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: [email protected] 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: [email protected]
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