Activated T Cell Exosomes Promote Tumor

Invasion via Fas Signaling Pathway

This information is current as
of May 10, 2015.

Zhijian Cai, Fei Yang, Lei Yu, Zhou Yu, Lingling Jiang,
Qingqing Wang, Yunshan Yang, Lie Wang, Xuetao Cao and
Jianli Wang
J Immunol 2012; 188:5954-5961; Prepublished online 9 May
2012;
doi: 10.4049/jimmunol.1103466
http://www.jimmunol.org/content/188/12/5954

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References

The Journal of Immunology

Activated T Cell Exosomes Promote Tumor Invasion via Fas

Signaling Pathway
Zhijian Cai,*,1 Fei Yang,*,1 Lei Yu,* Zhou Yu,* Lingling Jiang,* Qingqing Wang,*
Yunshan Yang, Lie Wang,* Xuetao Cao,* and Jianli Wang*

as (also known as CD95/Apo-1) is a transmembrane protein

belonging to the TNF/nerve growth factor receptor superfamily, which transmits an apoptotic signaling in susceptible cells once triggered by its natural ligand FasL (1). Fas-mediated
apoptosis plays important roles in various biological processes,
including activation-induced cell death in T lymphocytes or
cell-mediated cytotoxicity against virus infection (2, 3). Almost
all kinds of tumor cells express Fas, and the Fas/FasL system plays
an important role in controlling survival or growth of tumor cells
(4). Although Fas signaling was reported to induce tumor apoptosis and inhibit tumor growth in vivo when triggered by FasL
(57), accumulated evidence also demonstrated that, in the presence of certain levels of FasL, Fas signaling promotes, not
inhibits, tumor growth in Fas-resistant tumor cells (8, 9). In addition to inducing tumor cell proliferation, Fas ligation was proved
to advance cell cycle and increase tumor motility and invasiveness
(1014). All of these studies suggest that Fas signaling plays an
important role in tumor progress in Fas-resistant tumors.
*Institute of Immunology, School of Medicine, Zhejiang University, Hangzhou
310058, Zhejiang, China; and Zhejiang Cancer Hospital, Hangzhou 310022, Zhejiang, China
1

Z.C. and F.Y. contributed equally to this work.

Received for publication December 2, 2011. Accepted for publication April 5, 2012.
This work was supported by the National Key Basic Research Program of China
(2007CB512400), the National Natural Science Foundation of China (30671909 and
30972725), and the Natural Science Foundation of Zhejiang Province (Z2090042).
Address correspondence and reprint requests to Dr. Jianli Wang, Institute of Immunology, School of Medicine, Zhejiang University, Hangzhou 310058, China. E-mail
address: jlwang@zju.edu.cn
Abbreviations used in this article: c-FLIP, cellular FLICE inhibitory protein;
COX2, cyclooxygenase-2; DC, dendritic cell; EXO, exosome derived from activated CD8+ OT-I T cell; EXOcont, exosome derived from naive CD8+ OT-I T cell;
EXOgld, exosome derived from Con A-activated CD8+ T cell of gld mice; EXOwt,
exosome derived from Con A-activated CD8+ T cell of C57BL/6 mice; FasL, Fas
ligand; MMP, matrix metalloproteinase; OT-I, C57BL/6-Tg (Tcra Tcrb) 1100Mjb/J;
PDTC, ammonium pyrrolidinedithiocarbamate; siRNA, small interfering RNA;
WT, wild-type.
Copyright 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1103466

Activation of T cells is a pivotal step in the process of host antitumor immunity. During this course, T cells release large numbers
of membrane vesicular bodies termed exosomes (15), which were
initially described by Johnstone et al. (16). Exosomes are nanoscale membrane vesicles (50100 nm) released by various live
cells, which have a lipid bilayer structure (17). They are formed by
membrane budding into the lumen of an endocytic compartment,
leading to the formation of multivesicular bodies. Fusion of multivesicular bodies to the plasma membrane leads to the extracellular release of exosomes (18). The exosomes secreted by activated
human T cells express bioactive FasL and can induce the apoptosis
of Jurkat T cells (15). Exosomes purified from CD8+ T cells,
which are generated by cultivation of OVA-pulsed dendritic cells
(DCs) with naive CD8+ T cells derived from OT-I mice, also express FasL and can inhibit DCs to stimulate CD8+ CTL responses
(19). However, the potential effects of FasL in activated T cell
exosomes on Fas-resistant tumor cells are not well understood.
Matrix metalloproteinase (MMP)9 plays a critical role in the
breakdown of extracellular matrix in normal physiological processes, such as embryonic development, reproduction, and tissue
remodeling, as well as in disease processes, such as tumor metastasis (20). MMP9 was reported to be an important factor in
facilitating lung cancer invasion and metastases in the B16 melanoma model (21). It was also demonstrated that the activation of
tumor Fas signaling can induce the expression of MMP9 (22).
Therefore, we hypothesize that FasL in activated T cell exosomes
probably upregulates MMP9 expression and promotes the invasive
ability of Fas-resistant tumor cells, which may provide a novel
view for understanding tumor escape from the immune system.

Materials and Methods

Mice and cell lines
C57BL/6-Tg (Tcra Tcrb) 1100Mjb/J (OT-I) mice were obtained from the
Institute of Immunology, National Key Laboratory of Medical Immunology,
Second Military Medical University (Shanghai, China). Female C57BL/6J
(68-wk-old) mice were purchased from Joint Ventures Sipper BK
Experimental Animal (Shanghai, China). Tnfsf6gld mice on a C57BL/6

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Activated T cells release bioactive Fas ligand (FasL) in exosomes, which subsequently induce self-apoptosis of T cells. However,
their potential effects on cell apoptosis in tumors are still unknown. In this study, we purified exosomes expressing FasL from
activated CD8+ T cell from OT-I mice and found that activated T cell exosomes had little effect on apoptosis and proliferation of
tumor cells but promoted the invasion of B16 and 3LL cancer cells in vitro via the Fas/FasL pathway. Activated T cell exosomes
increased the amount of cellular FLICE inhibitory proteins and subsequently activated the ERK and NF-kB pathways, which
subsequently increased MMP9 expression in the B16 murine melanoma cells. In a tumor-invasive model in vivo, we observed that
the activated T cell exosomes promoted the migration of B16 tumor cells to lung. Interestingly, pretreatment with FasL mAb
significantly reduced the migration of B16 tumor cells to lung. Furthermore, CD8 and FasL double-positive exosomes from tumor
mice, but not normal mice, also increased the expression of MMP9 and promoted the invasive ability of B16 murine melanoma
and 3LL lung cancer cells. In conclusion, our results indicate that activated T cell exosomes promote melanoma and lung cancer
cell metastasis by increasing the expression of MMP9 via Fas signaling, revealing a new mechanism of tumor immune
escape. The Journal of Immunology, 2012, 188: 59545961.

The Journal of Immunology

5955

background (henceforth termed gld) were obtained from The Jackson

Laboratory, bred under specific pathogen-free conditions, and used at 68
wk of age. B16 melanoma cell line and 3LL Lewis lung cancer cell line
originating from C57BL/6 mice were purchased from American Type
Culture Collection (Manassas, VA).

Peptides, reagents, and Abs

OVA257264 peptide was synthesized by Chinese Peptide (Hangzhou,
China). Matrigel matrix basement membrane was from BD Biosciences
(San Diego, CA). DAPI was from BIOMOL Research Laboratories (NJ,
CA). INTERFERin siRNA transfection reagent was from Polyplus (NY,
CA). NF-kB inhibitor ammonium pyrrolidinedithiocarbamate (PDTC)
and ERK/MAPK inhibitor U0126 were from Sigma-Aldrich (St. Louis,
MO). Abs against FasL allophycocyanin (MFL3), CD8 PE (53-6.7),
CD9 PE (eBioKMC8), CD11c PE (N418), and TCR FITC (B20.1) were
from eBioscience (San Diego, CA). Abs against Tsg 101 (C-2), GRP 94
(H-212), P-ERK (E-4), ERK1/2 (MK1), MMP9 (M-17), FLIPs/L (G-11),
HSP70 (3A3), CD8 (YTS169.4), FasL (C-178), and Fas (5F9) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Abs against
phosphoNF-kB (Ser536) (93H1) and NF-kB (C22B4), as well as HRPlinked secondary Abs, were from Cell Signaling Technology (MI, CA).
+

Preparation and purification of CD8 T cell-released exosomes

Electron microscopy
For electron microscopy observations, exosome pellets were fixed in 4%
paraformaldehyde at 4C for 1 h. Then, the pellets were loaded onto
electron microscopy grids coated with formvar carbon, contrasted, and
embedded in a mixture of uranyl acetate and methylcellulose. Sections
were observed with a Philips Tecnai-10 transmission electron microscope operating at 80 kV (Phillips Electronic Instruments, Mahwah,
NJ).

FACS analysis of exosomes

For FACS analysis, exosomes were coated onto 4-mm-diameter aldehyde/
sulfate latex beads, using a previously described method (24). Briefly, 20
mg exosomes was incubated with 5 ml 4-mm-diameter aldehyde/sulfate
latex beads for 15 min at room temperature in PBS, with 20 ml final
volume. The mixture was then transferred to 1 ml PBS with gentle
shaking for 1 h. After centrifugation, the pellet was blocked by incubation with 20 ml FCS for 30 min. Exosome-coated beads were washed
thrice in PBS and resuspended in 50 ml PBS. Afterwards, beads were
incubated with corresponding fluorescent Abs for 1 h at room temperature in the dark. Beads were analyzed by flow cytometry using a FACSCalibur flow cytometer (Becton Dickinson, Mountain View, CA) and
FlowJo software.

Western blot
A total of 50 mg exosomes or crude proteins extracted from cell lysates
was separated by 12% SDS-PAGE and transferred onto polyvinylidene
difluoride membrane (Millipore, Billerica, MA). Membrane was blocked
with 5% BSA in TBST and then incubated with corresponding primary
Abs overnight at 4C. After incubating with HRP-coupled secondary Ab

Tumor invasion assay in vitro

After rehydration using a 6-fold volume of serum-free media, 50 ml
Matrigel was added on a 8-mm polycarbonate membrane in 24-well
Transwell plates, and the Matrigel was solidified at 37C. A total of 1 3
106 cells/ml was stimulated with exosomes for 2 h in cryovials under
constant rotation at 37C. Then 2 3 105 B16 or 2 3 104 3LL tumor cells in
100 ml serum-free media were seeded into the top chamber. The bottom
chamber was filled with 800 ml media containing 20% serum. Forty-eight
hours after culture at 37C, cells were fixed with methanol for 20 min and
washed with PBS thrice for 20 min each. The fixed cells were stained with
10 mg/ml DAPI for 30 min and washed with PBS. The stained cells were
examined using a fluorescence microscope. To confirm the role of Fas
signal, MMP9, NF-kB, and ERK signal in the tumor invasion, 20 mg/ml
anti-mouse FasL-neutralized Ab, 10 nM MMP9 inhibitor, 10 mM PDTC,
or 10 mM U0126 was added to the culture medium.

Detection of cell apoptosis

For detection of apoptosis induced by exosomes, thymocytes (1 3 106/ml)
were stimulated with 20 mg/ml EXOwt or EXOgld for 8 h. For detection of
B16 or 3LL cell apoptosis induced by exosomes, viable tumor cells (2 3
105/ml) were stimulated with 5, 10, or 20 mg/ml EXOcont or EXO for 24
h. The apoptotic cells were stained with FITC Annexin V (BD Pharmingen, San Diego, CA) and propidium iodide (Sigma-Aldrich) for 5 min at
4C in the dark. The stained cells were analyzed by FACS, as described
previously (25).

Cell proliferation assay

Cells were stimulated with 5, 10, or 20 mg/ml exosomes for 24, 48, or 72 h
in a 96-well plate, and 20 ml alamarBlue (Invitrogen) was added per well
for 6 h. The fluorescent intensity was detected using a DTX 880 multimode
detector (Beckman Coulter, Palo Alto, CA).

RNA interference assay

For transient silencing of cellular FLICE inhibitory protein (c-FLIP), 21-nt
sequences of small interfering RNA (siRNA) duplexes were synthesized
(GenePharma, Shanghai, China): 59-CCUCCUGGAUAGCUUAAGUTT-39
(sense) and 59-ACUUAAGCUAUCCAGGAGGTT-39 (antisense). 59-UUCUCCGAACGUGUCACGUTT-39 (sense) and 59-ACGUGACACGUUCGGAGAATT-39 (antisense) were synthesized as siRNA scramble control.
A total of 40 nM siRNA duplexes was transfected into cells (2 3 105/well)
using 3 ml INTERFERin siRNA transfection reagent on a 24-well plate. The
efficiency of c-FLIPL transient silencing was confirmed by Western blot.

Real-time PCR
Total RNA was extracted with TRIzol reagent (Invitrogen), according to the
manufacturers instructions. The specific primers for b-actin for real-time
PCR were 59-CGTTGACATCCGTAAAGACC-39 (sense) and 59-AACAGTCCGCCTAGAAGCAC-39 (antisense). The specific primers for MMP9
for real-time PCR were 59-CGGCACGCCTTGGTGTAGCA-39 (sense)
and 59-GGCGCACCAGCGGTAACCAT-39 (antisense). The following
PCR conditions were used: 1 cycle at 95C for 30 s and then 40 cycles
of 5 s at 95C and 34 s at 60C. Real-time PCR was performed on an
Applied Biosystems 7500 real-time PCR system (Foster City, CA).

Lung invasion assay of B16 tumor cells in vivo

A total of 5 3 105 B16 tumor cells with 30 mg EXOcont, EXO, 20 mg/ml
anti-FasLpreincubated EXO, or PBS was injected into 8-wk-old C57BL/6
mice via the tail vein. Two weeks after injection, mice were sacrificed, and
the lungs were detached. After the number of lung nodules was counted,
the excised lungs were photographed and fixed in 10% formaldehyde for
further H&E staining.

Purification of an in vivo counterpart of EXO from

tumor-activated CD8+ T cells
A total of 1 3 106 B16 tumor cells was injected s.c. into 8-wk-old C57BL/
6 mice. The mice were sacrificed 10 d after tumor inoculation. Lymphocyte
suspensions from normal and tumor mice were prepared by crushing the
lymph nodes and spleens between two pieces of ground glass. For preparation of tumor tissue suspensions, tumor tissues were detached and
dissociated enzymatically for 2 h with 1 mg/ml type I collagenase (SigmaAldrich, St. Louis, MO) in the presence of 50 U/ml RNase and DNase

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A total of 2 3 106 cells/ml naive lymphocytes derived from OT-I mice was
cultured in RPMI 1640 complete medium (containing 50 mM 2-ME, 1 mM
sodium pyruvate, 10 mM HEPES, and 10 U/ml IL-2) and stimulated or not
with OVA257264 peptide (2 mg/ml) for 48 h. Active CD8+ T cells were
purified using CD8+ Dynabeads (Invitrogen, Carlsbad, CA). Purified OVAspecific active CD8+ T cells were then cultured in exosome-free RPMI
1640 complete medium for 24 h. Exosomes in T cell culture supernatants
were isolated, as previously described (23). Briefly, collected culture supernatants were differentially centrifuged at 300 3 g for 10 min, 1,200 3 g
for 20 min, and 10,000 3 g for 30 min at 4C. The supernatant from the
final centrifugation was ultracentrifuged at 100,000 3 g for 1 h at 4C.
After removing the supernatant, the exosome pellets were washed in large
volume of ice-cold PBS and centrifuged at 100,000 3 g for another 1 h at
4C. To isolate the FasL+ exosomes released by CD8+ T cells of C57BL/6
and FasL-deficient gld mice, CD8+ T cells were stimulated with 50 mg/ml
Con A for 15 min and then washed and cultured in exosome-free RPMI
1640 complete medium for 1 h. Supernatant was collected, and exosomes
were purified, as previously described (23). Exosomes in the pellet were
resuspended in PBS. Exosomes from CD8+ T cells, stimulated or not with
OVA257264 peptide, were termed EXO or EXOcont; exosomes from Con
A-activated CD8+ T cells of C57BL/6 or gld mice were termed EXOwt or
EXOgld, respectively.

for 1 h, the membranes were scanned using Tanon 4500 (Shanghai, China),
according to the manufacturers instructions.

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ACTIVATED T CELL EXOSOMES PROMOTE TUMOR INVASION

(Sigma-Aldrich). Exosomes from all kinds of suspensions were filtrated

using a 0.22-mm-filter device and then purified by differential centrifugation, as mentioned above. For purification of exosomes from CD8+
T cells, the exosomes were sorted through CD8 + Dynabeads (Dynal;
Invitrogen). Briefly, 200 mg exosomes was mixed with 100 ml Dynabeads
at 4C by gentle shaking overnight, and the exosome-coated beads were
detached with 200 ml DETACHaBEAD at room temperature for 45 min.
Bead-free supernatant was colleted after precipitating beads via a magnet.
CD8+ exosomes were washed in a large volume of ice-cold PBS and
centrifuged at 100,000 3 g for 1 h.

Statistical analysis
Experimental data were analyzed using one-way ANOVA and the t test
using SPSS 11.5. Differences were considered significant when the p
values were , 0.05.

Results
FasL+ EXO showed no effect on B16 tumor cell apoptosis and
proliferation

FIGURE 1. FasL+ EXO showed no effect on B16

tumor cell apoptosis and proliferation. (A) Electron
micrograph of activated CD8+ T cell EXO. Scale bar,
200 nm. (B) For analysis of expression of surface
molecules, 20 mg of EXO was incubated with 5 ml of
4-mm-diameter aldehyde/sulfate latex beads and
stained with a panel of Abs (solid lines) or negative
control Abs (dotted lines). (C) Western blot analysis of
EXO/EXOcont and the whole-cell lysate of activated
CD8+ OT-I T cells/naive CD8+ OT-I T cells using the
Abs indicated. (D) A total of 1 3 106/ml thymocytes
was stimulated with 20 mg/ml exosomes, 20 mg/ml of
FasL-neutralized mAbs, or exosomes preincubated
with isotype mAbs (ISO) for 8 h. The apoptosis of
thymocytes was analyzed by FACS (n = 3). **p ,
0.01 versus Cont, EXOcont, or EXO+anti-FasL. (E) A
total of 1 3 106/ml thymocytes was stimulated with 50
mg/ml Con A-activated CD8+ T cell exosomes from
WT or gld mice for 8 h. The apoptosis of thymocytes
was analyzed by FACS (n = 3). **p , 0.01 versus
Cont or EXOgld. (F) B16 tumor cells were incubated
with a serial concentration of EXO or EXOcont for
24 h. The apoptosis of B16 tumor cells was analyzed
by FACS (n = 3). (G) B16 tumor cells were incubated
with a serial concentration of EXO or EXOcont
for 24, 48, or 72 h. The proliferation of B16 tumor cells
was measured by alamarBlue assay. Data are representative of three independent experiments.

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To evaluate the isolated EXO integrity from activated CD8+ T cells,

we inspected EXO morphology by electron microscopy. The observation showed that isolated EXO displayed a typical round
morphology, with a diameter of 50100 nm (Fig. 1A). Like their
parent CD8+ T cells, EXO also expressed CD8+ T cellspecific

molecules, including CD8 and TCR. In addition, they expressed

specific EXO markers, such as CD9, HSP70, and TSG101,
whereas endoplasmic reticulum-residing protein GRP94 was absent in EXO. Moreover, EXO, but not EXOcont, showed positive
expression of FasL (Fig. 1B, 1C). These results demonstrate the
integrity of isolated positive EXO from activated CD8+ T cells in
this study. With the successful isolation of Fas+ EXO, we first
examined whether EXO could induce apoptosis of thymocytes.
Apoptosis assay by FACS showed that EXO, but not EXOcont,
could significantly induce the apoptosis of thymocytes, and the
ability of EXO to induce the apoptosis of thymocytes was greatly
inhibited when preneutralized by FasL mAbs (Fig. 1D), which
suggest the biological activity of FasL in EXO. To further confirm
the relationship between apoptosis induction and FasL in EXO,
we isolated exosomes from activated CD8+ T cells of C57BL/6
and gld mice and confirmed their expression of FasL (data not
shown). After treatment with EXOwt or EXOgld, increasing apoptosis was found in EXOwt-treated, but not EXOgld-treated,
thymocytes (Fig. 1E). These results indicate that the apoptosisinducing function of EXO is FasL dependent. Then we detected
whether EXO could also induce the apoptosis of Fas-expressing
B16 or 3LL tumor cells. The results showed that FasL+ EXO has

The Journal of Immunology

little effect on B16 apoptosis (Fig. 1F) or 3LL apoptosis (data not
shown). Because it was demonstrated that Fas signaling can promote the proliferation of thyroid carcinomas (11), we next investigated whether EXO could promote the proliferation of B16 or
3LL tumor cells. The results showed that, with prolonged treatment time or increased concentration, EXO did not affect the
proliferation of B16 (Fig. 1G) or 3LL (data not shown) compared
with control.
EXO promoted tumor cell invasion via Fas signaling in vitro

EXO-mediated activation of Fas signaling induced

upregulation of MMP9 expression in tumor cells
It was reported that CD95L mediates the migration of myeloid
cells via MMP9 activation (26). We assume that the effect of EXO
on tumor cell invasion is also related to MMP9 activation. As
shown in Fig. 3A, after incubation with EXO, but not EXOcont,
the mRNA and protein levels of MMP9 in B16 were significantly
increased; the optimal concentration to stimulate was 10 mg/ml
(Fig. 3A, 3B). After blockage of Fas signaling by anti-FasL, the
expression of MMP9 induced by EXO decreased greatly (Fig.
3C), which provides solid evidence that upregulation of MMP9
induced by EXO is Fas/FasL dependent. To further confirm the
relationship between EXO-mediated promotion of tumor cell invasion and MMP9 upregulation, MMP9 inhibitor was added to
B16 tumor cell-invasiveness assays in vitro. A total of 10 nM of
MMP9 inhibitor completely abolished the ability of EXO to
promote tumor cell invasion (Fig. 3D). These results indicate that
the tumor cell invasion induced by FasL+ EXO was associated
with MMP9 expression.

EXO promoted MMP9 expression and tumor cell invasion

through ERK and NF-kB pathways
The stimulation of CD95L on tumor cells can activate the NF-kB
and ERK pathways (13), and the activation of NF-kB and ERK
increases MMP9 expression (8, 27). Similarly, EXO stimulation
activated the NF-kB and ERK pathways in B16 cancer cells,
which was demonstrated by the increased phosphorylation of NFkB and ERK in response to EXO stimulation but not EXOcont
stimulation (Fig. 4A). Nevertheless, it seems that the expression
of ERK and NF-kB is in parallel pathways, because when ERK
expression was disrupted by its specific inhibitor U0216, little
change in the expression of NF-kB was observed (data not
shown). To further reveal the roles of activated NF-kB and ERK in
MMP9 expression and tumor cell invasion, specific inhibitors for
the NF-kBand ERK-signaling pathways were used to treat B16
cancer cells before EXO stimulation; MMP9 expression was
subsequently examined. We found that both NF-kB and ERK
inhibitors (PDTC and U0126) markedly suppressed EXO-induced
MMP9 expression and tumor cell invasion (Fig. 4B, 4C). Collectively, these results showed that FasL+ EXO-activated NFkB and ERK-signaling pathways were responsible for the increased MMP9 expression and tumor cell invasion.
c-FLIP was critical to the increase in MMP9 expression and
tumor invasion induced by EXO
c-FLIP is an endogenous inhibitor of death receptor-induced apoptosis that acts through the caspase-8 pathway and is widely
expressed in tumors (28, 29). c-FLIP exists as a long (c-FLIPL)
or a short (c-FLIPS) variant (30, 31). c-FLIPL shows overall
structural homology to caspase-8, containing two death effector
domains that interact with Fas-associated death domain protein, as
well as an inactive caspase-like domain. c-FLIPS contains only the
two death effector domains and has lower antiapoptotic capacity
(32). In B16 tumor cells, we found that Fas engagement with EXO
led to the increased accumulation of c-FLIPL but not c-FLIPS (Fig.
5A). The increase in c-FLIPL was detected at 5 min after EXO
stimulation, and the high level was maintained for 24 h (Fig. 5A).
It was reported that c-FLIP can promote activation of the ERKand NF-kBsignaling pathways (32). Therefore, we investigated
whether their activation in B16 tumor cells is mediated by c-FLIPL
upon EXO stimulation. We inhibited c-FLIPL expression by
siRNA and confirmed the inhibitory efficiency by Western blot.
c-FLIPL expression in c-FLIPL siRNA-transfected B16 cells decreased 50% compared with c-FLIPL expression in control cells
(Fig. 5B). Then we wanted to know whether the knock down of
c-FLIPL leads to the apoptosis of B16 cells stimulated by EXO. We
found that the apoptosis of B16 cells with c-FLIPL knock down
showed few differences in comparison with control siRNA knock

FIGURE 2. EXO promoted tumor cell invasion via Fas signaling in vitro. (A) B16 or 3LL tumor cells were incubated with 10 mg of EXO, EXOcont, or
PBS for 2 h, and the cells were plated in the bottom chamber precoated with 50 ml of Matrigel. Forty-eight hours later, the number of cells on the bottom of
the Transwell filter was imaged and quantified. (B) Same experimental procedure as in (A), except that the cells were preincubated with 20 mg/ml of FasLneutralized mAbs or ISO for 2 h (n = 5). Data are representative of three independent experiments. *p , 0.05 versus Cont and EXOcont (A) or EXO+antiFasL (B).

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Despite the observation that FasL+ EXO showed little effect

on apoptosis and proliferation of B16 and 3LL tumor cells, we
further investigated whether EXO can cause other functional
changes in tumor cells. Because it was reported that activation
of Fas signaling can induce motility and invasion of apoptosisresistant tumor cells (13), we reasoned that EXO may increase the
invasive ability of tumor cells in vitro. To validate this, invasiveness assays in vitro were performed. In this assay, B16 or 3LL
tumor cells were placed in the top chamber in serum-free media
with stimulus (EXO, EXOcont, or PBS) for 2 h. The bottom
chamber contained 20% FCS. Forty-eight hours after culture, we
observed an increasing number of B16 or 3LL tumor cells in
the bottom chamber in the EXO group but not in the EXOcont or
PBS group (Fig. 2A). Interestingly, once preneutralized by FasL
mAbs, the ability of EXO to promote tumor cell invasion was
completely abrogated (Fig. 2B). These results suggest that EXO
may increase the invasive ability of tumor cells via Fas signaling
in vitro.

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ACTIVATED T CELL EXOSOMES PROMOTE TUMOR INVASION

FIGURE 3. EXO-mediated activation of Fas signaling induced upregulation of MMP9 expression in tumor cells. (A) B16 tumor cells were treated with
the indicated doses of EXO for 24 h or 10 mg/ml EXO for the indicated times. mRNA expression levels of MMP9 were assayed by quantitative PCR. (B)
Western blot analysis of MMP9 expression in B16 tumor cells stimulated with 10 mg/ml of EXO for the indicated times. (C) Western blot analysis of MMP9
expression in B16 tumor cells stimulated with 10 mg/ml of EXO, EXO preincubated with 20 mg/ml of FasL-neutralized mAbs, or isotype mAbs (ISO) for
2 h. (D) Invasive assay of B16 tumor cells after stimulation with 10 mg/ml of EXO or EXO plus MMP9 inhibitor (MMP9i) (n = 5). Data are representative
of three independent experiments. *p , 0.05, **p , 0.01.

EXO increased the tumor cell lung invasion via Fas signaling
in vivo
To further clarify the effect of EXO on tumor invasion, we performed tumor invasive analysis with the B16 tumor cell-induced
murine lung cancer model. We found that EXO greatly increased the tumor cell lung invasion, as indicated by gross morphology of the lungs (Fig. 6A, left panels) and the number of
invasive nodules (Fig. 6A, right panel). Histological assessment of
lungs showed few tumor colonies in EXOcont and PBS groups but
more numerous tumor colonies in EXO groups (Fig. 6B). After
preincubation with FasL mAbs, the ability of EXO to promote

tumor lung invasion was markedly decreased (Fig. 6). These

results strongly support that EXO increase tumor cell lung invasion via Fas signaling in vivo.
Identification of a counterpart of EXO in vivo from
tumor-activated CD8+ T cells
To further confirm the existence of CD8 and FasL+ exosomes and
their effects on tumor metastasis in tumor mice, we isolated CD8+
exosomes from spleens and draining lymph nodes of control and
tumor mice using CD8+ magnetic beads. The CD8+ exosomes
from tumor tissues were also isolated. As shown in Fig. 7A,
exosomes from 3LL and B16 tumor mice, but not control mice,
expressed FasL; its expression level in exosomes from tumor
tissues was greater than that in tumor lymphocytes. All exosomes
expressed CD8 molecules and exosomal mark proteins, such as
HSP70 and TSG101 (Fig. 7A). After stimulation with exosomes
isolated from tumor mice, but not control mice, 3LL and B16
tumor cells showed increase expression of MMP9 and enhanced
invasive ability. Consistent with their expression of FasL, exosomes from tumor tissue showed stronger stimulatory effect than
did those from tumor lymphocytes (Fig. 7B, 7C). Based on our
results, it can be predicted that the number of lung tumor nodules
in the B16 tumor model of wild-type (WT) mice will be greater
than that in gld mice. We examined the difference in the number
of lung tumor nodules between WT and gld mice; as expected, the
number was greater in WT mice (Fig. 7D). These results indicate
that CD8+ and FasL+ exosomes are naturally generated with tumor
progression and exert an exacerbated effect in tumor invasion.

FIGURE 4. EXO promoted MMP9 expression and tumor cell invasion through ERK and NF-kB pathways. (A) B16 tumor cells were stimulated with
10 mg/ml of EXO or EXOcont for the indicated times and harvested; the extracted crude proteins were subjected to Western blot with Abs against NF-kB,
phosphoNF-kB, ERK, or phospho-ERK. (B) B16 tumor cells were pretreated with NF-kBspecific inhibitor PDTC or ERK1/2-specific inhibitor U0126 for
30 min and then stimulated with 10 mg/ml EXO for 24 h. The expression of MMP9 in B16 tumor cells was detected by Western blot. (C) Using the same
experimental conditions as in (B), the number of cells that migrated through Matrigel and adhered to the bottom of Transwell filter was quantified (n = 5).
Data are representative of three independent experiments. **p , 0.01.

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down (Fig. 5C), which excludes the possibility that the changes
in signal pathways may be caused by increasing cell apoptosis.
Subsequent Western blot analysis showed that the phosphorylation
of ERK and NF-kB decreased significantly when the expression
of c-FLIPL was inhibited by siRNA (Fig. 5D). Moreover, cell
invasion and the expression of MMP9 were also inhibited when
c-FLIPL expression was knocked down (Fig. 5E, 5F). It is known
that NF-kB signals can induce the expression of c-FLIP (33),
which is related to the expression of MMP9 (8). We propose that
a similar pattern might also appear in Fas signaling-activated tumor cells induced by EXO. As shown in Fig. 5G, inhibition of the
NF-kBsignaling pathway decreased the expression of c-FLIPL.
Interestingly, the same result was observed when B16 tumor cells
were treated by specific inhibitors of ERK (Fig. 5G). These results
suggest that c-FLIPL plays an important role in the increase in
MMP9 and tumor invasion induced by EXO.

The Journal of Immunology

5959

Discussion
Stimulation of FasR normally induces an apoptotic death signal.
However, the interaction between Fas and FasL in tumor cells does
not always induce a death signal, and conversely, promotes tumorigenesis (11). Activated CD8+ T cells secrete FasL+ exosomes,
which can induce DC apoptosis and inhibit their ability to stimulate CD8+ CTL responses (19). Until now, the effect of FasL+
exosomes from activated CD8+ CTLs on Fas-resistant tumors was
unknown. In this study, we found that FasL+ EXO did not affect
the apoptosis and proliferation of tumor cells but accelerated their
invasion in vivo via upregulation of MMP9 expression. Moreover,
we identified a counterpart of EXO in tumor-bearing mice that

FIGURE 6. EXO increased tumor cell

lung invasion via Fas signaling in vivo.
(A) A total of 5 3 105 B16 tumor cells
with 30 mg of EXOcont, EXO, 20 mg/ml
of ISO-preincubated EXO, 20 mg/ml
of FasL-neutralized mAbs preincubated
EXO or PBS was injected into C57BL/6
mice through the tail vein. The invasion
of B16 tumor cells into lung (left panels)
and the number of lung nodules were
assessed (right panel) (n = 5). (B) The
invasion of B16 tumor cells into lung was
visualized in H&E-stained pathological
sections. Original magnification 3100.
Data are representative of three independent experiments. **p , 0.01 versus
PBS, EXOcont, and EXO+anti-FasL.

also possessed the ability to increase MMP9 expression and

cancer invasion.
Increasing the expression of FasL during activation is one of the
properties of T lymphocytes. Tumor Ag-specific CTLs and CD8+
T cells activated by tumor Ag are generally the most important
effector cells to attack tumor cells (34). Activated CD8+ T cells
are present within the tumor site, but after being educated by tumor cells, they are in an unresponsive state (35). Our results show
that CD8 and FasL+ EXO could be isolated from tumor tissues,
suggesting that, in the tumor environment, CTLs continuously
secrete FasL+ EXO to promote tumor growth rather than eliminate
tumor tissues (i.e., CTLs in the tumor site are converted from

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FIGURE 5. c-FLIP was critical to the increase in MMP9 expression and tumor invasion induced by EXO. (A) Western blot analysis of c-FLIPL and
c-FLIPS expression in B16 tumor cells stimulated with 10 mg/ml of EXO or EXOcont for the indicated times. (B) B16 cells were transfected with c-FLIPL
(Si) or control (Cont Si) siRNA duplexes for 24 h, and c-FLIPL expression in the cells was detected by Western blot. (C) The apoptosis of Si- or Cont Sitransfected B16 tumor cells treated by EXO were analyzed by FACS (n = 3). (D) After inhibition of c-FLIPL expression by siRNA, the phosphorylation of
NF-kB and ERK in EXO- or EXOcont-stimulated B16 tumor cells was detected by Western blot. (E) After inhibition of c-FLIPL expression by siRNA,
MMP9 expression was detected by Western blot in B16 cells stimulated with 10 mg/ml of EXO or EXOcont for 24 h. (F) After inhibition of c-FLIPL
expression by siRNA, the number of B16 tumor cells, stimulated with 10 mg/ml of EXO or EXOcont for 2 h, which migrated through Matrigel to the bottom
of the Transwell filter was quantified (n = 5). (G) Western blot analysis of c-FLIPL expression in EXO-stimulated B16 tumor cells pretreated with NF-kB
specific inhibitor PDTC or ERK1/2-specific inhibitor U0126. Data are representative of three independent experiments. **p , 0.01 versus Cont or Si+EXO.

5960

ACTIVATED T CELL EXOSOMES PROMOTE TUMOR INVASION

a guard into an accessory of tumor progression under such circumstances). In the FACS results in Fig. 1A, EXO is adsorbed
onto latex with an intact membrane structure, and if FasL can be
detected, it is probably membrane-bound. Moreover, in the results
from Western blotting, we detected a band at 40 kDa, which is
similar to the molecular mass of membrane-bound FasL, whereas
we detected no signal at 26 kDa, which is similar to the molecular
mass of soluble FasL (data not shown). CTLs themselves also
express FasL and may trigger Fas signal in tumor cells, which
probably promotes MMP9 expression and the invasive capability
of tumor cells. However, there are very limited numbers of CTLs
in tumor sites (35). Furthermore, unlike exosomes, whose diameters are ,100 nm, allowing them to freely pass through basement
membrane (36), immigration of CTLs from spleen and lymph
nodes into the tumor site is more complicated. It can be presumed
that more FasL+ EXO will accumulate in the tumor location than
CTLs, which will probably exert a stronger effect of invasive
promotion on tumors than their parent CTLs.
Many molecules were reported to be associated with tumor invasion. Among them, MMP2, MMP9, cyclooxygenase-2 (COX2),
b-catenin, and urokinase plasminogen activator, are considered
the primary tumor-invasive effectors (13, 20, 3739). In our study,
we found that, after stimulation by EXO, expression of the mRNA
levels of MMP2, MMP9, and COX2 was significantly elevated in
3LL and B16 tumor cells. However, we could not detect any
change in the protein expression of MMP2 in either cell type (data
not shown). Although the expression of COX2 mRNA increased in
both cells, almost no PGE2 secretion could be detected with or
without EXO stimulation in B16 tumor cells (data not shown).
Interestingly, urokinase plasminogen activator, which is reported
to be related to tumor invasion mediated by Fas signaling (13),
showed no increase in either cell type after EXO stimulation (data
not shown). Among those tumor-invasive effectors, we detected
only elevated mRNA and protein levels of MMP9 in both cancer
cell lines. It is likely that MMP9 is a universal effector molecule
that promotes tumor invasion induced by FasL in EXO.
Although increasing tumors have been found to possess Fas
resistance, the signaling mechanisms of tumor Fas resistance remain unknown. c-FLIP is an endogenous inhibitor of death
receptor-induced apoptosis, and it plays a critical role in tumor-

apoptosis resistance via the caspase-8 pathway (28, 40). In this

study, we found that, only 5 min after FasL+ EXO stimulation, the
amount of c-FLIPL increased markedly, and this was sustained for
$24 h. c-FLIP is extremely unstable and can be quickly degraded
by the ubiquitination pathway (41, 42). The early increase in cFLIPL probably results from the inhibition of its degradation by
ubiquitin. Although it was reported that protein kinase C-mediated
phosphorylation regulates c-FLIP ubiquitylation and stability
(43), the mechanism underlying the ubiquitylation inhibition of
c-FLIPL induced by EXO remains to be elucidated. As a result
of positive feedback, NF-kB and ERK activation triggered by
c-FLIPL probably promotes the synthesis of c-FLIPL, which may
explain why it remains at high levels for a long time after EXO
stimulation. Although the Fas-mediated apoptosis after c-FLIP
siRNA inhibition in B16 cells showed no difference compared
with control siRNA, we cannot exclude that c-FLIP plays a role in
the Fas resistance of B16 cells, because the effect of c-FLIP knock
down is mild, and the expression of c-FLIP decreases only 50%.
It was reported that activation of the ERK pathway and subsequent
induction of antiapoptotic proteins, such as Bcl-2, play important
roles in the survival of CD8+ T and NK cells (44, 45) In B16 tumor
cells stimulated by FasL+ EXO, we found that Bcl-2 was upregulated after EXO stimulation, and specific inhibitors of ERK and
NF-kB activation could decrease the expression of Bcl-2. But
there was no increasing apoptosis in the B16 cells treated with
Bcl-2 inhibitor compared with the group treated with DMSO or
control, which suggests that Bcl-2 may not be involved in the Fas
resistance of B16 tumor cells induced by FasL-expressed EXO
(data not shown). Tumor cells were also demonstrated to synthesize a protective protein to resistance apoptosis induced by Fas
signaling (46). A high level of a protein tyrosine phosphatase, Fasassociated phosphatase-1, which can interfere with transduction of
the apoptotic signal by Fas (47) upon interaction of its third PDZ
domain with the C-terminal three amino acids of Fas (48), has
been implicated as a possible agent causing preferential survival
of human pancreatic adenocarcinomas stimulated by FasL (46). It
is possible that Fas-associated phosphatase-1 contributes to the
Fas resistance in B16 tumor cells stimulated by EXO, but this
needs to be explored further.

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FIGURE 7. Identification of a counterpart of EXO in vivo from tumor-activated CD8+ T cells. CD8+ exosomes from spleens and draining lymph nodes of
control (EXOcln) and tumor mice (EXOtln), as well as tumor tissue of tumor mice (EXOts), were isolated by CD8+ magnetic beads. (A) Western blot
analysis of FasL, CD8, HSP70, and TSG101 expression in EXOcln, EXOtln, and EXOts. (B) Western blot analysis of MMP9 expression in tumor cells after
stimulation with 10 mg/ml of EXOcln, EXOtln, and EXOts for 24 h. (C) The number of invasive tumor cells stimulated with EXOcln, EXOtln, and EXOts
through Matrigel to the bottom of the Transwell filter was quantified (n = 5). *p , 0.05, **p , 0.01 versus Cont and EXOcln. (D) A total of 5 3 105 B16
tumor cells was injected into C57BL/6 (WT) or gld (GLD) mice via the tail vein. The number of lung nodules was measured (n = 5). Data are representative
of three independent experiments. **p , 0.01.

The Journal of Immunology

In conclusion, we report that CTLs are diverted from tumor
inhibition to tumor promotion through secreting FasL+ EXO. Our
data showed that EXO could activate c-FLIPL and ERK- and NFkBsignaling pathways to increase MMP9 expression in tumor
cells, which is Fas/FasL dependent. The upregulation of MMP9
leads to exacerbated tumor invasion, and these tumor-promoting
exosomes are proved to exist in tumor-bearing mice. Our results
reveal a novel mechanism of tumor immune escape and suggest
that strategies to inhibit, rather than to promote, Fas activity
should be considered during cancer therapy.

Disclosures
The authors have no financial conflicts of interest.

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