Establishment of in vitro 3D anti-tumor drug screening
systems using PDCs spheroids○ Takahiro Kimura1, Shinichiro Tsunesumi1, Misato Kakita1, Shigenori Enoki1,122Seiichi Katayama , Fumiko Chiwaki , Hiroki Sasaki
1: LSIM Safety Institute Corporation, 2: National Cancer Center Research Institute
Summary in JapaneseResults

PDC（Patient‒Derived Cell Line）は，がん患者の腹水より回収した細胞を低継代数で保存したものであり，抗がん剤の開発において臨床試験の薬剤予測率を高めるツールとして考えられている．また，がんの特徴を保持してその予測性を高める培養方法として，スフェロイド培養法（3 次元）がある．3 次元培養用の 96 穴プレートを用いて膵臓がん PDC，胃がん PDCそれぞれを播種したところ，胃がん PDC においてスフェロイド形成が認められた．さらにスフェロイド状の胃がん PDC にシスプラチンとドセタキセルをそれぞれ添加した．2 次元培養に比べて 3 次元培養下の胃がん
PDC のシスプラチンへの抵抗性は変わらなかったが，ドセタキセルへの抵抗性は上昇した．以上の結果から，3 次元培養下の胃がん PDCを用いた試験系は，生体内のがんの薬剤抵抗性を考慮して，抗がん剤の薬効を評価できる可能性がある．

Photo. Cell morphology in 2D and 3D culture
Pancreatic PDCs

NPC-7C

Gastric PDCs

NPC-20C

NSC-14C

NSC-22C

2D

Objective
3D
Scale bar = 403 µm

Fig 1. Cell proliferation rate and IC50 in 3D culture for 7 days
NPC-20C in 3D culture
Cell proliferation rate（%)

10050● PDCs

Pancreatic PDCsa) : NPC-7C, NPC-20C
Gastric PDCsa) :NSC-11C, NSC-14C, NSC-22C

1010050a) National Cancer Center (NCC) supplied.

0● Spheroid culture plate

11050150

B1004Cisplatinb) :0.391 to 100 µM (common ratio 2)
Docetaxelb) : 3.91 to 1000 pM (common ratio 2)b) Tokyo Chemical Industry Co., Ltd.

A

3Cell division times● Anticancer drugs

NPC-7C

4Seeding density

500 cells/well1000 cells/well5000 cells/well
10000 cells/well

210-1● Evaluation of cell viability

Day 4Day 7Day 11

3Cell division times

Cells were cultivated statically in a CO2 incubator set at 37.0℃ and5.0% CO2 under a humidiﬁed condition.

Day 14

110100

NPC-7C
NPC-20C
NSC-14C

2.6 N.C
72.5 41.6
2.4 16.1

NPC-7C
NPC-20C
NSC-14C

2D*

3D

0.194 N.C
>1.00 N.C
0.0245 N.C

N.C: Not Calculated

N.C: Not Calculated

1000

Docetaxel (pM)

NPC-20C

B

Seeding density

500 cells/well1000 cells/well5000 cells/well
10000 cells/well

Data represent the mean ± standard deviations (n = 3) .

These data show that NPC-7C and NPC-20C cells did notdivide even in a 14-day 3D culture.
Day 4Day 7

Day 11Day 14A

21Seeding density

500 cells/well1000 cells/well

0Measurment
Cell seeding-1

Day 4

Cell division times = Log2 {(D ‒ B) / (D4 ‒ B4)}

Day 4

Day 7

Day 11Day 14

NSC-11C (500 cells/well)

A

100

50

00.1

1IC50 (µM)

10B

NSC-14C (500 cells/well)

100

50

00.1

100

Cisplatin (µM)

150110Extending the duration of culture
from 7 to 11 days decreased IC50of Cisplatin against gastric PDCs.

100

Data represent the mean ± standarddeviations (n = 3) . The cell viability assay
was conducted three times. (Each lineshows each cell proliferation rate. )

50

00.1

100

Cisplatin (µM)

C

NSC-22C (500 cells/well)

1

10Cisplatin (µM)

100

NSC-14C

30

The method of 3D culture of gastric PDCs is determined. The gastric
PDCs proliferation increased gradually until 11 days after seeding(Fig 3). A seeding density of 500 cells/well resulted in increase of thenumber of dividing cells (Fig 3). These data show that the seeding
500 cells/well and the 3D culture duration of 11 days are appropriate
conditions for investigating anticancer eﬀect against gastric PDCs inthis study. To ﬁnd the method of 3D culture of pancreatic PDCs, further
study is required.

150

Cell proliferation rate（%)

Cell proliferation rate（%)

150● Optimization of 3D culture20

NSC-22C

NSC-11C
NSC-14C
NSC-22C

27.1

16.1
9.6

10
0.8

0

2.4

0.7

0.5

2D*

3D (Day 7)

2.2

1.0

3D (Day 11)

Scale bar = 500 µm

Fig 5. Cell proliferation rate and IC50 against Docetaxel for gastric PDCs in 3D culture for 11 days
200

Cell proliferation rate（%)

It has been reported that 3D culture models are more chemoresistantand clinically relevant than 2D (Seidlitz T et al. 2019; Nowacka M et al.
2021). Our results show that gastric PDCs had higher resistant toDocetaxel in 3D culture than in 2D culture (Fig 5D). On the other hand,
IC50 of Cisplatin against gastric PDCs in 3D culture were similar to theone in 2D culture (Fig 4D). These data show that the gastric PDCs
under 3D culture can maintain resistance to some kinds of anticancer
drug. Our drug screening system with gastric PDCs in 3D culture can bebetter at predicting anticancer drug eﬀect than a conventional drug
screening system with gastric cancer cell lines in 2D culture.

500 cells/well1000 cells/well

0-1

Day 14

Seeding density

Fig 4. Cell proliferation rate and IC50 of Cisplatin against gastric PDCs in 3D culture for 11 days

Conclusion● Anticancer drug against gastric PDCs in 3D culture

1

Scale bar = 403 µm

D: Luminescence intensity (LI) of Day 4, 7, 11 or 14
B: Mean LI value of the blank group
D4: Mean LI value of Day 4
B4: Mean LI value of the blank group of Day 4

IC50 was calculated by using SAS 9.4 [SAS Institute Japan Ltd., EXSUS
Version 8.1.0 (EPS Corporation)]

2

These data show that NSC-11C and NSC-22C cells successfully divided in 3D culture.
The optimal number of 3D culture days was thought to be 11 days for this drug eﬃcacy test.

● Calculation of cell division times, cell proliferation and IC50

T: LI of anticancer drug
B: Mean LI value of the blank group
C: Mean LI value of the control groupDay 11B

Data represent the mean ± standard deviations (n = 3) .

Day 11

Cell proliferation rate (%) = 100 × (T ‒ B) / (C ‒ B)

Day 7

3

Cell division times3

NSC-11C

NSC-22C* The data about 2D culture was reported at the 79th annual meeting of the Japanese cancer association.
(Title: Growth Inhibitory Eﬀect of Anti-cancer drug against Human Peritoneal Metastatic Pancreatic Cancer Cell Lines)
Cell proliferation rate of 2D culture (%) = 100 × [(T ‒ B4) ‒ D1] / [(C4 ‒ B4) ‒ D1]
T: LI of anticancer drug, C4: Mean LI value of the control group of Day 4, B4: Mean LI value of the blank group of Day 4,
B1: Mean LI value of the blank group of Day 1, C1: Mean LI value of the control group of Day 1, D1: LI of Day 1

150

A

NSC-11C (500 cells/well)

100
500

1200

10100

Docetaxel (pM)

B

150

NSC-14C (500 cells/well)

100

1000

500

Data represent the mean ± standard deviations(n = 3). The cell viability assay was conducted threetimes. (Each line shows each cell proliferation rate. )1200

Cell proliferation rate（%)

Day 7

4Cell proliferation rate（%)

Day 4

IC50 (nM)

Each IC50 in 3D culture was too high. We hypnotized that theduration of culture was too short to evaluate the eﬀect ofCisplatin and Docetaxel.

-1

Cell proliferation rate（%)

Day 03D

Data represent the mean ± standard deviations (n = 3) . The cell viability
assay was conducted three times. (Each line shows each cell proliferation rate.)

0NSC-11C

4Measurment
Cell seeding● Experimental design (2) : Cell proliferation assay
Anticancer drug or measurement
Anticancer drug

2D*Fig 3. Cell division of gastric PDCs

Cell division times

Day 14IC50 (µM)

D1

● Experimental design (1) : Cell division times
Day 11Cisplatin (µM)

2CellTiter-Glo 3D Cell Viability Assay (Promega Corporation)

Day 7

100

Table 2. IC50 of Docetaxel

Fig 2. Cell division of pancreatic PDCs

● Culture conditions

Day 4

10500

Prime Surface 96U plate (Sumitomo Bakelite Co., Ltd.)

Day 0

1100

1000Docetaxel (pM)

Table 1. IC50 of Cisplatin

100100Cisplatin (µM)

C00.1

Cell proliferation rate（%)

Cell proliferation rate（%)

1501(1000 cells/well)

150A00.1Materials and Methods

NSC-14C in 3D culture(10000 cells/well)

150

Cell proliferation rate（%)

Patient-Derived Cell Lines (PDCs) have been established from the ascites ofcancer patients and stocked after low-passage cultures (Tanaka Y, Chiwaki
F. et al. Nat Cancer 2021). Since PDCs maintain the characteristics of theoriginal cancers, they have been well-accepted as clinically reliable tools.
In vitro screening systems using PDCs may be useful for anticancer drug
screenings. In conventional in vitro studies, anticancer drugs are evaluatedby two-dimensional (2D) cell culture; however, the characteristics of cells in
2D are diﬀerent in some points from the original cancers. In this study, weaimed to develop drug screening systems with PDC-derived spheroids by
three-dimensional (3D) cell culture which can partly mimic the originalcancers. Gastric and pancreatic PDCs were seeded into microplates for 3Dculture, and it was found that gastric PDCs formed spheroids. Furthermore,
we evaluated anticancer drug eﬃcacy with spheroids consisted of cells
derived from gastric PDCs.

10100Docetaxel (pM)

1000

NSC-14C

C150

NSC-22C (500 cells/well)
Table 3. IC50 of Docetaxel

100
500110100

Docetaxel (pM)

1000

IC50 (nM)

2D*

3D

NSC-11C
NSC-14C
NSC-22C

0.02910.02450.0369

N.C
N.C
N.C

N.C: Not Calculated

NSC-22C

IC50 of Docetaxel against gastric PDCs
in 3D culture was higher than the one
in 2D culture.

Scale bar = 500 µm