Investigation of drug evaluation model using
Patient-Derived Cell (PDC)○ Misato Moriguchi1, Shinichiro Tsunesumi1, Shigenori Enoki1, Seiichi Katayama1,2
2
Fumiko Chiwaki , Hiroki Sasaki
1: LSIM Safety Institute Corporation, 2: National Cancer Center Research Institute
Summary in JapaneseResults

抗がん剤開発には，in vitro で長期継代されたがん細胞株を移植した担がんマウスが標準的な薬効評価モデルとして使用されている．しかし，これらのモデルで開発された新薬候補は，非臨床研究の結果と臨床研究の結果が一致しないものが多く存在することが課題となっている．そこで, 我々はより臨床予測性の高い抗がん剤評価モデルの確立を試みた.
本実験で用いたPDC（Patient‒Derived Cell）は，胃がんもしくは膵臓がん患者の腹水より回収した細胞で，国立がん研究センターから入手した．低継代数のPDCをマウスに移植して担がんモデルを作製し，既存の抗腫瘍薬を投与して腫瘍体積の変化により抗腫瘍効果を評価した．その結果，膵臓がん PDCモデルはゲムシタビン，胃がん PDCモデルはシスプラチンに対して，薬効に違いが認められた．
以上の結果から，PDCを用いた薬効評価モデルの確立は，既存の薬剤に抵抗性を示す癌の治療薬開発に有用であると考えられる．

NSC-10C

NSC-14C

As an example of the eﬃcacy andthe resistance against a single drug

As an example of a cachexia-like model andthe additive eﬀect of drug combination

Photos in vitro

Photos in vitro

HE staining of tumor(PANC-1 cells)

1200

CDDP 2 mg/kg (n=6)

800Control (n=6)

400
020

DTX 30 mg/kg (n=6)

***

0***

***

***

714Days after group allocation

10CDDP 2 mg/kg(n=6)**-10-20

1000

Control (n=6)

0DTX 30 mg/kg(n=6)

0Fig 3. Tumor growthin NSC-14C-bearing nude mice

***

***

Control (n=6)
CDDP 2 mg/kg(n=6)
Cape 150 mg/kg
(n=6)

800
600
400

****
(n=5)
#**

200

CDDP+Cape (n=6)***

7Days after group allocation

014Points: mean for 6 animals; vertical bars: standard error**: p<0.01, ***: p<0.001 vs the Control group (Studentʼs t test).

0Fig 4. Body weight Change
in NSC-14C-bearing nude mice

15
Change in body weight (%)

1600

Fig 2. Body weight Chnage
in NSC-10C-bearing nude mice

Tumor volume (mm3)

Fig 1. Tumor growthin NSC-10C-bearing nude mice
Change in body weight (%)

In anti-tumor drug discover y, establishedtumor cell lines are generally used for eﬃcacy
evaluation, but use of xenograf t models
inoculated with tumor cell lines cultured
in vitro for long-term often results in a poor
clinical prediction. A primary tumor consists of
not only malignant cells but also non-malignant cells including stromal,immune and vascular cells, while established cell lines form a tumor masscomposed of homogeneous cells (see the right ﬁgure). As a result,preclinical animal models show dissimilar properties to those of primarytumors, which is considered a cause of limited eﬃcacy of drug candidates
in clinical studies. To address this issue, we attempted to establish a drug
evaluation system using patient-derived cancer cell (PDC)-inoculated
models. PDCs from the ascites of patients with gastric or pancreaticcancer were obtained from the National Cancer Center (NCC), and were
subcutaneously inoculated into immunodeﬁcient mice in low-passage
culture condition (≤20 passages). Two lines each of gastric and pancreatic
PDCs were used in the eﬃcacy evaluation of pre-exiting anti-tumor drugs.
In addition, we investigated the expression of cancer stem cell markers,
CD44 and CXCR4 by immunohistochemistry (IHC).

Tumor volume (mm3)Objective

Gastric PDCs

7

14

Days after group allocation

non tumor-bearing (n=3)

5-5CDDP 2 mg/kg (n=6)†††††

Cape 150 mg/kg(n=6)-15-250Control (n=6)†††(n=5)*CDDP+Cape (n=6)

7Days after group allocation

14Points: mean for indicated animals; vertical bars: standard error†† : p<0.01, ††† : p<0.001 vs the non tumor-bearing group (Studentʼs t test).
*: p<0.05, **: p<0.01, ***: p<0.001 vs the Control group (Studentʼs t test).
#: p<0.05 vs the CDDP 2 mg/kg group (Studentʼs t test).

CDDP or saline was administered on Days 0 and 7 (i.v.).
DTX or saline was administered on Days 0 and 4 (i.v.).

CDDP or saline was administered on Days 0 and 7 (i.v.).
Cape or 0.5w/v% methyl cellulose was administered once a day from Day 0 to Day 11 (p.o).
An animal in the Cape 150 mg/kg group was reached humane endpoint because ofemaciation and euthanized on Day 7.

TGI (%) in the NSC-14C-inoculated modelTGI (%) in the NSC-10C-inoculated modelGroup
Control
CDDP 2 mg/kg
DTX 30 mg/kg

No. ofanimal

TGI (%)(Day 14)66
6-10.3
97.9

Group

No. ofanimal

TGI (%)(Day 11)

Control
CDDP 2 mg/kg
Cape 150 mg/kg
CDDP + Cape665620.7
34.4
46.0

Materials and Methods● PDCs

PDCs collected from the ascites of gastric cancer patients (NSC-10C, NSC-14Cand NSC-39C) and pancreatic cancer patients (NPC-7C and NPC-20C) wereobtained from the NCC. PDCs were incubated in vitro and prepared for
inoculation by suspending in DPBS and matrigel.

● Reagents

Pancreatic PDCs

● HE staining and IHC

Paraﬃn-embedded blocks were created from tumor immersed in 10%
formalin neutral buﬀer solution. Then, HE staining and IHC were performed.
Anti-CD44 (ab157107, abcam) and anti-CXCR4 (ab124824, abcam) antibodies
were used.

Discussion
In long-term 2D culture, tumor cell lines often lose their heterogeneity,accumulate further gene mutations and adapt to the artiﬁcial culture
condition. As a result, tumor cell line-inoculated xenografts show
dissimilar biological properties to those of primary tumors, which isconsidered a major hurdle for development of antitumor drugs. As a highpredictive model, patient-derived xenograft (PDX) is currently used for
drug discovery. PDX, which is produced by inoculation of a surgicallyexcised tumor from a patient to mice (or passage from PDX mice to otherm i ce), co n t a i n s n o n - m a l i g n a n t ce l l s a n d m a i n t a i n s t h e t u m o r
microenvironment. Therefore, PDX is highly likely to show a similar
response against drugs as primary cancers and is thus deemed to be a
beneﬁcial tool for drug discovery. On the other hand, unresectablemetastatic cancer is often associated with worse prognosis and is an
essential target of chemotherapy. We assumed that PDCs from metastatic
caners such as ascites carcinoma provide a new option for drug discovery.
Indeed, PDCs from the ascites of patients with gastric and pancreaticcancers in this study showed diﬀerential responses against cisplatin andgemcitabine, respectively. Therefore, it is suggested that PDC-inoculated
models are useful as good predictive models for antitumor drug discovery.

As an example of the tumor growth inhibition
by a single drug

60010Control (n=6)

4002000*07Gem 150 mg/kg (n=6)******14***

***

21

Days after group allocation***

Fig 6. Body weight Change
in NPC-7C-bearing SCID Beige mice

***

28

Control (n=6)

50Gem 150 mg/kg (n=6)-5071421

Days after group allocation28

Fig 7. Tumor growth
in NPC-20C-bearing SCID Beige mice

1200
900

Control (n=6)

600
3000*

Gem 150 mg/kg(n=6)

07142128

Days after group allocation

Fig 8. Body weight Change
in NPC-20C-bearing SCID Beige mice

15Change in body weight (%)

800

Fig 5. Tumor growth
in NPC-7C-bearing SCID Beige mice

Photos in vitro

Gem 150 mg/kg(n=6)

10Control (n=6)

50071421

Days after group allocation

Points: mean for 6 animals; vertical bars: standard error*: p<0.05, ***: p<0.001 vs the Control group (Studentʼs t test).

Points: mean for 6 animals; vertical bars: standard error*: p<0.05 vs the Control group (Studentʼs t test).

Gem or saline was administered on Days 0 , 3, 7, 10, 14 and 17 (i.v.).

Gem or saline was administered on Days 0 , 3, 7, 10, 14 and 17 (i.v.).

TGI (%) in the NPC-20C-inoculated modelTGI (%) in the NPC-7C-inoculated modelGroup

No. ofanimal

TGI (%)(Day 28)

Group

No. ofanimal

TGI (%)(Day 28)

ControlGem 150 mg/kg

6687.5ControlGem 150 mg/kg

6626.7

NSC-39C As an example that were not observed tumor engraftment
150

Tumor volume (mm3)

DV: the mean of tumor volume in the drug treatment groups,
CV: the mean of tumor volume in the control group

As an example of the tumor regression
by a single drug

Change in body weight (%)

Female nude mice and SCID Beige mice were used for the experiment ofgastric cancer and pancreatic cancer, respectively. All animals were purchasedfrom The Jackson Laboratory Japan, Inc. PDCs were subcutaneously
inoculated in the right abdominal region of each animal.
Tumor diameter was measured using caliper and the volume was calculatedby the following equation.
Estimated tumor volume (mm3) =1/2 × long diameter (mm) × short diameter (mm) × short diameter (mm)Tumor-bearing animals were assigned homogeneously to each test group by
the “stratiﬁed randomization method” on the basis of the tumor volume.
After group allocation, tumor diameter and body weight were measured
twice a week.
Tumor growth inhibition [TGI (%)] was calculated as follows:
TGI (%) = (1 ‒ DV / CV) × 100

NPC-20C

Photos in vitro

Tumor volume (mm3)● Animal experiment

NPC-7C

Tumor volume (mm3)

Cisplatin (CDDP), (Product name: Randa Injection, Nippon Kayaku Co., Ltd.)
Capecitabine (Cape), (Product name: Xeloda, Chugai Pharmaceutical Co., Ltd.)
Docetaxel (DTX), (Product name: ONETAXOTERE I.V. Infusion, Sanoﬁ K.K.)
Gemcitabine (Gem), (Product name: Gemzar Injection, Eli Lilly Japan K.K.)

The individual tumor volumeof NSC-39C-bearing nude mice.

1001234550

0071421

Days after tumor cell inoculation

2828