Interleukin-27 Inhibits the Growth of Pediatric Acute ... · 24/01/2012 · Purpose: Acute myeloid leukemia (AML) accounts for more than half of fatal cases in all pediatric leukemia

Cancer Therapy: Preclinical

Interleukin-27 Inhibits the Growth of Pediatric Acute MyeloidLeukemia in NOD/SCID/Il2rg�/� Mice

Alessia Zorzoli1, Emma Di Carlo5, Claudia Cocco1, Emanuela Ognio4, Domenico Ribatti6, Elisa Ferretti2,Carlo Dufour3, Franco Locatelli7,8, Daniela Montagna8, and Irma Airoldi1

AbstractPurpose: Acute myeloid leukemia (AML) accounts for more than half of fatal cases in all pediatric

leukemia patients; this observation highlights the need of more effective therapies. Thus, we investigated

whether interleukin (IL)-27, an immunomodulatory cytokine, functions as an antitumor agent against

pediatric AML cells.

Experimental Design: Expression of WSX-1 and gp130 on AML cells from 16 pediatric patients was

studied by flow cytometry. Modulation of leukemia cell proliferation or apoptosis upon IL-27 treatment

in vitro was tested by bromodeoxyuridine/propidium iodide (PI) and Ki67, or Annexin V/PI staining and

flow cytometric analysis. The angiogenic potential of AML cells treated or not with IL-27 was studied

by chorioallantoicmembrane assay and PCR array. In vivo studies were carried out using nonobese diabetic/

severe combined immunodeficient (NOD/SCID)/Il2rg�/� mice injected intravenously with five pediatric

AML cell samples. Leukemic cells engrafted in PBS and IL-27–treated animals were studied by immuno-

histochemical/morphologic analysis and by PCR array for expression angiogenic/dissemination-related

genes.

Results: We provided the first demonstration that (i) AML cells injected into NOD/SCID/Il2rg�/� mice

gave rise to leukemia dissemination that was severely hampered by IL-27, (ii) compared with controls,

leukemia cells harvested from IL-27–treated mice showed significant reduction of their angiogenic and

spreading related genes, and (iii) similarly to what was observed in vivo, IL-27 reduced in vitro AML cell

proliferation andmodulated the expression of different genes involved in the angiogenic/spreading process.

Conclusion: These results provide an experimental rationale for the development of future

clinical trials aimed at evaluating the toxicity and efficacy of IL-27. Clin Cancer Res; 18(6); 1–11.

�2012 AACR.

IntroductionInterleukin (IL)-27 is a heterodimeric cytokine belonging

to the IL-12 family (1). It is composed of the EBV-induced 3(EBI3) and p28 subunits that are homologs of the p40 andp35 chains of IL-12, respectively (2). IL-27 is produced byantigen-presenting cells, which protect their local microen-vironment from host pathogens, and it acts on different

cell types expressing the full receptor (R) complex (3, 4). TheIL-27R contains the unique receptor subunit WSX-1, alsoknown as IL-27Ra/TCCR, paired with the gp130 chain (5,6). Although IL-27R was found to be expressed on differenthuman hematologic malignancies (7, 8), no informationis available on the role played by IL-27 on pediatric AMLcells.

IL-27 exerts antitumor activity against different tumorsthrough indirect mechanisms, such as induction of naturalkiller (NK) and CTL response or inhibition of angiogenesisprimarily due to induction of CXCL10 and CXCL9 (3, 9–14). It has also been reported that IL-27 can directly inhibitthe proliferation of human melanoma cell lines (15), sup-presses tumorigenicity and invasiveness of human lungadenocarcinoma cell lines (16), and dampens multiplemyeloma cell growth in vivo, inhibiting osteoclast differen-tiation and function and inducing osteoblast proliferation(8). In addition, we recently reported that IL-27 inhibitsB-acute lymphoblastic leukemia (ALL) cell spreading inanimal models (7).

AML includes a heterogeneous group of hematologictumors deriving frommalignant transformation ofmyeloid

Authors' Affiliations: 1A.I.R.C. Laboratory of Immunology and Tumors,Department of Experimental and Laboratory Medicine; 2Laboratory ofOncology; 3Hematology Unit, Department of Oncology/Hematology,IRCCSG.Gaslini Institute; 4Animal Model Facility, IRCCSNational Institutefor Cancer Research, Genoa; 5Department of Oncology and Neuros-ciences, "G. d'Annunzio" University and Ce.S.I. Aging Research Center,"G. d'Annunzio" University Foundation, Chieti; 6Department of HumanAnatomy and Histology, University of Bari, Bari; 7Department of PediatricHematology and Oncology, IRCCS, Bambino Ges�u Hospital, Rome; and8Department of Pediatrics, University of Pavia, Pavia, Italy

Corresponding Author: Irma Airoldi, A.I.R.C. Laboratory of Immunologyand Tumors, Department of Experimental and Laboratory Medicine, G.Gaslini Institute,Genoa16147, Italy. Phone: 39-010-5636342; Fax: 39-010-3779820; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-11-2432

�2012 American Association for Cancer Research.

ClinicalCancer

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progenitors that proliferate and accumulate in the bonemarrow (BM; refs. 17, 18) that accounts for more than halfof fatal cases in all pediatric leukemia patients (19, 20). Therole of cytokines in the treatment of acute leukemias includ-ing AML has been investigated in several studies because oftheir immunologic and/or cycling properties (21). Cyto-kines such as IL-2, granulocyte-colony stimulating factor(G-CSF) and granulocyte macrophage-CSF (GM-CSF) havebeen mainly used as adjuvants of chemotherapy, as theymay stimulate immune cells (22) or limit the duration ofneutropenia andpromote cell cycling of quiescent leukemiacells, making them more sensitive to chemotherapeuticdrugs (21).

In this study, we investigated the ability of IL-27 torestrain pediatric AML cell growth both in vitro and in vivo,unraveling the potential mechanisms involved.

Materials and MethodsPatients

The study was approved by the Institutional Review Boardof IRCCS G. Gaslini Institute, Genoa and IRCCS FondazionePoliclinico San Matteo, Pavia, Italy. This investigation com-pliedwith the Principles ofHelsinkiDeclaration, and parents/legal guardians of the patients gave written informed consentto study participation. The initial diagnosis of AML, togetherwith subtype identification, was established according to theWHO and the French–American–British classification (18).Clinical characteristic of the children enrolled in the study aredetailed in Table 1. In the study, we employed aliquots ofpatient BM samples collected at disease onset for diagnosticpurposes, which contained at least 87% leukemia cells (medi-an92.5%, range: 87%–98%).Mononuclear cellswere isolatedfromBMbyFicoll-Hypaquedensity gradient (SigmaChemicalCo.), cells were collected and cryopreserved for later use.

Antibodies, reagents, and flow cytometryThe following antibodies (Ab) were used for flow cyto-

metric analyses: fluorescein isothiocyanate (FITC)-, PE-Tricolor-, APC-conjugated CD45, CD33, and anti-gp130monoclonal Abs (mAb) obtained from Invitrogen; PE- andFITC-conjugated anti-CXCR4 mAb, PE-conjugated anti-WSX1 mAb purchased from R&D System, Inc.; FITC-con-jugated CD3 (Immunotools GmbH) and PC7-conjugatedCD19 (Beckman Coulter). All of the above mAbs reactedonly with human cells. FITC-conjugated anti-human Ki67,a nuclear cell-cycle molecule marker of dividing cells at agiven time, and anti-bromodeoxyuridine (BrdUrd) mAbswere obtained fromDako and BD Biosciences, respectively.Cells were scored using a FACSCalibur analyzer (BD Bio-sciences) and data were processed using CellQuest software

Table 1. Clinical and laboratory features of pediatric AML patients

Patientsno.

FABclassification Sex

Age atdiagnosis

WBC/�109/Lat diagnosis

Cytogeneticanomalies

%WSX1þ

cells/MRFI

1 M6 M 12 y 13.240 None 80/192 M5 M 16 y 0.550 None 42/213 M4 M 12 y 62 �7 57/164 M1 M 15 y 300 None 19/13.25 M4 M 6 y 98 None 45/296 M2 F 8 y 29.2 None 14/12.87 t-AML F 4 y 16.5 None 34/4.98 t-AML M 14 y 31 Complex karyotype 41/79 M4 M 1 y 65 inv16 62/610 M3 M 8 y 2.1 t(15;17) 10/4.211 M4 M 10 mo 32 inv16 6/812 M2 F 19 y 14 t(6;9) 23/2513 M5 F 3 mo 174 None 74/2414 M3 F 2 y 3.1 None 70/1815 M2 M 9 y 16.5 �7 40/3316 M5 F 11 y 78 None 71/24

Abbreviations: t-AML, therapy-related AML; M, male; F, female.

Translational RelevanceAcute myeloid leukemia (AML) includes a group of

heterogeneous hematologic neoplasias that account formore than half of fatal cases in all pediatric leukemiapatients, highlighting the need of new, more effectivetherapies. In this view, here we provide the first evidencethat interleukin (IL)-27 dampens pediatric AML cellgrowth and spreading in NSG mice by acting directlyon leukemic cells andmodulated expression of differentgenes involved in angiogenic and spreading processes.Our results support the concept that IL-27may representa new antileukemic drug to be tested in future clinicaltrials.

Zorzoli et al.

Clin Cancer Res; 18(6) March 15, 2012 Clinical Cancer ResearchOF2




3.0 (BD Biosciences). Human recombinant (hr) IL-27 waspurchased from R&D Systems, Inc.

Signaling pathwayIL-27 signaling pathway was investigated in purified AML

cells from patients no. 3, 14, and 16 by flow cytometry.Signal transduction was investigated using the following:Alexa 488 fluorochrome–conjugatedmAbs: anti–phospho-STAT1 (clone 58D6), anti-STAT3 (cloneD3A7), anti-STAT5(clone C71E5; Cell Signaling Technology, Inc.), and fluo-rochrome-matched isotype antibodies (Beckman Coulter).Cells were resuspended in RPMI 10% AB serum and incu-batedwithmediumaloneorhrIL-27 for 30minutes at 37�C,5% CO2, and then processed following instructions of themanufacturer. Cells were scored by Gallios flow cytometry,104 events were acquired and analyzed using Kaluza soft-ware (BeckmanCoulter). Datawere expressed as percentageof phosho-STAT–positive cells and as mean relative offluorescence intensity (MRFI), calculated as fluorescenceintensity obtained with specific mAb/fluorescence intensityobtained with irrelevant isotype-matched mAb.

Cell proliferation and apoptosis assayNine pediatric AML cell samples (patients no. 1–9) were

cultured for 24, 48, and 60 hours, either in the presence orabsence of 50 ng/ml IL-27 (concentration selected accordingto previous studies; refs. 7, 8), and were tested for cellproliferation. All 16 AML cell samples were tested for apo-ptosis following the same treatment schedule used for pro-liferation assay. AML cell proliferation was studied by intra-cellular staining with anti-Ki67mAb using the Cytofix/Cyto-permKit (BDBiosciences) andbyBrdUrd staining. AML cellswere pulse-labeled with 10 mmol/L BrdUrd (Sigma) for 30minutes at 37�C temperature. Then, cells were washed andBrdUrd taken up was stained with FITC-conjugated anti-BrdUrd mAb and 5 mg/mL propidium iodide (PI; Sigma),according to the manufacturer protocol. Finally, flow cyto-metric analysis was carried out. Apoptosis was assessed usingthe rhAnnexin V/FITC Kit from Immunostep and apoptoticcells were identified as Annexin Vþ/PIþ cells.

Chorioallantoic membrane assayChorioallantoic membrane (CAM) assay was carried out

as previously reported (23). Sponges were loaded with 1 mLPBS (negative control), 1 mL PBS with 250 ng VEGF (R&DSystems, positive control), 1 mL conditioned medium fromunfractionated AML cell samples (patients no. 1, 9, 13, 14,and 16) cultured for 36 hours with or without IL-27 (50 ng/mL); IL-27 diluted in medium at the same final concentra-tionused to treat tumor cells; 250ngVEGFþIL-27diluted inmedium at the same final concentration used to treat tumorcells. All conditioned medium were tested in triplicate andmeans � SD were calculated. CAM were examined dailyuntil day 12 and photographed in ovo with a stereomicro-scope equipped with a camera and image analyzer system(Olympus). On day 12, the angiogenic response was eval-uated by the image analyzer system as the number of vesselsconverging toward the sponges.

In additional experiments, CAM were treated on day 8with sponges loaded with 1 mL of medium from AML cellstreatedwith IL-27 (patients no. 1, 9, 13, 14, and 16) alone orcontaining 0.5 mg/mL of an anti-human IFN-g mAb (EBios-ciences). Appropriate isotype-matched mAb were used ascontrols.

Differences in the number of vessels formed inCAMassayand differences in percentage and absolute numbers wereevaluated by Student t test with 99% confidence interval(99% CI; GraphPad Prism 3). All statistical tests were 2-tailed. A P value lower than 0.05 was considered to bestatistically significant.

Mice studiesFive/6-week-old nonobese diabetic/severe combined

immunodeficient/Il2rg-deficient (NSG) mice (JacksonLaboratory, Bar Harbor, ME), including both females andmales, were housed under specific pathogen-free condi-tions. All procedures involving animals were done inaccordance with national and international current reg-ulations (Italian D.L 27/01/1992, n.116, European Eco-nomic Community Council Directive 86/609, OJL 358,December 1, 1987). A total of 5 � 106 AML cells fromeach patient (nos. 1, 2, 3, 9, and 13) were injectedintravenously in 2 groups of 3 to 6 animals each. TheseAML samples were selected because they were highlyenriched (>95%) in neoplastic cells. One group of micewas treated intravenously with 2 doses per week of hrIL-27 (1 mg/mouse/dose) starting from 3 days after tumorcell injection. The other group of animals was treated withPBS (controls) according to the same time schedule. Tento 12 weeks after tumor cell inoculation, mice weresacrificed and the following tissues were collectedfrom each animal: spleen, peripheral blood (PB) fromthe retro-orbital vein, and BM obtained by femur flush-ing. An engraftment criterion of more than 1% of humanCD45þCD33þ cells in mononuclear cells from murinetissues assessed by flow cytometry was used as biologi-cally significant cut-off. Human leukemic cells were iden-tified as CD45þCD33þ cells.

Morphologic and immunohistochemical analysesTissue samples were fixed in 4% neutral buffered forma-

lin, embedded in paraffin, sectioned at 4 mm, and stainedwith hematoxylin and eosin (H&E). For immunohis-tochemistry, formalin-fixed, paraffin-embedded sectionswere immunostained with rabbit anti-human/mouse lam-inin Ab (Biogenex). After washing, sections were overlaidwith goat anti-rabbit immunoglobulin (Ig) conjugatedwithperoxidase-labeled dextran (EnVisionþ Peroxidase, rabbit/mouse; Dako) for 30minutes. Unbound Igwas removed bywashing and slideswere incubatedwithABC (avidin–biotincomplex)/alkaline phosphatase (Dako) for 30 minutes;then, sectionswere counterstainedwithH&E. Silver impreg-nation was done with Silver Impregnation For ReticulumStaining 04-040801 Kit (Bio-Optica Milano S.p.A), onparaffin-embedded sections according to manufacturer’sinstructions.

AML and IL-27

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PCR arrayTotal RNA was extracted using the RNeasy Micro Kit

(Qiagen,GmbH) from the following cell samples: (i) spleenand BM from 1 control and 1 IL-27–treated mouse injectedwith AML cells from patient no. 1, (ii) spleen and BMfrom 1 control and 1 L-27–treated animal injected withAML cells from patient no. 13, and (iii) AML cells frompatients no. 1 and 13 at diagnosis cultured 36 hours inthe presence or absence of hrIL-27. AML harvested frommurine tissues were purified using human CD45 Micro-Beads (Miltenyi Biotec). The purity of isolated CD45þ cellswas more than 95%, as assessed by flow cytometry.

Contaminant genomic DNA was removed by Dnasetreatment (Qiagen GmbH). RNA was retrotranscribed bythe RT2 First Strand cDNA Synthesis Kit (SABioscience).Human Angiogenesis (code #PAHS-024A) and Metastasis(code #PAHS-028A) RT2PCRArray andRT2 Real-Time SyBRGreen/ROX PCR Mix were from SABioscience. PCR wasdone on the ABI Prism 7700 Sequence Detector (AppliedBiosystems). Gene expression of IL-27–treated and controlsamples were analyzed separately in different PCR arrayplates. For each plate, results were normalized on themedian value of a set of housekeeping genes. Then, changesin gene expression between IL-27–treated and control sam-ples were calculated using the DDCt formula. Results fromthe different IL-27–treated and control samples, done induplicate, were pooled and analyzed by the software pro-vided by the manufacturer. A significant threshold of 4-foldchange in gene expression corresponded to P < 0.001.

ChemotaxisChemotaxis of human CD45þ cells purified from BM

(n ¼ 2 from controls and n ¼ 2 from IL-27–treated mice)and spleens (n ¼ 2 from controls and n ¼ 2 from IL-27–treated mice) was tested using transwell plates (5-mm poresize; Costar). A total of 2� 105 human cells were dispensedin the upper chamber and CXCL12 (300 ng/mL; R&DSystem) or medium alone was added to lower chamber.Plates were incubated 2 to 5 hours at 37�C. Cells from thelower compartments were collected and counted.

Statistical analysisDifferences in the number of vessels formed inCAMassay

were evaluated by Student t test; differences in STAT phos-phorylation, percentage, and absolute numbers were eval-uated by Mann–Whitney test with 99% CI (GraphPadPrism 3). All statistical tests were 2-tailed. A P value lowerthan 0.05 was considered to be statistically significant.

ResultsExpression and function of IL-27 receptor in pediatricAML cells

We initially investigated the expression of both chains ofIL-27R, namedWSX-1 and gp130, in primary leukemia cellsfrom 16 pediatric patients with a diagnosis of either de novoor therapy-related AML. To this end, 3 color staining of BMmononuclear cells fromAMLpatients for CD45, CD33, andWSX-1 or gp130 was done and cells were analyzed by flow

cytometry. WSX-1 and gp130 expression was analyzed ongated CD45þCD33þ AML cells.

Such analyses revealed that AML cells constitutivelyexpressed both chains of IL-27R (mean percentage ofgp130þ cells being 67%, range: 58%–81%; mean percent-age of WSX-1þ cells being 43%, range: 6%–80%), thusrepresenting a target of IL-27. Three representative experi-ments showing WSX-1 and gp130 expression are reportedin Fig. 1A, top and bottom panels, respectively. Data onMRFI and percentages of WSX-1 expression in blasts fromeach patient is reported in Table 1.

To elucidate the downstream pathway induced by IL-27,we analyzed the ability of the cytokine to induce activationof specific STAT molecules in AML cells, as reported forhuman tonsil B cells (24) andplasma cells (7). To this end, 3purified AML cell suspensions were treated withmedium orIL-27 and tested for intracellular phosphorylation (p) ofSTAT1, 3 and 5, by flow cytometry. These experimentsrevealed that IL-27 induced a significant phosphorylationof STAT1 (IL-27 vs. medium: MRFI 2.15 � 0.07 vs. 1.68 �0.25, P ¼ 0.0357; percentage of p-STAT1þ cells 35 � 11.31vs. 13� 8.48, P¼ 0.0027) and of STAT3 (IL-27 vs.medium:MRFI 2.25� 0.35 vs. 1.895� 0.27, P¼ 0.0079; percentageof p-STAT3þ cells 31.5 � 9.19 vs. 17 � 11.31, P ¼ 0.0035),but not of STAT5 in purified AML cells (Fig. 1B).

IL-27 inhibits pediatric AML cell growth in NSG miceThe antitumor activity of IL-27 against pediatric AML cells

was tested in vivo in NSG mice, which represent the mostsuitable model for efficient engraftment of primary neo-plastic cells (25). Thus, each AML cell suspension obtainedfrom 5 patients (patients no. 1, 2, 3, 9, and 13) was injectedintravenously in 2 groups of 3 to 6 animals each (totalanimals injected¼ 44); mice were sacrificed 10 to 12 weekslater when signs of poor health became evident in controls.PB, spleen, and BM from each animal were collected andsubjected to immunophenotypic evaluation and flow cyto-metric analysis. Human cells were identified using thehuman CD45 surface marker, in association with CD33.Such analyses revealed that AML cells efficiently engrafted inall control and treatedmice, as witnessed by the presence ofat least 1% human CD45þ cells, that consistently coex-pressed the CD33 marker, in PB, BM, and spleens (meanpercentage of CD45þCD33þ cells ¼ 94.5%, range: 89%–97% without significant differences between PB, BM, andspleen). Low levels of CD3þ or CD19þ cells within CD45þ

compartment from the same samples were observed (meanpercentage of CD45þCD3þcells: 3%, range: 1%–5%; meanpercentage of CD45þCD19þ cells: 2.5%, range: 1%–4%;not shown).

Notably, IL-27 significantly reduced the presence ofhuman CD45þCD33þ leukemia cells in PB (P < 0.0001),BM (P¼0.0015), and spleen (P<0.0001) as comparedwithcontrol mice (Fig. 1C). Representative CD45 stainings ofBM, spleen, and PB obtained from animals injected withAML cells from patient no 9 and treated with either PBS orhrIL-27 are shown in Fig. 1D. Cells obtained from patientsno. 3 and 9 and recovered from mice carried the same

Zorzoli et al.





cytogenetic anomaly detected at diagnosis, namely mono-somy 7 and inv (16), thus proving that leukemia cellsengrafted in the animal (not shown). This finding was inaccordance with previous results (26). Summary of humanCD45þCD33þ cells present in each animal is reportedin Table 2.

IL-27 modulated genes involved in angiogenesis andprogression of AML in NSG miceThe data from in vivo experiments prompted us to inves-

tigate the potentialmechanisms underlying the effects of IL-27 on leukemia cell growth in vivo. Morphologic andimmunohistochemical analyses of spleens collected fromcontrols (n ¼ 5) and IL-27–treated mice (n ¼ 5) revealedthat human AML cells gave rise to multiple and wide

neoplastic infiltrates (Fig. 2A, panel a). These infiltrateswere supplied with a well-developed microvascularization(Fig. 2A, panel b) and sustained by a rich stromal reticulinfiber network that seemed to be shaped to intratumoralmicrovascularization (Fig. 2A, panel c). By contrast, thespleen of IL-27–treated mice showed smaller and less fre-quent neoplastic infiltrates (Fig. 2A, panel d) and an almostabsent microvascularization and reticulin fiber support, asrevealed by both laminin staining (Fig. 2A, panel e) andsilver impregnation (Fig. 2A, panel f; refs. 27, 28).

Thus, neoplastic cells collected from (i) spleen and BMfrom 1 control and 1 IL-27–treated mouse injected withAML cells from patient no. 1 and (ii) spleen and BM from1 control and 1 IL-27–treated animal injected withAML cells from patient no. 13 were purified as human

Ab

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PB

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IL-2

7

PB

S

IL-2

7

PB

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IL-2

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0

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2

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412.5

22.5

Spleen BM PB

B

P < 0.0001

P = 0.0015

P < 0.0001

C

D

hrIL-27

hC

D45

PBS

79%

24%

60%

26%

35%

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A

hC

D45

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p-STAT5p-STAT3p-STAT1

* P = 0.0357** P = 0.0079

% P

osi

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lsp-STAT5p-STAT3p-STAT1

** P = 0.0027** P = 0.0035

WSX-1

gp130

Pt #1 Pt #5 Pt #12

Pt #5Pt #1 Pt #12

BM Spleen PB

CTR

IL-27

Figure 1. WSX-1 and gp130 expression in primary AML cells and IL-27 antitumor activity against AML cells into NSGmice. A, three representativeWSX-1 andgp130 stainings in gated CD33þ AML cells (patients no. 1, 5, and 12) are shown. Open profile: WSX-1 (top) and gp130 (bottom) staining. Dark profile: isotype-matched mAb staining. B, analysis of p-STAT1, p-STAT3, and p-STAT5 upon IL-27 stimulation in purified AML cells, as assessed by flow cytometry. Pooledresults from3different experiments are shown. Data are expressed asMRFI�SD (top) and as percentage of p-STAT–positive AML cells (bottom). C, absolutenumber of human CD45þCD33þ cells engrafted in spleens, BM, and PB of mice treated with PBS or hrIL-27. Each dot represents a single experiment.Horizontal lines indicated medians. D, 1 representative experiment of CD45 expression in BM, spleens, and PB harvested from PBS (top) or IL-27–treated(bottom) NSG mice injected with AML cells from patient no. 3 is shown. Pt #, patient number.

AML and IL-27





CD45þ (>95%) cells by immunomagnetic beads manipu-lation and used for evaluation of expression of a 84 angio-genic and 84 metastatic related genes, by PCR array. Asshown in Fig. 2B, AML cells infiltrating the spleens from IL-27–treatedmice showeddownregulationofdifferentproan-giogenic genes including angiopoietin (ANGPT)2 and 3,CXCL6andVEGF-C(Fig. 2B, left histogram), aswell as genesinvolved in the dissemination/spreading process (Fig. 2B,right histogram) such as CXCR4 and matrix metalloprotei-nase (MMP)7. However, it is of note that IL-27 also inducedupregulation of the angiostatic molecule tissue inhibitor ofmetalloproteinase (TIMP)2, indicating that IL-27–mediatedinhibition of angiogenesismay result from the regulation ofboth proangiogenic as well as angiostatic factors.

Similarly, the angiogenesis-related genes IL-6, CXCL1and 5 (Fig. 2C, left panel) or genes involved in the spreadingprocess (Fig. 2C, right panel), including CXCR4, MMP2,and 9 were found to be downregulated in human CD45þ

cells purified frommurine BM of IL-27–treated mice versuscontrols. The other genes included in the PCR array plateswere not significantly modulated by IL-27 (not shown).

IL-27 antitumor activity against pediatric AML cells invitro

The antitumor activity of IL-27 against pediatric AML cellswas tested in vitro in terms of (i) modulation of genes

involved in angiogenesis and leukemic progression, (ii)modulation of cell proliferation, and (iii) induction ofapoptosis. First, the angiogenic activity of 5 AML cell sus-pensions (patients no. 1, 9, 13, 14, and 16) cultured 36hours with or without 50 ng/mL of hrIL-27was investigatedusing the in vivo CAM system. CAM treated with spongesloaded with VEGF (positive control) or with conditionedmedium from AML cells were surrounded by allantoicvessels developing radially toward the implant in a"spoked-wheel" pattern. In the representative experimentshown in Fig. 3A, left panel, the mean number of vesselsformed in the presence of conditioned medium from AMLcells (patient no. 14)was 25� 4, whereas that formed in thepresence of VEGF was 30� 5 (not shown). When we testedthe conditioned medium from IL-27–treated cells from thesameAMLpatient in theCAMassay, a significant (P<0.001)reduction of the angiogenic response was appreciable(mean number of vessels 10 � 3; Fig. 3A, right panel), ascompared with the AML conditioned medium. No vascularreactionwas detected around the sponges upon exposure toIL-27 diluted in medium at the same final concentrationused to treat tumor cells (mean number of vessels 6 � 2 inthe presence or absence of IL-27, not shown). Finally,medium containing IL-27 did not inhibit angiogenesisinduced by VEGF (not shown). Similar results with thesame statistical significance (P <0.001)were obtainedwhen

Table 2. Absolute number of human CD45þCD33þ cells engrafted in spleens, BM, and PB from each NSGanimal injected intravenously with different AML cell samples

Spleen absolute number� 103

BM absolute number� 103

PB absolute number� 103

Patients no. PBS IL-27 PBS IL-27 PBS IL-27

1 2.200 180 360 60 150 201.700 210 250 120 110 20147 30 150 260 110 30200 20 280 110 228 10150 30 30 60 334 21120 100 200 50 187 24

2 150 80 90 40 230 7230 120 150 50 300 580 90 130 110 150 50

3 140 120 30 30 143 9170 60 90 30 122 112247 108 111 33 187 3.3110 65 169 46 278 28

9 356 22 278 108 298 16580 14 165 241 208 6130 132 297 103 167 5260 89 34 54 156 17980 126 222 43 193 21

13 1.950 165 134 98 284 362.050 201 378 53 222 81.222 178 54 26 256 191.488 93 109 28 270 33

Zorzoli et al.





conditioned medium from the 4 other pediatric AML cellsuspensions were tested. We checked by trypan blue stain-ing the viability of leukemic cells incubated with or withoutIL-27 before harvesting supernatants. There were no signif-icant differences in the proportions of viable cells betweencultures done with or without IL-27. AML cell suspensionsfrom patients no. 1 and 13 were analyzed by PCR array forexpression of genes involved in the angiogenic and meta-static processes. As shown in the left histogram of Fig. 3B,IL-27 upregulated significantly (P < 0.001) a set of anti-angiogenic genes including IFN-g , CXCL10, and TIMP2.Furthermore, IL-27 downregulated genes involved in tumorspreading such as CDH6 (Fig. 3B, right histogram). Theother genes included in the PCR array plates were notmodulated by IL-27 (not shown).Thus, to test whether IFN-g , the gene that was foundmost

upregulated in primary AML cells by PCR array, was a keymediator of the angiostatic activity exerted by IL-27, weanalyzed the angiogenic activity of IL-27–treated AML cellsupernatants (patients no. 1, 9, 13, 14, and 16) followingincubation with neutralizing antibodies to human IFN-g .These experiments revealed that neutralization of IFN-g didnot affect significantly the inhibition of angiogenesisobserved in supernatants from IL-27–treated leukemic cells(not shown).

Finally, the ability of IL-27 to affect proliferation andapoptosis of primary AML cells was assessed by Ki67 orBrdUrd/PI (patients no. 1–9) and Annexin V/PI (patientsno. 1–16) stainings, respectively. These analyses revealedthat IL-27 significantly (P ¼ 0.024) reduced the percentageof Ki67þ cells at 48 hours (Fig. 3C), but not at 24 hours (notshown). Cell-cycle analysis by BrdUrd/PI staining showedthat 48 and 60 hours of IL-27 treatment caused an increaseof percentage of AML cells in G1 and more marginally sub-G1 phase, paralleled by a decrease of cells in S and G2-Mphases (1 representative experiment is shown in Fig. 3D).IL-27 treatment did not affect apoptosis at any time pointtested (1 representative experiment is shown in Fig. 3E).

IL-27 downregulated CXCR4 surface expression in vivoCXCR4 represents the receptor for CXCL12 which was

found to be implicated in the growth and dissemination ofsolid and hematologic tumors (29, 30). Furthermore,CXCR4 antagonists inhibit ALL cell engraftment in the BM(30, 31). Because CXCR4 mRNA was consistently down-regulated by IL-27 treatment, we tested whether the corre-sponding protein was modulated at surface level in vivo. Tothis end, flow cytometric analyses was done on spleens, PB,and BM cell suspensions from NSG mice treated with PBS(spleens n ¼ 15, PB ¼ 15 and BM ¼ 15) or IL-27 (spleens

PBS IL-27

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Figure 2. Morphologic and immunohistochemical analyses of spleens collected fromNSGmice injectedwith primary AML cells and the potential mechanismsinvolved. A, AMLcells induced the development ofmultiple andwide neoplasticmasses (N) in the spleen (S: a) of controlmice. Thesemasseswere supplied bya richly developed microvascular (b) and reticular fiber network (c), as assessed, respectively, by laminin staining and silver impregnation (magnification:�400). By contrast, in IL-27–treated mice, neoplastic masses developed in the spleen were smaller (d) and lacking of microvascularization (e) andreticulin fiber network (f). Inset in panel b showsadetail of laminin staining (magnification:�630). BandC, gene expressionprofiling of humangenes involved intumor angiogenesis and dissemination, by PCR array in CD45þ AML cells purified from spleens (B) and BM (C) of NSG mice treated with PBS or IL-27.Results represent fold differences in individual mRNA expression between IL-27– and PBS-treated mice. A significant threshold of 4-fold changecorresponded to P < 0.001. All the other genes included in the PCR array not shown in the figure were not significantly regulated by IL-27. Pooled results from2 different experiments conducted in duplicate are shown in each histogram.

AML and IL-27





n¼ 15, PB¼ 15 and BM¼ 15) injected with AML cells frompatients no. 1, 3, 9, and 13. These experiments showed thatIL-27 treatment in vivo decreased the presence ofCD45þCD33þCXCR4þ cells homing in the different com-partments in which leukemic cells engrafted. Absolutenumbers of CXCR4 expressing leukemia cells are reportedin Fig. 4A. One representative CXCR4 staining of humanCD45þCD33þ leukemic cells harvested from BM, PB, andspleen of PBS and IL-27–treated mice is shown in Fig. 4Band C, respectively.

Finally, human CD45þ cells purified from BM andspleens obtained from NSG mice injected with 2 AML cellsamples (patients no. 1 and 3)were tested for their ability torespond to CXCL12, the ligand of CXCR4, by chemotacticassay. These experiments revealed that neoplastic cells iso-lated from the different murine compartments of both

controls and IL-27–treated animals did not migrate inresponse to CXCL12 (not shown).

DiscussionAML is a clinically and genetically heterogeneous disease

deriving from malignant transformation of hematopoieticprogenitors that accumulate in the BM (17, 18, 20).Although advances in the treatment of childhood AMLduring the last 2 decades have significantly increased theexpectancy of survival, AML still accounts formore thanhalfof fatal cases of pediatric leukemia. In view of these con-siderations, novel, more effective, and specific therapies forhigh-risk and relapsed patients are desirable. With thisbackground, we investigated whether IL-27, an immuno-modulatory cytokine (1, 32) with antitumor properties

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Figure 3. IL-27 antitumor activityagainst primary AML cells in vitro.A, angiogenic activity in the CAMassay of supernatant from 1representative AML sample(patient no. 14) of 5, cultured withmedium alone (left) or IL-27 (right).B, gene expression profile ofhuman angiogenesis andmetastatic related genes in primaryAML cells cultured in the presenceor absence of IL-27. Pooled results� SD from 2 experimentsconducted in duplicate are shown.Histogram represents folddifferences of individual mRNAbetween AML cells cultured inpresence or absence of IL-27.C, flow cytometry analysis ofproliferating cell fraction (Ki67þ)and cell cycle in primary AML cellscultured 48 hours with mediumalone or with IL-27. Pooled resultsof 9 experiments are shown.Whisker lines represent highestand lowest values, horizontal linesrepresent median values. D, flowcytometric analysis of BrDUrd/PIstaining of AML cells cultured 60hours with medium alone or in thepresence of IL-27. Onerepresentative experiment (patientno. 9) of the 9 conducted is shown.E, analysis of apoptotic cells,identified as Annexin Vþ/PIþ cells,in 1 representative AML samplecultured in the presence orabsence of 50 ng/mL IL-27 for 48and 60 hours.

Zorzoli et al.





against solid (3, 9, 33) andhematologicmalignancies (7, 8),may function as an antileukemic agent against pediatricAML using a preclinical model. Here, using both in vivo andin vitro approaches, we showed for the first time thatpediatric AML cells express functional IL-27R and that IL-27 represents an efficacious antitumor agent against AML.In this regard, it has to bementioned that functional geneticscreening of genes expressed in primary AML cells showedthat the IL-27R is frequently expressed on the surface ofthese cells, suggesting that this gene may possess hemato-poietic cell transforming properties (34). Although func-tional experiments on IL-27 activity on AML cells have notbeen carried out in a large cohort of patients, the latter workprovided results that apparently differ from that reported

here. In vivo experiments were conducted using NSG micethat represent an efficient animal model for engraftment oftumor cells and allowed us to evaluate the antitumoractivity of human IL-27 on human AML cells in vivo in theabsence of immune responses (25, 35, 36). We chose touse nonirradiated hosts and engrafted primary AML cellswere detected in all NSG animals, consistent with previousresults (26). This model allowed us to show that IL-27strongly reduced the presence of leukemia cells in the PBand inhibited leukemia spreading in the BM and spleens.The unambiguous neoplastic nature of human cellsengrafted in these mice upon injection with pediatric AMLcells was shown here and already reported by us (26). In thelatter study, it was clearly shown that human CD45þ cellsengrafted in NSG mice retained the ability to growth insecondary recipients and showed the same phenotype andpolymorphic short-tandem repeat loci detected at diagnosisin the original patient sample.

The inhibition of pediatric AML cell spreading is likelyrelated to the ability of IL-27 to function directly againsttumor cells through reduction of their angiogenic potential,modulation of genes involved in tumor spreading, and byinhibiting AML cell proliferation, as shown by our in vitroresults. Morphologic and immunohistochemical analysesdone on spleens explanted from control and treated ani-mals revealed that IL-27 caused a strong inhibition of theleukemia homing, tumor vascularization, and stromal reti-culin fiber network in the spleens. To gain more insightsinto this issue, we investigated the ability of IL-27 tomodulate the expression of different genes involved in theangiogenic and dissemination processes. In leukemia cellspurified from spleens and BM, we showed that IL-27 down-regulated different proangiogenic genes, including angio-poietins (37, 38), CXCL chemokines (39, 40), and IL-6 (41,42), as well as some genes involved in tumor progressionsuch as cadherins, MMP, and CXCR4 (43–45). However,AML cells harvested from the 2 murine compartmentsshowed a different pattern of genes modulated by IL-27,possibly because of the different microenvironments towhich they have been exposed in vivo. The only exceptionwas represented by CXCR4 that was consistently down-regulated by IL-27, at mRNA and surface levels, in leukemiccells from each compartment. However, these cells failed tomigrate in response to the CXCR4 specific ligand, that is,CXCL12. It is of note that, in acute leukemias, the CXCL12–CXCR4 axis represents much more than a traffic controller(29), as it is important for cell survival, proliferation, andrelease of soluble factors (29, 46–48). Thepotential involve-ment of this pathwayhasnot here been investigated becausetheCXCR4downregulationoccurred inAMLcells in vivobutnot in vitro, thus suggesting that this finding may be relatedto microenvironmental stimuli mediated by the cytokinemore than a specific direct activity of IL-27 on leukemiccells.

Similarly to observations in vivo, PCR array studies doneon primary AML cells treated in vitro revealed that IL-27modulated several genes involved in angiogenesis andleukemic dissemination. In particular, IL-27 strongly

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Figure 4. Reduction of the CXCR4þ leukemic cells by IL-27 treatmentin vivo. A, absolute number of CXCR4þCD45þ cells engrafted in spleens,BM, and PB from mice treated with PBS or hrIL-27. Pooled results areshown. Whisker lines represent highest and lowest values, horizontallines represent median values. B and C, 1 representative CXCR4 (lowerdot plots) and isotype-matched staining (upper dot plots) of gatedCD45þCD33þ cells harvested from BM (left dot plots in B and C), PB(middle dot plots) and spleen (right dot plots) from PBS (B) or IL-27–treated (C) NSGmice injected with AML cells from patient no. 1 is shown.

AML and IL-27





upregulated the angiostatic cytokine IFN-g and, although toa lesser extent, CXCL10 and TIMP2; moreover, it down-regulated not only the proangiogenic angiopoietin-4 andCXCL3 but also cadherin-6. Although human IL-27 is notspecies specific (2), the in vivo antitumor mechanismsoperated by IL-27 overlapped with those observed in vitroin leukemia cells, supporting the conclusion that the cyto-kine targeted AML cells directly. In a translational perspec-tive, we provided evidence that the cytokine exerts a strongantitumor activity against pediatric AML cells irrespective ofspecific leukemic subtypes. However, a large cohort ofpediatric AML patients should be included in future studiesto evaluate whether different AML subtypes may respondmore or less efficiently to IL-27.

Overall, our results support the concept that IL-27 mayrepresent a new antileukemic drug to be tested in futureclinical trials, as this cytokine may target pediatric AML byinhibiting their angiogenic and dissemination potentialdirectly. In this respect, it has been shown that IL-27enhances proliferation and differentiation of nonleuke-mia human CD34þ cells (49), thus suggesting that thiscytokine may have a favorable effect on normal hemato-poietic stem cells, a subset often highly compromised inleukemia patients. An additional argument in favor of thehypothesis of using IL-27 is the low toxicity shown by the

cytokine in animal models, likely in relation to thelimited induction of IFN-g in vivo (9). Finally, simulta-neous activation of in vivo antitumor effector mechanismsmediated by CTL, NK, and CD8þ T cell may be promotedby IL-27 (1).

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

AcknowledgmentsA. Zorzoli, E.D. Carlo, C. Cocco, E. Ferretti, and D. Ribatti carried

out research and analyzed data. E. Ognio carried out research. C. Dufour,F. Locatelli, and D. Montagna provided AML samples. I. Airoldi designedresearch, analyzed data, and wrote the manuscript. All the authors criticallyrevised the manuscript.

Grant SupportThis work was supported by grants from Associazione Italiana Ricerca sul

Cancro (A.I.R.C.)Milan, Italy (grant number 4014 to I. Airoldi), from ItalianMinistry of Health (RF, RC, 5/1000, Progetto Strategico Oncologico. 2006rif070701) as well as from the Special Grant 5 per mille from A.I.R.C. to F.Locatelli.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received September 21, 2011; revised December 20, 2011; acceptedJanuary 13, 2012; published OnlineFirst March 1, 2012.

References1. Trinchieri G, Pflanz S, Kastelein RA. The IL-12 family of heterodimeric

cytokines: new players in the regulation of T cell responses. Immunity2003;19:641–4.

2. PflanzS, Timans JC,CheungJ,RosalesR,KanzlerH,Gilbert J, et al. IL-27, a heterodimeric cytokine composed of EBI3 and p28 protein,induces proliferation of naive CD4(þ) T cells. Immunity 2002;16:779–90.

3. HisadaM,Kamiya S, Fujita K, BelladonnaML, Aoki T, Koyanagi Y, et al.Potent antitumor activity of interleukin-27. Cancer Res 2004;64:1152–6.

4. Stumhofer JS, Laurence A, Wilson EH, Huang E, Tato CM, JohnsonLM, et al. Interleukin 27 negatively regulates the development ofinterleukin 17-producing T helper cells during chronic inflammationof the central nervous system. Nat Immunol 2006;7:937–45.

5. Chen Q, Ghilardi N, Wang H, Baker T, Xie MH, Gurney A, et al.Development of Th1-type immune responses requires the type Icytokine receptor TCCR. Nature 2000;407:916–20.

6. Pflanz S, Hibbert L, Mattson J, Rosales R, Vaisberg E, Bazan JF, et al.WSX-1 and glycoprotein 130 constitute a signal-transducing receptorfor IL-27. J Immunol 2004;172:2225–31.

7. Canale S, Cocco C, Frasson C, Seganfreddo E, Di Carlo E, Ognio E,et al. Interleukin-27 inhibits pediatric B-acute lymphoblastic leukemiacell spreading in a pre-clinical model. Leukemia 2011;25:1815–24.

8. Cocco C, Giuliani N, Di Carlo E, Ognio E, Storti P, Abeltino M, et al.Interleukin-27 acts as multifunctional antitumor agent in multiplemyeloma. Clin Cancer Res 2010;16:4188–97.

9. Oniki S, Nagai H, Horikawa T, Furukawa J, BelladonnaML, YoshimotoT, et al. Interleukin-23 and interleukin-27 exert quite different antitumorand vaccine effects on poorly immunogenic melanoma. Cancer Res2006;66:6395–404.

10. ShimizuM,ShimamuraM,Owaki T, AsakawaM,Fujita K, KudoM, et al.Antiangiogenic and antitumor activities of IL-27. J Immunol 2006;176:7317–24.

11. Airoldi I, Di Carlo E, Banelli B, Moserle L, CoccoC, Pezzolo A, et al. TheIL-12Rbeta2 gene functions as a tumor suppressor in human B cellmalignancies. J Clin Invest 2004;113:1651–9.

12. Airoldi I, Di Carlo E, Cocco C, Caci E, Cilli M, Sorrentino C, et al. IL-12 can target human lung adenocarcinoma cells and normal bron-chial epithelial cells surrounding tumor lesions. PLoS One 2009;4:e6119.

13. Airoldi I, Di Carlo E, Cocco C, Taverniti G, D'Antuono T, Ognio E, et al.Endogenous IL-12 triggers an antiangiogenic program in melanomacells. Proc Natl Acad Sci U S A 2007;104:3996–4001.

14. Airoldi I, Cocco C, Giuliani N, Ferrarini M, Colla S, Ognio E, et al.Constitutive expression of IL-12R beta 2 on human multiple mye-loma cells delineates a novel therapeutic target. Blood 2008;112:750–9.

15. Yoshimoto T, Morishima N, Mizoguchi I, Shimizu M, Nagai H, Oniki S,et al. Antiproliferative activity of IL-27 on melanoma. J Immunol2008;180:6527–35.

16. Ho MY, Leu SJ, Sun GH, Tao MH, Tang SJ, Sun KH. IL-27 directlyrestrains lung tumorigenicity by suppressing cyclooxygenase-2-medi-ated activities. J Immunol 2009;183:6217–26.

17. Stone RM, O'Donnell MR, Sekeres MA. Acute myeloid leukemia.Hematology Am Soc Hematol Educ Program 2004;98–117.

18. Vardiman JW, Harris NL, Brunning RD. TheWorld Health Organization(WHO) classification of the myeloid neoplasms. Blood 2002;100:2292–302.

19. Kaspers GJ, Zwaan CM. Pediatric acute myeloid leukemia: towardshigh-quality cure of all patients. Haematologica 2007;92:1519–32.

20. ShahM,Agarwal B. Recent advances inmanagement of acutemyeloidleukemia (AML). Indian J Pediatr 2008;75:831–7.

21. Ravandi F. Role of cytokines in the treatment of acute leukemias: areview. Leukemia 2006;20:563–71.

22. Lange BJ, Yang RK, Gan J, Hank JA, Sievers EL, Alonzo TA, et al.Soluble interleukin-2 receptor alpha activation in a Children's Oncol-ogy Group randomized trial of interleukin-2 therapy for pediatric acutemyeloid leukemia. Pediatr Blood Cancer 2011;57:398–405.

23. Ribatti D, Gualandris A, Bastaki M, Vacca A, Iurlaro M, Roncali L, et al.New model for the study of angiogenesis and antiangiogenesis in thechick embryo chorioallantoic membrane: the gelatin sponge/chorio-allantoic membrane assay. J Vasc Res 1997;34:455–63.

Zorzoli et al.





24. Larousserie F, Charlot P, Bardel E, Froger J, Kastelein RA, DevergneO.Differential effects of IL-27 on human B cell subsets. J Immunol2006;176:5890–7.

25. Agliano A, Martin-Padura I, Mancuso P, Marighetti P, Rabascio C,Pruneri G, et al. Human acute leukemia cells injected in NOD/LtSz-scid/IL-2Rgamma null mice generate a faster and more efficientdisease compared to other NOD/scid-related strains. Int J Cancer2008;123:2222–7.

26. Ferretti E, Montagna D, Di Carlo E, Cocco C, Ribatti D, Ognio E, et al.Absence of IL-12Rbeta2 in CD33(þ)CD38(þ) pediatric acute myeloidleukemia cells favours progression in NOD/SCID/IL2RgammaC-defi-cient mice. Leukemia 2011;doi:10.1038/leu.2011.213.

27. Cr�aitoiu S. The morphopathological aspects of diabetic retinopathy.Oftalmologia 1992;36:141–8.

28. Garner A. Histopathology of diabetic retinopathy in man. Eye (Lond)1993;7:250–3.

29. Tavor S, Petit I. Can inhibition of the SDF-1/CXCR4 axis eradicateacute leukemia? Semin Cancer Biol 2010;20:178–85.

30. Burger JA, Kipps TJ. CXCR4: a key receptor in the crosstalk betweentumor cells and their microenvironment. Blood 2006;107:1761–7.

31. Burger JA, Peled A. CXCR4 antagonists: targeting the microenviron-ment in leukemia and other cancers. Leukemia 2009;23:43–52.

32. Villarino AV, Hunter CA. Biology of recently discovered cytokines:discerning the pro- and anti-inflammatory properties of interleukin-27. Arthritis Res Ther 2004;6:225–33.

33. Chiyo M, Shimozato O, Yu L, Kawamura K, Iizasa T, Fujisawa T, et al.Expression of IL-27 in murine carcinoma cells produces antitumoreffects and induces protective immunity in inoculated host animals. IntJ Cancer 2005;115:437–42.

34. Pradhan A, Lambert QT, Reuther GW. Transformation of hematopoi-etic cells and activation of JAK2-V617F by IL-27R, a component of aheterodimeric type I cytokine receptor. Proc Natl Acad Sci U S A2007;104:18502–7.

35. Castor A, Nilsson L, Astrand-Grundstrom I, Buitenhuis M, Ramirez C,Anderson K, et al. Distinct patterns of hematopoietic stem cell involve-ment in acute lymphoblastic leukemia. Nat Med 2005;11:630–7.

36. Quintana E, Shackleton M, Sabel MS, Fullen DR, Johnson TM, Morri-son SJ. Efficient tumour formation by single human melanoma cells.Nature 2008;456:593–8.

37. Loges S, Heil G, Bruweleit M, Schoder V, Butzal M, Fischer U, et al.Analysis of concerted expression of angiogenic growth factors in acutemyeloid leukemia: expression of angiopoietin-2 represents an indepen-dentprognostic factor foroverallsurvival.JClinOncol2005;23:1109–17.

38. Schliemann C, Bieker R, Thoennissen N, Gerss J, Liersch R, Kessler T,et al. Circulating angiopoietin-2 is a strong prognostic factor in acutemyeloid leukemia. Leukemia 2007;21:1901–6.

39. Strieter RM, Burdick MD, Mestas J, Gomperts B, Keane MP, BelperioJA. Cancer CXC chemokine networks and tumour angiogenesis. Eur JCancer 2006;42:768–78.

40. Kittang AO, Hatfield K, Sand K, Reikvam H, Bruserud O. The chemo-kine network in acute myelogenous leukemia: molecular mechanismsinvolved in leukemogenesis and therapeutic implications. Curr TopMicrobiol Immunol 2010;341:149–72.

41. Akashi K, Harada M, Shibuya T, Eto T, Takamatsu Y, Teshima T, et al.Effects of interleukin-4 and interleukin-6 on the proliferation of CD34þand CD34- blasts from acute myelogenous leukemia. Blood 1991;78:197–204.

42. Ferretti E, Di Carlo E, Cocco C, Ribatti D, Sorrentino C, Ognio E, et al.Direct inhibition of human acutemyeloid leukemia cell growth by IL-12.Immunol Lett 2010;133:99–105.

43. Koshiba T, Hosotani R, Miyamoto Y, Ida J, Tsuji S, Nakajima S, et al.Expression of stromal cell-derived factor 1 andCXCR4 ligand receptorsystem inpancreatic cancer: apossible role for tumorprogression.ClinCancer Res 2000;6:3530–5.

44. Shimazui T, Yoshikawa K, Uemura H, Hirao Y, Saga S, Akaza H. Thelevel of cadherin-6mRNA in peripheral blood is associatedwith the siteof metastasis and with the subsequent occurrence of metastases inrenal cell carcinoma. Cancer 2004;101:963–8.

45. ReikvamH,HatfieldKJ,OyanAM,KallandKH,KittangAO,BruserudO.Primary human acute myelogenous leukemia cells release matrixmetalloproteases and their inhibitors: release profile and pharmaco-logical modulation. Eur J Haematol 2010;84:239–51.

46. Jacobi A, Thieme S, Lehmann R, Ugarte F, Malech HL, Koch S, et al.Impact of CXCR4 inhibition on FLT3-ITD-positive human AML blasts.Exp Hematol 2010;38:180–90.

47. Tavor S, Eisenbach M, Jacob-Hirsch J, Golan T, Petit I, Benzion K,et al. The CXCR4 antagonist AMD3100 impairs survival of humanAML cells and induces their differentiation. Leukemia 2008;22:2151–5158.

48. Scupoli MT, Perbellini O, Krampera M, Vinante F, Cioffi F, Pizzolo G.Interleukin 7 requirement for survival of T-cell acute lymphoblasticleukemia and human thymocytes on bone marrow stroma. Haemato-logica 2007;92:264–6.

49. Seita J, Asakawa M, Ooehara J, Takayanagi S, Morita Y, Watanabe N,et al. Interleukin-27 directly induces differentiation in hematopoieticstem cells. Blood 2008;111:1903–12.

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Published OnlineFirst March 1, 2012.Clin Cancer Res Alessia Zorzoli, Emma Di Carlo, Claudia Cocco, et al.

Mice−/−Il2rgLeukemia in NOD/SCID/Interleukin-27 Inhibits the Growth of Pediatric Acute Myeloid

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Interleukin-27 Inhibits the Growth of Pediatric Acute ... · 24/01/2012 · Purpose: Acute myeloid leukemia (AML) accounts for more than half of fatal cases in all pediatric leukemia