High-Dose Compound Heat Map for 3D-Cultured Glioblastoma ...downloads.hindawi.com/journals/bmri/2017/7218707.pdf · a solenoid valve (The Lee Company, USA) for dispensing ... and

Research ArticleHigh-Dose Compound Heat Map for 3D-Cultured GlioblastomaMultiforme Cells in a Micropillar and Microwell Chip Platform

DongWoo Lee,1 Sang-Yun Lee,2 Il Doh,3 Gyu Ha Ryu,4 and Do-Hyun Nam2,5

1Department of Biomedical Engineering, Konyang University, Daejeon 35365, Republic of Korea2Department of Health Sciences and Technology, SAIHST, Sungkyunkwan University, Seoul 06351, Republic of Korea3Center for Medical Metrology, Korea Research Institute of Standards and Science (KRISS), Daejeon, Republic of Korea4Office of R&D Strategy & Planning, Samsung Medical Center, Seoul, Republic of Korea5Department of Neurosurgery, SamsungMedical Center, SungkyunkwanUniversity, School ofMedicine, Seoul 06351, Republic of Korea

Correspondence should be addressed to Gyu Ha Ryu; [email protected] and Do-Hyun Nam; [email protected]

Received 24 February 2017; Revised 9 June 2017; Accepted 20 June 2017; Published 18 October 2017

Academic Editor: Sara Piccirillo

Copyright © 2017 Dong Woo Lee et al.This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Glioblastoma multiforme (GBM) is recognized as the most common and lethal form of central nervous system cancer. To cureGBM patients, many target-specific chemotherapeutic agents have been developing. However, 2D monolayer cell-based toxicityand 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 themicroenvironments using a micropillar and microwell chip platform to draw a high-dose heat map of the cytotoxicity and efficacyof 70 compounds, with two DMSO controls. Moreover, the high-dose heat mapmodel compared the responses of four 3D-culturedpatient-derived GBM cells and astrocytes to high dosages of compounds with respect to efficacy and cytotoxicity, respectively, todiscern the most efficacious drug for GBM. Among the 70 compounds tested, cediranib (a potent inhibitor of vascular endothelialgrowth factor (VEGF) receptor tyrosine kinases) exhibited the lowest cytotoxicity to astrocytes and high efficacy to GBM cells in ahigh-dose heat map model.

1. Introduction

Glioblastoma multiforme (GBM) is the most common,aggressive, and lethal primary malignant brain tumor thatstems from astrocytes. These tumors are usually highlymalignant because the cells canmetastasize from the primarytumor without detection and invade the surrounding normalbrain tissue to form new tumor “satellites” that lead to tumorrecurrence [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 GBMa 12–14-month survival period after diagnosis [2, 3]. Despiteaggressive surgical resection and chemotherapy, almost allpatients with GBM present with tumor recurrence. Thus,many target-specific or general-chemotherapeutic agentshave been developed to cure patients with GBM. Althoughsome of the compounds exhibit good efficacy toward GBM,

the resulting cytotoxicity of normal glial cells in the centralnervous system has been an issue. To measure cytotoxicityof compounds in normal glial cells, neural stem cells orastrocytes are used [4, 5]. Astrocytes are the most abundantmember of the glial family and have a wide range of adaptivefunctions in the central nervous system (CNS). They interactwith neurons, provide structural, metabolic, and trophicsupport, participate in synaptic activity, mediate ionic andtransmitter homeostasis, and regulate blood flow [6, 7]. Sinceastrocytes 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 mapmodel comparing the responses of high-dose compounds onastrocytes and GBM cells is required to validate the mostefficacious drugs toward GBM. Previous high-dose heat mapmodels using 2D cell-based high-throughput screening arewell developed [9, 10]. However, because 2D cell-based assay

HindawiBioMed Research InternationalVolume 2017, Article ID 7218707, 7 pageshttps://doi.org/10.1155/2017/7218707

https://doi.org/10.1155/2017/7218707

2 BioMed Research International

does not fully reflect in vivo microenvironments (cell-to-celland cell-to-extracellular matrix interaction), a 3D cell-basedassay was used to screen compounds [11–15], including ourpreviously developed system [13–17]. Especially, 3D culturedastrocyte and GBMs show more in vivo like model [18–20]. Thus, assay based on 3D cultured astrocyte and GBMswith high-throughput manner may give new potential toscreen 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 2Dcell-based assay [13], gene [14], and clinical data [17]. Byapplying the abovementioned quantitative 3D-cultured cell-assay platform, astrocytes and patient-derived GBM cellswere 3D-cultured and screened to select themost representedcompounds that were not cytotoxic to normal brain cellsand were particularly efficient for patient-derived GBM cells.Figure 1 shows 3D cell-based high-throughput screeningchips culturing three-dimensionally four GBM cells andastrocyte. Since TMZ is a representative drug used in thetreatment of patients with GBM, it was used as a controlcompound to verify the high-dose heat map. By comparingTMZwith 69 other compounds, compounds in the high-doseheat map were tested for cytotoxicity and efficacy in GBMcells.

2. Materials and Methods

2.1. Astrocyte and Patient-Derived Cell Culture. 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 GBMpatients who underwent brain tumor removal surgery atthe Samsung Medical Center (Seoul, Korea). Informed con-sent was obtained from all patients. Following a previouslyreported procedure [13], surgical samples were enzymaticallydissociated into single cells. Four patient-derived cells wereobtained from four GBM patients. Dissociated GBM cellswere cultured in cell culture flasks (from Eppendorf, T-75)filledwithNeurobasal A (NBA) conditionedmedia.TheNBAconditionedmedia comprised N2 and B27 supplements (0.53each; Invitrogen) and human recombinant bFGF and EGF(25 ng/ml each; R&D Systems), hereafter, referred to as NBEcondition media. Cell flasks were placed in a humidified 5%CO2incubator (Sheldon Mfg., Inc.) at 37∘C. The cells were

routinely passed every 4 days at 70% confluence. For theexperiment, the cell suspensions were collected in a 50mlfalcon tube from the culture flask. GBM cells were then sus-pended in 5mL ofNBE conditionmedia. After centrifugationat 2000 rpm for 3min, the supernatant was removed, andthe cells were resuspended with NBA conditioned media toa final concentration of 10 × 106 cells/mL. The number ofcells in the NBA conditioned media was calculated with theAccuChip automatic cell counting kit (Digital Bio, Inc.). Therest of the cells were seeded at a concentration of 2 × 106 cellsin a T-75 flask containing 15mL of NBA conditioned media.

2.2. Chip Layout and Experimental Procedure. The basic lay-out of the micropillar and microwell chip for a 12-compoundscreening is shown in Figure 2.The microwell chip is dividedinto 72 lines, and each line has 6microwells for replicates. Forcompound analysis, approximately 100 cells (patient-derivedGBM cells) in 50 nL with a 0.75% alginate concentrationby volume (0.75w/w) were automatically dispensed ontoa micropillar chip by using ASFA� Spotter ST (Medical& Bio Device, South Korea). The ASFA Spotter ST usesa solenoid valve (The Lee Company, USA) for dispensingthe 50 nL droplets of the cell-alginate mixture and 1 𝜇L ofmedia or compounds. After dispensing the cells, as shown inFigure 1(b), the micropillar chip containing human cells inalginate was sandwiched (or “stamped”) with the microwellchip for 3D cell culture and compound efficacy tests. Asingle chip can screen 72 compounds with 6 replicatessimultaneously. A micropillar chip with alginate dispensedon each pillar spot and the microwell chip containing 72compounds are shown in Figure 2. The micropillar chip withcells dispensed is stamped together with its complementarymicrowell chip comprising 532 wells that are 1.2mm indiameter. One microliter of growth media was dispensedinto each microwell. The micropillar and microwell chip inthe combined form are shown in Figure 1(b). After 1 dayof incubation at 37∘C to stabilize the cells, the micropillarchip containing the cells was moved to a new microwell chipfilled with various test compounds. Next, the combined chipswere incubated for 3 days, as shown in Figure 1(b). Cellviability against the compounds was measured with CalceinAM live cell staining dye (4mM stock from Invitrogen),which stains viable cells with green fluorescence.The stainingdye solution was prepared by adding 1.0 𝜇L of Calcein AM(4mM stock from Invitrogen) to 8mL staining buffer (MBD-STA50, Medical & Bio Device, South Korea). To measurecell viability quantitatively after staining the alginate spots,cells on the micropillar chip were scanned. As shown inFigure 1(a), scanned images were obtained with an automaticoptical fluorescence scanner (ASFA Scanner ST, Medical &Bio Device, South Korea).

2.3. Workflow of High-Dose Compound Heat Map. The high-dose compound heat map model for measuring cytotoxicityand efficacy in GBM cells was quantified using a 3D cell-based screening using a micropillar and microwell chip.The workflow of high-dose compound heat map is shownin Figure 3. 72 compounds with high dosage, includingtwo DMSO controls, were screened against astrocytes andGBM cells for measuring toxicity and efficacy, respectively.Based on astrocyte cytotoxicity and efficacy of GBM cells,compounds were divided into four groups. For this heat map,we selected the most promising compounds that exhibitedless cytotoxicity toward astrocytes and high efficacy towardGBM cells.

3. Results and Discussion

To determine the cytotoxicity against normal glial cells, wetreated astrocytes (represented as normal glial cells) to high

BioMed Research International 3

Compound

Micropillar

Microwell

Astrocyte

PDC680T PDC464T

PDC096T PDC352T

GBMsin alginate

(a)

Loading cells 1 day before incubation Compound exposure Cell staining & analysis

ASFA spotter

(b)

Figure 1: 3D cell-based high-throughput screening chips. (a) Photo and schematic view of micropillar and microwell chip platform. Greendots are 3D-cultured astrocytes and glioblastoma multiforme (GBM) cells in alginate spot on the micropillar. (b) Schematic view of theexperimental procedure. Cells are dispensed and immobilized in alginate onto the top of the micropillars and dipped in the microwellscontaining growthmedia for 1-day culture by sandwiching themicropillar andmicrowell chips. Compounds are dispensed into themicrowellsand cells are exposed to the compounds by moving the micropillar chip to a new microwell chip. 3D-cultured cells are stained with CalceinAM, and the dried alginate spot on the micropillar chip is scanned for data analysis.

dosages (20𝜇M) of 72 compounds. Among the 72 com-pounds tested, TMZ is one of most popular drugs for GBM.The lipophilic nature of TMZ permits it to penetrate theblood-brain barrier and, thereby, allows it to be administeredorally. It has also been approved by the US FDA for use inthe treatment of refractory anaplastic astrocytoma in adultssince 1999 and in newly diagnosed adult patients with GBMsince 2005. Thus, we used TMZ as a control compound forcomparing cytotoxicity of compounds toward astrocytes. Weevaluated cytotoxicity of other compounds in comparison toTMZ and evaluated the efficacy of these compounds withpatient-derived GBM cells.

3.1. Toxicity of High-Dose Compounds in Astrocytes. The via-bility of astrocytes after exposure to 72 compounds (including2 DMSO control) after the 3- and 7-day treatment is shownin Figure 4. 16 compounds, including TMZ, exhibited highastrocyte viability and increased to >50% after the 3-daycompound treatment. On the seventh day, only 7 compoundsexhibited >50% astrocyte viability: cediranib, INCB28060,

nilotinib, LDE225, sotrastaurin, vismodegib, and amoralexhibited low toxicity (with astrocyte viability >50%). How-ever, TMZ, which is widely known for its low cytotoxicity,exhibited high toxicity (with astrocyte viability < 18%) afterthe 7-day compound treatment. 20 uM of TMZ is high doseand TMZ shows high toxicity for long-day culture (7 days).Thus, based on the 3-day compound treatment, we evaluatedthe cytotoxicity of the compounds by comparing the viabilityof astrocyte after exposure with other compounds and withTMZ (over 90%). Among 16 compounds, 7 exhibited similarastrocyte viability to TMZ, cediranib, INCB28060, ABT-888,dabrafenib, vismodegib, and amoral and therefore may begood candidates to test the efficacy against patient-derivedGBM cells.

3.2. Efficacy Test of 70 Compounds for Patient-Derived GBMCells. The viabilities of astrocyte and four patient-derivedGBM cells after the 3-day compound treatment are shown inFigure 5. Most of the targeted compounds exhibited high effi-cacy with 20 uMdosages. By comparing efficacy of GBM cells


Cel

l spo

tsCompound 1Compound 2Compound 3Compound 4Compound 5Compound 6Compound 7Compound 8Compound 9Compound 10Compound 11Compound 12Compound 13Compound 14Compound 15Compound 16Compound 17Compound 18Compound 19Compound 20Compound 21Compound 22Compound 23Compound 24Compound 25Compound 26Compound 27Compound 28Compound 29Compound 30Compound 31Compound 32Compound 33Compound 34Compound 35Compound 36

Compoundd 1Compoundd 2Compoundd 3Compoundd 4Compoundd 5Compoundd 6Compoundd 7Compoundd 8Compoundd 9Compoundd 10Compoundd 11Compoundd 12Compoundd 13Compoundd 14Compoundd 15Compoundd 16Compoundd 17Compoundd 18Compoundd 19Compoundd 20Compoundd 21Compoundd 22Compoundd 23Compoundd 24Compoundd 25Compoundd 26Compoundd 27Compoundd 28Compoundd 29Compoundd 30Compoundd 31Compoundd 32Compoundd 33Compoundd 34Compoundd 35Compoundd 36

Compound 37Compound 38Compound 39Compound 40Compound 41Compound 42Compound 43Compound 44Compound 45Compound 46Compound 47Compound 48Compound 49Compound 50Compound 51Compound 52Compound 53Compound 54Compound 55Compound 56Compound 57Compound 58Compound 59Compound 60Compound 61Compound 62Compound 63Compound 64Compound 65Compound 66Compound 67Compound 68Compound 69Compound 70Compound 71Compound 72

Figure 2: Chip layout for high-dose 72 (including 2 DMSO controls)-compound heat map. Each compound has seven replicates.

and cytotoxicity of astrocytes, most of the compounds exhib-ited nonspecific, high toxicity for both GBM cells and astro-cytes. Among seven nontoxic compounds, INCB28060, ABT-888, vismodegib, and amoral did not suppress the patient-derived GBM cells.Thus, they may be ineffective compoundsin treating GBM. TMZ, cediranib, and dabrafenib exhibitedlowGBMcell viability inmore than one patient-derivedGBMcell. In particular, cediranib exhibited high efficacy in allfour patient-derived GBM cells and exhibited no cytotoxicitytoward astrocytes, while TMZ showed high efficacy in only

#1 GBM cell among the four patient-derived GBM cells.Previous studies [21, 22] have shown that cediranib is apotent oral inhibitor of vascular endothelial growth factor(VEGF) receptors and demonstrates improved progression-free survival in an uncontrolled phase II study of patientswith recurrent glioblastoma. The drug is administered orallyand once daily and has a manageable side-effect profile.In addition, it has potent antiedema and steroid-sparingeffects that may improve the quality of life of patients withGBM. Several clinical trials are ongoing testing cediranib


Astrocyte cytotoxicityviability > 90%

Drugcandidate

No effectcompound

Nonspecifichigh toxicity compound

Nonanticancercompound

GBM efficacyviability <50%

GBM efficacyviability < 50%

Compounds

Promote to next step No-go decision Promote to next step No-go decision

Yes No

Yes No Yes No

Figure 3: High-dose compound heat map for selecting low cytotoxicity and high efficacy compound.

1_D

MSO

2_A

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8 (N

VP-

AEE

788)

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fatin

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6_D

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PF-0

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7_G

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(HKI

-272

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(Can

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11_C

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13_B

YL71

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16_A

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65 (S

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4540

9)19

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235

20_A

ZD53

6321

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22_C

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24_P

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HCl

25_S

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Mal

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26_T

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(MLN

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27_T

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28_R

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30_C

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35_P

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3329

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albo

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36_L

Y283

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37_A

ZD45

4738

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J398

(NV

P-BG

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b (T

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40_B

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41_D

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43_A

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44_T

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47_A

BT-1

99 (G

DC-

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(Vel

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49_A

UY9

22 (N

VP-

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22)

50_D

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E225

(NV

P-LD

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, Eris

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54_L

GK-

974

55_O

lapar

ib (A

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56_P

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59_S

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uraf

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61_V

ismod

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(GD

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49)

62_P

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6657

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oral

65_M

evas

66_A

mio

67_F

lu68

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70_A

stem

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63_T

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0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Rela

tive c

ell v

iabi

lity

Astrocyte_3 daysAstrocyte_7 days

Figure 4: Relative cell viability of astrocytes after exposure to 70 compounds after the 3- and 7-day compound treatment. Cell viabilities arecalculated from 3D cell size.

in patients with gliomas. Through this 3D cell-based high-dose heat map model, we could also identify cediranib as avalid compound for proof-of-concept of heat map model. So,this 3D cell-based high-dose heat map could narrow downdrug candidates for GMBs. As further study, we need to drawdose response curve to measure IC

50in GBMs about agents

showing low toxicity and high efficacy.

4. Conclusion

We used a 3D cell-based, high-throughput screening tech-nique using a micropillar and microwell chip platform todetermine the effects of high dosage compounds on thecytotoxicity and efficacy on astrocytes and GBM cells, whichwere graphically represented in a high-dose heat map model.


1_D

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5_Er

lotin

ib H

Cl

8_La

patin

ib

11_C

O-1

686

2_A

EE78

8 (N

VP-

AEE

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3_A

fatin

ib (B

IBW

2992

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6_D

acom

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b (P

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PF-

0029

9804

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(Ire

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9_N

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(HKI

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12_B

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17_P

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18_X

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19_B

EZ23

520

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5363

21_A

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24_P

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25_S

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Mal

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26_T

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(MLN

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27_T

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(AV-

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28_R

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deta

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30_C

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58)

40_B

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41_D

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(BM

S-35

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44_T

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47_A

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99 (G

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(Vel

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49_A

UY9

22 (N

VP-

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22)

50_D

abra

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(NV

P-LD

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, Eris

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55_O

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59_S

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62_P

HA-

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5263

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65_M

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66_A

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67_F

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0.00.10.20.30.40.50.60.70.80.91.01.11.2

1.0 0.0 0.0 0.0 0.31.0 0.0 0.0 0.1 0.71.0 0.1 0.0 0.5 0.51.1 0.0 0.0 0.3 0.61.0 0.0 0.0 0.2 0.6

Astrocyte#1#2#3#4

22_C

edira

nib

(AZD

2171

)

63_T

MZ

High-dose heat mapRe

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l via

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0.0 0.0 0.0 0.3 0.1 0.1 0.1 0.0 0.0 0.1 0.2 0.0 0.2 0.3 0.0 0.5 0.9 0.0 0.3 0.0 0.0 0.3 0.0 0.0 0.4 0.0 0.0 1.1 0.6 0.0 0.0 0.0 0.2 0.3 0.0 0.0 0.6 0.1 0.1 0.0 0.0 0.0 0.9 0.0 1.0 0.0 0.7 0.0 0.7 0.4 0.0 0.7 0.1 0.5 0.3 1.2 0.0 0.9 1.1 0.5 0.7 0.1 0.6 0.0 0.0 0.30.0 0.0 0.0 0.8 0.7 0.1 0.2 0.5 0.5 0.2 0.5 0.2 0.5 0.6 0.3 0.5 0.0 0.2 0.6 0.0 0.1 0.4 0.3 0.0 0.3 0.0 0.0 0.8 0.7 0.0 0.0 0.1 0.7 0.5 0.0 0.0 0.8 0.4 0.5 0.1 0.2 0.6 0.8 0.3 0.8 0.3 0.7 0.0 0.7 0.7 0.0 0.9 0.4 0.3 0.8 0.8 0.0 0.2 0.7 0.8 0.4 0.7 0.7 0.2 0.1 0.60.1 0.1 0.1 0.5 0.4 0.1 0.3 0.3 0.3 0.1 0.3 0.3 0.3 0.4 0.5 0.8 0.0 0.2 0.7 0.0 0.2 0.3 0.2 0.1 0.4 0.0 0.0 1.0 0.8 0.1 0.0 0.0 0.5 0.0 0.0 0.1 0.7 0.3 0.2 0.3 0.3 0.3 0.9 0.3 0.6 0.2 0.7 0.0 0.8 0.7 0.2 0.6 0.4 0.2 0.7 0.8 0.6 0.9 0.8 0.6 0.1 0.8 0.1 0.2 0.5 0.30.0 0.2 0.1 0.1 0.2 0.1 0.4 0.5 0.4 0.1 0.5 0.4 0.4 0.6 0.8 0.8 0.1 0.5 0.7 0.0 0.2 0.6 0.3 0.1 0.4 0.0 0.0 0.9 0.5 0.0 0.0 0.1 0.6 0.2 0.0 0.1 0.6 0.3 0.4 0.2 0.2 0.1 0.9 0.5 0.5 0.0 1.0 0.0 0.8 0.6 0.1 0.7 0.3 0.5 0.4 0.9 0.0 0.8 0.8 0.8 0.5 1.0 0.6 0.7 0.4 0.40.0 0.0 0.4 0.5 0.5 0.1 0.3 0.4 0.3 0.3 0.3 0.3 0.4 0.5 0.5 0.6 0.0 0.2 0.6 0.0 0.2 0.7 0.2 0.0 0.6 0.0 0.0 0.9 0.3 0.0 0.0 0.0 0.4 0.0 0.1 0.3 0.6 0.3 0.4 0.2 0.3 0.4 1.0 0.5 0.7 0.3 0.8 0.0 0.7 0.5 0.1 0.7 0.4 0.3 0.3 1.0 0.1 0.8 0.8 0.6 0.7 0.7 0.5 0.6 0.1 0.7

Astrocyte#1#2

#3#4

61_V

ismod

egib

(GD

C-04

49)

56_P

anob

inos

tat (

LBH

589)

32_C

rizot

inib

(PF-

0234

1066

)

23_I

mat

inib

(Gle

evec

)

4_BM

S-59

9626

(AC4

80)

Figure 5: High-dose compound heat map. Astrocytes and four patient-derived glioblastoma multiforme (GBM) cells were treated with 70compounds for 3 days. In the high-dose heat map (table over graph), red solid filling denotes astrocytes with viability >90% and blue solidfilling denotes GBM cells with viability <50%.

Although some compounds exhibited good efficacy towardGBM, cytotoxicity toward normal glial cells in the centralnervous system was an impending issue. Thus, we useda high-dose heat map model and considered both cyto-toxicity and efficacy by comparing the response of high-dose compound on 3D-cultured GBM cells and astrocytesto screen the most efficacious drugs for GBM. Seventycompounds with dosage of 20 uM, including specific-targetgeneral-chemotherapeutic agents, were used against fourpatient-derived GBM cells and astrocytes to compare thecytotoxicity and efficacy of the compounds. For simulatingan in vivo microenvironment, GBM cells and astrocytes wereencapsulated with alginate and 3D-cultured in a micropillarand microwell chip platform. Among the 70 compoundstested, cediranib exhibited the lowest cytotoxicity towardastrocyte and showed high efficacy toward GBM. Thus, inearly stage of the drug development, the micropillar andmicrowell chip platform could culture patient-derived GBMswith 3D manner and be used for screening toxicity andefficacy of many lead compounds targeted GBMs beforeanimal tests or clinical trials.

Conflicts of Interest

The authors declared no potential conflicts of interest withrespect to the research, authorship, and/or publication of thisarticle.

Acknowledgments

This research was supported by the Bio & Medical Technol-ogy Development Program of the NRF funded by the KoreanGovernment, MSIP (NRF-2015M3A9A7029740).

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