Proinflammatory Characteristics of SMAC/DIABLO-InducedCell Death in Antitumor Therapy
Perpetua U. Emeagi1, Sandra Van Lint1, Cleo Goyvaerts1, Sarah Maenhout1, Anje Cauwels3, Iain A. McNeish4,Tomas Bos2, Carlo Heirman1, Kris Thielemans1, Joeri L. Aerts1, and Karine Breckpot1
AbstractMolecular mimetics of the caspase activator second mitochondria-derived activator of caspase (SMAC) are
being investigated for use in cancer therapy, but an understanding of in vivo effects remains incomplete. Inthis study, we offer evidence that SMAC mimetics elicit a proinflammatory cell death in cancer cells thatengages an adaptive antitumor immune response. Cancer cells of different histologic origin underwentapoptosis when transduced with lentiviral vectors encoding a cytosolic form of the SMAC mimetic LV-tSMAC. Strikingly, treatment of tumor-bearing mice with LV-tSMAC resulted in the induction of apoptosis,activation of antitumor immunity, and enhanced survival. Antitumor immunity was accompanied by anincrease of tumor-infiltrating lymphocytes displaying low PD-1 expression, high lytic capacity, and high levelsof IFN-g when stimulated. We also noted in vivo a decrease in regulatory T cells along with in vitro activationof tumor-specific CD8þ T cells by dendritic cells (DC) isolated from tumor draining lymph nodes. Last, tumor-specific cytotoxic T cells were also found to be activated in vivo. Mechanistic analyses showed thattransduction of cancer cells with LV-tSMAC resulted in exposure of calreticulin but not release of HMGB1or ATP. Nevertheless, DCs were activated upon engulfment of dying cancer cells. Further validation ofthese findings was obtained by their extension in a model of human melanoma using transcriptionallytargeted LV-tSMAC. Together, our findings suggest that SMAC mimetics can elicit a proinflammatorycell death that is sufficient to activate adaptive antitumor immune responses in cancer. Cancer Res; 72(6);1342–52. �2012 AACR.
IntroductionDeregulated apoptosis is a hallmark of many cancers (1).
Several abnormalities have been described, including over-expression of inhibitor of apoptosis proteins (IAP; refs. 1–3)and failure of IAP antagonists to translocate from the mito-chondria to the cytosol (4). Second mitochondria-derivedactivator of caspases (SMAC), also known as direct inhibitorof apoptosis-binding protein with low pI (DIABLO) is an IAPantagonist that is a potentially interesting therapeutic target.Cleavage of the mitochondrial targeting signal of SMAC is
required for its translocation to the cytosol, in which SMACbinds to the baculovirus IAP repeat domain of IAPs (5). As suchSMAC competes with caspase-3 and -9 for binding to IAPs,resulting in their release, cleavage of their substrates, andinduction of apoptosis. This knowledge has led to the devel-opment of small molecules that mimic SMAC functions (6–9)and to the development of gene vectors encoding full-lengthSMAC (10), pro-SMAC (4, 11), or processed SMAC (tSMAC;ref. 12), illustrating that SMAC plays a pivotal role in the onsetof cancer cell apoptosis.
Dendritic cells (DC) in the tumor environment are mainlyimmature (13), as such ideally equipped to engulf andprocess dying cells (14). However, tumor-derived factorskeep DCs immature (13), trap them within the tumor (15)hence impair effective presentation of ingested antigens to Tcells. Moreover, these immature DCs have immune suppres-sive rather than stimulating properties (16). It has beensuggested that this blockade on antigen-presenting cellscan be overcome by proinflammatory signals such as path-ogen-derived factors (13). Because we showed that lentiviralvectors (LV) activate DCs through Toll-like receptors (TLR;refs. 17, 18), we evaluated whether lentiviral delivery oftSMAC (LV-tSMAC) to tumor cells induces apoptosis andboosts antitumor immunity. We report on the antitumorefficacy of this strategy, together with the immunologicmechanisms responsible for its outcome.
Authors' Affiliations: 1Department of Immunology-Physiology, Labora-tory of Molecular and Cellular Therapy; 2Laboratory of Hematology-Immunology, Vrije Universiteit Brussel, Jette; 3VIB Department forMolecular Biomedical Research, Research Unit for Molecular Patho-physiology and Experimental Therapy, University of Ghent, Ghent,Belgium; and 4Centre for Molecular Oncology and Imaging, BartsCancer Institute, Queen Mary University of London, United Kingdom
Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).
Corresponding Author: Karine Breckpot, Laboratory of Molecular andCellular Therapy, Department of Immunology-Physiology, Vrije UniversiteitBrussel, Laarbeeklaan 103/E, B-1090 Jette, Belgium. Phone: 0032-2-477-4565; Fax: 0032-2-477-4568; E-mail: [email protected]
Mice, cell lines, and tumor-infiltrating lymphocytesFemale 6- to 12-week-old C57BL/6, DBA/2, and C3H3 mice
were purchased fromHarlan. OT-Imice that carry a transgenicCD8þ T-cell receptor specific for the MHC I-restricted oval-bumin (OVA) peptide SIINFEKL were a gift from B. Lambrecht(University of Ghent). C57BL/6 TLR4 KOmice were a gift fromS. Akira (Hyogo College of Medicine). Animals were handledaccording to the Institutional Guidelines. Approval from theEthical Committee for use of laboratory animals of the VrijeUniversiteit Brussel was obtained.The mouse melanoma cell lines K1735-C4 (provided by
I.J. Fidler, University of Texas), B16F0 and MO4 (providedby K. Rock, University of Massachusetts Medical Center), andHEK293T cells [American Type Culture Collection (ATCC)]were cultured in Dulbecco's Modified Eagle Medium(Lonza), supplemented with 10% fetal bovine serum (Harlan),2 mmol/L L-glutamine (L-Glu; Lonza), 100 U/mL penicillin,and 100 mg/mL streptomycin (PS; Lonza). The mouse masto-cytoma cell line P815 (provided by C. Uytttenhove, Universit�eCatholique de Louvain), the human colon carcinoma cellline Caco-2 (ATCC), the human breast carcinoma cell lineMCF7 (ATCC), and the human melanoma cell lines 888-meland 1087-mel (provided by S.L. Topalian, Surgery Branch,National Cancer Institute) were cultured in RPMI-1640 medi-um (Lonza) supplemented with FBS, L-Glu, and PS. No fullauthentication was carried out. Cell lines were tested for theirknown characteristics including expression of antigens andMHC molecules by reverse transcriptase PCR or flow cytome-try. Their in vitro and in vivo growth characteristicswere closelymonitored.Tumor-infiltrating lymphocytes (TIL L2D8), recognizing
gp100 presented by HLA-A2 (provided by M. Dudley, SurgeryBranch, NIH) were maintained in RPMI-1640 containing1% human AB serum (PAA Laboratories), PS, L-Glu, and6,000 IU/mL IL-2 (Chiron).
Production and characterization of LVsThe packaging plasmid pCMVDR8.9 and the VSV.G encod-
ing plasmid pMD.G were a gift from D. Trono (Universityof Geneva). The plasmids pHR'-trip-CMV-luc2-Ires-tNGFR-SIN, pHR'-trip-CMV-Ires-tNGFR-SIN, pHR'-trip-CMV-Ires-eGFP-SIN, and pSIN-Thy1.1 were described (18, 19). Thesurvivin gene was obtained as a BamHI-BclI fragmentfrom the plasmid pcDNA3.1-survivin (a gift from E.Conway, Katholieke Universiteit Leuven) and clonedinto the BamHI linearized pHR'-trip-CMV-Ires-tNGFR-SIN. The human tyrosinase promoter (huTYR) was obtainedfrom pGL3-huTYR2E/P (a gift from D. Nettelbeck, GermanCancer Research Center; ref. 20) as a ClaI-BglII fragmentand used to replace the cytomegalovirus (CMV) promoterin pHR'-trip-CMV-Ires-eGFP-SIN, after restriction diges-tion with ClaI-BamHI. The sequence encoding tSMACwas excised from pcDNA3.1-tSMAC (21) using BglII-XbaIand cloned into the BamHI-SpeI digested pHR'-trip-CMV-Ires-tNGFR-SIN or pHR'-trip-huTYR-Ires-eGFP-SIN. Enzymes were purchased from Fermentas. The pro-
duction and characterization of LVs was described(18, 22).
Transduction of cells with LVsIn vitro transduction of 105 tumor cells was carried out at
the indicated multiplicity of infection (MOI). Where indi-cated tumor cells were treated with 1 mg/mL dexametha-sone (Organon) or 1 mg/mL mitoxantrone (Teva). Palpabletumors grown at the tailbase from 3 � 105 tumor cells wereinjected with 107 TU LVs. Where indicated mice weredepleted of CD8þ T cells by i.p. injection of 50 mg anti-CD8 antibody (clone 2.43.1, prepared in-house) 24 and2 hours before treatment, or treated with a s.c. injectionof 200 mg ZVAD-fmk (Bachem) or an i.p. injection of 5 U ofapyrase (Sigma-Aldrich), 1 hour before and 24 hours aftertreatment.
Western blotThe preparation of cell lysates and protein quantification
were described (23). Proteins (mg) were separated on a 15%SDS-PAGE and transferred to a nitrocellulose membrane.The following primary antibodies were used: polyclonalrabbit anti-mouse/human antibodies against panIAP (R&DSystems), survivin, caspase-3 and -9 (Santa Cruz Biotech-nology). An anti-rabbit immunoglobulin G (IgG) horseradishperoxidase (HRP)-conjugated antibody (Cell Signaling) wasused for detection. An HRP-conjugated b-actin antibody(Cell Signaling) was used for normalization. Antibody bind-ing was visualized with enhanced chemoluminescence(Pierce).
In vivo bioluminescence imagingIn vivo bioluminescence imaging was carried out as
described (24).
Preparation of single-cell suspensionSingle-cell suspensions were prepared after isolation of
tumors with the GentleMACS single cell isolation protocol(Miltenyi Biotec).
Flow cytometryStaining of cellular markers was previously described (22).
Mouse cells were stained with allophycocyanin (APC) conju-gated antibodies against CD11c, CD8, and Foxp3; fluoresceinisothiocyanate (FITC)-conjugated antibodies against CD86,CD11b, and CD4; phycoerythrin (PE)-conjugated antibodiesagainst Thy1.1, CD25, MHC II, and PD-1 (BD Biosciences), andbiotinylated antibodies against CD40, CD80, and Gr-1 (pre-pared in-house). The latter were detected by streptavidin-fluorochrome conjugates. Calreticulin (CRT) was detected bya rabbit anti-mouse CRT antibody and a polyclonal swine anti-rabbit IgG-APC (Abcam). Human cells were characterized withAPC-conjugated antibodies against CD11c and PE-conjugatedantibodies against CD80, CD83, CD86, and HLA-DR (BD Bio-sciences). Data were acquired on a FACSCanto flow cytometer(BD Biosciences) and analyzed with FACSDiva or FlowJosoftware.
Lentiviral Delivery of tSMAC to Tumor Cells
www.aacrjournals.org Cancer Res; 72(6) March 15, 2012 1343
In vitro cytotoxicity assayCD90þ T cells were isolated from single-cell suspen-
sions obtained from MO4 tumors by magnetic-activatedcell sorting (MACS). These T cells, containing on average81.2% � 1.5% CD8þ T cells (data not shown), were culturedin a 96-well round bottom plate at a 1:1 ratio with 2 � 105
B16F0 or MO4 cells labeled with 0.5 or 10 mmol/L carboxy-fluorescein diacetate succinimidine ester (CFSE), respec-tively. The following day, the cells were collected, pooledper condition, and analyzed by flow cytometry. A pool ofB16F0 and MO4 cells, which were not cocultured withT cells served as a control. The percentage specific lysiswas calculated as described in the work of M. Dullaers andcolleagues (25).
In vitro T-cell stimulationCD11cþ cells were isolated from the tumor draining lymph
node as described (18). OVA-specific CD8þ T cells wereobtained from the spleen of OT-I mice by positive selection(Miltenyi Biotec). These cells were cocultured at a stimulatorto responder ratio of 1:10. Supernatants were collected thefollowing day, to determine IFN-g secretion by ELISA(eBoscience). Proliferation of T cells was determined 4 dayslater by incorporation of 3H thymidine (22).
Mouse DCs (22) were cultured at 5 � 105 DCs/mL in thepresence of 50% supernatants of dying tumor cells or AnnexinVþ dying MO4 cells (MACS Dead Cell Removal Kit; MiltenyiBiotec). The following day, their phenotype was evaluatedand compared with immature DCs and DCs matured with10 mg/mL polyI:C (Sigma).
Human DCs from healthy HLA-A2þ donors (26) were cocul-tured at 5� 105 DCs/mL with GFPþHLA-A2� Annexin Vþ 888-mel cells. The following day, their phenotype was evaluated andcompared with immature DCs and DCs matured with inflam-matorycytokines (26).DCs that had engulfeddyingcells (eGFPþ,CD45þ, and CD11cþ) were FACS sorted (FACSAria; BD Bios-ciences) and used to stimulate TIL L2D8 in a 96-well roundbottom plate at a stimulator to responder ratio of 4:1. IFN-gsecretion was measured a day later by ELISA (eBioscience).
In vivo cytotoxicity assayTumor-bearing mice received an i.v. injection of 106
CD8þ OT-I cells 1 day before treatment. The in vivo cyto-toxicity assay was conducted 5 days posttreatment asdescribed (25).
High mobility group box 1 ELISA and ATP assayCellular release of HMGB1 and ATP was measured in
supernatants collected 24 to 72 hours after transduction,using the ELISA kit from Shino Test Corporation andthe luciferin-based ENLITEN ATP Assay from Promega,respectively.
Statistical analysisResults are expressed as mean � SEM. A one-way ANOVA
followed by a Bonferroni multiple comparison test was carriedout. Sample sizes and number of times experiments wererepeated are indicated in the figure legends. The number of
asterisks in the figures indicates the statistical significance asfollows: �, P < 0.05; ��, P < 0.01; ���, P < 0.001.
ResultsInduction of apoptosis by LV-tSMAC results in enhancedsurvival of tumor-bearing mice
To evaluate whether LV-tSMAC induces tumor cell death,we transduced MO4, K1735-C4, and P815 cells in vitro withLV-tSMAC or LV-tNGFR. Mock transduced cells served ascontrols. Cell viability was assessed 3 days later, showinginduction of cell death upon transduction with LV-tNGFR. Thelatter was enhanced when tSMAC was introduced (Fig. 1A–C).
Because LV-tSMAC induced cell death in vitro, we nextevaluated its therapeutic efficacy. When compared with PBS-or LV-tNGFR–treated mice, we observed a delayed tumorgrowth in mice treated with a single intratumoral injectionof LV-tSMAC in the MO4 and K1735-C4 model (Fig. 1D and E).Importantly, treatment of P815-bearing mice resulted in com-plete tumor regression (Fig. 1F).
To evaluate the mechanism behind this prolonged survival,we assessed the induction of apoptosis in vivo upon treatmentof MO4 tumors. Three days posttreatment, tumors were iso-lated and cell lysates prepared for Western blot analysis.We showed downregulation of survivin, upregulation ofcaspase-3 and -9 but no differences in IAP expression in micetreated with LV-tSMAC (Fig. 1G). To further assess the roleof survivin and caspases, we treated MO4-bearing mice withLV-tSMAC alone or combined with injection of LV-survivinor ZVAD-fmk. Mice treated with PBS or LV-tNGFR served ascontrols. Only mice treated with LV-tSMAC showed a delayedtumor outgrowth (Fig. 1H).
The therapeutic effect of LV-tSMAC involves effectorT cells
In vivo bioluminescence imaging carried out upon intra-tumoral injection of LV-luc2, showed modest transduc-tion of tumor cells in situ (Fig. 2A). The low in situ trans-duction efficiency was confirmed in flow cytometry carriedout on cells obtained from LV-Thy1.1–injected tumors (Sup-plementary Fig. S1A). These data suggest that it is unlikelythat induction of cell death is the sole mechanism explainingthe therapeutic potency of LV-tSMAC, leading us to inves-tigate whether LV-tSMAC induces antitumor immuneresponses.
We evaluated the presence of DCs, T cells, regulatory T cells(Treg), and myeloid-derived suppressor cells (MDSC) in theMO4 tumor environment by flow cytometry. We observed nodifferences in DC or MDSC numbers between the differentlytreated mice neither in the tumor nor in the spleen (data notshown). Importantly, we observed that LV-tSMAC–treatedtumors contained a higher number of TILs, which expressedlow PD-1 levels compared with TILs from PBS or LV-tNGFR–treated tumors. Moreover, a reduction in Treg numbers wasobserved in LV-tSMAC–treated tumors (Fig. 2B and C, Sup-plementary Fig. S1B).
To evaluate the function of CD8þ TILs, we transferred OT-Icells to MO4-bearing mice 1 day before treatment. Three days
Emeagi et al.
Cancer Res; 72(6) March 15, 2012 Cancer Research1344
later, CD8þ T cells were isolated from the tumor and cocul-tured with SIINFEKL-pulsed DCs. We observed that TILsisolated from LV-tSMAC–treated mice secreted high levels ofIFN-g (Fig. 2D).Moreover, these TILs showed high specific lysisof target cells in the in vitro cytotoxicity assay (Fig. 2E,Supplementary Fig. S1C).Subsequently, we evaluated the induction of OVA-
specific CTLs upon LV-tSMAC treatment. Therefore, CD8þ
OT-I cells were transferred to MO4-bearing mice 1 day beforetreatment. Five days later, an in vivo cytotoxicity assay wasconducted, showing potent lysis of target cells in mice treated
with LV-tSMAC (Fig. 3A and B). Depletion of CD8þ T cells(Fig. 3C) before treatmentwas carried out to confirm the role ofCTLs in the therapeutic effect of LV-tSMAC. Mice depleted ofCD8þ T cells and treated with LV-tSMAC showed a compa-rable tumor growth as control mice, whereas mice that werenot depleted of CD8þ T cells and treated with LV-tSMACshowed a reduced tumor growth (Fig. 3D).
To assess the generation of a memory immune response,DBA/2 mice that were cured of their tumor (Fig. 1F) wererechallenged with viable P815 cells. In contrast to naive mice,these mice did not develop a tumor (Fig. 3E).
Figure 1. Induction of apoptosis byLV-tSMAC results in reduced tumorgrowth. MO4 (A), K1735-C4 (B), andP815 (C) cells weremock transduced(CTRL) or transduced at a MOI 1 withLV-tNGFR or LV-tSMAC. Three dayslater, the cell viability was assayed byAnnexin V/7-AAD staining. Thegraphs summarize the results of 5experiments. D–F, tumor-bearingmice (5 mice per group) received anintratumoral injection of PBS (CTRL),107 TU of LV-tNGFR or LV-tSMAC,after which tumor growth wasmonitored. Mice with a tumorexceeding 3,000 mm3 wereeuthanized. The graphs show thegrowth curves of MO4 (D), K1735-C4(E), and P815 (F) tumors. G, 3 daysposttreatment, MO4 tumors wereisolated and Western blot analysisconducted to evaluate theexpression of caspases, surviving,and IAPs. b-Actin was used fornormalization. The resultsshown are representative for 3experiments. H, MO4-bearing mice(5 mice per group) were treated asdescribed in (D). In addition, micewere treated with LV-tSMACcombined with ZVAD-fmk or LV-survivin. The graph shows therespective growth curves.
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panIAP
Lentiviral Delivery of tSMAC to Tumor Cells
www.aacrjournals.org Cancer Res; 72(6) March 15, 2012 1345
DCs play a role in the immunologic effect of LV-tSMACWe next addressed whether DCs isolated from tumor drain-
ing lymph nodes can activate OVA-specific T cells. Three daysafter treatment, sorted CD11cþ cells were cocultured withCD8þ OT-I cells. When compared with CD11cþ cells fromcontrol mice, CD11cþ cells from LV-tSMAC–treated micestimulated CD8þ OT-I cells to produce high levels of IFN-g(Fig. 4A) and to strongly proliferate (Fig. 4B).
DCs are activated upon engulfment of LV-tSMACtransduced dying cells despite the absence ofimmunogenic cell death
To identify the mechanisms underlying the observedimmune activation, we evaluated the exposure of CRT and
release of HMGB1 and ATP by tumor cells 24 to 72 hours afterin vitro transduction. Exposure of CRT on tumor cells trans-duced with LV-tSMAC but not on tumor cells treated withPBS or LV-tNGFR was observed 48 hours after transduction(Fig. 5A). However, we did not observe any release of HMGB1or ATP by the in vitro transduced tumor cells, although bothwere released when tumor cells were treated with mitoxan-trone (Fig. 5B and C). To further address the involvement ofATP in the therapeutic effect of LV-tSMAC, we treated MO4-bearing mice with LV-tSMAC alone or in combination withthe enzyme apyrase to degrade ATP.Mice treated with PBS orLV-tNGFR served as controls. Mice treated with LV-tSMAC inthe presence of apyrase showed a similar delay in tumorgrowth as mice treated with LV-tSMAC (Fig. 5D).
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Figure 2. The therapeutic effect ofLV-tSMAC involves effector T cells.A, MO4-bearing mice were treatedwith 107 TU of LV-luc2. Micetreated with PBS served as acontrol (CTRL). In situ transductionof cells was evaluated by in vivobioluminescence imaging. Thebioluminescent overlay images of 1out of 3 experiments are shown.B–E, palpable MO4 tumors weretreated as described in the legendof Fig. 1D. Three days later, single-cell suspensions were prepared.B and C, flow cytometry wasconducted to evaluate thepercentage of CD4þ T-cells (B),their expression of PD-1 or CD25/Foxp3, and the percentage ofCD8þ T-cells and their expressionof PD-1 (C). The graphs summarizethe results. Each dot represents anindividual mouse. The horizontalline depicts the mean. D and E,CD8þ TILs were sorted andcultured with SIINFEKL-pulsedDCs to evaluate the production ofIFN-g (D). The results shown are asummary of 3 experiments. E,alternatively, theseCD8þTILswerecultured with B16F0 (CFSElow) andMO4 cells (CFSEhigh) to evaluatetheir lytic capacity. The graphsummarizes the results of 2experiments.
Emeagi et al.
Cancer Res; 72(6) March 15, 2012 Cancer Research1346
To further evaluate a role for HMGB1, we evaluated thephenotype of wild-type and TLR4 KO DCs incubated withtumor cell–conditioned media or Annexin Vþ cells from LV-tSMAC- or mitoxantrone-treated tumor cells. The phenotypeof both DC types was not altered when compared with imma-ture DCs incubated with media obtained from LV-tSMAC–transduced tumor cells. As expected, incubation of wild-typeDCs but not TLR4 KO DCs with medium obtained frommitoxantrone-treated cells induced upregulation of matura-
tion markers. When both DC types were cultured in thepresence of Annexin Vþ cells, we observed an upregulationof maturation markers. Of note, this was most pronouncedupon coculture with Annexin Vþ cells from LV-tSMAC–trans-duced tumor cells (Table 1).
As we could not evidence a role for ATP or HMGB1but showed that uptake of LV-tSMAC–transduced Annexin Vþ
cells by DCs results in their activation, we addressed whethermice could be protected from a subsequent challenge with
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Figure 3. The therapeutic effect of LV-tSMAC is dependent on CD8þ T cells. A and B, an in vivo cytotoxicity assay was conducted in MO4-bearing mice thatreceived CD8þ OT-I cells. A, the flow cytometry graphs show the specific lysis of OVA-pulsed targets (CFSEhigh) and are representative for 2 experiments.B, the graph summarizes the results of the in vivo CTL assays. Each dot represents an individual mouse. The horizontal line depicts the mean. C andD, MO4-bearing mice received an i.p. injection of PBS or 50 mg of an anti-CD8 antibody 24 and 2 hours before treatment (5 mice per group). C, depletion ofCD8þ T cells was verified by flow cytometry. The results shown are representative for 3 mice. D, the graph shows the growth curves of tumors injectedwith PBS (CTRL), LV-tNGFR, or LV-tSMAC in mice depleted or not of CD8þ T cells. E, to assess the presence of memory T cells, DBA/2 mice that werecured (Fig. 1C) were challenged with viable P815 cells. Naive mice served as a control. The graph shows the growth curves.
Lentiviral Delivery of tSMAC to Tumor Cells
www.aacrjournals.org Cancer Res; 72(6) March 15, 2012 1347
viable MO4 cells by immunization with Annexin Vþ LV-tSMAC–transduced MO4 cells. Naive mice served as a control.In contrast to naive mice, we observed that immunized miceshowed a delay in tumor growth (Fig. 5E).
Uptake of LV-tSMAC–transduced dying humanmelanoma cells enables DCs to activate T cells
To translate the mouse data to a human melanoma model,we first evaluated whether the expression of tSMAC can be
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Figure 5. Treatment of tumor cellswith LV-tSMAC results in exposureof CRT but not HMGB1 or ATPrelease. A–C,MO4, K1735-C4, andP815 cells were transduced asdescribed in the legend of Fig. 1A.Tumor cells treated withdexamethasone or mitoxantroneserved as controls. A, thehistogram overlays show theexposure of CRT 2 daysposttransduction. The resultsshown are representative for 3experiments. The graphs show theamount of HMGB1 (B) and ATPreleased by tumor cells treated asindicated (C). The results shownare representative for 3experiments. D,mice-bearingMO4tumors (5 mice per group) weretreated as described in the legendof Fig. 1D. In addition, mice weretreated with LV-tSMAC combinedwith apyrase. The graph shows thetumor growth. E, mice wereimmunized with Annexin Vþ LV-tSMAC–transduced MO4 cells orPBS (naïve). Five days later, micewere challenged with viable MO4cells, after which tumor growthwasmonitored. The graph shows thegrowth curves.
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Figure 4. DCs play a role in the immunologic effect of LV-tSMAC. MO4-bearing mice were treated as described in the legend of Fig. 1D. Three days later,CD11cþ cells were sorted from tumor draining lymph nodes and used to stimulate sorted CD8þOT-I cells. A, the following day, supernatants were tested forthe presence of IFN-g . B, proliferation of the stimulated CD8þOT-I cells was evaluated by incorporation of 3H thymidine on day 4 of coculture. The stimulationindex was calculated as the proliferation when stimulated divided by the steady state proliferation. The graphs summarize the results of 2 experiments.
Emeagi et al.
Cancer Res; 72(6) March 15, 2012 Cancer Research1348
Figure 6. Uptake of LV-tSMACtransduced Annexin Vþ humanmelanoma cells enables DCs toactivate TILs. A–E, 888-mel cellswere transduced at MOI 5 withtranscriptionally targeted LV-eGFPor LV-tSMAC. Mock-transducedcells served as a control (CTRL).Where indicated cells were treatedwith dexamethasone ormitoxantrone. A, 3 days later, cellswere stained for Annexin V/7-AAD.One experiment out of 3 is shown.The histogram overlays in (B) showthe exposure of CRT 2 daysposttransduction. The results shownare representative for 3 experiments.C and D, the graphs show theamount of HMGB1 (C) and ATP (D)released by tumor cells treated asindicated. The results shown arerepresentative for 2 experiments. Eand F, Annexin Vþ and GFPþ cellswere sorted and added to immatureDCs. Uptake of dying cells wasmonitored by flow cytometry. Thegraph depicts the percentage ofCD11cþ and GFPþ cells andsummarizes the results of 4independent experiments. E, flowcytometry was applied to evaluatethe expression of maturationmarkers on DCs that engulfed dyingcells. Immature DCs and DCsactivated with proinflammatorycytokines served as controls. Theresults shown are a summary of 3experiments. F, DCs (HLA-A2þ) thatengulfed Annexin Vþ cells (HLA-A2�)were sorted and cocultured with TILL2D8. TILs cocultured withnonmanipulated DCs and 1087-mel(HLA-A2þ) served as a negative andpositive control, respectively. Thegraph depicts the production ofIFN-g by TILs. The results shown arerepresentative for 3 experiments.
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Lentiviral Delivery of tSMAC to Tumor Cells
www.aacrjournals.org Cancer Res; 72(6) March 15, 2012 1349
restricted to melanoma cells with the huTYR promoter. UsingGFP as a reporter, we showed that the huTYR promoterrestricted the expression to tyrosinaseþ cells (SupplementaryFig. S2A). However, the expression of GFP was low whencompared with the expression upon transduction withCMV-containing LVs. Therefore, we increased the MOI usedfor transduction, showing high GFP expression when anMOI of 5 was used (Supplementary Fig. S2B). Importantly,transduction of melanoma cells with targeted LVs encodingboth tSMAC and GFP at MOI 5 resulted in induction ofapoptosis (Fig. 6A). We next evaluated the exposure of CRTand release of HMGB1 and ATP by 888-mel cells transducedwith LV-tNGFRor LV-tSMAC.Mock-transduced cells served asa control. Similar to our observations with mouse tumor cells,we observed expression of CRT (Fig. 6B) but no release ofHMGB1 (Fig. 6C) or ATP (Fig. 6D) after transduction with LV-tSMAC.
Subsequently,we showed theuptake of dying 888-mel cells bya significant fraction of immature HLA-A2þ DCs (Supplemen-tary Fig. S2C, Fig. 6E). Phenotypic analysis of these DCs showedan enhanced expression of CD80 and CD83 (Fig. 6F). Finally, weshowed that these DCs stimulate production of high levels ofIFN-g by HLA-A2/gp100-specific T cells (Fig. 6G).
DiscussionIn this study, we report on the potency of LV-tSMAC to
induce apoptosis of tumor cells of different histological origin,i.e. melanoma and mastocytoma cells. We demonstrated theanti-tumor efficacy of this strategy together with the under-lying immunologic mechanisms. To our knowledge, we are thefirst to demonstrate that LV-tSMAC induced cell death resultsin anti-tumor immunity, which is mediated by a crosstalkbetween DCs and T-cells.
Previously McNeish and colleagues (12) showed thattransduction of ovarian cancer cells with adenoviral vectorsencoding tSMAC resulted in induction of tumor cell death.Our data extend these findings to melanoma and mastocy-toma cells, which is of importance as it has been suggestedthat the role of SMAC can vary depending on the cell type
(27). Of note, we achieved substantial induction of cell deathusing an MOI as low as 1. We hypothesize that the highefficiency we observed is attributed to the use of LVs as adelivery system. We previously showed that LVs activateTLR3 (17, 18), which is important as it has been described inmelanoma that TLR3 engagement leads to a TRIF-depen-dent activation of caspase-8 hence results in induction ofapoptosis (16). We indeed observed that transduction ofmelanoma cells with LVs encoding a reporter had an effecton melanoma cell death. Importantly, it was described formelanoma (16) and other cancer types (28) that SMACmimicry and TLR3 triggering synergize to induce apoptosis.This could explain the potency of LV-tSMAC in our tumormodels. Moreover, a single injection of 107 TU of LV-tSMACwas sufficient to prolong the survival of tumor-bearing mice.Herewith, we confirm the feasibility of delivering LVs totumor cells in situ to induce tumor cell death, a strategy thatwas previously explored by others, delivering among otherssuicide genes (29, 30).
On the basis of the low in situ transduction efficiency, wehypothesized that the prolonged survival might be due to animmunologic component resulting from the LV-tSMAC–induced cell death. A first observation that corroborates thishypothesis is the infiltration of effector T cells within tumorstreated with LV-tSMAC. Moreover, these T cells showed a lowexpression of PD-1, a receptor expressed on so calledexhausted T cells (31). Our observation that these T cells incontrast to the highly PD-1þTcells fromcontrol animals, couldbe stimulated in vitro to produce high amounts of IFN-g and killtarget cells confirms that PD-1 is critical in the suppression ofTILs (32, 33). In addition, we observed only a low percentage ofCD4þ CD25þ Foxp3þ T cells. These Treg actively contribute toinhibition of effector T cells and APCs through variousmechanisms (34–36). Importantly, both the infiltration ofCD8þ T cells (37) and the "loss" of Treg (38) in the tumorenvironment have been shown to be predictive for therapeuticoutcome. We next showed that CD11cþ cells from tumordraining lymph nodes stimulate T cells that recognize themodel antigen OVA, which is expressed by MO4 cells. Thisobservation suggests that DCs take up dying tumor cells,
Table 1. DCs are activated upon incubation with Annexin Vþ cells
NOTE: Supernatants or Annexin Vþ cells obtained from LV-tSMAC–transduced or mitoxantrone-treated MO4 cells were added toimmature wild-type or TLR4 KODCs. Flow cytometry was carried out 48 hours later to evaluate the expression of CD40, CD80, CD86,andMHC II. ImmatureDCs andpoly I:C–activatedDCs served as controls. The table shows the fluorescence intensity of themarkers asmean � SEM of 2 experiments.
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Cancer Res; 72(6) March 15, 2012 Cancer Research1350
migrate to draining lymphnodes, process tumor antigens, andacquire a mature phenotype that enable them to activateantigen-specific T cells. Although Bonnotte and colleagues(39) succeeded in showing the presence of DCs that hadengulfed FITCþ dying tumor cells in draining lymph nodes,attempts to visualize trafficking of DCs that had taken upGFPþ dying MO4 cells using a similar experimental set upfailed in our hands. Consequently, we were unable toanalyze the phenotype of DCs that engulfed dying cells invivo. However, in vitro analysis of wild-type and TLR4 KODCs that were exposed to dying cell–conditioned media orAnnexin Vþ cells showed that DCs are activated only whendying cells are engulfed. This suggests that LV-tSMAC doesnot induce immunogenic cell death as defined by theexposure of CRT (40), release of HMGB1, and ATP (41), asthe latter would result in DC activation through interactionwith TLR4 and/or the receptor P2X7. Indeed, we onlyobserved exposure of CRT, although we did not observerelease of HMGB1 or ATP (42).Using a human melanoma model, we showed that human
DCs engulf dying cells, resulting in their maturation andpresentation of the tumor cell–derived antigen gp100 to estab-lish TILs. This observation strengthens our view that theinduction of antitumor immune responses is dependent onthe capacity of DCs to engulf dying cells, mature, and migratetoward T cells to present tumor antigen–derived peptides. Afinal observation that strengthens this view is the induction ofCTLs in vivo.Our data show the potential of LV-tSMAC as a strategy to
target the tumor from within. Despite its promise, the clinical
application of this type of therapy must be approached withcaution (43). Undoubtedly a prerequisite for the translationof LV-based strategies to the clinic is restricting transgeneexpression to target cells. As shown, the latter can beaccomplished by the use of target cell–specific promoters.Moreover, strategies to direct the transduction pattern of LVsusing cell-specific nanobodies will further improve the safetyand potential efficacy of LVs as an off-the-shelf therapeutic(44, 45).
In conclusion, LV-tSMAC–induced tumor cell death elicitsantitumor immune responses mediated by a cross-talkbetween DCs and T cells.
Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.
AcknowledgmentsThe authors thank Jurgen Corthals, Xavier De Baere, Petra Roman, Elsy
Vaeremans, and Angelo Willems for their technical assistance and T. Lahouttefor the use of the imaging facility.
Grant SupportThiswork was supported by the Research foundation Flanders (FWO-V, grant
number #G023411N), the Agency of Innovation by Science and Technology, theInteruniversity Attraction Poles Program, the "Stichting tegen Kanker", andBelgian State-Belgian Science Policy. C. Goyvaerts, J.L. Aerts, and K. Breckpotare funded by the FWO-V.
The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received July 18, 2011; revised January 5, 2012; accepted January 11, 2012;published OnlineFirst February 29, 2012.
References1. Vucic D, Fairbrother WJ. The inhibitor of apoptosis proteins as
therapeutic targets in cancer. Clin Cancer Res 2007;13:5995–6000.2. Grossman D, McNiff JM, Li F, Altieri DC. Expression and targeting of
the apoptosis inhibitor, survivin, in humanmelanoma. J Invest Derma-tol 1999;113:1076–81.
3. Vucic D, Stennicke HR, Pisabarro MT, Salvesen GS, Dixit VM.ML-IAP,a novel inhibitor of apoptosis that is preferentially expressed in humanmelanomas. Curr Biol 2000;10:1359–66.
4. Ruttinger D, Li R, Poehlein CH, Haley D, Walker EB, Hu HM, et al.Increased susceptibility to immune destruction of B16BL6 tumor cellsengineered to express a novel pro-Smac fusion protein. J Immunother2008;31:43–51.
5. Martinez-Ruiz G, Maldonado V, Ceballos-Cancino G, Grajeda JP,Melendez-Zajgla J. Role of Smac/DIABLO in cancer progression.J Exp Clin Cancer Res 2008;27:48.
6. Arnt CR, Chiorean MV, Heldebrant MP, Gores GJ, Kaufmann SH.Synthetic Smac/DIABLO peptides enhance the effects of chemother-apeutic agents by binding XIAP and cIAP1 in situ. J Biol Chem2002;277:44236–43.
7. Li L, Thomas RM, Suzuki H, De Brabander JK, Wang X, Harran PG. Asmall molecule Smac mimic potentiates TRAIL- and TNFalpha-medi-ated cell death. Science 2004;305:1471–4.
8. Bockbrader KM, Tan M, Sun Y. A small molecule Smac-mimiccompound induces apoptosis and sensitizes TRAIL- and
Table 1. DCs are activated upon incubation with Annexin Vþ cells (Cont'd.)
etoposide-induced apoptosis in breast cancer cells. Oncogene2005;24:7381–8.
9. Vince JE,WongWW,KhanN, FelthamR,ChauD, Ahmed AU, et al. IAPantagonists target cIAP1 to induce TNFalpha-dependent apoptosis.Cell 2007;131:682–93.
10. McNeish IA, Bell S, McKay T, Tenev T, Marani M, Lemoine NR.Expression of Smac/DIABLO in ovarian carcinoma cells induces apo-ptosis via a caspase-9-mediated pathway. Exp Cell Res 2003;286:186–98.
11. Li R, Ruttinger D, UrbaW, Fox BA, Hu HM. Targeting and amplificationof immune killing of tumor cells by pro-Smac. Int J Cancer 2004;109:85–94.
12. McNeish IA, Lopes R, Bell SJ, McKay TR, Fernandez M, Lockley M,et al. Survivin interacts with Smac/DIABLO in ovarian carcinoma cellsbut is redundant in Smac-mediated apoptosis. Exp Cell Res 2005;302:69–82.
13. Fricke I, Gabrilovich DI. Dendritic cells and tumor microenvironment: adangerous liaison. Immunol Invest 2006;35:459–83.
14. Zitvogel L, Kepp O, Kroemer G. Decoding cell death signals in inflam-mation and immunity. Cell 2010;140:798–804.
15. Feijoo E, Alfaro C, Mazzolini G, Serra P, Penuelas I, Arina A, et al.Dendritic cells delivered inside human carcinomas are sequestered byinterleukin-8. Int J Cancer 2005;116:275–81.
16. Weber A, Kirejczyk Z, Besch R, Potthoff S, Leverkus M, Hacker G.Proapoptotic signalling through Toll-like receptor-3 involves TRIF-dependent activation of caspase-8 and is under the control of inhibitorof apoptosis proteins in melanoma cells. Cell Death Differ 2010;17:942–51.
17. Breckpot K, Emeagi P, Dullaers M, Michiels A, Heirman C, ThielemansK. Activation of immaturemonocyte-derived dendritic cells after trans-duction with high doses of lentiviral vectors. Hum Gene Ther 2007;18:536–46.
18. Breckpot K, Escors D, Arce F, Lopes L, Karwacz K, Van Lint S, et al.HIV-1 lentiviral vector immunogenicity ismediated byToll-like receptor3 (TLR3) and TLR7. J Virol 2010;84:5627–36.
19. Zeissig S, Dougan SK, Barral DC, Junker Y, Chen Z, Kaser A, et al.Primary deficiencyofmicrosomal triglyceride transfer protein in humanabetalipoproteinemia is associated with loss of CD1 function. J ClinInvest 2010;120:2889–99.
20. NettelbeckDM, JeromeV,Muller R. A dual specificity promoter systemcombining cell cycle-regulated and tissue-specific transcriptionalcontrol. Gene Ther 1999;6:1276–81.
21. Baird SK, Aerts JL, Eddaoudi A, Lockley M, Lemoine NR, McNeish IA.Oncolytic adenoviralmutants induce a novelmode of programmed celldeath in ovarian cancer. Oncogene 2008;27:3081–90.
22. Breckpot K, Dullaers M, Bonehill A, van Meirvenne S, Heirman C, deGreef C, et al. Lentivirally transduced dendritic cells as a tool for cancerimmunotherapy. J Gene Med 2003;5:654–67.
23. Aerts-ToegaertC,HeirmanC,TuyaertsS,Corthals J, Aerts JL, BonehillA, et al. CD83 expression on dendritic cells and T cells: correlationwitheffective immune responses. Eur J Immunol 2007;37:686–95.
24. Keyaerts M, Verschueren J, Bos TJ, Tchouate-Gainkam LO, PelemanC, Breckpot K, et al. Dynamic bioluminescence imaging for quantita-tive tumour burden assessment using IV or IP administration of D:-luciferin: effect on intensity, time kinetics and repeatability of photonemission. Eur J Nucl Med Mol Imaging 2008;35:999–1007.
25. Dullaers M, Van Meirvenne S, Heirman C, Straetman L, Bonehill A,Aerts JL, et al. Induction of effective therapeutic antitumor immunity bydirect in vivo administration of lentiviral vectors. Gene Ther 2006;13:630–40.
26. Tuyaerts S,NoppeSM,Corthals J, BreckpotK, HeirmanC,DeGreefC,et al. Generation of large numbers of dendritic cells in a closed systemusing Cell Factories. J Immunol Methods 2002;264:135–51.
27. Yu J,Wang P, Ming L,WoodMA, Zhang L. SMAC/Diablo mediates theproapoptotic function of PUMA by regulating PUMA-induced mito-chondrial events. Oncogene 2007;26:4189–98.
28. Friboulet L, GourzonesC, TsaoSW,Morel Y, Paturel C, TemamS, et al.Poly(I:C) induces intense expression of c-IAP2 and cooperates with anIAP inhibitor in induction of apoptosis in cancer cells. BMC Cancer2010;10:327.
29. Petrigliano FA, Virk MS, Liu N, Sugiyama O, Yu D, Lieberman JR.Targeting of prostate cancer cells by a cytotoxic lentiviral vectorcontaining a prostate stem cell antigen (PSCA) promoter. Prostate2009;69:1422–34.
30. Gerolami R, Uch R, Faivre J, Garcia S, Hardwigsen J, Cardoso J, et al.Herpes simplex virus thymidine kinase-mediated suicide gene therapyfor hepatocellular carcinoma using HIV-1-derived lentiviral vectors. JHepatol 2004;40:291–7.
31. Wherry EJ. T cell exhaustion. Nat Immunol 2011;131:492–9.32. Zhou Q, Xiao H, Liu Y, Peng Y, Hong Y, Yagita H, et al. Blockade of
programmed death-1 pathway rescues the effector function of tumor-infiltrating T cells and enhances the antitumor efficacy of lentivectorimmunization. J Immunol 2010;185:5082–92.
33. Fourcade J, Sun Z, BenallaouaM,GuillaumeP, Luescher IF, Sander C,et al. Upregulation of Tim-3 and PD-1 expression is associated withtumor antigen-specificCD8þ T cell dysfunction inmelanoma patients.J Exp Med 2010;207:2175–86.
34. Jarnicki AG, Lysaght J, Todryk S, Mills KH. Suppression of antitumorimmunity by IL-10 and TGF-beta-producing T cells infiltrating thegrowing tumor: influence of tumor environment on the induction ofCD4þ and CD8þ regulatory T cells. J Immunol 2006;177:896–904.
35. Larmonier N, Marron M, Zeng Y, Cantrell J, Romanoski A, Sepassi M,et al. Tumor-derived CD4(þ)CD25(þ) regulatory T cell suppression ofdendritic cell function involves TGF-beta and IL-10. Cancer ImmunolImmunother 2007;56:48–59.
37. Zhang L, Conejo-Garcia JR, Katsaros D, Gimotty PA, Massobrio M,Regnani G, et al. Intratumoral T cells, recurrence, and survival inepithelial ovarian cancer. N Engl J Med 2003;348:203–13.
38. Curiel TJ, Coukos G, Zou L, Alvarez X, Cheng P, Mottram P, et al.Specific recruitment of regulatory T cells in ovarian carcinoma fostersimmune privilege and predicts reduced survival. Nat Med 2004;10:942–9.
39. Bonnotte B, Favre N, Moutet M, Fromentin A, Solary E, Martin M, et al.Role of tumor cell apoptosis in tumor antigen migration to the draininglymph nodes. J Immunol 2000;164:1995–2000.
40. Gardai SJ, McPhillips KA, Frasch SC, Janssen WJ, Starefeldt A,Murphy-Ullrich JE, et al. Cell-surface calreticulin initiates clearanceof viable or apoptotic cells through trans-activation of LRP on thephagocyte. Cell 2005;123:321–34.
41. Kepp O, Galluzzi L, Martins I, Schlemmer F, Adjemian S, MichaudM, et al. Molecular determinants of immunogenic cell deathelicited by anticancer chemotherapy. Cancer Metastasis Rev2011;30:61–9.
42. Apetoh L, Ghiringhelli F, Tesniere A, Obeid M, Ortiz C, Criollo A, et al.Toll-like receptor 4-dependent contribution of the immune system toanticancer chemotherapy and radiotherapy. Nat Med 2007;13:1050–9.
43. Escors D, Breckpot K. Lentiviral vectors in gene therapy: their currentstatus and future potential. Arch Immunol Ther Exp (Warsz) 2010;58:107–19.
44. Goyvaerts C, De Groeve K, Dingemans J, Van Lint S, Robays L, et al.Development of the Nanobody display technology to target lentiviralvectors to antigen-presenting cells. Gene Ther. 2012 Jan 12. [Epubahead of print].
45. Breckpot K, Aerts JL, Thielemans K. Lentiviral vectors for cancerimmunotherapy: transforming infectious particles into therapeutics.Gene Ther 2007;14:847–62.
Emeagi et al.
Cancer Res; 72(6) March 15, 2012 Cancer Research1352
2012;72:1342-1352. Published OnlineFirst February 29, 2012.Cancer Res Perpetua U. Emeagi, Sandra Van Lint, Cleo Goyvaerts, et al. Death in Antitumor TherapyProinflammatory Characteristics of SMAC/DIABLO-Induced Cell
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