A Simple, Low-Cost Staining Method for Rapid-Throughput Analysis of Tumor Spheroids

Eckerdt, Frank; Alvarez, Angel A.; Bell, Jonathan B.; Arvanitis, Constadina; Iqbal, Asneha; Arslan, Ahmet Dirim; Hu, Bo; Cheng, Shi Yuan; Goldman, Stewart; Platanias, Leonidas C.

doi:10.2144/000114372

Cited by 11 publications

(21 citation statements)

References 13 publications

(15 reference statements)

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“…Next we investigated whether PIM inhibition can block GSC growth. 83Mes GSCs were grown as neurospheres, treated with SGI-1776 and BYL-719 individually and in combination, and subjected to the neurosphere formation assay as previously described [31]. While only minimal effect on neurosphere size by SGI-1776 or BYL-719 alone was observed, combination of SGI-1776 and BYL-719 resulted in greatly impaired neurosphere growth as indicated by a substantial decrease in neurosphere size (Figure 5D).…”

Section: Resultsmentioning

confidence: 90%

“…Neurosphere assays were performed as described previously [31] with the exception that we used 83Mes (mesenchymal) patient-derived glioma stem cells [24]. In brief, 83Mes cells were plated into a round bottom 96-well plate at 500 cells per well.…”

Section: Methodsmentioning

confidence: 99%

“…Subsequently, cells were treated with indicated inhibitors. After seven days, neurospheres were stained with Acridine Orange (0.1 μg/ml) and subjected to microscopic analysis to determine neurosphere cross-section area as described previously [31]. …”

Section: Methodsmentioning

confidence: 99%

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Targeting of glioblastoma cell lines and glioma stem cells by combined PIM kinase and PI3K-p110α inhibition

et al. 2016

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The PIM family of proteins encodes serine/threonine kinases with important roles in protein synthesis and cancer cell metabolism. In glioblastoma (GBM) cell lines, siRNA-mediated knockdown of PIM kinases or pharmacological inhibition of PIM kinases by SGI-1776 or AZD-1208 results in reduced phosphorylation of classic PIM effectors and also elements of the PI3K/mTOR pathway, suggesting interplay between PIM and mTOR signals in GBM cells. Combination of PIM kinase inhibitors with BYL-719, an inhibitor specific for the PI3K catalytic isoform p110α, results in enhanced antineoplastic effects in GBM cells. Additionally, pharmacologic inhibition of PIM kinases impairs growth of patient-derived glioma sphere cells, suggesting an important role for PIM kinases in cancer stem cell (CSC) function and survival. Such effects are further enhanced by concomitant inhibition of PIM kinase and p110α activities. Altogether these findings suggest that pharmacological PIM targeting in combination with PI3K inhibition may provide a unique therapeutic approach for the treatment of heterogeneous tumors containing populations of therapy-resistant CSCs in GBM.

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Section: Resultsmentioning

confidence: 90%

Section: Methodsmentioning

confidence: 99%

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Targeting of glioblastoma cell lines and glioma stem cells by combined PIM kinase and PI3K-p110α inhibition

et al. 2016

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“…After 7 days, cells were stained with 0.1 μg/mL acridine orange as previously described (29). Neurospheres were imaged using the Cytation 3 Cell Imaging Multi-Mode Reader with a 4X objective.…”

Section: Methodsmentioning

confidence: 99%

MNK Inhibition Disrupts Mesenchymal Glioma Stem Cells and Prolongs Survival in a Mouse Model of Glioblastoma

Bell

Eckerdt

Alley

et al. 2016

Molecular Cancer Research

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Glioblastoma multiforme (GBM) remains the deadliest malignant brain tumor, with glioma stem cells (GSCs) contributing to treatment resistance and tumor recurrence. We have identified MAPK-interacting kinases (MNKs) as potential targets for the GSC population in GBM. Isoform-level subtyping using The Cancer Genome Atlas (TCGA) revealed that both MNK genes (MKNK1 and MKNK2) are upregulated in mesenchymal GBM as compared to other subtypes. Expression of MKNK1 is associated with increased glioma grade and correlated with the mesenchymal GSC marker, CD44; and co-expression of MKNK1 and CD44 predicts poor survival in GBM. In established and patient-derived cell lines, pharmacological MNK inhibition using LY2801653 (merestinib) inhibited phosphorylation of the eukaryotic translation initiation factor 4E (eIF4E), a crucial effector for MNK induced mRNA translation in cancer cells and a marker of transformation. Importantly, merestinib inhibited growth of GSCs grown as neurospheres as determined by extreme limiting dilution analysis (ELDA). When the effects of merestinib were assessed in vivo using an intracranial xenograft mouse model, improved overall survival was observed in merestinib-treated mice. Taken together, these data provide strong preclinical evidence that pharmacological MNK inhibition targets mesenchymal GBM and its GSC population. Implications These findings raise the possibility of MNK inhibition as a viable therapeutic approach to target the mesenchymal subtype of GBM.

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“…After one week incubation at 37 C in 5% CO 2 , spheres were stained with acridine orange at 0.1 μg/mL as previously described (26). Spheres were then imaged using a Cytation 3 Cell-Imaging Multi-Mode Reader and analyzed using the Gen5 software (BioTek).…”

Section: Methodsmentioning

confidence: 99%

Differential Response of Glioma Stem Cells to Arsenic Trioxide Therapy Is Regulated by MNK1 and mRNA Translation

Bell

Eckerdt

Dhruv

et al. 2018

Molecular Cancer Research

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Mesenchymal (MES) and proneural (PN) are two distinct glioma stem cell (GSC) populations that drive therapeutic resistance in glioblastoma (GBM). We screened a panel of 650 small molecules against patient-derived GBM cells to discover compounds targeting specific GBM subtypes. Arsenic trioxide (ATO), an FDA-approved drug that crosses the blood–brain barrier, was identified as a potent PN-specific compound in the initial screen and follow-up validation studies. Furthermore, MES and PN GSCs exhibited differential sensitivity to ATO. As ATO has been shown to activate the MAPK-interacting kinase 1 (MNK1)-eukaryotic translation initiation factor 4E (eIF4E) pathway and subsequent mRNA translation in a negative regulatory feedback manner, the mechanistic role of ATO resistance in MES GBM was explored. In GBM cells, ATO-activated translation initiation cellular events via the MNK1–eIF4E signaling axis. Furthermore, resistance to ATO in intracranial PDX tumors correlated with high eIF4E phosphorylation. Polysomal fractionation and microarray analysis of GBM cells were performed to identify ATO’s effect on mRNA translation and enrichment of anti-apoptotic mRNAs in the ATO-induced translatome was found. Additionally, it was determined that MNK inhibition sensitized MES GSCs to ATO in neurosphere and apoptosis assays. Finally, examination of the effect of ATO on patients from a phase I/II clinical trial of ATO revealed that PN GBM patients responded better to ATO than other subtypes as demonstrated by longer overall and progression-free survival. Implications These findings raise the possibility of a unique therapeutic approach for GBM, involving MNK1 targeting to sensitize MES GSCs to drugs like arsenic trioxide.

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A Simple, Low-Cost Staining Method for Rapid-Throughput Analysis of Tumor Spheroids

Cited by 11 publications

References 13 publications

Targeting of glioblastoma cell lines and glioma stem cells by combined PIM kinase and PI3K-p110α inhibition

Targeting of glioblastoma cell lines and glioma stem cells by combined PIM kinase and PI3K-p110α inhibition

MNK Inhibition Disrupts Mesenchymal Glioma Stem Cells and Prolongs Survival in a Mouse Model of Glioblastoma

Differential Response of Glioma Stem Cells to Arsenic Trioxide Therapy Is Regulated by MNK1 and mRNA Translation

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