-Habr et al 2017), and cell spheroids generated by seeding 2500 cells/100μL 0.4% methylcellulose (Sigma) in a U-bottom well (96 wells plate, Falcon).…”

Section: Methodsmentioning

confidence: 99%

“…Cell invasion was assessed using spheroid-in-collagen assay (Guyon et al 2020) and Matrigel-coated transwells (Renault-Mihara et al 2006). Spheroids generated as described above, were seeded in collagen (1mg/mL culture media, Corning), and cell invasion assessed 24 hours later.…”

Section: Methodsmentioning

confidence: 99%

Human glioblastoma cell motility depends on the activity of the cysteine metabolism enzyme 3-Mercaptopyruvate sulfurtransferase

Saurty-Seerunghen

Daubon

Bellenger

et al. 2022

Preprint

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Cancer cells in similar functional states are found in all glioblastoma, despite the genomic heterogeneity observed between and within these brain tumors. Metabolism being downstream of all signaling pathways regulating cell behaviors, we looked for metabolic weaknesses in link with motility, a key functional state for glioblastoma aggressiveness. A signature-driven data reduction approach highlighted motile cells present in thirty tumors from four independent single-cell transcriptomic datasets. Analyses integrating trajectory modeling disclosed, as characteristic of motile cells, enhanced oxidative stress coupled with mobilization of the cysteine metabolism enzyme 3-Mercaptopyruvate sulfurtransferase (MPST). The soundness of this prediction was verified using migration and invasion assays with patient-derived cells and tissue organoids. Pharmacological and genetic manipulations showed that enhanced ROS production and MPST activity are required for glioblastoma cell motility. Biochemical assays indicated that MPST acts by protecting protein cysteine residues from dismal hyperoxidation. In vivo, MPST knockdown translated in reduced tumor burden, and a robust increase in mice survival. These results show that enhanced oxidative stress coupled with MPST mobilization plays a key role in glioblastoma cell motility.

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A 3D Spheroid Model for Glioblastoma

Abstract: An easy to use invasion assay for glioblastoma is described; -The assay is suitable for glioblastoma stem-like cells; -A macro in FiJi software is described for easy quantification of invasion, migration and proliferation.

Cited by 16 publications

References 11 publications

Role of Furin in Colon Cancer Stem Cells Malignant Phenotype and Expression of LGR5 and NANOG in KRAS and BRAF-Mutated Colon Tumors

Role of Furin in Colon Cancer Stem Cells Malignant Phenotype and Expression of LGR5 and NANOG in KRAS and BRAF-Mutated Colon Tumors

Refining the Role of Pyruvate Dehydrogenase Kinases in Glioblastoma Development

Human glioblastoma cell motility depends on the activity of the cysteine metabolism enzyme 3-Mercaptopyruvate sulfurtransferase

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