LPA4-mediated vascular network formation increases the efficacy of anti-PD-1 therapy against brain tumors Daisuke Eino,1,2,3,6 Yohei Tsukada,1,6 Hisamichi Naito,1 Yonehiro Kanemura,4 Tomohiro Iba,1 Taku Wakabayashi,1 Fumitaka Muramatsu,1 Hiroyasu Kidoya,1 Hideyuki Arita,2 Naoki Kagawa,2 Yasunori Fujimoto,2 Kazuhiro Takara,1,5 Haruhiko Kishima,2 and Nobuyuki Takakura1,*

1Department of Signal Transduction, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan; 2 Department of Neurosurgery, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan; 3 Department of Radiation Oncology, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan; 4 Division of Regenerative Medicine, Institute for Clinical Research, Osaka National Hospital, National Hospital Organization, 2-1-14 Hoenzaka, Chuo-ku, Osaka 540-0006, Japan; 5 Research Unit/Frontier Therapeutic Sciences Sohyaku, Innovative Research Division, Mitsubishi Tanabe Pharma Corporation, 1000, Kamoshida-cho, Aoba-ku, Yokohama 227-0033, Japan; 6These authors contributed equally to this work *Correspondence: [email protected] [N.T.]

Supplementary Figure 1. Coverage of pericyte and the basal membrane by LPA. Expression of LPA4 in several tumors.

Supplementary Figure 2. Survival of wild-type and LPA4-KO mice harboring LLC brain tumors treated with or without LPA in the presence or absence of 5-FU.

Supplementary Figure 3. Junctional protein expressioon in vascular endothelial cells by LPA.

Supplementary Figure 4. LPA induces ICAM-1 expression but doesn’t alter the expression of an HEV marker protein on endothelial cells

Supplementary Figure 5. Investigation of LPA mediated VCAM-1 expression in MS-1 cells by using several inhibitors.

Supplementary Figure 6. PD-L1 expression in GL261 brain tumors and LLC subcutaneous tumors. HE staining of the brain after anti-PD-1 antibody and LPA treatment. Supplementary Figure 1. A B C D PDGFRβ CD31 Merge CD31 Col IV Merge n.s. n.s. 100 100 ) l l % o o ( r r t V )

80 I 80 n o % on t ( c age c pe r 60 y 60 e v age o r c e 40 40 v e agen t t o l y c A o A i c 20 r C 20 P L P e L P 0 0 control LPA control LPA E F G LPA4 CD31 Merge LPA4 CD31 Merge LPA4 CD31 Merge

e e p p y y t t d d l i l i W W O O K K 4 A P PA 4 L L

H control LPA VPC I Wild type

250 LPA4 KO e )

p ** y t

d 200 mm ² l i / ** (

W y

i t 150

den s

O 100 K la r 4 A

sc u 50 P a L V 0 control LPA VPC

Supplementary Figure 1. Coverage of pericyte and the basal membrane by LPA. Expression of LPA4 in several tumors. (A) Representative images of GL261 brain tumor. Sections were stained with anti-PDGFR-β mAb (green), and anti-CD31 mAb (red). Scale bar, 200 μm. (B) Quantification of PDGFR-β+ vessels (n = 12 tumors per group). (C) Representative images of GL261 brain tumor. Sections were stained with anti-collagen type IV mAb (red) and anti-CD31 mAb (green). Scale bar, 200 μm. (D) Quantification of collagen type IV+ vessels (n = 12 tumors per group). (E) GL261 brain tumor sections from wild type (upper panels) and LPA4 KO (lower panels) mice were stained with anti-LPA4 pAb green) and anti-CD31 mAb (red). Scale bar, 50 µm. (F) Representative image of human GBM sample. Sections were stained with anti-LPA4 pAb (green) and anti-CD31 mAb (red). Dashed white boxes in the upper panels are shown magnified in the lower panels. Scale bar, 500 µm (upper panels) and 100 µm (lower panels). (G) LLC brain tumor sections from wild type and LPA4 KO mice were stained with anti-LPA4 pAb (green) and anti-CD31 mAb (red). Scale bar, 40 µm. (H, I) LLC brain tumor sections from wild type and LPA4KO mice treated with vehicle, LPA (3 mg/kg) or VPC31144(S) (1 mg/kg) daily from day 7 to day 14. Representative images of sections stained with anti-CD31 mAb (green) (H). Quantification of vascular density. (n=4 per group) (I). All experiments were repeated at least twice. Error bars indicate mean ± SEM. **p<0.01. Supplementary Figure 2.

A B C

1.0 1.0 Wild type LPA4 KO 0.8 0.8 e e t t a a r r l 0.6 l 0.6 a a i v i v v v r 0.4 control r control u u 0.4 S LPA * S LPA 0.2 5-FU 5-FU 0.2 n.s. LPA/5-FU ** LPA/5-FU 0.0 0.0 0 5 10 15 20 25 0 5 10 15 20 25 Days after inoculation Days after inoculation D E 350 ** vehicle LPA 5-FU LPA/5-FU ) 300

mm ² n.s. / (

250 y i t 200 *** den s

150

la r 100 sc u

a 50 V 0 control LPA 5-FU LPA/5-FU

Supplementary Figure 2. Survival of wild-type and LPA4-KO mice harboring LLC brain tumors treated with or without LPA in the presence or absence of 5-FU. (A) Representative image of Evans Blue (150 mg/kg) penetration into the tumor 60 min after intraperitoneal injection. Sections were stained with anti-CD31 mAb (green). Dashed line indicates the tumor edge. Scale bar, 500 μm. (B, C) Survival of wild type (B) and LPA4 KO (C) mice harboring LLC brain tumors. Mice were treated with intraperitoneal injection of vehicle or LPA (3 mg/kg) daily from day 7 with or without weekly 5-FU (100 mg/kg) from day 7. (D, E) Tumor vascular morphological change after LPA and/or 5-FU treatment. Representative images of tumor sections stained with anti-CD31 mAb (green) (D). Quantification of vascular density at tumor center (n=4 tumors per group) (E). Scale bar, 500 µm. All experiments were repeated at least twice. Error bars indicate mean ± SEM. *p<0.05, **p<0.01, ***p<0.001. Supplementary Figure 3. A Merge VE-cadherin CD31 Merge le eh i c v A P L

B 150 100 C 300 100 control CD31 LPA

1 CD31 125 1 3 250 ca d 80 80 ca d

VE-cad - -

D VE-cad E E C CD 3

V V

f 100 200

o 60 o f

60 o f o f e

u 75

ue 150 l ue ue a 40 40 v va l

va l 50 100 va l

y y y y a r 20 20 G 25 Gr a 50 Gr a Gr a 0 0 0 0 0 1 2 3 4 5 6 7 8 9 110… 0 1 2 3 4 5 claudin 5 CD31 Merge occludin CD31 Merge D E l l o r o r on t on t c c A A P P L L

ZO1 CD31 Merge F G 1.8 control LPA 1.6 l H

o 1.4 r D P

on t 1.2 c G A 1 o t 0.8 ve i t

a 0.6 l e 0.4 R A

P 0.2 L 0 claudin 5 occludin ZO1 VEcad PECAM Supplemenatry Figure 3. Junctional protein expressioon in vascular endothelial cells by LPA. (A-C) Localization of VE-cadherin in ECs of GL261 brain tumors treated with vehicle or LPA. Sections were stained with anti-CD31 mAb (red) and anti-VE-cadherin mAb (green). Nuclei were labelled with TOPRO3 (blue). Five tumors per group were analyzed and 4 vessels per tumor section randomly chosen from tumor center. Representative images are shown in (A). The dashed white box in the left-side panel is magnified in the three right-hand panels in each group. Scale bar, 50 µm (left panels) and 10 µm (high-power fields). The fluorescence intensity profiles of VE-cadherin (green line) and CD31 (red line) in the ECs are indicated by the white lines in the high-power fields of (A) (B,C). (D-F) Representative images of endothelial tight-junction marker, claudin 5 (D, green), occludin (E, green) and ZO1 (F, green), in GL261 brain tumors treated with vehicle or LPA. Sections were stained with anti-CD31 mAb (red). (G) Quantita- tive real-time PCR analysis of interendothelial junction markers of ECs in GL261 brain tumors. Tumor-bearing mice were treated with vehicle or LPA (n=4 tumors per group) and mRNA expression in sorted ECs from tumors was analyzed. All experiments were repeated at least twice. Error bars indicate mean ± SEM. Supplementary Figure 4.

A B C 30 control control LPA 80 n.s. d

LPA l ll s e e i f c

20 60 / l

a t ll s o t e

40 c f o

10 +

% 20

0 CD 11 b 0 CD3+ CD3+ B220+ CD11c+ control LPA CD4+ CD8+ CD11b+

D ICAM-1 CD31 Merge E

80 ro l ) * % ( on t

l c 60 ss e e

v 40 + 1 -

M 20 A A C P I L 0 control LPA

F CD31 MECA79 Merge(+TOPRO3) G 6 n.s.

) ro l % ( on t area /

c 4 + 9 area 7 + A 1

C 2 E M CD 3 A

P 0 L control LPA

Supplementary Figure 4. LPA induces ICAM-1 expression but doesn’t alter the expression of an HEV marker protein on endothelial cells. (A) Quantification of immunocyte infiltration into normal lymph node by flow cytometric analysis in mice treated with or without LPA. (n = 5 mice per group). (B) Representative images of GL261 brain tumor generated in wild type mice treated with vehicle or LPA. Sections were stained with anti-CD11b mAb (green) and TOPRO-3 (blue). Scale bar, 50 μm. (C) Quantification of CD11b+ cells as indicated in (B) (n = 4 tumors per group). (D) ICAM-1 expression on ECs in GL261 brain tumor treated with vehicle or LPA. Representative images of tumor sections stained with anti-ICAM-1 mAb (green) and anti-CD31 mAb (red). Scale bar, 100 µm. (E) Quantification of the percentage of ICAM-1 expressing vessels among total blood vessels (n=5 tumors per group). Error bars indicate mean ± SEM. *p<0.05,. (F) GL261 brain tumor sections were stained with anti-MECA79 (green) mAb, anti-CD31 mAb (red), and TOPRO3 (blue). Representative images of GL261 tumors treated with LPA 3 times per week from day 7 to day 14. Scale bar, 50 µm. (G) Quantification of the MECA79-positive area normalized by the CD31-positive area at the tumor center (n=4 tumor per group). Error bars indicate mean ± SEM. Supplementary Figure 5.

control 2’5’DDA SQ22536 YM254890 NF023 A le eh i c v A P L

VPC 10µM (min)

B control 1 5 15 30 60 180 360 D control PDTC p-Erk1/2 le eh i c

Erk1/2 v

p-AktSer473 A

Akt P L

GAPDH

C DMSO PD-98059 U0126 SB202190 VX702 SP600125 LY294002 wartmannin le eh i c v A P L

Supplementary Figure 5. Investigation of LPA mediated VCAM-1 expression in MS-1 cells by using several inhibitors (A) Representative images of MS-1 cells pretreated with 2’5’DDA (100 μM, 60 min), SQ22536 (10 μM, 30 min), YM254890 (10 μM, 5 min), or NF023 (25 μM, 15 min) and subsequently stimulated with 100 μM LPA or vehicle for 24 hr. (B) Western blotting for detecting phosphorylated Erk1/2 and Akt. Confluent MS-1 cells were stimulated with 10 μM VPC31144(S). (C) Representative images of MS-1 pretreated with various inhibitors shown in Fig. 5F. (D) Representative images of MS-1 pretreated with PDTC as described in Fig.5I. (A, C and D) MS-1 cells were stained with anti-VCAM-1 mAb (green) and nuclei were labeled with TOPRO3 (blue). Scale bar, 100 µm. All experiments were repeated at least twice. Supplementary Figure 6.

GL261-EGFP PD-L1 LLC-EGFP PD-L1 A TOPRO3 B TOPRO3 C

control : 85% LPA : 84% Unstained oun t C

0 101 102 103 104 PD-L1 D Post PD-1/LPA therapy E F No treatment

Supplementary Figure 6. PD-L1 expression in GL261 brain tumors and LLC subcutaneous tumors. HE staining of the brain after anti-PD-1 antibody and LPA treatment. (A, B) Representative images of tumors generated by intracranial inoculation of GL261-EGFP (A) and subcutaneous inoculation of LLC-EGFP (B) cells. Cell were stained with anti-PD-L1 antibody (green). Nuclei were labeled with TOPRO3 (blue). White arrowheads indicate tumor cells expressing PD-L1. Scale bar, 20 μm. (C) Representative flow cytometric analysis of PD-L1 fluorescence intensity and the percentage of PD-L1+ cells relative to GL261-EGFP cells (n=5 tumor per group). (D, E) HE staining of GL261 brain tumor after combination therapy with anti-PD-1 antibody and LPA. Dashed box in (D) is magnified in (E). Scale bar, 2 mm in (D) and 100 μm in (E). (F) Representative HE stainig of GL261 brain tumor with no treatment for comparison with (D). Scale bar, 100 μm.