Supplementary Information
Intraperitoneal administration of p53-armed oncolytic adenovirus
inhibits peritoneal metastasis of diffuse-type gastric cancer cells
Naoto Hori1, Hiroshi Tazawa1,2, Yuncheng Li1, Tomohiro Okura1,Satoru Kikuchi1, Shinji Kuroda1, Toshiaki Ohara1,3, Kazuhiro Noma1,
Masahiko Nishizaki1, Yasuo Urata4, Shunsuke Kagawa1,5, Toshiyoshi Fujiwara1
1 Department of Gastroenterological Surgery, and 3 Department of Pathology andExperimental Medicine, Okayama University Graduate School of Medicine, Dentistry
and Pharmaceutical Sciences, Okayama 700-8558, Japan. 2 Center for Innovative
Clinical Medicine and 5 Clinical Cancer Center, Okayama University Hospital,Okayama 700-8558, Japan. 4 Oncolys BioPharma, Inc., Tokyo 105-0001, Japan.

Materials and Methods
Supplementary Figure S1.
Schematic diagrams of structures of the replication-deficient and conditionally replicating
adenoviral vectors.
Supplementary Figure S2.
Expression of CAR protein on the surface of human GC cells.
Supplementary Figure S3.
Suppression of FGFR2 in KATOIII cells after treatment with OBP-301.
Supplementary Figure S4.
Induction of apoptosis in the sphere-forming human GC cellsafter treatment with Ad-p53 or OBP-702.
Supplementary Figure S5.
Frequency of hemorrhagic ascites in mice with peritoneally metastatic NUGC-4 tumors
after treatment with Ad-p53 or OBP-702.

Supplementary Materials and Methods
Flow cytometric analysis
To analyze the expression of CAR protein, cells were incubated with anti-CAR antibody (Upstate
Biotechnology, Lake Placid, NY, USA) or isotype control IgG for 60 min on ice. The cells werelabeled with fluorescein isothiocyanate–conjugated rabbit anti-mouse IgG secondary antibody(Invitrogen, Carlsbad, CA, USA) for 30 min and analyzed using FACS array (BD Biosciences). The
mean fluorescence intensity (MFI) for each cell line was determined by calculating the difference
between the MFI of the antibody-treated and isotype control IgG–treated cells.
Fluorescence assay of apoptotic cells using cleaved caspase-3
Cells were seeded in a CorningTM 96-well, round-bottom, ultra-low attachment microplate at a densityof 5 × 103 cells/well. Twenty-four hours later, the cells were treated with Ad-p53 or OBP-702 at a
multiplicity of infection (MOI) of 0, 1, 10, or 100 plaque-forming units (PFU)/cell. Cells withactivated caspase-3 were evaluated on day 6 after virus infection using CellEventTM Caspase-3/7
Green reagent according to the manufacturer’s protocol. Cells that formed spheroids were imaged
using a BZ-X700 fluorescence microscope system (Keyence, Osaka, Japan).

A

hTERT-p

E1A IRESOBP-301
ITRB

hTERT-pC

ITR▲△E1CMV-pp53

ITR▲△E1Ad-p53

Ad5▲
△E1

E1A IRES

OBP-702

E1B

ITR

Egr1-p

E1B

Ad5p53pA▲△E3ITR▲△E3ITRpA

Ad5

Supplementary Figure S1. Schematic diagrams of structures of the replication-deficient andconditionally replicating adenoviral vectors. A, OBP-301 is a telomerase-specific, replicationcompetent oncolytic adenovirus in which the hTERT promoter drives expression of the E1A and E1B
genes, which are linked by an IRES. B, OBP-702 is a p53-armed OBP-301 variant in which the CMV
promoter drives expression of the human wild–type p53 gene. C, Ad-p53 is non-replicative E1/E3deleted adenovirus expressing the human wild–type p53 gene. Ad5, adenovirus serotype 5; CMV,cytomegalovirus; hTERT, human telomerase reverse transcriptase; IRES, internal ribosome entry site;
ITR, inverted terminal repeat.

GCIY(MFI = 5617)

KATOIII(MFI = 1697)

CountsNUGC-4(MFI = 5446)

CAR
Anti-CAR Ab

Control IgG

Supplementary Figure S2. Expression of CAR protein on the surface of human GC cells.
Expression level of CAR on NUGC-4, GCIY, and KATOIII cells as assessed using flow cytometry. The
mean fluorescence intensity (MFI) of each cell line was determined by calculating the difference
between the MFI of anti-CAR antibody–treated and isotype control IgG–treated cells.

KATO III
OBP-301 (MOI)01

5 10 50 100

FGFR2b-actin

Supplementary Figure S3. Suppression of FGFR2 in KATOIII cells after treatment with OBP301. Expression of FGFR2 in KATOIII cells infected for 72 h with OBP-301 at the indicated MOI. bActin was assayed as a loading control.

Ad-p53Control

1 MOI

10 MOI

OBP-702
100 MOI

1 MOI

10 MOI

100 MOINUGC-4GCIY

KATOIII

Hemorrhagic ascites (%)

Supplementary Figure S4. Induction of apoptosis in the sphere-forming human GC cells aftertreatment with Ad-p53 or OBP-702. Cells were seeded in ultra-low attachment, round–bottom 96well plates at a density of 5 × 103 cells/well 24 h before virus infection. Apoptosis induction wasdetermined using CellEventTM Caspase-3/7 Green reagent on day 6 after infection with Ad-p53 orOBP-702 at the indicated MOI.

1009/9

7/7

8060
40

2/9

200

Supplementary Figure S5. Frequency of hemorrhagic ascites in mice with peritoneally metastaticNUGC-4-RFP tumors after treatment with Ad-p53 or OBP-702.