High-Dose Compound Heat Map for 3D-Cultured Glioblastoma Multiforme Cells in a Micropillar and Microwell Chip Platform

Glioblastoma multiforme (GBM) is recognized as the most common and lethal form of central nervous system cancer. To cure GBM patients, many target-specific chemotherapeutic agents have been developing. However, 2D monolayer cell-based toxicity and efficacy tests did not efficiently screen agents due to the pool reflection of in vivo microenvironments (cell-to-cell and cell- to-extracellular matrix interaction). In this study, we used a 3D cell-based, high-throughput screening method reflecting the microenvironments using a micropillar and microwell chip platform to draw a high-dose heat map of the cytotoxicity and efficacy of 70 compounds, with two DMSO controls. Moreover, the high-dose heat map model compared the responses of four 3D-cultured patient-derived GBM cells and astrocytes to high dosages of compounds with respect to efficacy and cytotoxicity, respectively, to discern the most efficacious drug for GBM. Among the 70 compounds tested, cediranib (a potent inhibitor of vascular endothelial growth factor (VEGF) receptor tyrosine kinases) exhibited the lowest cytotoxicity to astrocytes and high efficacy to GBM cells in a high-dose heat map model.

1.Introduction
Glioblastoma multiforme (GBM) is the most common, aggressive, and lethal primary malignant brain tumor that stems from astrocytes. These tumors are usually highly malignant because the cells can metastasize from the primary tumor without detection and invade the surrounding normal brain tissue to form new tumor “satellites” that lead to tumor recurrence [1]. The current standard of care is surgical resec- tion coupled with ionizing radiation (IR) and the chemother- apeutic agent temozolomide (Temodar®, Temodal®, TMZ) [2, 3]. However, this treatment only provides patients with GBM a 12–14-month survival period after diagnosis [2, 3]. Despite aggressive surgical resection and chemotherapy, almost all patients with GBM present with tumor recurrence. Thus, many target-specific or general-chemotherapeutic agents have been developed to cure patients with GBM. Although some of the compounds exhibit good efficacy toward GBM,

the resulting cytotoxicity of normal glial cells in the central nervous system has been an issue. To measure cytotoxicity of compounds in normal glial cells, neural stem cells or astrocytes are used [4, 5]. Astrocytes are the most abundant member of the glial family and havea wide range of adaptive functions in the central nervous system (CNS). They interact with neurons, provide structural, metabolic, and trophic support, participate in synaptic activity, mediate ionic and transmitter homeostasis, and regulate blood flow [6, 7]. Since astrocytes play an important role in the CNS, treatment- induced toxicity of the CNS remains a major cause of mor- bidity in patients with cancer [8]. Thus, a high-dose heat map model comparing the responses of high-dose compounds on astrocytes and GBM cells is required to validate the most efficacious drugs toward GBM. Previous high-dose heat map models using 2D cell-based high-throughput screening are well developed [9, 10]. However, because 2D cell-based assay

does not fully reflect in vivo microenvironments (cell-to-cell and cell-to-extracellular matrix interaction), a 3D cell-based assay was used to screen compounds [11–15], including our previously developed system [13–17]. Especially, 3D cultured astrocyte and GBMs show more in vivo like model [18– 20]. Thus, assay based on 3D cultured astrocyte and GBMs with high-throughput manner may give new potential to screen GBM target agents. Our previous system [13–17] shows successfully data of 3D cell-based assays with high- throughput manner by comparing their own data with 2D cell-based assay [13], gene [14], and clinical data [17]. By applying the abovementioned quantitative 3D-cultured cell- assay platform, astrocytes and patient-derived GBM cells were 3D-cultured and screened to select the most represented compounds that were not cytotoxic to normal brain cells and were particularly efficient for patient-derived GBM cells. Figure 1 shows 3D cell-based high-throughput screening chips culturing three-dimensionally four GBM cells and astrocyte. Since TMZ is a representative drug used in the treatment of patients with GBM, it was used as a control compound to verify the high-dose heat map. By comparing TMZ with 69 other compounds, compounds in the high-dose heat map were tested for cytotoxicity and efficacy in GBM cells.

2.Materials and Methods
We pur- chased NHA-astrocyte AGM (LONZA, Cat. number cc- 2565). Astrocyte was cultured with ABM Basal media (LONZA, Cat. number cc-3187) added with AGM Single- Quot Kit Suppl.&Growth Factors (LONZA, Cat. number cc- 4123). Patient-derived GBM cells were obtained from GBM patients who underwent brain tumor removal surgery at the Samsung Medical Center (Seoul, Korea). Informed con- sent was obtained from all patients. Following a previously reported procedure [13], surgical samples were enzymatically dissociated into single cells. Four patient-derived cells were obtained from four GBM patients. Dissociated GBM cells were cultured in cell culture flasks (from Eppendorf, T-75) filled with Neurobasal A (NBA) conditioned media. The NBA conditioned media comprised N2 and B27 supplements (0.53 each; Invitrogen) and human recombinant bFGF and EGF (25 ng/ml each; R&D Systems), hereafter, referred to as NBE condition media. Cell flasks were placed in a humidified 5% CO2 incubator (Sheldon Mfg., Inc.) at 37∘C. The cells were routinely passed every 4 days at 70% confluence. For the experiment, the cell suspensions were collected in a 50 ml falcon tube from the culture flask. GBM cells were then sus- pended in 5 mL of NBE condition media. After centrifugation at 2000 rpm for 3 min, the supernatant was removed, and the cells were resuspended with NBA conditioned media to a final concentration of 10 × 106 cells/mL. The number of cells in the NBA conditioned media was calculated with the AccuChip automatic cell counting kit (Digital Bio, Inc.). The rest of the cells were seeded at a concentration of 2 × 106 cells in a T-75 flask containing 15 mL of NBA conditioned media.

The basic lay- out of the micropillar and microwell chip for a 12-compound screening is shown in Figure 2. The microwell chip is divided into 72 lines, and each line has 6 microwells for replicates. For compound analysis, approximately 100 cells (patient-derived GBM cells) in 50 nL with a 0.75% alginate concentration by volume (0.75 w/w) were automatically dispensed onto a micropillar chip by using ASFA Spotter ST (Medical & Bio Device, South Korea). The ASFA Spotter ST uses a solenoid valve (The Lee Company, USA) for dispensing the 50 nL droplets of the cell-alginate mixture and 1