Therapeutic Targeting of Tetraspanin8 in Epithelial Ovarian Cancer

Invasion and Metastasis

Running title: TSPAN8 as a therapeutic target in epithelial ovarian cancer

Chang Sik Park1,†,§, Taek-Keun Kim1,†, Han Gyul Kim2, Youn-Jae Kim3, Mee Hyun Jeoung1,

Woo Ran Lee1, Nam Kyung Go1, Kyun Heo2,*, Sukmook Lee1,*

1Laboratory of Molecular Cancer Therapeutics, Scripps Korea Antibody Institute, 1

Gangwondaehak-gil, Chuncheon-si, Gangwon-do, 200-701, Korea; 2New Experimental

Therapeutics Branch, Research Institute, National Cancer Center, 323 Ilsan-ro, Ilsandong-gu,

Goyang-si, Gyeonggi-do, 410-769, Korea; 3Specific Organs Cancer Branch, Research

Institute, National Cancer Center, 323 Ilsan-ro, Ilsandong-gu, Goyang-si, Gyeonggi-do, 410-

769, Korea

†These authors contributed equally to this work.

*To whom correspondence should be addressed:

Dr. Sukmook Lee, Laboratory of Molecular Cancer Therapeutics, Scripps Korea Antibody

Institute, Hyoja-2-dong, Chuncheon-si, Gangwon-do, 200-701, Korea, Tel: 82-33-250-8096;

Fax: 82-33-250-8088; E-mail: lees@skai.or.kr or Dr. Kyun Heo, New Experimental

Therapeutics Branch, Research Institute, National Cancer Center, 323 Ilsan-ro, Ilsandong-gu,

Goyang-si, Gyeonggi-do, 410-769, Korea, Tel: 82-31-920-2430; Fax: 82-31-920-2542; E-

mail: hk@ncc.re.kr

1

§Current address: Eco & Bio Convergence Team, Korea Institute of Ceramic Engineering and

Technology, 101 Soho-ro, Jinju-si, Gyeongsangnam-do, 661-031, Korea

Abstract2

Epithelial ovarian cancer (EOC) invasion and metastasis are complex phenomena that

result from the coordinated action of many metastatic regulators and must be overcome to

improve clinical outcomes for patients with these cancers. The identification of novel

therapeutic targets is critical because of the limited success of current treatment regimens,

particularly in advanced-stage ovarian cancers. In this study, we found that tetraspanin 8

(TSPAN8) is overexpressed in about 52% (14/27) of EOC tissues and correlates with poor

survival. Using siRNA-mediated TSPAN8 knockdown and a competition assay with purified

TSPAN8 large extracellular loop (TSPAN8-LEL) protein, we identified TSPAN8-LEL as a

key regulator of EOC cell invasion. Furthermore, monotherapy with TSPAN8-blocking

antibody we developed shows that antibody-based modulation of TSPAN8-LEL can

significantly reduce the incidence of EOC metastasis without severe toxicity in vivo. Finally,

we demonstrated that the TSPAN8-blocking antibody promotes the internalization and

concomitant downregulation of cell surface TSPAN8. Collectively, our data suggest TSPAN8

as a potential novel therapeutic target in EOCs and antibody targeting of TSPAN8 as an

effective strategy for inhibiting invasion and metastasis of TSPAN8-expressing EOCs.

Keywords

TSPAN8, Therapeutic target, Epithelial ovarian cancer, Invasion, Metastasis

3

Introduction

Epithelial ovarian cancer (EOC) is the most common type of ovarian cancer and is the

fifth leading cause of cancer-related deaths among women worldwide. This cancer arises

from epithelial cells of the ovary, which are important for hormonal regulation of female

reproduction (1). Because of a lack of characteristic symptoms and early detection strategies,

most ovarian cancer patients are diagnosed at stages III and IV, after the cancer has already

metastasized to other organs (2). The high mortality rate associated with this cancer is largely

explained by the fact that the majority (around 75%) of patients present at advanced stages

with widely metastatic disease within the peritoneal cavity. These cancers grow rapidly,

metastasize early, and have a very aggressive disease course (3). Thus, ovarian cancer

invasion and metastasis still represent a major hurdle that must be overcome to improve

patient outcomes.

Over the course of several decades, a number of chemotherapeutic agents that target DNA

and microtubule structures have been developed for treating ovarian cancer. Despite their

clinical efficacy, these agents are not targeted therapies and result in widespread cytotoxicity

and side effects, including vomiting, diarrhea, hair loss, bleeding, and bone marrow

suppression (4). Furthermore, the 5-year survival rates for stages III and IV ovarian cancer

patients are extremely low, at 21.9% and 5.6%, respectively (5). Recently, bevacizumab, a

humanized antibody targeting vascular endothelial growth factor (VEGF), received European

Medicines Agency (EMA) approval as a first-line therapy for advanced ovarian cancer and

recurrent, platinum-resistant ovarian cancer, in combination with chemotherapy. However,

bevacizumab only increases progression-free survival by approximately 3–4 months,

compared to standard chemotherapeutic agents, including paclitaxel, carboplatin, and

4

gemcitabine (6). Accordingly, there remains a need to identify novel therapeutic targets for

ovarian cancer therapy.

Tetraspanins are a family of small proteins that consist of four transmembrane domains,

two extracellular domains, including the small and large extracellular loops (SEL and LEL),

and three cytosolic domains. They form complexes by interacting with themselves and a

variety of other transmembrane and cytosolic proteins, building a network of interactions

referred to as tetraspanin webs or tetraspanin-enriched microdomains (TEMs) (7). These

TEMs provide a signaling platform that is involved in many important cellular functions and

malignant processes (8).

Tetraspanin 8 (TSPAN8), a member of the tetraspanin superfamily, is a tumor-associated

antigen. It is highly overexpressed during the progression of colorectal, liver, pancreatic, and

gastric cancers (9-11), and its increased expression promotes liver and lung metastasis (12-

14). TSPAN8 may also act as an adaptor molecule, forming a complex with various

membrane proteins, including CD151, EpCAM, claudin-7, E-cadherin, and CD44v6, that has

been shown to promote cancer progression and metastasis (15, 16). However, the relevance

and role of TSPAN8 are yet to be investigated in EOC.

In this study, we examined the relevance and function of TSPAN8 in EOC in vitro and in

vivo. We showed that antibody targeting of TSPAN8 reduced EOC invasion and metastasis by

internalizing and concomitant downregulation of cell surface TSPAN8. Thus, these findings

indicate that targeting of TSPAN8 may potentially be effective against TSPAN8-expressing

EOCs. Therefore, TSPAN8 is not only a prognostic biomarker of EOCs, but also a

therapeutic target for antibody therapy.

5

Results

Analysis of TSPAN8 Expression in EOC Patient Samples - We performed

immunohistochemistry to compare TSPAN8 expression between normal ovarian and EOC

tissues (Figures 1a and b). Normal ovarian tissue (n = 4) lacked TSPAN8 expression, whereas

high expression of TSPAN8 (over 2-fold) was observed in about 52% (14/27) of EOC tissues.

Furthermore, using EOC patient gene expression profiling data (GSE14764) obtained from

the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus

(GEO), bioinformatics-based survival analysis also indicated that there was a statistically

significant association between high TSPAN8 expression and poor survival. The 5-year

overall survival in the low-expression group (n = 25) of the cohort was 63.7% compared to

32.1% in the high-expression group (n = 49) (Figure 1c). Collectively, these results suggest

that TSPAN8 may be closely associated with EOCs.

Identification of TSPAN8-LEL as a Key Target in EOC Invasion - We examined the

role of TSPAN8 in EOC invasion at the molecular level by silencing TSPAN8 in the SK-OV3

EOC cell line using siRNA (Figure 2a). A tumor cell invasion assay demonstrated that

TSPAN8 knockdown reduced SK-OV3 cell invasion by 50.71% with statistical significance

(Figure 2b). To investigate the functional relationship between the two TSPAN8 extracellular

domains (TSPAN8-SEL and TSPAN8-LEL) in EOC invasion, we generated Fc-fusion

proteins, including TSPAN1-LEL-Fc, TSPAN8-SEL-Fc, and TSPAN8-LEL-Fc, and

determined their effects on SK-OV3 cell invasion (Figure 2c). With a competitive blocking

experiment, we found that SK-OV3 cell invasion was specifically and significantly inhibited by

TSPAN8-LEL-Fc but not by TSPAN8-SEL-Fc; TSPAN1-LEL-Fc, used as a negative control,

was also without any inhibitory effect, suggesting that TSPAN8-LEL-Fc may interrupt the 6

TSPAN8-LEL-mediated interactions in SK-OV3 cell invasion. Taken together, these data

suggest that the TSPAN8-LEL domain may play a key role in the regulation of TSPAN8-

mediated EOC invasion.

Effect of TSPAN8-LEL IgG on Inhibition of EOC Invasion and Metastasis - Using

phage display technology, we generated a novel human antibody specific to TSPAN8-LEL

(TSPAN8-LEL IgG) that has a dissociation constant (Kd) of approximately 0.35 nM

(Supplementary Figures 1a-d, available online). To investigate the effect of TSPAN8-LEL

IgG on EOC invasion, we evaluated TSPAN8 expression in SNU-8, SNU-251, and SK-OV3

EOC cell lines. TSPAN8 was expressed specifically in all of these cell lines (Figure 3a).

Here, HUVECs were used as negative cells that TSPAN8 does not express. Next, we

performed Transwell invasion assays using these cell lines in the absence or presence of

TSPAN8-LEL IgG. The antibody significantly inhibited the invasion of all three EOC cell

lines to a similar extent (Figures 3b-d), suggesting a generalized inhibitory effect of the

TSPAN8-LEL antibody on EOC invasion, whereas bevacizumab does not significantly

inhibited SK-OV3 cell invasion (Supplementary Figure 2, available online).

To investigate the effect of TSPAN8-LEL IgG on EOC metastasis, we established an EOC

metastasis animal model. Control IgG or TSPAN8-LEL IgG was then injected intravenously

twice weekly, starting from one day prior to, and continuing for 42 days after, SK-OV3-luc

cell injection (Figure 4a). Metastasis was monitored using bioluminescence imaging (Figure

4b). The incidence of cell metastasis was determined as the number of mice with a detectable

luminescence signal in removed organs, including the ovary, pancreas, colon, heart, liver,

spleen, and kidney. We found that in the TSPAN8-LEL IgG-treated group (15/30), the SK-

OV3-luc cell metastasis was observed in 15 of 30 mice injected with the SK-OV3 cells,

whereas in the control IgG-treated group (24/31), the cell metastasis was observed in 24 of 31 7

mice injected with the SK-OV3 cells. The results indicated that the incidence of SK-OV3-luc

cell metastasis was suppressed significantly by approximately 35%, with a single dose in the

TSPAN8-LEL IgG-treated group, compared with the control IgG-treated group (Figure 4c).

These results suggest that the targeting of TSPAN8 may be effective in the suppression of

EOC metastasis in vivo.

Influence of TSPAN8-LEL IgG on In Vitro or In Vivo Toxicity - To evaluate the in

vitro cytotoxicity of TSPAN8-LEL IgG, we determined the viability of HUVECs and

TSPAN8-overexpressing COS-7 cells after treatment with TSPAN8-LEL IgG. We found that

TSPAN8-LEL IgG had no cytotoxic effect on all of these cells, whereas 5-fluorouracil (5-FU)

significantly reduced the viability of HUVECs and TSPAN8-overexpressing COS-7 cells

(Figure 5a and b). Here, TSPAN8-overexpressing COS-7 cells were representative of other

TSPAN8-expressing cells and were used to further confirm that the TSPAN8-LEL IgGs had

little effect on the viability of other TSPAN8-expressing cells. We also evaluated HUVEC

morphology in the absence or presence of TSPAN8-LEL IgG using immunocytochemistry.

TSPAN8-LEL IgG did not alter the morphology of HUVECs (Figure 5c). To investigate the

effect of TSPAN8-LEL IgG on endothelial cell activation—an initial inflammatory response

to harmful stimuli—we treated HUVECs with TSPAN8-LEL IgG and monitored HUVEC

activation by measuring the expression of endothelial cell activation markers, including

vascular cell adhesion molecule-1 (VCAM-1) and intercellular cell adhesion molecule-1

(ICAM-1). We used human tumor necrosis factor- (hTNF) as a positive control for

endothelial cell activation. TSPAN8-LEL IgG had little effect on HUVEC activation, whereas

hTNF, as expected, induced HUVEC activation (Figure 5d).

To evaluate the in vivo toxicity of the antibody, we performed our immunohistochemistry-

based tissue cross-reactivity study and found that TSPAN8-LEL IgG specifically bound to 8

ovarian cancer tissues but had weak or no affinity for normal ovarian or other tissue types

(Supplementary Figures 3a and b, available online). We also administered control IgG or

TSPAN8-LEL IgG into mice via intravenous injection and then monitored the liver and

kidney function and body weight both prior to, and 42 days after, antibody injection. Liver

function was determined by measuring serum concentrations of glutamic-oxaloacetic

transaminase (GOT), glutamic pyruvic transaminase (GPT), and total bilirubin (TBIL); and

kidney function was determined by measuring blood urea nitrogen (BUN) and creatinine

(CRE) concentrations. No significant changes in liver function, kidney function, or body

weight were observed (Figure 5e). Collectively, these data suggest that the TSPAN8-LEL

antibody is not significantly toxic in vitro or in vivo.

Effect of TSPAN8-LEL IgG on Internalization and Downregulation of Cell Surface

Expression of TSPAN8 - To determine the effect of TSPAN8-LEL IgG on the

downregulation of TSPAN8 expression on EOC cells, we performed a cell ELISA, using

horseradish peroxidase (HRP)-conjugated TSPAN8-LEL IgG, to measure TSPAN8

expression on the surface of SK-OV3 cells following TSPAN8-LEL IgG treatment. TSPAN8-

LEL IgG significantly downregulated the surface expression of TSPAN8 in a time-dependent

manner, whereas control IgG had no effect (Figure 6a). The time-dependent downregulation

of TSPAN8 expression was also confirmed by immunoblot analysis (Figures 6b and c). To

exclude the possibility that the lower signal could be attributed to steric hindrance, we treated

SK-OV3 cells in the absence or presence of HRP-conjugated TSPAN8-LEL IgGs or naked

TSPAN8-LEL IgGs, respectively, and then monitored the cell surface TSPAN8 on SK-OV3

cells using cell ELISA. The results indicated that HRP-conjugated TSPAN8-LEL IgGs and

naked TSPAN8-LEL IgGs could also induce the down-regulation of cell surface TSPAN8 on

SK-OV3 cells, suggesting specific down-regulation of TSPAN8 by TSPAN8-LEL IgGs 9

(Supplementary Figure 4). To verify antibody-induced internalization of TSPAN8, we treated

SK-OV3 cells with fluorescein isothiocyanate (FITC)-labeled TSPAN8-LEL IgG and then

monitored internalization by immunocytochemistry. LysoTracker was also used to label

lysosomes. TSPAN8-LEL IgG rapidly colocalized with lysosomes in SK-OV3 cells,

demonstrating rapid internalization and lysosomal targeting of TSPAN8-LEL IgG (Figure

6d). These data indicate that the TSPAN8-LEL antibody induces rapid internalization and

concomitant downregulation of TSPAN8 on the surface of EOC cells.

10

Discussion

EOC cell invasion and metastasis are complicated processes regulated by the coordinated

action of multiple metastatic regulators (17). Despite the availability of a number of cancer

therapeutics, invasion and metastasis still occur at high frequency and are major hurdles that

must be overcome to improve outcomes for patients with ovarian cancers (2, 3). To this end,

it is important to identify potential therapeutic targets and therapeutics for the treatment of

ovarian cancer. In this study, we propose TSPAN8 as a novel therapeutic target for antibody

therapy, and antibody targeting of TSPAN8 as an effective strategy for inhibiting invasion

and metastasis of TSPAN8-expressing EOCs.

Cancer biomarkers are indicators of the severity or presence of cancer and are useful for

evaluating the efficacy of therapeutic regimens. Human epidermal growth factor 2 (HER2),

which is overexpressed in around 18–20% of breast cancer patients, is currently a useful

biomarker for identifying patients who could benefit from treatment with trastuzumab, a

humanized antibody that targets HER2 (18-20). Another biomarker, wild-type KRAS, is used

to identify patients with epidermal growth factor receptor (EGFR)-positive colorectal cancers

who could benefit from cetuximab, a chimeric antibody that targets EGFR (21, 22). However,

an EOC biomarker is yet to be identified. Our results showed a high expression of TSPAN8

in around 52% of EOC patients and its correlation with poor survival, suggesting that

TSPAN8 might be a useful biomarker for EOC and thus could be exploited for therapeutic

targeting.

Despite the development of many ovarian cancer therapeutics, the 5-year survival rate for

patients remains relatively low (4, 5), reinforcing the importance of developing novel

therapeutics. A number of lines of evidence suggest that TSPAN8-LEL IgG may have

therapeutic potential. We verified that the IgG antibody binds specifically, and with 11

subnanomolar affinity, to TSPAN8-LEL. Our in vitro and in vivo efficacy testing showed that

TSPAN8-LEL IgG suppressed the invasion and metastasis of TSPAN8-expressing EOC. In

addition, we demonstrated that the IgG antibody had little effect on the HUVEC viability,

morphology, and activation. in vivo, the IgG antibody did not induce any changes in liver or

kidney function, or body weight in a mouse model. Furthermore, our immunohistochemistry-

based tissue cross-reactivity study suggests the specific targeting of the IgG antibody to

ovarian cancer tissues in vivo. Thus, the TSPAN8-LEL antibody may specifically inhibit the

invasion and metastasis of TSPAN8-expressing EOCs without causing severe toxicity to

normal tissue.

TSPAN8 is a tumor-associated antigen that forms complexes with itself and with other

factors involved in intracellular signal transduction (7, 15, 16). Using immunocytochemistry,

we showed that TSPAN8-LEL IgG induced rapid internalization of TSPAN8 from the surface

of SK-OV3 cells, along with TSPAN8 translocation to lysosomes, which are cellular

organelles involved in protein degradation. ELISA and immunoblot analyses showed that

treatment with TSPAN8-LEL IgG also significantly downregulated TSPAN8 in SK-OV3 cells

in a time-dependent manner. Thus, the binding of TSPAN8-LEL IgG to TSPAN8 on the

surface of EOC cells leads to rapid internalization of TSPAN8 and a consequent reduction in

its surface expression, thereby suppressing TSPAN8-mediated signaling that promotes EOC

metastasis. In this context, Ailane et al. recently reported that a mouse monoclonal antibody

to TSPAN8 suppresses the growth of TSPAN8-expressing colorectal cancer cell lines in vivo

(23). Collectively, these observations suggest that antibody-based modulation of TSPAN8

may suppress TSPAN8-mediated signaling in tumor cells.

Bevacizumab was the first therapeutic antibody available for treating patients with

ovarian cancers (6). Previously, several groups reported that, although bevacizumab could

prolong life in a peritoneal model of human ovarian cancer by inhibiting tumor growth, it did 12

not suppress the incidence of tumor metastasis (24, 25). Intriguingly, in the current study, we

found that TSPAN8-LEL IgG alone reduced the incidence of metastasis in a peritoneal model

of human ovarian cancer. We also found that TSPAN8-LEL IgG, but not bevacizumab,

inhibited SK-OV3 cell invasion in vitro. Therefore, these results lead us to speculate that the

TSPAN8-LEL antibody suppresses more efficiently the invasion and metastasis of EOC, with

a different mode of action from that of bevacizumab. Finally, we also suggest that, for better

clinical outcome in ovarian cancer therapy, the TSPAN8-LEL antibody may be used not only

in combination with chemotherapeutic agents, including paclitaxel and carboplatin, but also

as an antibody platform for an antibody–drug conjugate or radioimmunotherapy, although

additional studies are required. Taken together, these findings support the therapeutic

potential and possible application of the TSPAN8-LEL antibody in the treatment of EOC.

In conclusion, we have shown that TSPAN8 is a novel therapeutic target in EOC, and

antibody targeting of TSPAN8 may be an effective strategy for suppressing the invasion and

metastasis of TSPAN8-expressing EOC. On the basis of currently available evidence, we

suggest a mode of action whereby the TSPAN8-LEL antibody binds to TSPAN8 on the

surface of EOC cells and rapidly induces TSPAN8 internalization and translocation to

lysosomes, resulting in a reduction in TSPAN8 surface levels and suppression of TSPAN8-

mediated signaling implicated in EOC invasion and metastasis. In future studies, we plan to

investigate the mechanism of action of the TSPAN8-LEL antibody in more detail and

evaluate its in vivo efficacy, in combination with chemotherapeutic agents, against TSPAN8-

mediated EOC metastasis.

13

Materials and methods

Immunohistochemistry - Immunohistochemistry was performed as described previously

(26). Briefly, tissue slides printed with normal ovarian or ovarian cancer tissues were

purchased from SuperBioChips Laboratories. The slides were incubated first with rabbit anti-

TSPAN8 antibody (1:200; Abcam, ab70007) and then with biotinylated goat anti-rabbit IgG

(1:200; Vector Laboratories, BA1000). Immunoreactive proteins were visualized using

VECTASTAIN ABC Reagent (Vector Laboratories). For chromogenic reactions, slides were

incubated with a fresh 3.3'-diaminobenzidine tetrahydrochloride solution (Vector

Laboratories). All samples were counterstained with Meyer’s hematoxylin (Vector

Laboratories). TSPAN8 expression was observed by light microscopy using an Olympus

BX51 microscope (Olympus). TSPAN8 expression was quantified by acquiring RGB images

from TSPAN8-stained images using Paint Shop Pro X software (Corel) and measuring

density with Image J software version 1.48v (National Institutes of Health), after performing

background subtraction.

Survival analysis - EOC patient gene expression profiling data (GSE14764) were

obtained from National Center for Biotechnology Information (NCBI) Gene Expression

Omnibus (GEO). The EOC patients were classified into two groups, according to their

TSPAN8 expression level. Kaplan–Meier analysis and log-rank test were performed using the

R survival package.

Cell culture - COS-7 cells were cultured in Dulbecco’s Modified Eagle Medium

(DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1%

penicillin/streptomycin (Gibco). All human EOC cell lines, including SNU-8, SNU-251 and 14

SK-OV3 (Korean Cell Line Bank), were maintained in RPMI-1640 medium (Gibco) with the

same supplements. HUVECs (Lonza) were maintained in endothelial growth medium-2

(EGM-2; Lonza). All cells were maintained at 37°C in a humidified incubator with 5% CO2

(Panasonic Healthcare Company). Expi293 cells were cultured in Expi293 expression

medium (Invitrogen) in a humidified Multitron incubator shaker (Infors HT, Basel,

Switzerland) at 37°C with 8% CO2.

Transfection - COS-7 and SK-OV3 cells were grown to 50-80% confluence and

transiently transfected with TSPAN8 cDNA or ON-TARGET plus Smart pool siRNA

(Thermo Scientific) specific to TSPAN8 using Lipofectamine 2000 transfection reagent

(Invitrogen), according to the manufacturer’s instructions. HEK293F cells were transfected

with an expression plasmid for TSPAN8 using polyethylenimine (Polysciences, Inc.). For

protein overproduction, Expi293 cells were transiently transfected with expression plasmids

encoding control IgG, TSPAN8-LEL antibody, or Fc-fusion proteins using ExpiFectamine

(Invitrogen). SK-OV3 cells overexpressing firefly luciferase were generated by transfecting

SK-OV3 cells with the pGL4.51 [luc2/CMV/Neo] firefly luciferase reporter plasmid

(Promega) using Lipofectamine (Invitrogen) and culturing in the presence 500 µg/ml of G418

to select positive clones. The activity of firefly luciferase was determined using a Dual-

Luciferase Reporter Assay System (Promega) and TD-20/20 Luminometer (Turner Designs).

Immunoblot analysis - Proteins (30 µg) in cell lysates from scrambled- or TSPAN8

siRNA-transfected SK-OV3 cells, HUVECs, human EOC cell lines, or control IgG- or

TSPAN8-LEL IgG-treated SK-OV3 cells were resolved by sodium dodecyl sulfate-

polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto nitrocellulose

membranes using a wet transfer system (GE Healthcare Life Sciences). After blocking in 15

TTBS (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.05% [v/v] Tween 20) containing 5%

(w/v) skim milk, the membranes were incubated first with rabbit anti-TSPAN8 polyclonal

antibody (1:1,000; Abcam, ab70007) or mouse anti-β-actin monoclonal antibody (1:3,000;

Santa Cruz Biotechnology, Inc., SC47778) at 4°C overnight and then with horseradish

peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:5,000; Santa Cruz Biotechnology, Inc.,

SC2004) or goat anti-mouse IgG antibody (1:5,000; Santa Cruz Biotechnology, Inc.,

SC2031).

Cell invasion assay - Cell invasion assays were performed as described previously (13).

For investigating the effect of TSPAN8 knockdown in SK-OV3 cells, scrambled- or TSPAN8

siRNA-transfected cells (5 × 104) in serum-free medium were loaded into the upper part of a

Transwell insert pre-coated with Matrigel (BD Biosciences), and the lower chamber was

filled with complete medium containing 10% FBS as a chemoattractant. The role of

TSPAN8-LEL in SK-OV3 cell invasion was assessed by incubating 5 × 104 SK-OV3 cells in

the presence of 25 µg/ml of TSPAN1-LEL-Fc, TSPAN8-SEL-Fc, TSPAN8-LEL-Fc, or Fc.

The effect of TSPAN8-LEL IgG on the invasion of ovarian epithelial cancer cell lines was

evaluated by incubating SK-OV3 (5 × 104), SNU-251 (5 × 104) or SNU-8 (1 × 105) cells in

the presence of 20 µg/ml of control IgG, TSPAN8-LEL IgG, or bevacizumab. After non-

invading cells were removed by wiping the upper surface of the membrane with a cotton

swab, the membrane was fixed with 4% paraformaldehyde (PFA) and stained with 0.2%

crystal violet. The degree of cell invasion was quantified by counting the number of cells in

the membrane in three random fields (200× magnification) per filter.

In Vivo Mouse Experiments and Analysis - Seven-week-old female BALB/c-nude mice

(SLC Inc., Seoul, Korea) were housed under specific pathogen-free conditions and 16

maintained in the animal facility of the National Cancer Center (an accredited unit of the

National Cancer Center Research Institute; unit number, NCC-13-163B) in accordance with

the AAALAC International Animal Care Policy. Mice were injected intraperitoneally with

luciferase-overexpressing SK-OV3 cells (SK-OV3-luc) (2 × 106) cells. Starting from one day

prior to SK-OV3-luc cell inoculation, 10 mg/kg of control IgG or TSPAN8-LEL IgG was

injected intravenously twice weekly until 42 days post-inoculation. The incidence of ovarian

cancer cell metastasis was monitored by bioluminescence imaging using an IVIS Lumina

series III system (Perkin Elmer, Waltham, MA, USA).

Cell viability assay - HUVECs (5 × 103) or COS-7 cells (1 × 104) were plated in each

well of a 96-well plate and then incubated in the absence or presence of 20 µg/ml of control

IgG or TSPAN8-LEL IgG for 48 hr. As a positive control, a subset of cells was incubated in

the presence of 36 µg/ml of 5-fluoruricil (5-FU) for 48 hr. Cell viability was determined

using a Cell Counting Kit-8 (Dojindo Laboratories) according to the manufacturer’s

instructions. The final absorbance was measured at 450 nm using a spectrophotometer

(VICTOR X4).

Flow cytometry - Flow cytometry was performed as described previously (27). Effects of

TSPAN8-LEL IgG on endothelial cell activation were evaluated by incubating 2 × 105

HUVECs in the absence or presence of 20 ng/ml hTNF (Millipore) and 20 µg/ml of control

IgG, TSPAN8-LEL IgG, or bevacizumab for 24 hr. After blocking with PBS containing 1%

BSA for 1 hr at room temperature, cells were incubated first with 20 µg/ml anti-VCAM-1 or

anti-ICAM-1 polyclonal antibody (27) for 1 hr at 37°C, and then with an Alexa Fluor 488-

conjugated anti-rabbit antibody (1:1,000; Invitrogen, A11008) for 1 hr at 37°C. The samples

were analyzed by flow cytometry (BD FACSCalibur; BD Bioscience) with the aid of FlowJo 17

software (TreeStar).

Immunocytochemistry - Immunocytochemistry was performed as described previously

(28). Effects of TSPAN8-LEL IgG on HUVEC morphology were monitored by incubating

cells in the absence or presence of 20 µg/ml of TSPAN8-LEL IgG for 24 hr at 37°C. Cells

were fixed with 4% PFA, blocked by incubating with PBS containing 5% BSA and 0.1% TX-

100 for 1 hr at 37°C, and then incubated with 1 unit/well of rhodamine-phalloidin (Molecular

Probes) and 0.1 µg/ml Hoechst (Sigma-Aldrich) for 1 hr.

For detection of TSPAN8-LEL IgG-mediated internalization of TSPAN8 in SK-OV3

cells, cells were grown on poly-L-lysine–coated glass coverslips (Marienfeld-Superior) at

37°C for 18 hr, then incubated with 20 µg/ml of FITC-labeled TSPAN8-LEL IgG for 0.5, 1,

or 2 hr at 37°C. After two washes with ice-cold PBS, cells were fixed by incubating with 4%

PFA for 10 min at room temperature, and then incubated with 200 nM LysoTracker Red

DND-99 (Molecular Probes) for 1 hr at 37°C. Images were acquired using a Zeiss LSM 510

laser-scanning confocal microscope (Carl Zeiss).

In vivo toxicity testing - Seven-week-old female BALB/c-nude mice (n = 4) were

injected intravenously twice weekly with 10 mg/kg of control IgG or TSPAN8-LEL IgG, and

body weight was monitored every week. After 42 days, animals were sacrificed and blood

samples were collected. The blood samples were then centrifuged at 7,000 rpm for 20 min at

4°C, and the serum was stored at -80°C for evaluation of biochemical parameters. Serum

levels of GOT, GPT, BUN, CRE, and TBIL were measured using a Fuji Dri-Chem 3500

biochemistry analyzer (Fujifilm).

Cell ELISA - Cell ELISAs were performed as described previously (27) with minor 18

modifications. Briefly, 1 × 104 SK-OV3 cells were plated in wells of a 96-well plate and then

incubated in the presence of 20 µg/ml of control IgG or TSPAN8-LEL IgG for 0, 0.5, 1, 1.5,

2, 2.5, 3 or 4 h at 37°C. Following fixation with 4% PFA, TSPAN8 expression in SK-OV3

cells was detected by incubating with 2 µg/ml of HRP-conjugated TSPAN8-LEL IgG. After

three washes with ice-cold PBS, the cells were incubated with TMB solution. Optical density

was measured at 450 nm using a microtiter plate reader.

ELISA - ELISAs were performed as described previously (27) with minor modifications.

The specificity of TSPAN8-LEL IgG was confirmed using 96-well plates in which each well

was coated with 0.1 µg of TSPAN8-LEL-Fc, TSPAN8-SEL-Fc, TSPAN1-LEL-Fc, or Fc. The

binding site of TSPAN8-LEL IgG on TSPAN8-LEL was confirmed using 96-well plates in

which each well was coated with 0.1 µg of TSPAN8-LEL wild-type or deletion-mutant

protein. After incubation at 37°C overnight and blocking with PBST (PBS with 0.05% [v/v]

Tween 20) containing 3% (w/v) bovine serum albumin (BSA) for 1 hr, the plate was

incubated with 0.1 µg of HRP-conjugated control IgG or TSPAN8-LEL IgG for 2 hr at 37°C.

Following three washes with PBST, 3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution

(BD Biosciences) was added to each well. The reaction was stopped by the addition of an

equal volume of 1N H2SO4 to the microplate. Optical density was measured at 450 nm using

a microplate reader (VICTOR X4; Perkin Elmer).

Statistical Analysis - Data were analyzed with GraphPad Prism 5.0 (GraphPad Software,

La Jolla, CA, USA), using a two-tailed Student’s t-test for comparison between two groups

and a one-way analysis of variance with Bonferroni’s correction for multiple comparison. All

data represented the means ± SEM. P-values < 0.05 were considered statistically significant.

Tumor incidence data were evaluated using the Fisher’s exact test calculator 19

(http://www.socscistatistics.com/ tests/fisher/ default2.aspx).

20

Conflict of interest

The authors have declared that no conflict of interest exists

Acknowledgments

This work was supported by a research grant (10TS03) from the Scripps Korea Antibody

Institute.

Supplementary Information accompanies the paper on the Oncogene website

(http://www.nature.com/onc)

21

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24

Figure legends

Figure 1. Histological examination of TSPAN8 and survival analysis with epithelial

ovarian cancer (EOC) patient samples. (a) Histological examinations of TSPAN8 were

performed on normal ovarian and EOC tissues by immunohistochemistry using an anti-

TSPAN8 polyclonal antibody. (b) TSPAN8 expression was quantified and expressed as a bar

graph. Tissues with significant upregulation of TSPAN8 (>2-fold that is the mean level for

the four normal ovarian tissue samples (N1-N4); dotted line) are designated with an arrow.

(c) Kaplan–Meier plot for overall survival of EOC patients classified by TSPAN8 expression

(red: high expression group, n = 49; blue: low expression group, n = 25). P=0.03.

Figure 2. Effects of siRNA-mediated TSPAN8 knockdown and TSPAN8-LEL-Fc on

SK-OV3 cell invasion. (a) Immunoblot analysis showing downregulation of TSPAN8 in SK-

OV3 cells by TSPAN8 siRNA. (b) The numbers of invading scrambled siRNA-transfected

and TSPAN8 siRNA-transfected SK-OV3 cells were compared and expressed as a percentage

of invading control cells. (c) The number of invading SK-OV3 cells in the presence of

purified TSPAN1-LEL-Fc, TSPAN8-SEL-Fc, TSPAN8-LEL-Fc, or Fc protein was counted

and expressed as a percentage of invading control cells. All experiments were performed in

triplicate. All data represented the means ± SEM. ***P < 0.001, relative to scrambled siRNA-

or Fc-treated cells, using Student’s t-test in (b) and one-way ANOVA with Bonferroni’s

multiple comparison test in (c). All tests were two-sided. LEL = large extracellular loop; SEL

= small extracellular loop; TSPAN = tetraspanin.

Figure 3. Effect of TSPAN8-LEL IgG on TSPAN8-mediated invasion of epithelial

ovarian cancer (EOC) cell lines. (a) Immunoblot analysis showing TSPAN8 expression in 25

HUVECs and EOC cell lines. The number of invading SNU-8 (b), SNU-251 (c), and SK-

OV3 (d) cells was measured in the presence of control IgG or TSPAN8-LEL IgG and

expressed as a percentage of invading control cells. All experiments were performed in

triplicate. All data represented the means ± SEM. **P < 0.01, ***P < 0.001, relative to

control IgG-treated cells, using Student’s t-test in (B–D). All tests were two-sided. IgG =

immunoglobulin G.

Figure 4. Effect of TSPAN8-LEL IgG on SK-OV3 cell metastasis. (a) Schematic depiction

of the treatment protocol. (b) The peritoneal metastasis model of EOC was established by

injecting mice with SK-OV3-luc cells. Following treatment with control IgG (n = 31) or

TSPAN8-LEL IgG (n = 30), the incidence of SK-OV3-luc metastasis was monitored by

bioluminescence imaging. (c) The incidence of SK-OV3-luc metastasis represents the

number of mice with detectable luminescence signals in removed organs and is expressed as

a percentage of SK-OV3-luc metastasis in controls. This experiment was analyzed by

Fisher’s exact test. *P < 0.05. luc = luciferase.

Figure 5. Evaluation of TSPAN8-LEL IgG toxicity in vitro and in vivo. HUVECs (a), and

MOCK- (■) or TSPAN8-transfected (□) COS-7 cells (b) were incubated in the presence of

control IgG, TSPAN8-LEL IgG, or 5-FU for 2 days. Cell viability was assessed by measuring

absorbance at 450nm. (c) HUVECs cultured in the absence or presence of TSPAN8-LEL IgG

were stained with rhodamine–phalloidin and Hoechst, and examined by confocal microscopy.

Scale bars represent 20 µm. (d) Following the culture of HUVECs in the absence (dashed

line) or presence (solid line) of hTNF, control IgG, TSPAN8-LEL IgG, or bevacizumab,

cells were stained with anti-ICAM-1 (upper) or anti-VCAM-1 (lower) polyclonal antibodies

and analyzed by flow cytometry. Results are representative of three independent experiments. 26

(e) BALB/c-nude mice were injected intravenously twice weekly with 10 mg/kg of control

IgG (■) or TSPAN8-LEL IgG (□). In vivo toxicity reflects changes in the body weight of

mice and serum concentrations of GOT, GPT, BUN, CRE, and TBIL measured 42 days after

antibody injection. All data represented the means ± SEM. from three independent

experiments. ***P < 0.001, relative to control IgG-treated cells or mice, using one-way

ANOVA with Bonferroni’s multiple comparison test in (a, b) and Student’s t-test in (e). All

tests were two-sided. BUN = blood urea nitrogen; BW = body weight; CRE = creatinine; 5-

FU = 5-fluorouracil; hTNF = human tumor necrosis factor alpha; ICAM = intercellular

adhesion molecule; GOT = glutamic oxaloacetic transaminase; GPT = glutamic pyruvic

transaminase; TBIL = total bilirubin; VCAM = vascular cell adhesion molecule.

Figure 6. Effects of TSPAN8-LEL IgG on the internalization and downregulation of

TSPAN8 in SK-OV3 cells. (a) The time-dependent downregulation of TSPAN8 in SK-OV3

cells in the presence of control IgG (■) or TSPAN8-LEL IgG (□) was measured by cell

ELISA. (b) Immunoblot analysis showing the effects of control IgG and TSPAN8-LEL IgG

on TSPAN8 downregulation in SK-OV3 cells. Results are representative of three independent

experiments. (c) Line plot of band densities. (d) Following treatment with FITC-labeled

TSPAN8-LEL IgG, SK-OV3 cells were fixed and stained with LysoTracker Red DND-99.

The localization of TSPAN8-LEL IgG was examined using confocal microscopy (600×

magnification). All data represented the means ± SEM from three independent experiments.

Scale bars represent 20 µm. *P < 0.05, **P < 0.01, ***P < 0.001, relative to control IgG-

treated cells, using Student’s t-test used in (A, C). All tests were two-sided. FITC, fluorescein

isothiocyanate.

27

Supplementary figure legends

Supplementary Figure 1. Generation and in vitro characterization of TSPAN8-LEL IgG.

(a) TSPAN1-LEL-Fc (▨), TSPAN8-SEL-Fc (▧), TSPAN8-LEL-Fc (■), or Fc (□) were

coated onto wells of a 96-well microtiter plate. Binding specificity of the antibodies to

TSPAN8-LEL was determined by ELISA. (b) The specificity of antibody binding to

TSPAN8-transfected HEK293F cells (solid line) was verified by flow cytometry; cells

transfected with empty vector (MOCK, dashed line) were used as a control. (c) Antibody

binding to TSPAN8 on SK-OV3 cells in the absence (dashed line) or presence (solid line) of

TSPAN8-LEL IgG was measured by flow cytometry. (d) The affinity of TSPAN8-LEL IgG

binding to TSPAN8-LEL was measured using a biolayer interferometry assay and the Octet

RED96 system. Values represent means ± S.D. of triplicate measurements from one of three

independent experiments.

Supplementary Figure 2. Effects of TSPAN8-LEL IgG and bevacizumab on SK-OV3

cell invasion. Following treatment of SK-OV3 cells with control IgG, TSPAN8-LEL IgG, or

bevacizumab, cell invasion assays were performed. The number of invading SK-OV3 cells

was expressed as a percentage of invading control cells. All experiments were performed in

triplicate. The data represented the means ± SEM from three independent experiments. ***P

< 0.001, relative to control IgG cells, using one-way ANOVA with Bonferroni’s multiple

comparison test. All tests were two-sided.

Supplementary Figure 3. Tissue cross-reactivity of TSPAN8-LEL IgG.

Immunohistochemical staining was performed using biotinylated TSPAN8-LEL IgG and

horseradish peroxidase-conjugated streptavidin to detect TSPAN8 expression in normal 28

ovarian and EOC tissues (a) and in the indicated normal tissues (b). Scale bars represent 200

µm.

Supplementary Figure 4. Effects of TSPAN8-LEL IgGs on cell surface TSPAN8 down-

regulation on SK-OV3 cells. The cell surface TSPAN8 down-regulation on SK-OV3 cells in

the absence or presence of TSPAN8-LEL IgG was measured by a cell ELISA. (a) After the

generation of horseradish peroxidase (HRP)-conjugated TSPAN8-LEL IgGs (HRP-TSPAN8-

LEL IgGs), SK-OV3 cells were treated with 10 μg/ml HRP-TSPAN8-LEL IgGs for 2 hr at

37°C. To measure the initial amount of cell surface TSPAN8 on SK-OV3 cells, the cells were

first fixed and then incubated with 10 μg/ml HRP-TSPAN8-LEL IgGs for 2 hr at 37°C. (b)

SK-OV3 cells were treated with naked 20 μg/ml TSPAN8-LEL IgGs for 2 hr at 37°C.The

cells were fixed and incubated with HRP-conjugated anti-human Fab (1:3000) for 2 hr at

37°C. To measure initial amount of cell surface TSPAN8, the cells were fixed and incubated

with 20 μg/ml HRP-TSPAN8-LEL IgG for 2 hr at 37°C. Optical density was measured at 450

nm using a microtiter plate reader. Data represented the means ± SEM from three

independent experiments. **P < 0.01, ***P < 0.001.

29