Abstract
Background
Glioblastoma (GBM) is the most common and lethal adult brain tumour. Tumours typically contain large numbers of binuclear and multinucleated cells, and a subpopulation of relatively quiescent glioma stem-like cells (GSC) that are thought to be responsible for treatment resistance and tumour recurrence. GSC display robust G2/M arrest following ionising radiation (IR) yet are highly prone to aberrant cell division and are sensitive to mitotic spindle checkpoint inhibitors. The aim of this study was to investigate the therapeutic activity of mitotic inducers in preclinical models of GBM.
Material and Methods
Bioinformatic analysis of mRNA expression data was used to confirm the relevance of mitotic activity following irradiation of GSC in a 3D patient-derived GBM stem cell model. Immunofluorescence, clonogenic survival and cell viability assays were used to evaluate the therapeutic potential of mitotic inducers (ME-344; Wee1 inhibitor AZ1775) in 2D and 3D U87MGLuc and E2, G1, G7, S2 and R15 patient-derived GSC cell culture models. either alone or in combination with IR. In vivo validation was determined in U87-MGLuc orthotopic GBM mouse model.
Results
Radiation induced downregulation of mRNA expression of several mitotic genes was observed in G7 and E2 cell lines confirming mitotic relevance. In cell viability and clonogenic survival assays, AZ1775 and ME-344 showed potent cytotoxicity against all GSC cell lines in both 2D and 3D, with EC50 values of 0.2 to 0.4 μM for AZ1775 and 0.003 to 0.02 μM for ME-344. ME-344 and AZ1775 triggered profound morphological and cell cycle effects including mitotic induction and arrest, increased mitotic fraction, reduced nuclear tubulin in mitotic cells and, induced mitotic catastrophe in the most sensitive cell lines U87MGLuc and E2. These events were apoptosis-independent. Combination with IR increased GSC cell death in the two GSC models tested to date. Accumulation of cells in mitosis following ME-344 treatment was recapitulated in orthotopic GBM xenografts in vivo, although few mitotic catastrophe events were observed 24 h after treatment. ME-344 demonstrated therapeutic efficacy as a single agent in U87MGLuc2 orthotopic xenografts by extending mouse survival compared to vehicle (p=0.043).
Conclusion
Two agents that induce mitosis through different mechanisms have promising single agent activity against all GBM cell lines tested in vitro and in vivo. Further preclinical evaluation in combination with IR and/or temozolomide is underway. Results indicate that this therapeutic strategy for GBM has clinical potential.
Glioblastoma (GBM) is the most common and lethal adult brain tumour. Tumours typically contain large numbers of binuclear and multinucleated cells, and a subpopulation of relatively quiescent glioma stem-like cells (GSC) that are thought to be responsible for treatment resistance and tumour recurrence. GSC display robust G2/M arrest following ionising radiation (IR) yet are highly prone to aberrant cell division and are sensitive to mitotic spindle checkpoint inhibitors. The aim of this study was to investigate the therapeutic activity of mitotic inducers in preclinical models of GBM.
Material and Methods
Bioinformatic analysis of mRNA expression data was used to confirm the relevance of mitotic activity following irradiation of GSC in a 3D patient-derived GBM stem cell model. Immunofluorescence, clonogenic survival and cell viability assays were used to evaluate the therapeutic potential of mitotic inducers (ME-344; Wee1 inhibitor AZ1775) in 2D and 3D U87MGLuc and E2, G1, G7, S2 and R15 patient-derived GSC cell culture models. either alone or in combination with IR. In vivo validation was determined in U87-MGLuc orthotopic GBM mouse model.
Results
Radiation induced downregulation of mRNA expression of several mitotic genes was observed in G7 and E2 cell lines confirming mitotic relevance. In cell viability and clonogenic survival assays, AZ1775 and ME-344 showed potent cytotoxicity against all GSC cell lines in both 2D and 3D, with EC50 values of 0.2 to 0.4 μM for AZ1775 and 0.003 to 0.02 μM for ME-344. ME-344 and AZ1775 triggered profound morphological and cell cycle effects including mitotic induction and arrest, increased mitotic fraction, reduced nuclear tubulin in mitotic cells and, induced mitotic catastrophe in the most sensitive cell lines U87MGLuc and E2. These events were apoptosis-independent. Combination with IR increased GSC cell death in the two GSC models tested to date. Accumulation of cells in mitosis following ME-344 treatment was recapitulated in orthotopic GBM xenografts in vivo, although few mitotic catastrophe events were observed 24 h after treatment. ME-344 demonstrated therapeutic efficacy as a single agent in U87MGLuc2 orthotopic xenografts by extending mouse survival compared to vehicle (p=0.043).
Conclusion
Two agents that induce mitosis through different mechanisms have promising single agent activity against all GBM cell lines tested in vitro and in vivo. Further preclinical evaluation in combination with IR and/or temozolomide is underway. Results indicate that this therapeutic strategy for GBM has clinical potential.
| Original language | English |
|---|---|
| Pages (from-to) | iii217-iii218 |
| Number of pages | 2 |
| Journal | Neuro-Oncology |
| Volume | 20 |
| Issue number | Suppl 3 |
| DOIs | |
| Publication status | Published - 19 Sept 2018 |
| Event | 13th Meeting of the European Association of Neurooncology - Stockholm, Sweden Duration: 10 Sept 2018 → 14 Oct 2018 |
Keywords
- mitosis
- glioblastoma
- therapeutic
- lethal adult brain tumour
- apoptosis
- cell culture techniques
- cell cycle
- cell growth
- stem cells
- cell death
- cell survival
- down regulation
- RNA
- neoplasms
- bioinformatics
- cytotoxcity