Transduction of Dendritic Cells with Bcl-xL Increases TheirResistance to Prostate Cancer-Induced Apoptosis andAntitumor Effect in Mice 1
Georgi Pirtskhalaishvili,* Galina V. Shurin, † Andrea Gambotto,† Clemens Esche,†
Madeline Wahl,† Zoya R. Yurkovetsky,‡ Paul D. Robbins,‡ and Michael R. Shurin2†
We have shown that prostate cancer (PCa) causes apoptosis of dendritic cells (DC), which might block the development of specificantitumor immune responses. Analysis of murine prostatic carcinoma tissues revealed the significant decrease in intratumoral DCnumber during tumor progression. We demonstrated that the cytokine-mediated increase in DC survival was accompanied by anelevated expression of the anti-apoptotic protein Bcl-xL. Next, we evaluated the resistance to tumor-induced apoptosis and theantitumor efficiency of genetically engineered DC overexpressing Bcl-xL. DC were transduced with an adenoviral vector encodingthe murine Bcl-xL gene and injected intratumorally. Data analysis revealed that treatment of PCa-bearing mice with Bcl-xL-transduced DC resulted in significant inhibition of tumor growth compared with the administration of nontransduced DC. Thus,our data suggest that the protection of DC from PCa-induced apoptosis might significantly increase the efficacy of DC-basedtherapies in cancer even in the absence of available tumor-specific Ags.The Journal of Immunology,2000, 165: 1956–1964.
Prostate cancer (PCa)3 remains the most common malig-nancy in American men, and the second most commoncause of cancer-related death (1). Although more men are
diagnosed at the early stages of the disease, when radical treatmentis feasible, one-third of them will recur. PCa, in general, is resis-tant to chemotherapy, which may be due to its intrinsic low pro-liferative index (2). The development of new immunotherapeuticapproaches that do not require a high proliferative rate of tumorcells has a strong rationale in the treatment of PCa. For the tumorto survive and grow, it should overcome immunologic defensemechanisms of the host. In many cases tumor cells achieve thiseither by suppressing the key elements of the host immune re-sponse and thus disrupting the antitumor reaction, or by promotingthe tolerance of the immune system toward the tumor. PCa tumorsare low in immunogenicity due to the lack of MHC class I mol-ecules in the majority of cases (3). However, tumor-induced im-munosuppression was well documented in PCa patients (4, 5). Ithas been recently demonstrated that tumor causes apoptotic deathof key immunocompetent cells, including the major APCs, den-dritic cells (DC) (6, 7). DC, first discovered in 1973 (8), originatein the bone marrow and migrate to the different lymphoid andnonlymphoid tissues. They recognize, uptake, and process Ag(s),
including tumor Ags, and then present it to naive T cells, stimu-lating their proliferation (9). Elimination of DC from the tumorenvironment significantly diminishes the initiation of specific im-munologic responses. In fact, it has been demonstrated that PCa isalmost devoid of DC, and DC number further decreases in highergrade tumors (10, 11). The importance of DC in the induction ofspecific antitumor immunity has been recently documented in avariety of preclinical models (9), and several DC-based clinicaltrials have been initiated, including those with PCa patients (12,13). The reported limited efficacy of these trials may be due to thefact that PCa cells cause significant suppression of human DCsurvival (14). Therefore, effective protection of DC from PCa-induced apoptosis may significantly improve the efficacy of DC-based therapies in cancer clinical trials.
There are several things that can increase the survival of DC.Activation of DC with cytokines is the most common method. Forinstance, IL-12, a pleotropic proinflammatory cytokine describedin 1989 (15), has been shown to enhance the survival of hemo-poietic progenitor cells (16, 17) and DC (18, 19). The strong an-titumor activity of IL-12, demonstrated in a variety of immuno-therapy and immune gene therapy models (20–24), may be at leastin part mediated by the activation of the DC system (25). In fact,we have recently reported that administration of IL-12 resulted inincreased generation of DC in vivo (26). It is likely that the stim-ulation of DC survival by IL-12 is due to the increased expressionof the anti-apoptotic protein Bcl-xL (14). Bcl-xL is a known anti-apoptotic protein of a Bcl-2 family, capable of suppressing apo-ptosis in various cell types (7, 27) and playing an important role inthe regulation of hemopoiesis and the survival of cells of the im-mune system (28, 29). The first aim of this study was to charac-terize PCa-induced apoptosis of DC. We have demonstrated thatthe progression of murine PCa was accompanied by decrease inthe number of tumor-infiltrating DC and that PCa-induced apopto-sis of DC was not mediated by a Fas/FasL interaction. The secondaim of this study was to test the hypothesis that increased survivalof DC results in a more efficient induction of antitumor immunityand a higher efficacy of DC-based therapies for cancer. We haveevaluated the antitumor activity of DC transduced to overexpress
Departments of *Urology,†Surgery, and‡Biochemistry and Molecular Genetics, Uni-versity of Pittsburgh Medical Center, Pittsburgh, PA 15213
Received for publication February 7, 2000. Accepted for publication June 2, 2000.
The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby markedadvertisementin accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by Grants RO1CA80126 (to M.R.S.) and RO1CA84270(to M.R.S.), the Pittsburgh Foundation for Medical Research (to M.R.S. and G.P.),Pilot Project Program of the Prostate and Urological Cancer Center of the Universityof Pittsburgh Cancer Institute (to M.R.S. and G.P.), and Department of DefenseDAMD17-00-1-0099 (to M.R.S.).2 Address correspondence and reprint requests to Dr. Michael R. Shurin, Universityof Pittsburgh Cancer Institute, Surgical Oncology, 3471 Fifth Avenue, 300 KaufmannBuilding, Pittsburgh, PA 15213. E-mail address: [email protected] Abbreviations used in this paper: PCa, prostate cancer; DC, dendritic cells; PI,propidium iodide; GFP, green fluorescent protein; mBcl-xL, mouse Bcl-xL; EGFP,enhanced GFP; FasL, Fas ligand.
IL-12 or Bcl-xL in a murine PCa model and demonstrated thatintratumoral administration of DC transduced with thebcl-xL generesulted in a strongest inhibition of tumor growth compared withthat in other groups.
Materials and MethodsMice and cell lines
Male C57BL/6 mice, 6–8 wk old, were obtained from Taconic Farms(Germantown, NY). MRL/MpJ mice carrying thelpr mutation (lpr/lpr )and their parental wild-type controls were purchased from The JacksonLaboratory (Bar Harbor, ME). Animals were maintained at the CentralAnimal Facility at the University of Pittsburgh according to standardguidelines. RM-1, a murine prostate cancer cell line, was a gift from Dr.Timothy C. Thompson (Baylor College of Medicine, Houston, TX). Tumorcells were maintained in RPMI 1640 medium supplemented with 5% heat-inactivated FBS, 2 mML-glutamine, 100 U/ml penicillin, 100mg/ml strep-tomycin, 1 mM sodium pyruvate, and 0.1 mM nonessential amino acids(Life Technologies, Grand Island, NY).
Generation of murine DC
Murine DC cultures were established as described previously (30). Briefly,bone marrow cells were depleted of RBC with lysing buffer (155 mMNH4Cl in 10 mM Tris-HCl buffer, pH 7.5, 25°C) for 2–3 min. The single-cell suspensions were then incubated with a cocktail of Abs for 1 h at4°C,followed by incubation with rabbit complement for 30 min at 37°C todeplete cells expressing lymphocyte Ags B220, CD4, and CD8. Treatmentwith Abs (partially purified supernatant of hybridoma cell cultures TIB-146, TIB-207, and TIB-105 for B220, CD4, and CD8, respectively; Amer-ican Type Culture Collection, Manassas, VA) and rabbit complement (LifeTechnologies) removed detectable B and T lymphocytes from the cell sus-pensions. Cells were then incubated overnight (37°C, 5% CO2) in six-wellplates (Falcon, Franklin Lakes, NJ) at a concentration of 106 cells/ml incomplete medium, consisting of RPMI 1640, 2 mML-glutamine, 50mg/mlgentamicin sulfate, 10 mM HEPES, 10% FBS, 0.1 mM nonessential aminoacids, 1 mM sodium pyruvate (Life Technologies), 1mg/ml indomethacin(Sigma), and 50mM N-methyl-L-arginine (Alexis, San Diego, CA). Thenonadherent cells were then collected by gentle pipetting and resuspendedat a concentration of 105 cells/ml in complete medium supplemented with1000 U/ml recombinant murine GM-CSF and recombinant murine IL-4(both provided by Schering-Plough, Kenilworth, NJ). Cells were culturedin six-well plates (4 ml/well) for 7 days at 37°C in 5% CO2. NonadherentDC were collected by gentle pipetting, characterized as described previ-ously (30), and used for further studies. In some experiments cultured DCwere treated with recombinant murine IL-12 (Roche, Indianapolis, IN) at afinal concentration of 100 ng/ml. IL-12 was added to DC on the sixth dayof culture. Cells were collected 24 h later, washed, ad lysed, and expressionof proteins was assessed by Western blot as described below.
Flow cytometry for Fas and FasL expression
Cells were washed in FACS medium (HBSS containing 0.1% BSA and0.1% NaN3) and stained with appropriately diluted Abs directly conjugatedwith PE according to the manufacturer’s recommendations. Hamster anti-mouse Abs against CD95 (Fas) and FasL (CD95L) were used (PharMin-gen, San Diego, CA), and 10,000 cells were analyzed by flow cytometrywith the CellQuest 1.0 software package (Becton Dickinson, San Jose,CA). For intracellular FasL detection, cells were fixed in cold 1% para-formaldehyde and methanol and permeabilized using 0.01% saponin (Sig-ma, St. Louis, MO) in FACS medium. The staining procedure was thesame as that described above.
Immunohistochemistry
Tissue samples were embedded in OCT compound (Miles, Elkhart, IN),snap-frozen on dry ice, and stored at280°C. Cryostat sections (6mm)were air-dried and fixed in ice-cold acetone. Slides were washed in PBSand incubated for 1 h atroom temperature with the following Abs: NLDC-145 (DEC 205; Serotec, Raleigh, NC; dilution, 1/5) and CD11c (N418;Serotec; dilution, 1/800). Biotinylated mouse anti-rat IgG (Jackson Immu-noResearch Laboratories, West Grove, PA; 1/500 dilution) and anti-hamster IgG (Vector, Burlingame, CA; dilution, 1/1000) were used as sec-ondary Abs and were applied for 45 min. After developing using theperoxidase chromogen kit (3-amino-9-ethylcarbazol, Biomega, FosterCity, CA) for 8 min, counterstaining was performed with hematoxylin.Two investigators analyzed the slides independently to determine the num-ber of positive cells per area. At least 10 different areas were analyzed.Negative controls included the staining with irrelevant isotype Abs.
Apoptosis assays
DC were harvested after coincubation with tumor cells and were fixed onmicroscope slides for morphologic evaluation using a Cytospin centrifuge(Shandon Lipshaw, Pittsburgh, PA). After drying for 5 min, cells werefixed and stained with a LeukoStat Stain Kit (Fisher Scientific, Pittsburgh,PA), and the percentage of apoptotic cells was assessed quantitatively us-ing morphologic criteria, which include condensation of chromatin, reduc-tion in nuclear size, shrinkage of total cell volume, increase in cell density,and formation of apoptotic bodies (31).
For annexin V binding assay and propidium iodide (PI) uptake evalu-ation, DC were collected and double stained with FITC-conjugated annexinV (PharMingen) and/or PI (Sigma). Annexin V was added according to themanufacturer’s recommendations. PI was used at a final concentration of10mg/ml. All annexin V-positive cells were considered apoptotic, and theirpercentage was calculated among the total number of cells. Cells takingvital dye PI were considered dead. Samples (10,000 cells) were analyzedby FACScan. When green fluorescent protein (GFP)-transfected cells wereanalyzed, only PI staining was used for cell death assessment.
The cell viability in the cultures after transfection with different ad-enovectors was evaluated using trypan blue (0.2%; Life Technologies).Trypan blue-positive cells were considered dead, and their percentageamong the total cell number was calculated.
Adenoviral vectors and cell transfection
Adenovirus (Ad5) vectors were constructed through Cre-Lox recombina-tion with reagents generously provided by Somatix (Almenda, CA). Con-struction and characterization of the Ad5 vector expressing mIL-12 havepreviously been described (23). In brief, pAdCMV-mIL-12 was con-structed by transferring aBglII/BamHI fragment containing the p40 subunitof mIL-12 into theBamHI site of pAdCMV-B. A BamHI fragment con-taining the p35-encoding gene was subsequently cloned in theBamHI sitebehind the p40 subunit. This resulted in a polycistronic message expressingboth subunits of mIL-12. Virus generated by recombination between theshuttle vector and the Ad DNA was propagated on 293 cells and purifiedfrom infected cells 2 days after infection by three freeze-thaw cycles fol-lowed by three successive bandings on CsCl gradients.
For the construction of Bcl-xL encoding adenoviral vector (Ad5Bcl-xL),the mousebcl-xL gene was cloned from a BALB/c mouse total spleen usingthe PCR technique. Primers were designed according to the published se-quence (GenBank accession no. U51278), in which additional restrictionenzyme target sequences were added. The forward sequence was 59-TCAGAG CTC ATG TCT CAG AGC AAC AGG GAG-39, and the reversesequence was 59-CTA GGC GGC CGC GTC TGG TCA CTT CCG ACTGAAG-39. Mouse Bcl-xL cDNA was subcloned into expression plasmidpCR3.1 (Invitrogen, Carlsbad, CA) and subsequently sequenced (GenBankaccession no. AF060226). To prove that plasmid contained the correctinsert sequence, pAdlox-mBcl-xL was digested withEcoRI-restricted en-zyme. The solution containing 5ml of EcoRI buffer, 2ml of EcoRI enzyme,5 ml of pAdlox-mBcl-xL, and 38ml of dH2O was incubated for 2 h at37°Cand then loaded on an 1% agarose gel at the appropriate concentrationfollowed by electrophoresis. ASalI-NotI fragment containing Bcl-xLcDNA derived from pCR3.1-mBcl-xL was inserted in the shuttle vectorpAdlox (Fig. 1A). An E1-substituted recombinant adenovirus was gener-ated by cotransfection ofc5 helper virus DNA andSfiI-digested pAdlox-mBcl-xL into the adenoviral packaging cell line CRE8, propagated, andpurified as previously described (32).
Control adenoviral vector encoding the enhanced GFP gene (Ad5EGFP)was constructed by inserting aSnaBI-HpaI fragment containing part of theCMV promoter, the EGFP cDNA, and part of the SV40 poly(A) derivedfrom pEGFPNI (Clontech, Palo Alto, CA) (33) into the shuttle vector pAd-lox. Remaining steps for the construction of Ad5EGP were similar to thosefor Ad5Bcl-xL construction.
For the transfection, murine bone marrow-derived DC were harvestedon day 5, washed twice in HBSS, and incubated at 37°C with the corre-sponding adenoviral vectors. Virus was used at a dose of 200 multiplicityof infection (MOI). Complete medium supplemented with mouse recom-binant GM-CSF and mouse recombinant IL-4 was added 2 h later, and cellswere allowed to recover for 24 h. Then cells were washed twice in HBSSand injected intratumorally at a dose of 106 DC/mouse/injection.
Western blot
The level of expression of the anti-apoptotic protein Bcl-xL was examinedusing a Western blot technique. Briefly, cells were collected, washed inPBS, and homogenized in lysing buffer. The homogenate was centrifuged
1957The Journal of Immunology
at 12,0003 g for 15 min at 4°C. The protein concentration in the super-natant was determined by the Bradford method using the Bio-Rad kit (Bio-Rad, Hercules, CA). Each sample was denatured for 5 min at 100°C insample buffer. Equal amounts of protein were loaded for each sample in alllanes and electrophoretically separated on 16.5% SDS-PAGE followed bytransfer to a nitrocellulose membrane. The membrane was blocked with0.2% nonfat milk and 0.1% Tween-20 (Fisher, Fairlawn, NJ) in 20 mMTris-HCl buffer, pH 7.2. Bcl-xL was detected using specific rabbit primaryAbs (Oncogene Research Products, Cambridge, MA) with a final concen-tration of 2.5mg/ml, and donkey anti-rabbit secondary Abs (1/2000 dilu-tion; Amersham Pharmacia Biotech, Piscataway, NJ). The membrane wasprocessed and treated with chemiluminescence reagents (Tropix, Medford,MA). The bands were visualized on Kodak film (Eastman Kodak, Roch-ester, NY) exposed to the membrane to detect chemiluminescence signals.
Experimental design: in vitro
Five- to 6-day-old cultured DC were harvested and coincubated with themurine prostate cancer cell line RM-1 in six-well plates. DC and tumorcells were separated using membrane inserts with 0.4-mm pore size, whichexcluded direct cell-to-cell contact, but allowed free exchange of solubletumor-derived factors. Specifically, 5–103 105 DC were placed in six-well plates in 3 ml of medium. Two million PCa cells resuspended in 2 mlof medium were placed into the inserts on the top of each well. As controls,DC were coincubated with murine splenocytes ormedium alone placed ininserts. DC were harvested 48 h later, and apoptosis was assessed usingthe morphological criteria and an annexin Vbinding assay. For thepositive control, DC were irradiated (25,000 rad), and apoptosis wasassessed after 16 h.
To determine DC survival in cultures after transfection, cells were leftin six-well plates without fresh medium and cytokines, and the number ofdead cells was counted every other day using trypan blue. Each experimentwas repeated twice, and combined data are presented.
Experimental design: in vivo
RM-1 tumor cells (20,000 cells/100ml) were inoculated s.c. in the rightflank of C57BL/6 mice, and tumor establishment was determined by pal-pation. Measuring the perpendicular tumor diameters with a Vernier caliper(Electron Microscopy Sciences, Ft. Washington, PA) assessed tumor size.Tumor volume was calculated using the formula of rotational ellipsoid: m1
2
3 m2 3 0.5236, where m1 represents the shorter axis, and m2 the longeraxis (34). Treatment groups consisted of five mice per group. Mice weresacrificed when they exhibited signs of distress or when total tumor volumeexceeded 3000 mm3. Experiments were independently repeated threetimes. Combined data from these experiments are presented.
Statistical analysis
A x2 analysis was performed to evaluate the significance of differencesbetween the experimental groups in the annexin V and PI staining assayswhen discrete data were presented. For a single comparison of two groups,Student’st test was used. If the data distribution was not normal, the Mann-Whitney rank-sum test was employed for the nonparametric analysis. Two-way ANOVA using the Student-Newman-Keuls method was employed forcomparison of tumor size in mice after the different types of treatment. Forall analyses, the level of significance was set atp , 0.05. All statisticalcalculations were performed using the SigmaStat statistical software pack-age (SPSS, Chicago, IL). Data are presented as the mean6 SEM.
ResultsRM-1 cells induce apoptotic death of murine DC in vitro
Murine DC were generated from bone marrow precursors, andRM-1 cells were added on day 5 in cell inserts (0.4-mm pore size)for an additional 48 h. Based on morphological characteristics, thelevels of apoptotic DC were 13.486 2.16 and 31.136 3.43% aftercoincubation of DC with splenocytes or RM-1 cells, respectively( p , 0.001). The results of the annexin V binding assay revealedthat the percentages of apoptotic DC were 17.716 2.23 and53.666 6.06% among DC coincubated with splenocytes or RM-1cells, respectively (p , 0.001; Fig. 1A). In irradiated DC cultures,which served as a positive control, the level of apoptotic cells was66.746 5.47%. Thus, these data suggested that PCa cells releasefactors that cause apoptotic death of DC.
To elucidate the mechanism of regulation of tumor-induced DCapoptosis and to determine whether the family of Bcl-2 proteins is
involved in these pathways, DC were collected after coincubationwith splenocytes or RM-1 cells for 48 h, washed, and lysed, andproteins were extracted. Western blot was used to assess the ex-pression of Bcl-xL. Since equal amounts of protein were loaded inall lanes, the results presented in Fig. 1B suggest that coincubationof DC with RM-1 cells, but not splenocytes, significantly de-creased the expression of anti-apoptotic protein Bcl-xL in DC.Taken together, these data demonstrated that PCa-derived solublefactors caused apoptosis of DC in vitro, and that decreased ex-pression of Bcl-xL in DC might mediate this effect. These data alsoraise the question of whether DC might undergo apoptosis withinthe tumor microenvironment in vivo.
Number of tumor-infiltrating DC decreases with the tumorprogression in vivo
To determine the infiltration of PCa tissues by DC, 106 RM-1 cellswere injected into the shaved right flank of C57BL/6 mice. Micewere sacrificed at different time points, tumor was measured andharvested, and immunohistochemical staining was performed us-ing murine DC markers NLDC-145 (DEC 205) and CD11c(N418). DC were readily identifiable within the tumor tissues onday 7, but their numbers significantly decreased within 10-day-oldtumors, and tumor-infiltrating DC virtually disappeared within 20-day-old tumor tissues (Fig. 2). Importantly, similar results wereobtained when both DC-specific markers were used to evaluate thelevels of DC infiltration within the tumor tissues. Thus, these data
FIGURE 1. Prostate cancer cells induced apoptotic death of DC, whichwas mediated by decreased expression of the anti-apoptotic protein Bcl-xL.Coincubation of RM-1 tumor cells with cultured bone marrow-derived DCresulted in significantly increased levels of death of DC as assessed bymorphological analysis and annexin V binding assay.A, DC were coincu-bated with RM-1 tumor cells separated through a 0.4-mm pore size mem-brane for 48 h, harvested, cytospan, and evaluated for apoptosis based onmorphological criteria, which included condensation of cytoplasm and nu-clei, degranulation, shrinkage of cytoplasm, and formation of apoptoticbodies. Splenocytes and tumor medium, used instead of tumor cells, servedas controls. Annexin V binding was performed as described inMaterialsand Methods. As shown, RM-1 cells caused significantly higher levels ofDC apoptosis compared with splenocytes, which was confirmed by bothmorphologic evaluation and annexin V assay. Data are shown as themean6 SEM. Three independent experiments were performed with sim-ilar results, and combined data are presented.B, The results of Western blotanalysis suggested that the coincubation of cultured bone marrow-derivedDC with RM-1 cells for 48 h caused the marked decrease in expression ofBcl-xL protein in DC compared with that in control DC.p, p , 0.001.
1958 ANTITUMOR EFFECT OF DENDRITIC CELLS PROTECTED FROM APOPTOSIS
suggest that the progression of prostate carcinoma in vivo wasaccompanied by the marked decrease in DC infiltration within thetumor tissue. Although RM-1 cells are MHC class I negative (datanot shown) and might be considered nonimmunogenic, the factthat DC were identified at the site of the tumor at the beginning oftumor growth and disappeared later during tumor progressiontaken together with our in vitro findings supports the conclusionthat RM-1 cells markedly inhibit DC survival and/or accumulationwithin the tumor tissue.
Role of Fas/FasL interaction in inhibition of DC survival byRM-1 cells
To evaluate the role of the Fas/FasL (CD95/CD95L) interaction inapoptosis of DC induced by prostate cancer cells, MRL/MpJ micewith functional mutations of Fas (lpr/lpr ) (35) were used in thenext experiments. Wild-type control MRL/MpJ (1/1) andC57BL/6 mice were used as controls. FACScan analysis of FasLexpression on RM-1 cells revealed moderate levels (;30–35% ofcells) of expression. Expression of Fas on DC generated in vitrofrom wild-type MRL/MpJ and C57BL/6 mice reached 70.66 and61.83% of that in positive cells, respectively, while DC generated
from lpr/lpr mice demonstrated a low level (2.78%) of Fas ex-pression (Fig. 3). Next, DC obtained from different mouse strainswere coincubated with RM-1 cells or splenocytes for 48 h, andapoptosis was assessed by annexin V and PI staining. Two inde-pendent experiments were performed with identical results, andcombined data are presented. As expected, the level of apoptosis incontrol C57BL/6 mice-derived DC cultures was 16.516 3.06%,while RM-1 cells caused the death of 44.476 4.78% of these DC( p , 0.01). In wild-type MRL mice-derived DC cultures, RM-1caused an elevation of the apoptotic rate from 32.276 0.91% incontrol DC to 45.566 4.41% (p , 0.05). Fas-deficient DC havedemonstrated the similar sensitivity to PCa-induced apoptosis; co-incubation with RM-1 cells led to an increase in the apoptotic ratefrom 22.966 6.69 to 33.866 2.45% (p , 0.05). Although thelevel of spontaneous apoptosis in DC cultures obtained from MRLmice was higher than that in C57BL/6-derived DC, and Fas-defi-cient DC appeared to be somewhat less sensitive to apoptosis,there was no significant difference between the PCa-induced apo-ptotic rate among DC derived from Fas-deficient or wild-type mice(1.47- and 1.41-fold increases, respectively). Furthermore, the in-crease in levels of RM-1-induced DC apoptosis was statistically
FIGURE 2. The growth of prostate carcinomas in vivo was accompanied by a significant decrease in the number of tumor-infiltrating DC. Immuno-histochemical analysis of tumor-infiltrating DC was performed with two DC-specific Abs, NLDC-145 (DEC205;A–C) and CD11c (N418;D–F). Mor-phometric analysis of data suggested that tumor-infiltrating DC (brown cells) were readily identifiable within the RM-1 tumor tissues after 7 days of growthin vivo (A andD). The number of DC was significantly decreased within 10-day-old tumors (B andE), and DC almost completely disappeared in 20-day-oldtumor tissues (C andF).
FIGURE 3. Expression of Fas (CD95) on cultured murine DC. FACScan analysis of Fas expression on cultured DC obtained from wild-type MRL/MPJ(a), C57BL/6 (b), or MRL/MPJlpr/lpr (c) mice revealed that DC derived from both wild-type MRL/MPJ and control C57BL/6 mice demonstrated a strongexpression of Fas molecules, while DC generated from MRL/MPJlpr/lpr mice had no Fas expression.
1959The Journal of Immunology
significant in both groups. Thus, these data suggest that the Fas/FasL interaction probably does not play a crucial role in PCa-induced DC apoptosis in a murine system.
Increase in DC survival by overexpression of IL-12 and Bcl-xL
in vitro
Since IL-12-encoding adenoviral vector was characterized previ-ously(23), in this study we have evaluated transduction of DC with
murine bcl-xL gene and transgene expression (Fig. 4). For thispurpose, murine cultured DC were collected on day 5 and trans-duced with IL-12 or thebcl-xL gene using adenoviral vectors. Con-trol transfection was performed with the GFP-encoding gene. Inmost experiments, the transfection efficacy varied between 60 and80%, as determined by a FACScan analysis of GFP-transfectedDC (data not shown). Western blot performed on Bcl-xL-trans-duced DC showed significant elevation of Bcl-xL protein expres-sion compared with that in control cells (Fig. 4), suggesting a hightransfection efficacy of the method used.
We have recently shown that the treatment of human culturedDC with human IL-12 protein in vitro results in prolonged DCsurvival and increased resistance to tumor-induced apoptosis (14).Since our previous studies also indicated that prolonged survival ofhuman DC was accompanied by increased expression of Bcl-xL,we first confirmed these results in a murine system. As shown inFig. 5, stimulation of murine bone marrow-derived DC with mouseIL-12 protein (100 ng/ml) for 24 h resulted in increased expressionof the anti-apoptotic protein Bcl-xL as assessed by Western blot.Taken together, these data suggest that IL-12-induced increasedsurvival of both human and murine DC is mediated by enhancedexpression of the anti-apoptotic protein Bcl-xL in these cells. Thisallowed us to hypothesize that the increased concentrations ofIL-12 in the local DC environment or stimulation of Bcl-xL syn-thesis might result in prolonged DC survival. To test this hypoth-esis, we have evaluated whether overexpression of IL-12 or Bcl-xL
by DC might affect their survival in long-term cultures and theirsensitivity to prostate cancer-induced apoptosis in vitro.
First, we determined the survival of DC in cultures using thetrypan blue excretion method (Fig. 6). Murine DC were generatedin cultures with GM-CSF and IL-4 added on day 1 and day 3.Transfection was performed on day 5, and DC were further cul-tured without cytokines and growth factors. The analysis of cellviability demonstrated that starting from day 11, the percentage ofdead cells among nontransfected or GFP-transfected DC cultures(38.336 1.67 and 45.986 10.69%, respectively) was significantlyhigher compared with that in IL-12- and Bcl-xL-transfected DCcultures (20.106 3.43 and 13.896 2.78%, respectively;p ,0.05). Interestingly, starting from day 15, the survival of IL-12-transduced DC also declined, whereas the death rate of Bcl-xL-transduced DC remained significantly lower compared with thosein all other groups (p , 0.01). Almost 50% of DC overexpressingBcl-xL were alive in 20-day-old cultures supplemented with nogrowth factors (Fig. 6b). These results suggest that DC geneticallyengineered to produce IL-12 performed prolonged survival for1 wk in growth factor-deprived cultures, whereas overexpressionof an anti-apoptotic protein Bcl-xL resulted in significantly pro-longed survival for at least 2 wk during growth factor withdrawal.
FIGURE 4. Construction of the Bcl-xL-encoding vector and transduc-tion of DC.A, Schematic diagram of Ad5Bcl-xL vector with thebcl-xL geneincorporated into the adenovirus. The details of the construction are de-scribed in Materials and Methods.B, Digestion of mouse Bcl-xL (mBcl-xL)-encoding plasmid withEcoRI-restricted enzyme followed by electro-phoresis demonstrated an appropriately sized band (lane 1) thatcorresponded to the calculated size of Bcl-xL, as assessed by size markers(lane 2). C, Western blot analysis of proteins in DC demonstrated signif-icantly higher levels of Bcl-xL expression in DC transduced withAd5Bcl-xL vector than in GFP-transduced (control) DC.
FIGURE 5. IL-12 increased the expression of Bcl-xL protein in DC.Murine cultured bone marrow-derived DC were treated with murine IL-12protein (100 ng/ml) for 24 h, harvested, and washed, and extracted proteinswere analyzed by Western blot as described inMaterials and Methods.Equal amounts of proteins were loaded in all lanes. A high level of Bcl-xL
protein expression in IL-12-stimulated DC was demonstrated, suggesting apossible mechanism of prolonged survival of DC treated with IL-12protein.
1960 ANTITUMOR EFFECT OF DENDRITIC CELLS PROTECTED FROM APOPTOSIS
In the next series of experiments we examined whether trans-fection of DC with IL-12 orbcl-xL genes protects them from tu-mor-induced apoptosis. DC were transduced on day 5, recoveredfor 24 h, and then coincubated with RM-1 cells on day 6 for 48 has described above. DC were then stained with PI, and determi-nation of the number of PI-positive (dead) cells was assessed byFACScan. Analysis of these results suggested that IL-12- or Bcl-xL-transfected DC were characterized by increased resistance toPCa-induced apoptosis compared with nontransduced or GFP-transduced DC. For instance, the levels of PI-positive cells amongnontransfected and GFP-transfected DC cultures coincubated withRM-1 cells were 30.336 5.55 and 33.476 3.35%, respectively,whereas the transduction of DC with IL-12 orbcl-xL genes signif-icantly decreased levels of PCa-cancer-induced cell death up to20.806 0.69 and 10.986 1.78%, respectively (p , 0.01; Fig.6C). Thus, these data suggest that overexpression of IL-12 orBcl-xL in murine DC results in their prolonged survival in culturesand increased resistance to PCa-induced apoptotic death in vitro.
In summary, our in vitro data demonstrate that the adenoviraltransfection of murine bone marrow-derived DC with differentgenes is a highly efficient procedure resulting in high levels ofprotein expression. Transfection of DC with IL-12 andbcl-xL
genes led to the enhanced survival in cultures and increased resis-tance to PCa-induced apoptosis. These results raise the question ofwhether the enhanced resistance of DC to tumor-induced cell deathwould be accompanied by an increased antitumor efficacy ofthese DC.
Antitumor activity of IL-12- or Bcl-xL-transduced DC in vivo
In the next series of experiments we examined the antitumor ef-ficacy of IL-12- and Bcl-xL-transfected DC in a murine PCa tumormodel. On day 1, mice were injected with 23 105 RM-1 tumorcells into the right flank. Syngeneic DC were generated from thebone marrow and transfected with adenovector encoding IL-12,bcl-xL, or GFP gene. Cells were allowed to recover for 24 h, har-vested, and injected intratumorally (106 cells in 100ml of HBSS)
CB
A
FIGURE 6. Transduction of DC with IL-12 orbcl-xL genes resulted in significantly increased survival of DC in long-term cultures and increasedresistance of DC to PCa-induced apoptosis in vitro. A, Following the adenoviral transfection of DC with GFP (control), IL-12, or Bcl-xL on day 5, cellswere maintained in cultures without cytokines and growth factors. The morphologic characteristics of DC are shown for the 20th day. Nontransfected DC(1) and GFP-transfected DC (2) cultures were practically lacking cell structures; IL-12-transfected DC were mostly apoptotic (3), whereas at least 50% ofBcl-xL-transfected DC were still alive (4). B, The dynamics of the death rate of transfected and control DC were assessed in cultures by trypan blue uptake.Cells were maintained without cytokines and growth factors. The results of survival analysis suggested that starting from day 11, the percentage of deadcells among nontransfected and GFP-transfected DC was significantly higher than the percentage of dead cells among IL-12- or Bcl-xL-transfected DC.Starting from day 15, the survival of IL-12-transfected DC decreased as well. Thus, DC overexpressing Bcl-xL demonstrated the highest level of survivalin long-term cultures. Data represent the mean6 SEM from two independent experiments.C, Cultured DC were incubated with RM-1 cells for 48 h, stainedwith PI (10 mg/ml), and analyzed by FACScan. DC transfected with IL-12 orbcl-xL genes demonstrated the lowest death rate, suggesting their highestresistance to tumor-induced apoptosis. Data are shown as the mean6 SEM from two independent experiments.
1961The Journal of Immunology
on day 5. Five groups of animals (five mice per group) were usedin each experiment. Group 1 was injected with IL-12-transfectedDC, group 2 with Bcl-xL-transfected DC, group 3 with GFP-trans-fected DC (control for the adenovirus), group 4 with nontrans-fected DC, and group 5 with HBSS. All injections were repeatedon day 12. Animals were sacrificed 20–21 days after tumor cellinjection when the size of the tumor in the control group becamelarge enough to be distressing for the animals. Experiments wererepeated three times with similar results, and the combined data arepresented on Fig. 7.
The mean tumor volume of the mice treated with HBSS injec-tion alone was 2650.66 506.3 mm3 on day 19. The mean tumorvolumes in mice treated with DC alone or with GFP-transfectedDC were 1690.36 269.3 and 1566.86 205.4 mm3, respectively.Both these values were significantly (p , 0.05) smaller than tumorsizes in mice treated with HBSS. Treatment of mice with IL-12- orBcl-xL-transfected DC resulted in further inhibition of tumorgrowth and a decrease in tumor size (1025.96 252.2 and 816.56174.0 mm3, respectively). Tumor sizes in both these groups weresignificantly (p , 0.05) smaller than tumor sizes in other groupsof animals. Although the mean tumor size was smallest in mice
treated with Bcl-xL-transfected DC in all independent experiments,the difference between IL-12- and Bcl-xL-transfected DC did notreach statistical significance. Thus, these data suggest that over-expression of IL-12 or Bcl-xL in DC significantly improved theantitumor efficacy of DC-based immunotherapy in mice.
In summary, treatment of PCa-bearing mice with DC protectedfrom tumor-induced apoptosis appears to be an effective approachfor cancer immunotherapy even in the case of nonimmunogenic,fast growing, and aggressive tumors.
DiscussionDuring the last several years immunotherapy has been employedmore frequently in both preclinical studies and human clinical tri-als. DC-based therapy represents a relatively new approach to treattumor-bearing hosts. DC pulsed with isolated or synthetic tumor-associated peptides have been shown to induce an effective anti-tumor immune response in animal models (13, 36) and cancerpatients (12, 37). DC can also be pulsed with a tumor lysate (38)or tumor RNA (39) to induce a specific immune response againsttumors. For instance, it has been recently demonstrated that humanDC transfected with RNA encoding prostate-specific Ag stimulateprostate-specific CTL responses in vitro (40). However, most ofthese approaches have certain limitations when considered forclinical use in humans, since the preparation of tumor lysates orextraction of tumor Ags requires a large amount of solid tumor,which may not be suitable in all cases. Thus, development of DC-based therapies that do not require tumor Ags is highly justifiedand timely.
In this study we have evaluated the delivery of genetically mod-ified DC directly to the tumor site. The working hypothesis wasthat tumor-resistant DC would recognize and pick up tumor Ag(s),emigrate from the tumor mass, traffic to the regional lymph nodesto present the Ag(s) to T cells, and induce a specific antitumorresponse. Since we did not pulse DC with tumor Ag in this study,the important question was whether administered DC could rec-ognize and process endogenous tumor Ags at the site of the tumor.Although synthetic or stripped Ags, tumor lysates, tumor apoptoticbodies, or tumor-derived RNA or DNA ensure relative specificity,they cannot provide protection of DC from tumor-induced apopto-sis per se. Furthermore, as was demonstrated in our studies, tumorexpansion was associated with the local elimination of DC withinthe tumor, and the coincubation of DC with RM-1 tumor cellsresulted in the massive apoptotic death of DC. Importantly, PCa-induced apoptosis in DC was accompanied by a decreased expres-sion of the anti-apoptotic protein Bcl-xL. Therefore, we have ex-amined whether genetic modification of DC increases theirresistance to tumor-induced apoptosis and their potential to induceantitumor immune responses. Based on our previous results, IL-12andbcl-xL genes were chosen for this purpose.
IL-12 is a potent proinflammatory cytokine, which is producedby DC in response to CD40 ligation (41–43). IL-12, in turn, stim-ulates the expression of CD154 (CD40 ligand) on T cells (44) andIFN-g production by NK and T cells (15, 45, 46) and inhibits orreverses anergy of T cells (47, 48). In addition to its immunopo-tentiating and antitumor effects, human IL-12 enhances resistanceof human DC to prostate cancer-induced cell death (14), and asshown here, IL-12-transfected murine DC demonstrated signifi-cantly higher levels of survival in both long-term cultures andtumor microenvironment in vitro. An IL-12 cDNA-encoding ad-enoviral vector has been successfully used in the treatment of var-ious tumors (22–24), including prostate cancer in mice (49). DCtransfected with IL-12 using retroviral vector were also effective(50), although the antitumor properties of adenovirally transducedDC, overexpressing IL-12, have not yet been evaluated.
FIGURE 7. Intratumoral administration of IL-12- or Bcl-xL-transducedDC resulted in significant inhibition of tumor growth in vivo. Murine PCacells were injected in mice as described inMaterials and Methods. Non-transduced DC, transduced DC, or HBSS were injected at the tumor site ina volume of 50ml, and the tumor sizes were recorded twice per week.A, Treatment with nontransfected or GFP-transfected DC had a significanteffect on tumor growth compared with HBSS administration. Treatment ofPCa-bearing mice with IL-12- or Bcl-xL-transfected DC resulted in signif-icant inhibition of tumor growth compared with that in mice treated withHBSS, nontransfected DC, or GFT-transfected DC (p , 0.05). Data rep-resent the mean6 SEM from three independent experiments.B, Repre-sentative examples of PCa-bearing mice sacrificed on day 20. An HBSS-treated mouse (1) exhibits the largest size of RM-1 tumor, a nontransfectedDC-treated mouse has a smaller tumor (2), and a mouse treated with Bcl-xL-transfected DC bears the smallest tumor (3).
1962 ANTITUMOR EFFECT OF DENDRITIC CELLS PROTECTED FROM APOPTOSIS
In this study we used an adenoviral vector approach for thegenetic engineering of DC. Gene expression of an adenoviral vec-tor is transient, since the transferred gene is not incorporated intothe target cell chromosomal DNA. In our experiments this fact wasnot an issue, because the long-term expression of transfected geneswas not required. Another concern that was considered to be aproblem by some authors (51) is immunogenicity of adenoviralvectors. However, Bramson et al. recently demonstrated that thepre-existing immunity against the adenovirus reduces virus dis-semination, but not the antitumor effect of therapy (52). In addi-tion, adenovirus-mediated transfection of various cell types provento be a highly efficient procedure. We have confirmed these resultsfor murine DC and demonstrated here that the level of transfectionin GFP-transduced DC was between 60 and 80%, as assessed byflow cytometry. Murine DC transfected with IL-12-encoding vec-tor produced high levels of IL-12 protein, as determined by ELISA(data not shown). As demonstrated in this study, these cells causeda significant inhibition of PCa growth in mice when injected intothe site of the tumor even without prior pulse with a tumor Ag. Itis likely that both a direct effect of IL-12 on different immune cellsand stimulation of DC survival contributed to the observed anti-tumor effect of IL-12-transfected DC in a murine PCa model.
It is of a great interest that the intratumoral administration ofBcl-xL-transfected DC induced a stronger antitumor effect than thetreatment with IL-12-transfected DC, although the difference wasnot statistically significant. It suggests that an increased survival ofDC may be a key mechanism of the induction of antitumor im-mune responses initiated by the intratumoral administration of IL-12-transfected DC. It is possible that DC, overexpressing Bcl-xL,also produce IL-12. Furthermore, it is likely that the overexpres-sion of Bcl-xL is a stronger survival factor than the overexpressionof IL-12. Although we observed a significant difference betweenthe survival of Bcl-xL- and IL-12-transfected DC only after day 15in cultures, it is quite possible that the viability of transfected cellsin the tumor microenvironment in vivo might be different from thecell survival in vitro.
Bcl-xL belongs to the Bcl-2 family of proteins. Members of thisfamily with anti-apoptotic properties, including Bcl-2 and Bcl-xL,have been shown to protect cells from NO-mediated apoptosis (53)and markedly inhibit caspase activation (54–56) and Fas/FasL(CD95/CD95L)-mediated killing of cells (57, 58).
The expression of FasL by different tumor cells and their abilityto cause apoptosis of Fas-expressing T cells has been proposed asan important mechanism of tumor escape from immune surveil-lance (59). Since DC have been reported to express Fas and besusceptible to Fas-mediated apoptosis (58), it has been suggestedthat Fas/FasL-mediated tumor-induced apoptosis of DC is an ad-ditional mechanism that allows tumor cells to escape immune rec-ognition and elimination (60). Here, we have demonstrated a highlevel of FasL expression by RM-1 PCa cells and evaluated the roleof the Fas/FasL pathway in the induction of DC apoptosis by pros-tate cancer using Fas-deficient cells obtained fromlpr/lpr knock-out animals. Our data suggested that Fas-mediated pathway wasnot involved in RM-1-induced DC apoptosis, since we did notdetect a significant difference in the sensitivity to apoptosis be-tween Fas-deficient and wild-type DC. Interestingly,lpr/lpr DCappeared to be slightly less sensitive to apoptosis, although thestatistical analysis did not reveal a significant difference comparedwith control DC. Furthermore, neutralizing anti-FasL Ab failed toblock tumor-induced DC apoptosis as well. Thus, it is likely thatother tumor cell-derived factors are involved in DC apoptosis inthe case of the murine prostate cancer cell line RM-1. These resultsconfirm our early findings suggesting that B16 melanoma-inducedapoptosis of DC was not mediated byFas/FasL interaction (19).
Further studies are required to identify the DC-killing factorsproduced by different tumor cell lines and primary tumors aswell as to determine mechanisms of antitumor activity ofBcl-xL-transfected DC.
In conclusion, we have shown here that murine PCa causes sig-nificant inhibition of DC survival both in vivo and in vitro, and thatDC viability can be significantly augmented in vitro by an in-creased expression of the anti-apoptotic protein Bcl-xL. In vivotreatment of PCa-bearing mice with IL-12- or Bcl-xL-transfectedDC demonstrated a strong antitumor activity of intratumorally ad-ministered DC that have not been pulsed with tumor-associatedAg. Taken together, these data suggest that protection of DC fromtumor-induced apoptosis and enhanced survival of DC in the tu-mor microenvironment are key factors that may significantly im-prove DC-based therapies of cancer patients.
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