Investigating Programmed Cell Death and Tumor Invasion in a Three-Dimensional (3D) Microfluidic Model of Glioblastoma

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.

Keywords: apoptosis; autophagy; cell phenotype; glioblastoma; invasion; tumor on a chip.

Figure 1

Concept of glioblastoma on chip (GoC). Schematic representation of glioblastoma multiforme (GBM) and the developed tumor-on-a-chip model. The GoC was comprised of a tumor and tumor-associated stroma compartments and side channels that delivered nutrients and drugs to the cells. The bottom right image shows the actual image of the fabricated model.

Figure 2

Characterization of GoC. (a) Image sequences showing the diffusion of fluorescein isothiocyanate-Dextran (FITC-dextran) (20 kDa) into the extracellular matrix (ECM)-like matrix (collagen concentration of 3 mg/mL). (b) The distribution of FITC-dextran along the collagen channel at different time points. (c) Live/dead staining of the U251 and U87 cells after 72 h of culture. (d) Quantitative viability of the U251 and U87 cells after 0 and 72 h of culture. (e) Immunofluorescence images of the U251 and U87 cells stained with glial fibrillary acidic protein (GFAP). (f) Bright field image sequences of the invasion of the U87 cells over 72 h. (g) Distribution profile of the invaded U87 cells across the channel at different time points. Data are expressed as mean ± standard deviation (n = 3). ** p < 0.01, **** p < 0.0001. In (a), (c), and (f), scale bars are 500 µm; in (e), scale bar is 50 µm.

Figure 3

Drug sensitivity of the GoC and 2D culture systems. Viability and morphological analyses of the U251 and U87 cells after 72 h of treatment with different concentrations of temozolomide (TMZ) and Simva in the 2D culture and GoC. (a) Viability of the U251 cells treated with TMZ. (b) Viability of the U251 cells treated with Simva. (c) Viability of the U87 cells treated with TMZ. (d) Viability of the U87 cells treated with Simva, (a–d). Viability was calculated by live staining and normalizing each treatment with its control group. (e) Bright field images of the U251 cells treated with TMZ. (f) Bright field images of the U251 cells treated with Simva. (g) Bright field images of the U87 cells treated with TMZ. (h) Bright field images of the U87 cells treated with Simva. (i) Cytoskeleton of the U251 and U87 cells in the GoC treated with TMZ; actin (green), and DAPI (4′,6-diamidino-2-phenylindole) (blue). (j) Cytoskeleton of the U251 and U87 cells in the GoC treated with Simva; actin (green), and DAPI (blue). Data are expressed as mean ± standard deviation (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. All scale bars are 50 µm.

Figure 4

Drug-induced apoptosis in glioma cells in the 2D culture and GoC. (a) Immunofluorescence staining of the U251 cells with cleaved PARP after 72 h of treatment with TMZ (100 µM) and Simva (1 µM) in the 2D culture and GoC model. (b) Comparison of the expression level of the cleaved PARP (percentage of the number of cleaved PARP to nuclei) between the 2D and GoC models for low drug concentrations (TMZ 100 µM, Simva 1 µM). (c) Immunofluorescence staining of the U251 cells with cleaved PARP after 72 h of treatment with TMZ (250 µM) and Simva (2.5 µM) in the GoC model. (d) Expression level of the cleaved PARP in the U251 and U87 cells in the GoC model for high drug concentrations (TMZ 250 µM, Simva 2.5 and 5 µM). (e) Immunofluorescence staining of the U251 cells with cleaved Caspase-3 after 60 h of treatment with TMZ (100 µM for 2D and 250 µM for microfluidic model) and Simva (1 µM for 2D and 2.5 µM for GoC model). (f) Expression level of cleaved Caspase 3 (percentage of the number of cleaved Caspase 3 to nuclei) in both the 2D and 3D models. Data are expressed as mean ± standard deviation (n = 3). *** p < 0.001, **** p < 0.0001. All scale bars are 50 µm.

Figure 5

Drug-induced autophagy in the cells in the GoC. (a) Immunofluorescence staining of the U251 cells with LC3β (green) and SQSTM1/p62 (red) after 72 h of treatment with TMZ (250 µM) and Simva (2.5 µM) in the 3D model. (b) Expression level of LC3β (ratio of the number of LC3β puncta to the number of nuclei). (c) Ratio of the number of co-localized LC3β puncta and p62 to the number of nuclei. Data are expressed as mean ± standard deviation (n = 3). * p < 0.05, ** p < 0.01. Scale bar is 50 µm.

Figure 6

Analysis of the effect of autophagy on the viability of the U87 and U251 cells in response to treatment with TMZ and Simva in the 2D culture and GoC. For each case, the Atg7 knocked down (U251 KD and U87 KD) and scrambled (U251 SC and U87 SC) cells have been compared. The 2D and 3D viability were calculated using PrestoBlue and live/dead staining assays, respectively. (a) U25—TMZ, (b) U251—Simva, (c) U87—TMZ, (d) U87—Simva. Data are expressed as mean ± standard deviation (n = 3), * p < 0.05.

Figure 7

Effect of the drug treatment on tumor invasion in the GoC. (a) Z-stacked DAPI/actin immunofluorescence images of the U251 and U87 cells invading to the side compartment under treatment with different concentrations of temozolomide (0–500 µM). Number of invaded cells per mm width of the chip for the U251 and U87 cells treated with TMZ (0–500 µM) and Simva (0–10 µM): (b) U251 cells, TMZ; (c) U251 cells, Simva; (d) U87 cells, TMZ; and (e) U87 cells, Simva. Data are expressed as mean ± standard deviation (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Scale bar is 200 µm.

Figure 8

Effect of drugs on tumor invasiveness. (a) Immunofluorescence staining of the U251 and U87 cells with vimentin after 72 h of treatment with TMZ (250 µM) and Simva (2.5 µM for U251 and 5 µM for U87) in the microfluidic tumor model. (b) Expression level of vimentin (average area coverage by vimentin per nuclei). Data are expressed as mean ± standard deviation (n = 3). ** p < 0.01. Scale bar is 50 µm.