Research Article Addressing the ‘‘Fas Counterattack’’ Controversy: Blocking Fas Ligand Expression Suppresses Tumor Immune Evasion of Colon Cancer In vivo Aideen E. Ryan,1 Fergus Shanahan,1,2 Joe O’Connell,1,2 and Aileen M. Houston1,2 1Department of Medicine, Cork University Hospital, and 2Alimentary Pharmabiotic Centre, National University of Ireland Cork, Cork, Ireland Abstract factor (TNF) family (2). FasL interacts with its receptor, Fas Fas ligand (FasL/CD95L) is a transmembrane protein (CD95/APO-1), and can trigger a cascade of subcellular events belonging to the tumor necrosis factor superfamily that culminating in the apoptotic cell death of sensitive cells (3). This can trigger apoptotic cell death following ligation to its interaction plays an important role in immune homeostasis (4) receptor, Fas (CD95/APO-1). Expression of FasL may help to and in the maintenance of immune privilege in sites such as the maintain tumor cells in a state of immune privilege by eye (5) and testis (6). In these sites, constitutive expression of inducing apoptosis of antitumor immune effector cells—the FasL may limit inflammatory responses and maintain relative ‘‘Fas counterattack.’’ However, the ability of FasL to mediate immunosuppression by inducing apoptosis in infiltrating proin- flammatory lymphocytes. tumor immune privilege is controversial due to studies that indicate FasL has both pro- and anti-inflammatory activities. The discovery that FasL is also expressed by a variety of tumor To resolve this controversy and functionally define the role of cells raised the possibility that FasL may mediate immune privilege FasL in tumor immune evasion, we investigated if suppres- in human tumors (7–9). Activated T cells express Fas and are sion of endogenously expressed FasL in colon tumor cells sensitive to Fas-mediated apoptosis (10). Thus, up-regulation of resulted in reduced tumor development and improved FasL expression by tumor cells may enable the tumor cells to kill antitumor immune challenge in vivo. Specifically, FasL infiltrating antitumor lymphocytes. Numerous studies have shown expression in CMT93 colon carcinoma cells was down- that tumor-expressed FasL is capable of killing Fas-bearing, regulated following stable transfection with a plasmid sensitive cells in vitro (7, 9), whereas expression of FasL by human encoding antisense FasL cDNA. Down-regulation of FasL tumors is associated with apoptosis and loss of tumor-infiltrating expression had no effect on tumor growth in vitro but lymphocytes (TIL) in vivo (11, 12). significantly reduced tumor development in syngeneic im- However, the role of FasL in tumor-mediated immunosuppres- munocompetent mice in vivo. Tumor size was also signifi- sion is controversial. In addition to its well-defined role in cantly decreased. Reduced FasL expression by tumor cells led apoptosis, Fas/FasL interactions may mediate other responses to increased lymphocyte infiltration. The overall level of such as cytokine secretion and inflammation. In fact, genetically neutrophils present in all of the tumors examined was low, engineered overexpression of FasL in allografts of tissues and with no difference between the tumors, irrespective of FasL tumor cells in many instances targeted these cells for rapid expression. Thus, down-regulation of FasL expression by rejection in vivo (13–15). Rejection was mediated primarily by colon tumor cells results in an improved antitumor immune neutrophils. These findings contrast with others showing an challenge in vivo, providing functional evidence in favor of immunoprotective effect of FasL (16–18). Reasons proposed to the ‘‘Fas counterattack’’ as a mechanism of tumor immune account for these discrepancies include differences in the level of evasion. (Cancer Res 2005; 65(21): 9817-23) FasL expressed by the allografts (17, 19), sensitivity of the transduced cells to apoptosis (20), overexpression of membrane- bound FasL (21), and/or the moderating effects of the local Introduction microenvironment (22). These studies indicate that FasL can be Tumor escape is a major obstacle to successful immunotherapy either immunoprotective or immunodestructive, and question the (1). Despite compelling evidence that immunogenic tumors can ability of FasL to mediate tumor immune privilege in vivo. be rejected by the immune system under optimum conditions, a Overexpression of some proteins can lead to atypical con- large number of tumors continue to grow and evade immune- sequences. In addition, significant neutrophil recruitment and mediated elimination. During carcinogenesis, tumors develop inflammation has not been observed in tumors endogenously multiple mechanisms to evade the host immune response. Up- expressing FasL in vivo. Thus, to functionally investigate the role of regulation of Fas ligand (FasL/CD95L) expression may represent FasL in tumor immune escape and to avoid any potential artifacts one such mechanism. FasL was first identified in 1993 as a 40 kDa of overexpression systems, we investigated whether inhibition of type II transmembrane protein belonging to the tumor necrosis native FasL expression resulted in reduced tumor development and improved antitumor immune challenge in vivo. In the present study, we specifically inhibited FasL expression in murine colon tumor cells using antisense RNA. Relative to controls, we found Requests for reprints: Aileen M. Houston, Department of Medicine, Clinical that tumor formation by FasL antisense–transfected cells was Science Building, Cork University Hospital, Wilton, Cork, Ireland. Phone: 353-21-490- significantly reduced following s.c. injection into syngeneic C57BL/ 1226; Fax: 353-21-434-5300; E-mail: [email protected]. I2005 American Association for Cancer Research. 6 mice. In those tumors that did develop, leukocyte infiltration was doi:10.1158/0008-5472.CAN-05-1462 significantly increased. Interestingly, despite strong expression of www.aacrjournals.org 9817 Cancer Res 2005; 65: (21). November 1, 2005 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2005 American Association for Cancer Research. Cancer Research constitutive FasL in the control tumors, neutrophil recruitment did incorporates into the plasma membrane, allowing the identification of not occur. Taken together, our results provide for the first time target cells (23). Aliquots of the target cells (500 AL) were incubated in definitive, functional evidence in favor of a role for FasL in tumor triplicate for 16 hours with the effector cells at the indicated effector-to- immune evasion. target cell ratios. To block the Fas pathway, neutralizing anti-FasL monoclonal antibody (MFL3, 10 Ag/mL; PharMingen, San Diego, CA) was preincubated on the monolayer for 1 hour at 37jC, followed by the addition Materials and Methods of the labeled Jurkat T cells. Cells were harvested, washed in PBS, and Mice. Four- to six-week-old female C57BL/6 mice were obtained from stained with Annexin V-FITC (BD PharMingen). The percentage of PKH-26/ Harlan UK Ltd., (Oxon, United Kingdom) and maintained in the animal Annexin V dual positive cells was determined by flow cytometry using a facility of University College Cork. Animal experiments were carried out in FACSCalibur flow cytometer and Cell Quest software (Becton Dickinson, accordance with institutional guidelines, using an Animal Research San Jose, CA). Committee–approved protocol. The mice were maintained in standard Detection of soluble Fas ligand. Soluble FasL was detected in the environmental conditions with free access to food and water and were culture supernatant of CMT93 cells (transfected and nontransfected) by allowed to adapt to their environment for 2 weeks before commencement ELISA. Briefly, after 72 hours, the culture supernatant was removed, of the experiment. centrifuged to remove particulate matter, and analyzed by ELISA according Cells and culture conditions. CMT93, a mouse colon cancer cell line, to the instructions of the manufacturer (R&D Systems, Minneapolis, MN). was kindly provided by Dr. Stephen Todryk (Oxford University, United Proliferation assays—[3H]thymidine incorporation and 3-(4,5- Kingdom). Cells were maintained in DMEM supplemented with 2 mmol/L dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cells glutamine, 100 Ag/mL streptomycin, 100 units/mL penicillin, and 10% FCS were seeded at 2 Â 104 cells/mL in quadruplicate in 24-well plates 3 in a humidified 5% CO2 atmosphere. and grown for 48 hours. Fresh medium containing [ H]thymidine (1 ACi/mL; Generation of stable CMT93 FasLLow/À cell line. A cDNA fragment Amersham, Buckinghamshire, United Kingdom) was then added to each complementary, when transcribed, to nucleotides 35 to 371 of the mouse well. After 6 hours, the cells were washed with ice-cold PBS and FasL mRNA sequence (GeneBank accession no. U06948) was amplified by harvested for liquid scintillation counting. Alternatively, after 48 hours PCR using the following set of primers: forward, 5V-TGGAGCAGTCAGCGT- culture, 20 AL of 5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra- CAGAGTT-3V, and reverse, 5V-CCACCGGTAGCCACAGATTTG-3V. The frag- zolium bromide (MTT; Sigma) in PBS were added to each well. After ment spans the translational start site of the mouse FasL gene and was 4 hours at 37jC, the medium was carefully removed from the wells and cloned into the StuI site of pBlast expression vector (Invivogen, San Diego, the remaining formazan crystals were dissolved in DMSO. The CA). Ligation products were transformed into chemically competent XL2- absorbance