Proc. Natl. Acad. Sci. USA
Vol. 94, pp. 6420–6425, June 1997
Medical Sciences

Expression of Fas ligand in liver metastases ofhuman colonic adenocarcinomas(tumor escapeyimmune surveillanceycolonization)

KATSUYA SHIRAKI*, NAOKI TSUJI*†, TOSHI SHIODA‡, KURT J. ISSELBACHER*‡, AND HIROSHI TAKAHASHI*§*Gastrointestinal Unit and Department of Medicine, Harvard Medical School and Massachusetts General Hospital, Boston, MA 02114; and ‡Tumor Biology
Laboratory, Massachusetts General Hospital Cancer Center, Charlestown, MA 02129

Contributed by Kurt J. Isselbacher, March 27, 1997

We found that FasL was strongly expressed in hepatic
metastatic tumors of colonic adenocarcinomas. Since thisexpressed FasL was functional, it may be important for theelimination of activated T lymphocytes that attempt to attack
tumor cells and it may support the belief that FasL plays apivotal role in the escape of tumor cells from destruction by thehost’s immune system (13–17). In addition, the constitutive
expression of FasL in hepatic metastatic tumors suggests thatit may also play a critical role in the liver colonization of coloncancer cells.

ABSTRACTFas ligand (FasL) plays a pivotal role inlymphocyte cytotoxicity and the maintenance of immunological homeostasis. Since FasL has been implicated in theexistence of immunologically privileged body sites by inducing
apoptosis of activated T lymphocytes, we investigated theexpression of FasL in human colon cancers. We found that two
out of seven primary tumors and all four hepatic metastatic
tumors of surgically obtained colonic adenocarcinoma expressed FasL mRNA and protein, detected by reverse transcription-coupled PCR and by immunohistochemical staining, respectively. Expression of FasL was not detected innormal colonic epithelial cells. FasL mRNA was also expressed in some human colonic adenocarcinoma cell lines
including SW480, SW1116, and LS180 cells. Cell-surfaceassociated FasL was detected in these human colon cancer
cells by f luorescence immunocytochemical staining. In addition, the expressed FasL was demonstrated to be functional,
since coculture experiments using FasL-expressing SW480
cells resulted in apoptosis of Jurkat T leukemia cells that are
sensitive to Fas-mediated apoptosis, and this process was
specifically inhibited by the neutralizing anti-human FasL
antibody. Thus, our findings and other data suggest analternative mechanism that enables tumors to evade immune
destruction by inducing apoptosis in activated T lymphocytes.
Furthermore, constitutive expression of FasL in hepatic metastatic tumors suggests that FasL may also be important intheir colonization in the liver through induction of apoptosis
in the surrounding Fas-expressing hepatocytes.

MATERIALS AND METHODS
Cell Lines. Human colonic adenocarcinoma cell lines LS180,
HT-29, Caco-2, SW480, SW403, SW1116, SK-CO-1,
COLO320DM, and COLO320HSR and the Jurkat human T
leukemia cell line were obtained from American Type Culture
Collection. KM12C cells were kindly provided by Isaiah J.
Fidler (University of Texas, M.D. Anderson Cancer Center,
Houston) (18). Jurkat cells, COLO320DM, and COLO320HSR
were maintained in RPMI 1640 medium supplemented with 10%
heat-inactivated fetal bovine serum with penicillin (100 unitsyml)
and streptomycin (100 mgyml); all other colon cancer cell lines
were maintained in DMEM.
To establish tumor xenografts of human colonic adenocarcinomas, 4- to 6-week-old BALByc nude mice (Taconic
Farms) were injected subcutaneously in the left shoulder with
1 3 106 SW480 cells or KM12C cells in 0.1 ml of sterile 20 mM
phosphate-buffered saline (PBS), pH 7.2. When they reacheda size of approximately 10 mm in diameter, these tumors were
snap-frozen in liquid nitrogen, embedded in OCT compound(Miles Scientific), and stored at 280°C for histologic study.
Human Colonic Adenocarcinoma Tissues. Primary and hepatic metastatic tumors of colon adenocarcinoma, as well asadjacent normal colonic mucosa counterparts, were obtained
fresh at the time of surgery, immediately frozen in liquid
nitrogen, and stored at 280°C. Cryostat sections were cut fromthese snap-frozen tissues, stained with hematoxylinyeosin, andexamined histologically to identify and separate tumor andnonneoplastic areas for RNA extraction and immunohistological study.
Detection of FasL Transcript. The expression of FasLmRNA was determined by reverse transcription (RT) of total
RNA followed by PCR analysis (RT-PCR) (19). Approximately 107 cells of human colonic adenocarcinoma cells or
50–100 mg of colon adenocarcinoma tissue from primary andhepatic metastatic tumors was homogenized with 1 ml of
Ultraspec RNA reagent (Biotecx Laboratories, Houston) by

The Fas ligand (FasL) and its receptor (Fas, CD95) are a setof regulatory components in the immune system (1–4). Activation of the Fas by FasL results in apoptosis of many cell types(5, 6), and this process has been shown to play a critical rolein the maintenance of immunological homeostasis and peripheral tolerance by deletion of activated T lymphocytes (7–10).
In addition, it has been found that cells in immunologicallyprivileged sites, such as Sertoli’s cells of the testis and parenchymal cells of the anterior chamber of the eye, express FasL.
Any activated T cell bearing Fas that enters such a site would
encounter cells expressing FasL and receive a death signal,
thereby preventing an immune response (11, 12).
It has recently been demonstrated that FasL is expressed inhuman melanoma (13), hepatocellular carcinoma (14), and acolon cancer cell line (15). These studies suggested that theexpression of FasL may play an important role in establishing
immunologically privileged environments that allow tumors toescape the host’s immune surveillance. We therefore investigated the expression of FasL in primary colon cancers, liver
metastases, and colon cancer cell lines.

Abbreviations: FasL, Fas ligand; RT, reverse transcription; MTT, 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide.
†N.T. substantially contributed to this work and should also be
considered a first author.
§To whom reprint requests should be addressed at: Gastrointestinal
Unit, Massachusetts General Hospital, Fruit Street, Boston, MA
02114. e-mail: Takahashi.Hiroshi@MGH.HARVARD.EDU.

The publication costs of this article were defrayed in part by page charge
payment. This article must therefore be hereby marked ‘‘advertisement’’ inaccordance with 18 U.S.C. §1734 solely to indicate this fact.
© 1997 by The National Academy of Sciences 0027-8424y97y946420-6$2.00y0

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using a Mini-Beadbeater (Biospec Products, Bartlesville, OK),and total RNA was isolated according to the manufacturer’s
protocol. cDNAs were synthesized by extension of (dT)12–18
primers with 200 units of SuperScript II reverse transcriptase(Life Technologies) in a mixture containing 2 mg of total RNA
digested by RNase-free DNase I (2 unitsymg of RNA) (Promega) for 30 min at 37°C. PCR of the cDNA was performed
in a final volume of 50 ml containing all four dNTPs (each at
200 mM), 2.5 mM MgCl2, 2.5 units of AmpliTaq Gold (Perkin–
Elmer), and each primer at 0.4 mM. The amplification cycles
were 94°C for 30 s, 55°C for 1 min, and 72°C for 1.5 min.
The PCR products were separated by electrophoresis on a
2% agarose gel after 35 cycles (231-bp human FasL fragment)or on a 1.5% agarose gel after 30 cycles (838-bp human b-actin
fragment) and visualized by ethidium bromide staining. Amplification of human b-actin served as a control for sample
loading and integrity. Primers used for amplification were
human FasL sense primer corresponding to nucleotides 483–
503 (59-CTGGGGATGTTTCAGCTCTTC-39), and antisense
primer complementary to nucleotides 713– 693 (59-CTTCACTCCAGAAAGCAGGAC-39), franking introns 1, 2,
and 3 (20, 21), and b-actin sense primer corresponding to
nucleotides 578 – 609 (59-ATCTGGCACCACACCTTCTACAATGAGCTGCG-39), and antisense primer complementary to nucleotides 1415–1384 (59-CGTCATACTCCTGCTTGCTGATCCACATCTGC-39). Total RNA was obtained fromhuman peripheral T lymphocytes stimulated with ionomycin
and phorbol 12-myristate 13-acetate for 24 hr as described (22)and served as a positive control for detection of FasL mRNA.
Detection of FasL by Immunof luorescence Cell Staining.
Human colon adenocarcinoma cells were grown for 48 hr ona sterile circular cover glass (25 mm diameter) placed in thebottom of a six-well culture plate. These live tumor cells wereincubated for 1 hr at 4°C with rabbit polyclonal IgG antibody
against the carboxyl terminus (anti-FasL C-20) or against theamino terminus (anti-FasL N-20) of human FasL (Santa Cruz
Biotechnology) at 1.0 mgyml. The cells were then incubatedwith fluorescein isothiocyanate-conjugated goat anti-rabbit
IgG secondary antibody (Sigma) at 2.0 mgyml for 30 min at
4°C, and visualized with a fluorescence microscope after
fixation with 2% paraformaldehyde. Similar experiments were
performed with permeabilized cells fixed with 2% paraformaldehyde for 1 hr at 4°C and treated with 0.1% saponin in
PBS for 15 min at room temperature. Staining with isotypematched purified immunoglobulin (rabbit IgG; Sigma) thatwas isolated from pooled normal rabbit serum was performed
as a negative control, and all primary antibodies were dissolved
in PBS containing 1.5% normal goat serum.
Immunohistochemical Staining. Cryostat sections of surgically excised human colon cancers or tumor xenografts ofcancer cell lines grown in nude mice were cut from snap-frozen
tissues embedded in OCT compound (Miles), air-dried onto
glass slides at room temperature, and fixed in 100% acetone at
220°C for 5 min. These tissue sections were incubated with
anti-FasL N-20 rabbit IgG antibody (0.05 mgyml) overnight at
4°C. Bound primary antibodies were visualized by the avidinbiotin complex immunoperoxidase method, using the Vectastain ABC Elite peroxidase kit (Vector Laboratories) and
3,39-diaminobenzidine as chromogen, and counterstained withmethyl green.
Apoptosis Assay. The FasL-expressing SW480 colon cancer
cells were subconfluently seeded in 6- or 96-well tissue culture
plates (Falcon) and allowed to grow to confluence. The cells
were then washed once with sterile PBS and fixed with 2%paraformaldehyde at 4°C for 1 hr. After the cells were washed
two times with serum-free RPMI 1640 medium, 2 ml or 200 ml
of Jurkat cell suspension (5 3 105 cells per ml of complete
RPMI 1640 medium) was added to each 6- or 96-well, respectively.

Proc. Natl. Acad. Sci. USA 94 (1997)

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After 72 hr of coculture, floating Jurkat cells were collectedfrom the six-well tissue culture plates after gentle rocking of
the culture plate. After centrifugation, the cell pellets were
resuspended in 1.0 ml of hypotonic fluorochrome solution(propidium iodide at 50 mgyml in 0.1% sodium citrate and0.1% Triton X-100) and incubated at 4°C for 4 hr in the dark
(23). For detection of apoptosis, 20,000 events were measured
per sample, using a FACScan flow cytometer (Becton Dickinson). The propidium iodide was excited with 488 nm of argon
laser light and was collected as red fluorescence by using a
560-nm dichroic mirror and a 600-nm band pass filter. Percentage of apoptotic cells was determined by evaluating hypodiploid nuclei. The FasL-negative KM12C cells were confluently grown in six-well tissue culture plates, fixed with 2%paraformaldehyde, and used as negative control effector cells
for FasL-expressing SW480.
To quantitatively assess the proportion of dead Jurkat cells,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT) assay was also performed (24). Briefly, MTT stock
solution (2.5 mgyml in H2O) was added to 96-well culture
plates (10 ml per well) at the end of coculture and incubatedfor 4 hr at 37°C. After removing culture supernatant by
centrifugation, a blue formazan product formed in the live cellsby oxidation was solubilized by acid isopropanol (0.04 M HCl
in isopropanol) and the absorbance of each well was measured
with a microculture plate reader (model 7520 microplate
reader, Cambridge Technology, Cambridge, MA) at 590 nm.
To block FasL-induced apoptosis, Jurkat cells were cocultured with fixed SW480 cells in the presence of hamsteranti-human FasL IgG neutralizing mAb (clone 4H9; MBL
International, Watertown, MA) at 1 mgyml (25) or isotypematched irrelevant hamster mAb (anti-trinitrophenol IgG
mAb, PharMingen). As a control for Fas-mediated apoptosis,
Jurkat cells were cultured in the presence of mouse anti-humanFas IgM mAb (clone CH11, MBL International) at 100 ngyml(26), or irrelevant mouse IgM mAb derived from myeloma
cells (Sigma). All antibodies were treated with immobilized
endotoxin affinity ligand (END-X endotoxin neutralization
resin, Associates of Cape Cod) for overnight at 4°C to remove
any possible endotoxin contamination.

RESULTS
FasL mRNA Expression in Human Colonic Adenocarcinoma Cell Lines and Tumor Tissues. To investigate theexpression of FasL mRNA in human colonic adenocarcinoma
cell lines and tumor tissues, total RNA was extracted fromsubconfluent cell cultures or snap-frozen primary tumors andhepatic metastases and analyzed by RT-PCR. As shown in Fig.
1, a 231-bp RT-PCR fragment of FasL transcript was detectedby ethidium bromide in LS 180, SW480, and SW1116 humancolonic adenocarcinoma cell lines and very weakly detected in
HT-29 and SK-CO-1 cells. SW480 cells expressed FasL transcript more strongly than other colon cancer cell lines. FasL
transcript was not detected in Caco-2, COLO320 MD,
COLO320 HSR, SW403, and KM12C cells.
There was strong expression of FasL mRNA in all (4 of 4)
hepatic metastatic tumors (Fig. 2, lanes 1–4). In contrast, FasL
transcript was detected only in two of seven primary coloncancers (Fig. 2, lanes 7 and 11), and its intensity was less thanin the hepatic metastatic tumors (Fig. 2).
Cell Surface Expression of FasL in Colon Cancer Cell Lines.
Immunofluorescence cell staining for FasL expressed in human colon cancer cell lines were performed with rabbit
polyclonal antibodies against the carboxyl terminus (anti-FasL
C-20) and amino terminus (anti-FasL N-20) of FasL. To
determine the orientation of FasL protein expressed on thecell surface of human colon cancer cell lines, nonpermeabilized live SW480 cells were stained by these antibodies. FasL
was detected only by anti-FasL C-20 antibody (Fig. 3A) but not6422

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Proc. Natl. Acad. Sci. USA 94 (1997)

FIG. 1. Detection of FasL mRNA in human colonic adenocarcinoma cell lines by RT-PCR. A 231-bp human FasL-specific sequence (A) andan 838-bp b-actin sequence (B) were amplified from total RNA isolated from human colon cancer cell lines, separated by agarose gel electrophoresis,
and visualized by ethidium bromide staining.

by anti-FasL N-20 antibody or control rabbit IgG (data notshown). Staining was particularly strong in the outer linings ofthese nonpermeabilized cells, indicating cell surface localization of FasL. In contrast, when SW480 cells were permeabilized by saponin, their FasL was detected by staining with bothanti-FasL C-20 and N-20 antibodies and the staining showeda diffuse cytoplasmic pattern (Fig. 3B). These results wereconsistent with the previous observation that FasL is a type IImembrane protein (amino-terminal is cytoplasmic) (2, 4, 5,
21). Other human cancer cell lines that expressed FasL mRNA
(Fig. 1) demonstrated similar staining patterns, but FasL
protein was not detected in FasL mRNA-negative KM12C
cells by either anti-FasL C-20 or N-20 antibodies (data notshown).
To exclude the possibility that FasL expression in humancolonic adenocarcinoma cell lines was a consequence of in vitro
culture conditions, FasL expression was examined in the
xenograft of SW480 and KM12C cells grown subcutaneously innude mice by immunoperoxidase staining using anti-FasL
N-20 antibody. FasL was detected in solid tumors of SW480
cells (Fig. 3C) but not in tumors of KM12C (Fig. 3D). No
staining was observed with a nonspecific control antibody(data not shown).
Expression of FasL in Colon Cancer Tissues. Expression of
FasL protein in liver metastases and primary colon cancer
tissues was examined by immunoperoxidase staining using
anti-FasL N-20 antibody. Figs. 3 E and F show typical staining
patterns of hepatic metastatic tumors for FasL. The staining of
FasL was limited to the colon cancer cells and demonstrated
a diffuse cellular staining pattern (Fig. 3 E and F). All (4 of 4)
liver metastases expressed FasL protein. In contrast, themajority (5 of 7) of the primary tumors expressed minimal, if
any, FasL protein (Fig. 3G). There was very little or no staining
in normal colon mucosa (Fig. 3H).
Induction of Lymphocyte Apoptosis. To determine whether
or not FasL expressed in colonic adenocarcinoma is functional,
coculture was performed between FasL-expressing SW480
cells and Jurkat cells of Fas-expressing T cell leukemia cells(10, 14, 15, 27). As demonstrated in Fig. 4A, untreated Jurkat
cells showed a typical diploid DNA peak. Jurkat cells cocultured with FasL-positive SW480 cells showed a significant
reduction of the number of nuclei with diploid DNA content
with a marked increase in the hypodiploid DNA peak, indicating apoptosis of Jurkat cells (14, 23). This shift in the
nuclear content of DNA was not observed when Jurkat cells
were cocultured with FasL-negative KM12C cells.
The cytotoxicity of SW480 cells to Jurkat cells was also
quantified by MTT assay. Viability of Jurkat cells decreased
notably when they were cocultured with SW480 cells (Fig. 4B).
This cytotoxicity was mediated by FasL, because it was inhibited by a neutralizing anti-human FasL mAb, but not by anirrelevant control mAb (Fig. 4B).

DISCUSSION
FasL is a member of the tumor necrosis factor family (2, 4, 5,21) and mediates apoptosis of many cell types, including bothtransformed and nontransformed cells (1, 5, 6, 28). FasL has
been shown to play a pivotal role in lymphocyte cytotoxicity(29–31) and is also important in mediating autocrine suicide in
activated T lymphocytes (3, 7–10, 32). Since tumor progression
and the metastatic process may require an evasion of host

FIG. 2. Detection of FasL mRNA in primary and hepatic metastatic human colonic adenocarcinoma tissues by RT-PCR. Agarose gelelectrophoresis and ethidium bromide staining of 231-bp FasL RT-PCR products (A) and 838-bp b-actin RT-PCR products (B). Liver Mets, hepatic
metastatic tumor tissues of human colonic adenocarcinoma; Primary Colon CA, primary tumor tissues of human colonic adenocarcinoma; P, human
FasL cDNA synthesized from total RNA of human peripheral blood T lymphocytes stimulated with ionomycin and phorbol 12-myristate 13-acetate(positive control) (22).

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Proc. Natl. Acad. Sci. USA 94 (1997)

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FIG. 3. Expression of FasL protein in human colonic adenocarcinoma. (A) Immunofluorescence staining of live SW480 human colonicadenocarcinoma cells with anti-FasL C-20 polyclonal antibody (31,000). (B) Immunofluorescence staining of permeabilized SW480 cells with
anti-FasL N-20 polyclonal antibody (31,000). (C and D) Immunoperoxidase staining of cryostat sections of SW480 (C) and KM12C (D) xenograft
tumors with anti-FasL N-20 polyclonal antibody (3400). (E and F) Immunoperoxidase staining of hepatic metastatic lesion of human colonicadenocarcinoma tissue (case 4 in Fig. 2) with anti-FasL N-20 polyclonal antibody (3200). F is a higher magnification (3400) of E. Note that especiallyas seen in F, every tumor cell strongly expressed FasL protein. (G and H) Immunoperoxidase staining of primary tumor of human colonicadenocarcinoma tissue (G) and normal human colon mucosa (H) with anti-FasL N-20 polyclonal antibody (3200).

immune surveillance, we investigated whether colon cancers
express FasL. We found that FasL mRNA was expressed inseveral human colon cancer cell lines (3 of 10) and primarytumors (2 of 7). Surprisingly all (4 of 4) liver metastases
expressed FasL mRNA strongly. Protein expression of FasL
was confirmed by immunoperoxidase staining. This observation contrasts with the lack of expression of Fas in liver

metastases (data not shown). It was also demonstrated that theexpressed FasL is functional; FasL-expressing SW480 humancolonic adenocarcinoma cells induced apoptosis of Jurkat T
cells, and this process was specifically inhibited by treatment
with a neutralizing anti-human FasL antibody. These findings
are consistent with those reported by O’Connell et al. (15) that
SW620 human colon cancer cells express functional FasL.

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Proc. Natl. Acad. Sci. USA 94 (1997)

FIG. 4. Apoptosis of Jurkat cells induced by SW480 cells. (A) FACScan analysis of propidium iodide staining of nucleus of Jurkat cells cocultured
with SW480 cells (Jurkat 1 SW480) or with KM12C cells (Jurkat 1 KM12C). After coculture (72 hr), Jurkat cells were isolated and stained withpropidium iodine. The DNA content of Jurkat cells was examined by flow cytometry. (B) MTT assay of Jurkat cells alone or cocultivated with
SW480 cells in the presence of irrelevant mouse IgM mAb (irrelevant IgM), mouse anti-human Fas IgM mAb (anti-Fas IgM), hamsteranti-trinitrophenol mAb (irrelevant IgG), or hamster anti-human FasL IgG neutralizing mAb (anti-FasL IgG).

It has been shown previously that the number of tumorinfiltrating lymphocytes in primary colorectal carcinomas decreases in parallel with tumor progression and metastasis (33).
This observation supports the hypothesis that local anti-tumor
immunity conferred by the infiltrating lymphocytes may play
an important role in controlling progression and metastasis(34, 35). Furthermore, it has recently been proposed that FasL
expressed on tumor cells may be responsible for the elimination of tumor-infiltrating lymphocytes in vivo (13), promoting
progression and metastasis through the evasion of the hostimmune response. In this regard, the production of FasL bymurine T cell lymphoma cells transfected with FasL gene has
been show to induce a CD81 T cell-mediated protective
immunity against subsequent challenge with FasL-negative
parental tumor cells in vivo (36); it will be interesting todetermine whether or not FasL-expressing T cell lymphoma
cells escape from this protective immunity by induction of
apoptosis in CD81 T cells.
We propose that FasL expressed on the surface of colonicadenocarcinoma cells may have an additional role in malignancy; namely, FasL may be important in the colonization ofcolon cancer cells in the liver, a major target of metastasis (37).
The metastatic cascade is a complex series of processes

including angiogenesis, intravasation of tumor cells, transport
by the circulation, adhesive interaction with endothelial cells,
extravasation, and colonization of the target organ (38–41).
Only a small subpopulation of cells from heterogeneous
primary tumors appears able to form metastatic colonies (42,43). The survival and proliferation of these metastatic cells
depend on various biological properties, such as resistance tohost defense mechanisms, regulation of adhesion molecules,
and enzymes that degrade basement membranes (39, 40, 43,44). In addition, liver colonization competence is a critical
determinant in colon cancer metastasis (41). FasL expression
on colon cancer cells may enhance liver colonization competence through induction of apoptosis in the Fas-expressing
hepatocytes (45–47). This concept was indirectly supported by
our finding that FasL was expressed strongly in all the hepatic
metastatic tumors tested (4 of 4), wherease less than 30% of
the primary colon cancer cells expressed FasL. Thus, FasL
expression on tumor cells may be important in enhancing livercolonization through the induction of apoptosis of the hepatocytes at metastatic foci.
Fig. 5 summarizes a proposed working hypothesis on the
FasL-mediated colon cancer–host interactions, as derived
from our present data and from the previous literature.
FasL-expressing colon cancer cells may induce apoptosis oftumor-infiltrating lymphocytes as well as of hepatocytes but
not of cancer cells themselves, which are generally resistant toFas-mediated apoptosis (15, 48). Thus, FasL in colon cancer
cells may play a pivotal role in escaping the host’s immune
system and promoting liver colonization of colonic adenocarcinoma. Further evaluation of these possible roles of FasL andthe regulation of FasL expression in malignant cells should becritically important for the development of new strategies forcontrolling the growth of malignant cells that escape hostimmune surveillance and spread to the liver.
This work was supported by Grants CA57584 and NIDDK43351
from the National Institutes of Health and an Educational Grant from
Toray Industries, Inc. (Japan).
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FIG. 5. Hypothetical models of FasL-expressing human colonicadenocarcinomas in tumor escape and liver colonization.

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