Abstract
Glioblastoma multiforme (GBM) is a rapidly progressive and deadly form of brain tumor with a median survival rate of ~15 months. GBMs are hard to treat and significantly affect the patient’s physical and cognitive abilities and quality of life. Temozolomide (TMZ)—an alkylating agent that causes DNA damage—is the only chemotherapy choice for the treatment of GBM. However, TMZ also induces autophagy and causes tumor cell resistance and thus fails to improve the survival rate among patients. Here, we studied the drug-induced programmed cell death and invasion inhibition capacity of TMZ and a mevalonate cascade inhibitor, simvastatin (Simva), in a three-dimensional (3D) microfluidic model of GBM. We elucidate the role of autophagy in apoptotic cell death by comparing apoptosis in autophagy knockdown cells (Atg7 KD) against their scrambled counterparts. Our results show that the cells were significantly less sensitive to drugs in the 3D model as compared to monolayer culture systems. An immunofluorescence analysis confirmed that apoptosis is the mechanism of cell death in TMZ- and Simva-treated glioma cells. However, the induction of apoptosis in the 3D model is significantly lower than in monolayer cultures. We have also shown that autophagy inhibition (Atg7 KD) did not change TMZ and Simva-induced apoptosis in the 3D microfluidic model. Overall, for the first time in this study we have established the simultaneous detection of drug induced apoptosis and autophagy in a 3D microfluidic model of GBM. Our study presents a potential ex vivo platform for developing novel therapeutic strategies tailored toward disrupting key molecular pathways involved in programmed cell death and tumor invasion in glioblastoma.
Highlights
Glioblastoma multiforme (GBM) is a grade 4 astrocytoma, a brain cancer which accounts for 47.1% of malignant tumors in the central nervous system (CNS) [1,2]
Since the infiltrative behavior of GBM plays a significant role in the progression and expansion of tumor cells, the invasion of the cells has been thoroughly studied in GoC, considering the effects of chemotherapy agents on the invasiveness of the cells in the model
A polydimethylsiloxane (PDMS) elastomer kit was purchased from Ellsworth Adhesives Co. (Germantown, WI, USA); SU-8 100 and its developer were purchased from Kayaku Advanced Materials, Inc. (Westborough, MA, USA); a high glucose Dulbecco’s modified eagle medium (DMEM) with L-glutamine, penicillin streptomycin (Pen Strep, 10,000 units/mL penicillin and 10,000 μg/mL streptomycin), fetal bovine serum (FBS), 0.5% trypsin-EDTA, temozolomide, simvastatin, live/dead staining viability kit, puromycin dihydrochloride, fluorescein isothiocyanate-Dextran (FITC-Dextran), Dulbecco’s phosphate buffered saline (DPBS), and poly-D-lysine were purchased from Millipore Sigma (Oakville, ON, Canada)
Summary
Glioblastoma multiforme (GBM) is a grade 4 astrocytoma, a brain cancer which accounts for 47.1% of malignant tumors in the central nervous system (CNS) [1,2]. GBM is highly malignant, and grows and spreads rapidly in the CNS [3]. The current standard of care for newly diagnosed GBM is surgical resection to the extent feasible, followed by 6 weeks of radiotherapy with concurrent chemotherapy with temozolomide (TMZ). It has been shown that statins could improve chemotherapy response in many cancers, including glioblastoma [5]. It has been demonstrated that lipophilic statin, simvastatin (Simva), which can cross the blood–brain barrier [6,7], significantly improved the survival of GBM patients who have been using TMZ [8]
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