Effective Activity of Cytokine-Induced Killer Cells against … · 2013. 7. 25. · Cancer Therapy: Preclinical Effective Activity of Cytokine-Induced Killer Cells against Autologous

Cancer Therapy: Preclinical

Effective Activity of Cytokine-Induced Killer Cells againstAutologous Metastatic Melanoma Including Cells withStemness Features

Loretta Gammaitoni1, Lidia Giraudo1,5, Valeria Leuci4,5, Maja Todorovic1,5, Giulia Mesiano1,5,Franco Picciotto2, Alberto Pisacane3, Alessandro Zaccagna2, Maria Giuseppa Volpe1, Susanna Gallo1,5,Daniela Caravelli1, Elena Giacone2, Tiziana Venesio3, Antonella Balsamo3, Ymera Pignochino4,5,Giovanni Grignani4, Fabrizio Carnevale-Schianca1, Massimo Aglietta1,5, and Dario Sangiolo1,5

AbstractPurpose:We investigate the unknown tumor-killing activity of cytokine-induced killer (CIK) cells against

autologous metastatic melanoma and the elusive subset of putative cancer stem cells (mCSC).

Experimental Design: We developed a preclinical autologous model using same patient-generated

CIK cells and tumor targets to consider the unique biology of each patient/tumor pairing. In primary

tumor cell cultures, we visualized and immunophenotypically defined a putative mCSC subset using a

novel gene transfer strategy that exploited their exclusive ability to activate the promoter of stemness

gene Oct4.

Results: The CIK cells from 10 patients with metastatic melanoma were successfully expanded (median,

23-fold; range, 11–117). Primary tumor cell cultures established and characterized from the same patients

were used as autologous targets. Patient-derived CIK cells efficiently killed autologousmetastaticmelanoma

[up to 71% specific killing (n¼ 26)]. CIK cells were active in vivo against autologousmelanoma, resulting indelayed tumor growth, increased necrotic areas, and lymphocyte infiltration at tumor sites. The metastatic

melanoma cultures presented an average of 11.5% � 2.5% putative mCSCs, which was assessed by Oct4promoter activity and stemnessmarker expression (Oct4, ABCG2, ALDH,MITF). Expression was confirmed

on mCSC target molecules recognized by CIK cells (MIC A/B; ULBPs). CIK tumor killing activity against

mCSCs was intense (up to 71%, n ¼ 4) and comparable with results reported against differentiatedmetastatic melanoma cells (P ¼ 0.8).Conclusions: For the first time, the intense killing activity of CIK cells against autologous metastatic

melanoma, including mCSCs, has been shown. These findings move clinical investigation of a new

immunotherapy for metastatic melanoma, including mCSCs, closer. Clin Cancer Res; 1–12. �2013 AACR.

IntroductionThe incidence of malignant melanoma in western popu-

lations has increased in recent decades. Although surgicalresection of primary lesions has high cure rates, metastaticmelanoma remains largely refractory to conventional ther-apies with a dismal prognosis (1, 2). Targeted strategiesagainst key molecules such as B-RAF and MEK (3–5), andimmune system antitumor activity restoration to block the

CTLA4 (6, 7) or PD-1 molecules (8) are two recent break-throughs that have positively impacted metastatic melano-ma treatment.Despite some survival advantage, results havealmost always been short-lived; it seems that as hypothe-sized for other tumors, metastatic melanoma includes asmall cell subpopulation endowed with the stemness fea-tures that sustain drug resistance and disease relapse (9).This challenging clinical scenario demands new therapeuticapproaches, ideally the ones that are able to target mela-noma cancer stem cells (mCSC).

Much promise lies in adoptive immunotherapy for thetreatment of metastatic melanoma as supported by resultsreported with ex vivo expanded tumor infiltrating lympho-cytes (TIL; ref. 10) or T cells engineered with melanomaantigen-specific T-cell receptors (TCR; refs. 11, 12). Theextended clinical application of TIL is, however, limited byavailable, suitably sized resectable tumor lesions, whereasspecific human leukocyte antigen (HLA) restriction, com-plex expansion conditions, and stringent regulatory require-ments confound the TCR transfer approach.

Authors' Affiliations: Units of 1Stem Cell Transplantation and Cell Ther-apy, 2Surgical Dermatology, 3Pathology, and 4Sarcoma, Fondazione delPiemonte per l'Oncologia, I.R.C.C.S.; 5Department ofOncology, Universityof Torino Medical School, Candiolo, Torino, Italy

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Dario Sangiolo, Cancer Cell Therapy Laboratory,I.R.C.C.S., Provinciale 142Candiolo 10060, Torino, Italy. Phone: 390-1199-33503; Fax: 390-1199-33522; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-13-0061

�2013 American Association for Cancer Research.

ClinicalCancer

Research

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Cytokine-induced killer (CIK) cells are ex vivo expandedT-natural killer (NK) lymphocytes potentially able toaddress some of the issues currently limiting clinicalapplication of other immunotherapies (13, 14). CIK cellscan be massively expanded from peripheral blood mono-nuclear cells (PBMC) cultured with the timed addition ofIFN-g , Ab-anti-CD3, and interleukin (IL)-2 through sim-ple standardized culture conditions (15–18). CIK cellactivity is not dependent on HLA restriction; it is mediatedmostly by the interaction of its NKG2D receptor withstress-inducible molecules [MHC class I-related chain Aand B (MIC A/B) and UL-16–binding proteins (ULBPs)]on tumor targets (19, 20). Initial clinical trial resulting invarious tumor settings are encouraging, but data are gen-erally absent on CIK cell potential activity against autol-ogous metastatic melanoma (13, 14, 21, 22), let alonework that might take into account the unique biologicand immunogenic features of a specific tumor in a specificpatient.

Furthermore, whether or not the tumor-killing ability ofCIK cells affects the subpopulation of mCSCs is completelyunexplored. Currently, clear identification of mCSCs isintensely debated. Several membrane molecules or geneshave been proposed as putative markers, but agreementremains elusive. Quintana and colleagues recently failed toidentify any marker that robustly distinguished mCSCsdespite examining 85 markers, including ATP-binding cas-sette B5 (ABCB5), CD271 (also known as nerve growthfactor receptor; NGFR), and CD133 (23, 24).

Several facts germane to this topic are commonly accept-ed. First, expression of human embryonic stem cell plur-ipotency markers SOX2, Klf4, and Oct4 indicate high plas-ticity (25, 26). Second, inhibition of microphthalmia-associated transcription factor (Mitf), the master regulatorof melanocyte differentiation, increases the tumorigenicpotential of melanoma cells and upregulates stem cellmarkers Oct4 and Nanog as shown by Cheli and colleagues(27). Third and recently reposted is that forced expressionofthe Oct4 gene promoted dedifferentiation of melanomacells toward mCSCs with decreased expression of melano-

cytic markers, acquisition of multipotent differentiationcapacity, membrane expression of ABCB5 and CD271,resistance to chemotherapy, hypoxia, and increased tumor-igenic capacity (28).

Here, we first report preclinical activity of patient-derivedCIK cells against autologous metastatic melanoma includ-ing putative mCSCs. To highlight the mCSCs, we intro-duced a gene transfer strategy whereby bulk melanoma cellcultures were transduced with a lentiviral vector encodingthe enhanced eGFP under expression control of the Oct4promoter. We visualized mCSCs exploiting their exclusiveability to activate theOct4 promoter and sorted themon thebasis of eGFP expression. CIK cells efficiently killed autol-ogous melanoma targets regardless of their stemness ordifferentiated phenotype.

Materials and MethodsCIK culture and expansion

Human peripheral blood samples were obtained fromsubjectswith histologically confirmed stage IVmelanoma atthe Fondazione del Piemonte per l’Oncologia-–Institute forCancer Research and Treatment (FPO-IRCC; Candiolo,Torino, Italy). All individuals provided their informedconsent.

Cultures were started with PBMCs collected from samepatients with metastatic melanoma and isolated by densitygradient centrifugation using cell separation media Lym-phoprep (SentinelDiagnostic). For some experiments, freshPBMCs collected from volunteer donors were used. PBMCswere cultured overnight in cell culture flasks at a cell densityof 1.5 � 106/mL RPMI (Gibco BRL) supplemented with10% FBS (Sigma) in 1,000 U/mL IFN-g (PeproTech). After18 to 24 hours in culture at 37�C and 5% CO2, 50 ng/mLanti-CD3 antibody (OKT3, PharMingen) and 300 U/mLrecombinant human IL-2 (Proleukin, Aldesleukin, ChironCorporation) were added. Fresh medium with IL-2 wasadded as needed.

Establishment of primary melanoma cell culturesHuman melanoma tissues were obtained from surgical

specimens; patients provided consent under institutionalreview board–approved protocols. Human melanoma tis-sues were cut into 3 mm3 pieces and processed for cellisolation. Tumor tissue was processed by mechanical andenzymatic dissociation (Collagenase Type I, Invitrogen) for3 hours and then subsequently for an additional 12 hours.Cells were then resuspended in KnockOut Dulbecco’smod-ified Eagle medium: nutrient mixture F-12 medium (KODMEM:F12 medium Gibco BRL) with the addition ofpenicillin (50 U/mL), streptomycin (50 mg/mL), Glutamax100X (all from Gibco BRL); cells could be seeded either inserum-free conditions with the addition of N2 supplement(Gibco BRL) or in 10% heat-inactivated FBS (Euroclone).Cells were plated at clonal density (104–105 cells/cm2)either in ultra-low attachment multi-well plates for suspen-sion cell culture applications or in multi-well plates treatedfor anchorage-dependent cultures (Corning/Costar).

Translational RelevanceThis work is the first report of effective antitumor

activity of patient-derived cytokine-induced killer (CIK)cells against autologousmetastaticmelanoma, includingputative cancer stem cells (CSC). The translational rel-evance of this work is borne out in two ways. First, theautologous model (same patient CIK cells and tumortargets) uniquely considers the intrinsic biology ofeach patient—tumor, and second, chemoresistant- andrelapse-associated putative melanoma CSCs are identi-fied and targeted. The ability of CIK cells to kill bothdifferentiated melanoma cells and CSCs may promotethem into clinical trials, either alone or in associationwith emerging molecular targeted therapies.

Gammaitoni et al.

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In vivo tumorigenesisFour-week-old nonobese diabetic/LtSz-scid/scid [nonob-

ese diabetic/severe combined immunodeficiency (NOD/SCID)] (Charles River) female mice were injected subcuta-neously with 106 cells from melanoma primary culturesresuspended in sterile PBS1X and BD Matrigel BasementMembrane Matrix (Becton Dickinson), 1:1. Tumor growthwasmonitored weekly with calipers; volumewas calculatedusing formula, V ¼ 4/3 � p � (l/2)2 � (L/2), where L isthe length and l the width diameter of the tumor. When thetumor volume reached 2 cm in diameter, the animal waseuthanized, the tumor was recovered and fixed overnight in4% paraformaldehyde, then dehydrated, paraffin-embed-ded, sectioned (5 mm), and stained with hematoxylin andeosin (H&E; Bio.Optica).

hOct4.eGFP lentiviral vector generationVSV-G pseudotyped third-generation lentiviral vectors

were produced by transient four-plasmid cotransfectioninto 293T cells as described by Follenzi and colleagues(29). The transfer vector pRRL.sin.PPT.hPGK.EGFP.Wpre(LV-PGK.EGFP) was kindly provided by Dr. Elisa Vigna(Gene Transfer and Therapy, IRCC Candiolo, Torino, Italy)and has been described elsewhere (29).The phOCT4.EGFP1 vector (30)was provided byWei Cui

(IRDB, Imperial College London, London, United King-dom). The pRRL.sin.PPT.hOct4.eGFP.Wpre (LV-Oct4.eGFP) was obtained by replacing the expression cassettehPGK.eGFP into LV-PGK.eGFP with the hOct4-eGFP1cleaved from the phOct4-eGFP1 vector by insertion intothe SalI and XhoI restriction enzyme sites. Physical titers forlentiviral vector stocks were determined on the basis of p24antigen content (HIV-1 p24 ELISA kit; PerkinElmer).

Melanoma primary cell transductionFor each lentiviral vector transduction, melanoma pri-

mary cells were resuspended in fresh KODMEM-F12 with10%FBS. Virus-conditionedmediumwas added at a dose of400 ng P24/100,000 cells. After 16 hours, cells were washedtwice and grown for a minimum of 10 days to reach steady-state eGFP expression and to rule out pseudotransductionbefore flow cytometry analysis. As a transduction efficiencycontrol, the same melanoma primary cells were transducedwith LV-PGK.eGFP. Murine embryonic cells and PBMCswere transduced with LV-Oct4.eGFP as positive and nega-tive expression controls.

Analysis of LV-Oct4.eGFP presence in eGFP-positiveand -negative cell fractionsDetection of the presence of LV-Oct4.eGFP in both frac-

tions of freshly sorted cells was verified by PCR-basedamplification of the expression cassette Oct4.eGFP. Geno-mic DNA was extracted separately from eGFPþ and eGFP�

cells using a commercial kit (Qiagen). PCR was conductedusing 100 ng of gDNA per sample and Phusion High-Fidelity DNA Polymerase (Thermo Scientific) according tothe manufacturer’s protocol.

Annealed on the lentiviral vector backbone sequencesboth upstream and downstream the expression cassettewere primers LV forward primer 50-AGG CCCGAAGGAA-TAGAAGA-30 and LV reverse primer 50-CCACATAGCG-TAAAAGGAGCA -30.

The PCR products were separated by electrophoresis on1% agarose gel.

In vitro proliferation assay and PKH26 stainingTo evaluate the proliferation rate of eGFPþ versus eGFP�

cell sorted fractions, cells had been labeled with lipophylicdye PKH26, for which fluorescence intensity decreased byhalf at each cell division per kit protocol (PKH26GL kit,Sigma). Briefly, an adequate quantity of Diluent C labelingvehicle was added to the previously washed cell pellet (es.1� 106 cells/0.5 mL) to obtain a 2� single-cell suspension.A 2� (4 mmol/L) PKH26dye solution was prepared andadded to 2� single-cell suspension. Cell membrane dyeuptake was stopped by adding an equal volume of heat-inactivated serum. PKH26-stained cells were then washedtwice with culture medium added with 10% heat-inacti-vated serum (5 minutes at 400 � g). An aliquot of labeledand counted cells was read on a Flow Cytometry Cyan(Cyan ADP, Dako) and analyzed using Summit Softwareto set the baseline fluorescence level. The remaining cellswere seeded in culture under optimal conditions as previ-ously described. After 7, 14, and 21 days, the reduction influorescence was quantified by flow cytometry.

Cytotoxicity assayCIK tumor-killing ability was assessed against an alloge-

neic cell line (DettMel) and melanoma primary tumor cells.The allogeneic melanoma cell line DettMel was a kind giftfrom Dr V. Russo (Cancer Gene Therapy Unit, ScientificInstitute S. Raffaele, Milan, Italy), derived from metastasesof malignant melanomas as described elsewhere (31). Theeffector cells were assayed against both autologous andallogeneic tumor targets (32). A nonradioactive stain ofmelanoma target cells was used from PKH26 kit (Sigma-Aldrich) to conduct in vitro assays. Their immune-mediatedkillingwas analyzedbyflowcytometry (CyanADP,Dako)bypropidiumiodidepermeability of target cells (PKH26þ gate).CIK cells were coculturedwith either autologous or allogenicmelanoma primary cells with a 40:1, 20:1, 10:1, and 5:1effector:target ratio for 2 to6hours in200mLofmediumwithIL-2at a concentrationof300U/mLat37�C/5%CO2. Tumorcells, in the absence of CIK cells, were used as a control toassess spontaneous mortality. The percentage of tumor-spe-cific lysis for each effector/target ratio was calculated accord-ing to the following formula: (experimental�spontaneousmortality/100�spontaneous mortality) � 100.

In vivo activity assayNOD/SCID (Charles River) female mice were subcuta-

neously injected with an 8 mm3 tumor fragment from apatient-derived melanoma biopsy (mMel2). Starting 1week after tumor implantation, mice received 8 weeklyintravenous infusions with 1� 107 mature autologous CIK

CIK Cells Kill Autologous Melanoma and mCSCs

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cells resuspended in 1� PBS (200 mL total volume injected).Mice injected with PBS alone were used as the untreatedcontrol. Tumor growth was monitored weekly with calipersand volume calculated according to the formula: V¼ 4/3�p � (l/2)2 � (L/2), where L is the length and l the widthdiameter of the tumor. Animals were euthanized when thetumor size reached 2 cm in itsmain diameter. The recoveredtumor was fixed overnight in 4% paraformaldehyde, dehy-drated, paraffin-embedded, sectioned (5 mm), and finallystained with H&E (Bio.Optica). Immunohistochemicalassay was conducted with human anti-CD5 and anti-CD56(NCMA) antibodies (Novocastra, Leica Biosystem).A certified pathologist evaluated the necrotic areas.

Statistical analysisStatistical analysis was conducted using software Graph-

Pad Prism 5. A descriptive statistical analysis of CIK andmelanoma primary cell culture median values and ranges,ormean� SEM, was used as required. Subgroup phenotypeand necrotic area extension were compared with theunpaired, two-tailed t test. The mixed model ANOVA wasused to assess CIK cytotoxic activity curves in vitro and tocompare tumor volumes in vivo. Statistical significance hasbeen expressed as true P value and all less than 0.05 wereconsidered statistically significant.

ResultsEstablishment and characterization of autologousmelanoma primary cell cultures

Autologous melanoma cell lines were successfully estab-lished from 10 patients with stage IV melanoma. Thecharacteristics of the 10 patients are shown in Table 1.

Primary cell cultures were generated from tumor tissuebiopsies onmetastatic sites in 4 to 12weeks. All cell culturesdisplayed morphologic features consistent with the pathol-ogy evaluation of the corresponding tumor; a representativepicture of the primary tumor cell cultures is shown inSupplementary Fig. S1.

We analyzed each cell culture for expression of previous-ly described main melanoma surface antigens: CD271,melanoma-associated chondroitin sulfate proteoglycan(MCSP), CD34, and VEGF receptor 1 (VEGFR1). All testedtumors (Table 2) showed heterogeneous expression ofCD271 and MCSP at medians of 55% (range: 15%–93%)and 78% (range: 2%–99%), respectively (SupplementaryFig. S2A and S2B). CD34þwas expressed at a median of 7%(range: 2%–25%) and VEGFR1bright at 10% (range: 2%–19%), whereas CD34 and VEGFR1 were coexpressed in 4%of the cells (range: 2%–21%; Supplementary Fig. S2E).mMel6 and mMel10 were the only two melanoma cellcultures found to express CD117 (also known as c-kit) at89% and 70%, respectively (Supplementary Fig. S2K).

Some of the principal molecules reported as associatedwithmCSCphenotypewere also evaluated.Oct4,ATP-bind-ing cassette G2 (ABCG2), and aldehyde dehydrogenase(ALDH) were detected in all samples that averaged expres-sion of 10.9% � 0.9%, 11.1% � 1.4%, and 9.7% � 2.3%,respectively (Supplementary Fig. S2F–S2H).Melanomacellsnegative for Mitf expression were 10.8% � 1.5% (Supple-mentary Fig. S2I). All data are expressed as mean � SEM.

Each melanoma cell culture was analyzed for expressionof the principal known ligands recognized by the NKG2Dreceptor on CIK cells (MIC A/B, ULBP1, ULBP2, andULBP3). MIC A/B and ULBP2 were found expressed in

Table 1. Main characteristics of melanoma patients and corresponding samples

Subject number Age/sex Status Lesion siteaPrimary cellcultureb

CIK cellexpansionc

Autologouscytotoxicity assayd

mMel1 67/M PD SC Ye 115 YmMel2 64/F PD SC Ye,f 133 YmMel3 69/F PD SC Ye,f 419 YmMel4 54/M PD LN Ye 314 YmMel5 67/M PD LN Ye,f 1870 YmMel6 78/M PD LN Ye,f 109 YmMel7 79/F PD LN Y 125 YmMel8 87/F PD SC Y 1387 YmMel9 74/F PD LN Y 49 YmMel10 67/M PD LN Y 195 Y

Abbreviations: Y, yes; mMel, metastatic melanoma.aBiopsy was conducted at different metastatic sites including subcutaneous (SC) or lymph node (LN) sites.bIn vitro primary cell cultures derived from tumor biopsies.cCD3þ/CD56þ cell number-fold increase after 3 weeks of expansion.dCytotoxicity assay with CIK cells versus autologous tumor target cells.emMel samples assessed for tumorigenic capacity in vivo in NOD/SCIDmice. The samemMel sampleswere transducedwith LV-Oct4.eGFP to visualize mCSC and subsequently were sorted on the basis of eGFP expression.fmMel samples used for cytotoxicity assay with CIK cells versus LV-Oct4-eGFP–transduced target cells sorted on the basis of eGFPexpression.

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every tumor tested to varying levels with medians of 24%(range: 7%–90%) and 61% (range: 40%–98%), respective-ly. ULBP 1 and 3 expression was negligible except inmMel10 (16.57% ULBP3þ cells; Supplementary Fig. S2Cand S2D).All melanoma cell cultures, except two (mMel9 and

mMel10), retained membrane expression of HLA class Imolecules (>99%HLA-ABCþ).Melanoma cell cultures gen-erated from selected representative patients (n ¼ 6) weresubcutaneously inoculated in NOD/SCID mice to showtheir tumorigenicity. All mice inoculated with 6 differentmelanoma primary cell cultures produced tumor growthstarting between 1 and 8 weeks after injection (Fig. 1).

Expansion and phenotype of CIK cellsThe ex vivo expansion of CIK cells was evaluated in the

same 10 patients from whom we had generated melanomaprimary cultures. CIK cells were classically expanded fromfresh or cryopreserved PBMCs cultured with the timedaddition of IFN-g , Ab-anti-CD3, and IL-2. CIK cells fromall patients were successfully expanded within 3 to 4 weeksof culture.Median expansion of bulk CIK cells was 23-fold (range

11–117-fold) after 3 weeks of culture, whereas 252-foldexpansionwasobtained for theCD3þCD56þ fraction(range49–1870-fold). The presence of pure NK (CD3�CD56þ)cells was negligible at less then 5% in all cases at the end ofexpansion. The subset of mature CIK cells coexpressingCD3 and CD56molecules (CD3þCD56þ) was present witha median of 49% (range 23%–80%) while 78% (59–91%)of CD3þ cells were also CD8þ (Supplementary Fig. S3).The median membrane expression of the NKG2D recep-

tor, which is the main molecule responsible for tumorrecognition, on expanded CD3þ CIK cells was 84% (range:57%–93%).A summary of patient characteristics and relative CIK

expansion data is reported in Table 3.

Killing activity of CIK cells against melanoma cell lineTo test the antitumor activity of CIK cells expanded from

the 10 patients, we evaluated their ability to kill in vitro amelanoma cell line (DettMel). The cytotoxicity test was

conducted at the end of ex vivo expansion and showedefficient killing that varied among patients. The averagespecific tumor killing was 63% � 4%, 52% � 5%, 39%� 5%, and 28% � 5% (mean � SEM) at 40:1, 20:1, 10:1,and 5:1 effector/target ratio, respectively (n ¼ 18, Fig. 2Aand B).

In vitro and in vivo killing activity of CIK cells againstautologous metastatic melanoma cells

Patient-derived CIK cells efficiently killed in vitro autol-ogous metastatic melanoma targets with an average specifickilling of 71%�2%, 61%� 3%, 49%� 3%, and37%� 3%(mean � SEM) at a 40:1, 20:1, 10:1, and 5:1 effector/targetratio, respectively (n ¼ 26). The intensity of killing againstautologous targets was comparable (P ¼ 0.9991) with thatobservedwith allogeneicCIK cells assessed inparallel versusthe same tumor cells with an average specific killing of 70%� 4%, 61� 4%, 49%� 5%, and 35%� 4% (mean� SEM)at a 40:1, 20:1, 10:1, and 5:1 effector/target ratio, respec-tively (n ¼ 20). A summary of cytotoxicity in vitro againstautologous or allogeneic tumor targets is reported in Fig. 2A.

We evaluated also the activity of patient-derived CIK cellsin vivo against autologous metastatic melanoma. NOD/SCID mice (n ¼ 12) were subcutaneously implanted withan 8 mm3 tumor fragment from a patient-derived melano-ma biopsy (mMel2). One week after tumor implantation, agroup of implanted mice (n ¼ 8) were infused weeklyby tail vein injection withmature autologous CIK cells (1�107/week for 6 weeks). When tumor growth in untreatedmice (n¼ 4) wasmore than 2 cm in at least one dimension,all animals were euthanized. Tumors were excised andanalyzed for the presence of lymphocytic infiltration andthe extension of necrotic tissue areas. At the end of theexperiment, tumors fromanimals treatedwith CIK cells hadsignificantly larger necrotic areas compared with untreatedcontrols (Fig. 2B; P ¼ 0.0255), and we could confirmthe presence of CIK cells infiltrating the autologous tumor(Fig. 2C).Moreover, a significant delay in the tumor growthcurve was observed in treated mice compared with untreat-ed controls (P ¼ 0.0305) after two-way ANOVA analysis(Fig. 2D).

Table 2. Phenotype characterization of primary melanoma cell cultures

Subjectnumber

% MICA/B

%ULBP1

%ULBP2

%ULBP3

%NGFR

%MCSP

%Oct4

% ALDHbright

%ABCG2

%Mitf-

% VEGFR1bright

%CD34

%CD117

%HLA-ABC

mMel1 33.7 0 62.8 0 74.2 64.7 15.5 8.3 11.8 16.8 19.3 7.6 neg 99.9mMel2 15.2 1.6 40.7 1.9 58.3 71.4 11.5 8.7 12.4 16.9 10.0 18.0 neg 99.5mMel3 90.1 1.6 97.9 0 93.0 81.5 8.3 28.9 12.2 14 12.5 25.1 neg 99.6mMel4 64.9 0 60.6 7.4 19.9 95.0 10.1 8.2 1.3 10.7 10.6 6.3 neg 99.2mMel5 7.3 1.1 40.3 1.3 82.4 88.9 10.7 11.6 7.3 6.8 12.3 12.3 neg 99.7mMel6 32.4 0 55.0 0 50.8 95.1 7.3 2.8 11.2 6.5 10.7 6.4 88.8 99.1mMel7 20.5 0 64.5 0 39.1 78 10.7 8.9 14.1 9.8 5.0 3.1 30.1 99.4mMel8 53.5 0 52.7 0 28.9 74.2 11.4 3.3 17.5 15 4.4 3.1 neg 99.6mMel9 7.4 0 65.5 0 82.3 99.7 8.3 6.0 10.2 3.4 2.7 2.1 neg negmMel10 14.2 1.17 62.1 16.6 33.6 73.6 15.2 9.9 13 8.3 5.6 7.0 70.3 neg






Activity ofCIK cells against autologousputativemCSCsVisualization of putative mCSCs was accomplished by

stably transducing the primary melanoma cell culturesusing a lentiviral vector that carried eGFP controlled by thepromoter regulatory element of the Oct4 gene (LV-Oct4.eGFP; Fig. 3A and B). The average eGFP expression, 7 daysafter transduction, was 11.5%� 2.5%. As a positive control,a murine embryonic cell line expressing Oct4 (mES) wassuccessfully transduced with LV-Oct4.eGFP up to 90.5% ofeGFP expression (Fig. 3B), whereas no eGFP expression wasdetected on differentiated PBMCs from healthy donorstransduced with the same vector (LV-Oct4.eGFP). As anadditional control, we confirmed that both primary mela-noma cell cultures (Fig. 3B) and mES could be transducedefficiently (>90% of eGFP expression; data not shown)when the strong ubiquitous promoter (Phospho GlyceratoKinase, PGK, regulatory element) was used in place of theOct4 promoter to control eGFP expression (Fig. 3B).

On the basis of eGFP expression, transduced melanomacells were sorted into two fractions (eGFPþ and eGFP�) thatserved as targets to assess separately the antitumor activity ofpatient-derived CIK cells against their own putative mCSCs

(eGFPþ) and bulk eGFP� melanoma cells (SupplementaryFig. S4A–S4D). As further evidence of stem cell enrichmentwithin the eGFPþ fraction, we transduced bulk primarymelanoma cells with LV-Oct4.eGFP vector and culturedthem in anchorage-independent and serum-free conditionsat low cell concentration (104 cells/cm2) to observe theformation of mainly fluorescent (eGFPþ) spheroids (Sup-plementary Fig. S4E).

The integration of LV-Oct4.eGFP was confirmed by PCRin both eGFPþ and eGFP�melanoma cell subsets (Fig. 3A).Additional evidence that the eGFPþmelanoma cell fractionwas enriched in putative mCSCs, LV-Oct4.eGFP–trans-duced cells were evaluated on the basis of ABCG2 expres-sion. The average cell percentage expressing ABCG2 was10.6% � 2.3%, 72.4% of which coexpressed eGFP (Sup-plementary Figs. S5 and S6A).

We then measured the distribution of the main NKG2Dligands (MIC A/B, ULBP2) expressed in the target cells foreGFP expression. The percentage of MIC A/Bþ or ULBP2þ

cells were equally represented in eGFPþ and eGFP� frac-tions without a statistically significant difference (P ¼0.5181 and 1.000, respectively; Supplementary Fig. S6).

A mMel3mMel2mMel1

mMel6mMel5mMel4

Weeks after tumor cell inoculation (1 ×× 106)

Tu

mo

r vo

lum

e (c

m3 )

1W2 W3 W4 W5 W6 W7 W8 W9 W10 W

11 W

12 W

13 W

0

1

2

3

4

5mMel1mMel2

mMel3

mMel4

mMel5

mMel6

Melanoma primary cell growth curves in NOD/SCID mice B

Figure 1. Primary melanomacultures successfully generatetumor xenografts in vivo. A,primary metastatic melanoma cellcultures were successfully provedfor their tumorigenic activity in vivo.Representative H&E-stainedparaffin-embedded sections fromtumor generated aftersubcutaneous injection in NOD/SCID mice (1 � 106 metastaticmelanoma cells): mMel1, mMel2,mMel3, mMel4, mMel5, mMel6.Tumor xenograft morphology wasconsistent with the original humantumor as confirmed by a pathologyreview.Scalebars, 12mm.B, tumorgrowth curves of the melanomacell lines transplanted into theNOD/SCID mice.

Gammaitoni et al.





Through a functional assay, we evaluated the prolifera-tion ability of putative mCSCs. Melanoma cells transducedwith LV-Oct4.eGFP were stained with PKH26 dye to distin-guish the rare quiescent/slowly dividing cells of putativeCSCs from themore differentiated fast-growing population.Cell fluorescence was acquired initially and weekly for 3weeks. Putative eGFPþ mCSCs displayed a proliferativepotential in vitro that was, on average, three times less thantheir eGFP� counterparts after 21 days of culture (n ¼ 5),showing a slow-growing phenotype typical of CSCs; arepresentative histogram is reported in Fig. 3C.Patient-derived CIK cells efficiently killed in vitro autol-

ogous eGFPþ melanoma cells with an average specifickilling of 71% � 5%, 56% � 7%, 44% � 7%, and 40%� 6% (mean� SEM) at a 40:1, 20:1, 10:1, and 5:1 effector/target ratio, respectively (n¼ 4). Comparable (P¼ 0.8224)tumor killing intensity was reported against autologouseGFP� targets with an average specific killing of 66% �6%, 54%� 8%, 44%� 10%, and 36%� 8% at a 40:1, 20:1,10:1, and 5:1 effector/target ratio, respectively (n ¼ 4). Thekilling activity of CIK cells remained equally effectiveagainst both eGFPþ and eGFP� autologous melanoma cells(n¼ 5; P¼ 0.6286) even when the assay was conducted ontotal tumor cells without preemptive sorting of putativeeGFPþ mCSCs. A summary of cytotoxicity against autolo-gous tumor targets is reported in Fig. 3D.

DiscussionThe present work addresses two main issues regarding

the activity of immunotherapy in preclinical models:first, to show tumor-killing activity towards autologoussolid tumor cells; second, to show effective killing ofautologous putative cancer stem cells hitting one of thereservoirs responsible for tumor resistance to standardtreatments.

For the first time, we report the strong preclinical activityof patient-derived CIK cells against autologous metastaticmelanoma, with insight on their potential to target putativemCSCs. CIK cells represent promise for cancer adoptiveimmunotherapy and carry biologic features that comparepositively with other immunotherapies for reliable andeffective clinical translation.

The intense expansibility is the first of such features (13).Our data confirmed that CIK cells from patients withmetastatic melanoma were expanded at clinically relevantlevels. The simplicity and relative low expense of the expan-sion protocol, already validated in good manufacturingpractice–controlled conditions (33, 34), may positivelyimpact the clinical perspective. Recent clinical trials withCIK cells have reported encouraging observations in chal-lenging settings like metastatic lung, renal, and gastrointes-tinal tumors (13, 21, 35–37). However, such trials lackformal demonstration of CIK cell antitumor activity againstautologous metastatic targets; indeed, no data are currentlyavailable for metastatic melanoma.

For 10 different patients, CIK cells efficiently killed autol-ogous metastatic melanoma cells. Such findings are bothnovel and potentially clinically relevant. They overcomeimportant limitations linked to the use of commerciallyavailable allogeneic tumor cell lines, avoiding confoundingresults based on alloreactive HLA mismatches and allowedfull appreciation of the unique biology of each tumor.Furthermore, within each patient, it is possible to hypoth-esize the existence of additional and important biologicdifferences between metastases and the primitive tumor,supporting the importance of the observed killing of CIKcells against autologous metastatic melanoma.

Our in vitro cytotoxicity assays were evaluated conserva-tively, within a 6-hour experimental timeframe, to favorkilling specificity of the extremely delicate autologoustumor targets. Results are indicative of CIK killing capacity

Table 3. Expansion rate and phenotype characterization of CIK cells

Subjectnumber

FIcellsa

%iCD3b

%fCD3c FId CD3

%iCD3/CD56b

%fCD3/CD56c FId CD3/CD56

%iCD3/CD8b

%fCD3/CD8c

%iCD3/NKG2Db

%fCD3/NKG2Dc

mMel1 11 69 99 16 6 60 115 25 91 10 93mMel2 21 74 99 28 4 25 133 20 69 10 74mMel3 41 60 99 67 4 41 419 15 73 17 85mMel4 55 93 99 58 4 23 314 20 82 20 57mMel5 117 73 99 159 5 80 1870 11 76 14 91mMel6 11 50 94 20 5 50 109 17 80 11 91mMel7 22 68 100 32 10 58 125 37 69 37 79mMel8 27 49 95 53 1 51 1387 4 59 16 83mMel9 14 76 99 18 14 48 49 59 80 50 93mMel10 13 57 98 22 3 45 195 15 79 14 79

aFold increase [FI ¼ (cell number T ¼ week 3)/(cell number T ¼ 0)] of total cell number after 3 weeks of expansion.bPercentage of cells expressing different surface antigens at the basal time (T ¼ 0).c Percentage of cells expressing different surface antigens after 3-week expansion.dFold increase [FI = (cell number T ¼ week 3)/(cell number T ¼ 0)] of absolute cell number after 3 weeks of expansion calculated for everysubpopulation of cells expressing different surface antigens.






but a linear projection and quantification of prospectiveclinical efficacy is difficult to be predicted. In vivopersistenceof patient-infused CIK cells is expected to be about 2 weeksand multiple infusions will be possible based on theirintense ex vivo expansion and production simplicity.

A selected experiment to assess the in vivo activity ofpatient-derived CIK cells against autologous melanomatargets engrafted into NOD/SCID mice has shown delayedtumor growth, along with increased extension of necroticareas and infiltration of CIK cells at tumor sites. Thisadditional work was intended to provide proof of in vivoactivity of CIK cells; however, a deeper and more definitivein vivo analysis will require a dedicated study.

The second important feature of CIK cells is their HLA-unrestricted tumor killing, which extends to virtually allpatients the possibility to benefit from this approach,regardless of the expression of specific tumor-associatedantigen (TAA) restricted by precise HLA haplotypes. Themechanistic investigation of CIK cell tumor killing was notthe aim of our study; however, we showed the expression of

target molecules, recognized by NKG2D receptor, on allmetastatic melanoma primary cell cultures from ourpatients. The ULPB2 molecule was most consistently repre-sented while more variability among patients was observedfor MIC A/B.

Direct expression of MIC A/B molecules has been des-cribed on both primary andmetastaticmelanomas (38, 39).The possible upregulation of NKG2D ligands in varioustypes of treatment (e.g., chemotherapy, statins, doxycycline),with a consequent increased susceptibility toMHC-indepen-dent immune-mediated lysis, has been described in variousexperimental models (40–42) and may provide intriguingprospective synergies with immunotherapy.

Downregulation of MHC expression is one of the mainimmune-escape mechanisms developed by tumor cells. Inour study, CIK cells were effective against two MHC class Inegative melanoma samples, which confirmed their poten-tial in melanomas with immunogenicity alterations.

Melanoma primary cell cultures were derived frompatient tumor biopsies. These cultures retained original

Effector:Target ratio

% o

f T

um

or-

spec

ific

lysi

s

40:1

20:1

10:1 5:

10

20

40

60

80CIKs vs. autologous melanoma

CIKs vs. DettMel

Effector:Target ratio

% o

f T

um

or-

spec

ific

lysi

s

40:1

20:1

10:1 5:

10

20

40

60

80CIKs vs. allogenic melanoma

CIKs vs. DettMel

CIK in vitro tumor killing activityA

C

Anti-CD56Anti-CD5

Are

a o

f ti

ssu

e n

ecro

sis

(%)

Trea

ted

(aut

olog

ous C

IK)

Untre

ated

0

10

20

30

*

B

D

Weeks of treatment

Tu

mo

r vo

lum

e (c

m3 )

1 2 3 4 5 6 70

1

2

3

Untreated

CIK treated (1×107 CIK/mouse)

Figure 2. In vitro and in vivo activity of CIK cells against autologous melanoma. A, patient-derived CIK cells efficiently killed in vitro all bulk autologousmelanoma targets (n ¼ 35; left); results were comparable with those obtained with allogeneic CIK cells assessed in parallel versus the same tumor cells(n ¼ 20; right). Tumor killing was evaluated by flow cytometry assay conducted after coculturing mature CIK cells with PKH-26–stained targets for 4 hours.B,NOD/SCIDmice (n¼12)were subcutaneously implantedwith an8mm3 tumor fragment of patient-derivedmelanomabiopsy (mMel2).Oneweekafter tumorimplantation, 107 CIK cells were infused weekly by tail vein injection (n ¼ 8). Percentage of tissue necrosis on tumor growth was calculated at the endof the experiment on paraffin-embedded histologic sections. The results were expressed by mean� SEM and the extension of necrotic areas was analyzedby an unpaired, two-tailed t test (�, P ¼ 0.0255). C, at the end of the infusions, infiltration of CIK cells at tumor sites were shown by immunohistochemistryusing antibodies against CD5 and CD56. Scale bars, 12 mm. D, a significant delay in tumor growth was observed in NOD/SCID–treated mice comparedwith the controls (n ¼ 4). Tumor volume increments were expressed as mean � SEM and CIK activity was analyzed by two-way ANOVA (P ¼ 0.0308).

Gammaitoni et al.





tumor characteristics and displayed great immunopheno-typic heterogeneity among samples. Most differentiationantigens detected on metastatic melanoma cells showedvariable levels of expression, as others have described (23,24). In contrast, the expression and average levels of puta-tive stemness markers, Oct4 and ABCG2, as well as the lackof Mitf expression, were quite comparable among differentmelanoma samples (27, 43–45). Together, these data sug-gest that the cell fraction endowed with stemness features isstably detectable and retained in different samples. This isconsistent with the decade-old CSC theory that tumorscontain a subset of cells that both self-renew and generatedifferentiated progeny (9, 46, 47). CSCs are, therefore, thedriving force of the tumor.Truthfully, the identification of molecular and pheno-

typic markers for CSCs still remains partially unsolved. Infact, CSCs seem to have a dynamic phenotype, more likelyas expression of a functional state rather than a precisecellular entity (48, 49). The expression of the Oct4 geneseems able to be reliably associated with cancer cells ofvarious histotypes endowed with stemness features (50–55). Recently, Oct4 expression was correlated to dediffer-entiation of melanoma cells, reacquisition of stem pheno-type, increased tumorigenic capacity, and resistance tochemotherapy (28). We exploited these observations by

designing a gene transfer strategy to detect mCSCs andassess their susceptibility to CIK-mediated killing. Bulk,patient-derived metastatic melanoma cells were transducedwith a lentiviral vector that encodes eGFP under control ofthe humanOct4 regulatory element, with the idea that onlymCSCs are able to activate the Oct4 promoter to expresseGFP, which allows their specific killing by CIK cells to betracked and evaluated.

This approach uncovered a small subpopulation ofeGFPþ putative mCSCs, consistent with the expected rateof mCSCs given detected Oct4 protein. This small fractionappeared to preferentially coexpress the stemness markerABCG2 relative to its eGFP� counterpart. To identify func-tionally rare quiescent/slowly dividing CSCs, a lipophilicfluorescent dye, PKH26, was used to visualize relativelyquiescent cells within a proliferating population (56).Indeed, eGFP� cells encountered up to 5 cell divisionsduring a 3-week culture period, whereas eGFPþ cellsencountered a maximum of 2 cell divisions in the sameelapsed time. Moreover, as CSCs can withstand anoikis,they proliferate/differentiate in anchorage-independentconditions and give rise to clonal spheroids. Melanomaprimary cells after LV-Oct4.eGFP transduction were thencultured in anchorage-independent and serum-free condi-tions. Only a small fraction of cells retained the ability to

T=0eGFP– T=+21 days

eGFP+ T=+21 days

PKH26 Dye fluorescence

C Proliferation assay

mM

el10

O

ct4.

eGF

P+

mM

el10

Oct

4.eG

FP

-

mM

el3

Oct

4.eG

FP

+

mM

el3

Oct

4.eG

FP

-

mM

el2

Oct

4.eG

FP

+

mM

el2

Oct

4.eG

FP

-

LV-Oct4.eGFPExpression cassette

A

LV-Oct4.eGFP–Transduced

melanoma cells

LV-Oct4.eGFP–Transduced

mES cells

LV-PGK.eGFP–Transduced

melanoma cells

B

Effector:Target ratio%

of

Tu

mo

r-sp

ecif

ic ly

sis

40:1

20:1

10:1 5:

10

20

40

60

80CIKs vs. eGFP+ sorted cellsCIKs vs. eGFP– sorted cells

CIK in vitro tumor killing activityD

Figure 3. CIK cells efficiently killed autologous melanoma putative mCSC. A, schematic representation of lentiviral vector LV-Oct4.eGFP used to visualizeputative mCSC. The presence of integrated LV-Oct4.eGFP was confirmed by PCR in both eGFPþ and eGFP� fractions of freshly sorted cells. Pictureshows representative PCR electrophoresis gel (100 ng gDNA/each sample) with primers annealing on the lentiviral vector backbone upstream anddownstream the Oct4.eGFP expression cassette. B, representative eGFP expression in melanoma primary cell cultures transduced with LV-Oct4.eGFPor LV-PGK.eGFP; as positive transduction control, mES cells transduced with LV-Oct4-eGFP (scale bars, 15 mm). C, proliferation assay in vitro wasevaluated by staining LV-Oct4.eGFP-transduced tumor cells with the vital dye PKH26 and assessing the fluorescence intensity decrement over time. Arepresentative experiment is reported in figure. D, the antitumor activity of patient-derived CIK cells was equally intense against autologous Oct4.eGFPþ

mCSC (n ¼ 4); results were comparable with those observed against Oct4.eGFP� bulk melanoma cells.






grow, and spheroidswere generated exclusively fromeGFPþ

cells that maintained the fluorescence even when hetero-geneously distributedwithin the spheres. CIK cells intenselykilled the autologous eGFPþ-sorted fraction at a lysis ratecomparable with that observed against eGFP� tumortargets.

Killing the truemelanoma stem cells currently remains anideal concept. Yet, our data suggest that CIK cells kill asubset of autologous metastatic melanoma cells able toactivate Oct4 that, based on current knowledge, reliablydefines a subpopulation of tumor cells with stemness fea-tures (28). Dedicated studies are required and currentlyongoing to investigate deeply the functional and tumori-genic characteristics of eGFPþ mCSCs. Nevertheless, ourfindings provide new and additional weight to the potentialof cancer immunotherapy with CIK cells. Data from Kumarand colleagues confirmed that the Oct4 expression corre-lates with putative CSC features and that Oct4-expressingcells display a significantly higher chemotherapy agentresistance (28). Indeed, the observed killing ability of CIKcells against putative mCSCs may reveal other valuableperspectives on the potential of this immunotherapy strat-egy. Moreover, the elevated safety profile of CIK cells doesnot preclude their use in associationwith other approaches.One appealing possibility would be to explore their poten-tial synergism with conventional chemotherapies or evenmolecular-targeted treatments. This strategy could reduceresistance occurrence by improving the odds of targeting thecrucial CSC subset from which tumor regrowth is specu-lated to start.

The MHC-unrestricted tumor killing mechanism of CIKcells showed in this study may advantage it over otherimmunotherapy approaches because it addresses the diffi-cult quest of targeting mCSCs and also HLA-negativetumors. We showed that the membrane expression ofNKG2D ligands is maintained on putative mCSCs. Viceversa, it is still unknown whether or not such a peculiartumor subpopulation retains the same antigenic features(specific TAA or MHC molecule expression), as do othertumor cells.

Overall, shown here, for the first time, is the intensetumor killing activity of CIK cells against autologous met-astatic melanoma, including putative mCSCs. These data

point toCIK cells as favorable candidates for clinical trials inpatients withmelanoma. The biologic basis is set for furtherpreclinical and clinical investigations on the prospectivepotential of targeting mCSCs with CIK cells, either inde-pendently or in synergismwith other therapeutic strategies.

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

Authors' ContributionsConception and design: L. Gammaitoni, F. Picciotto, G. Grignani, M.Aglietta, D. SangioloDevelopment of methodology: L. Gammaitoni, V. Leuci, F. Picciotto, A.Pisacane, T. Venesio, A. Balsamo, Y. Pignochino, D. SangioloAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): L. Gammaitoni, L. Giraudo, V. Leuci, M. Todoro-vic, G. Mesiano, A. Pisacane, A. Zaccagna, M.G. Volpe, S. Gallo, D. Caravelli,E. Giacone, T. Venesio, A. Balsamo, G. GrignaniAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): L. Gammaitoni, L. Giraudo, V. Leuci, M.Todorovic, G. Grignani, D. SangioloWriting, review, and/or revision of the manuscript: L. Gammaitoni, L.Giraudo, V. Leuci, M. Todorovic, G. Mesiano, F. Picciotto, A. Zaccagna, E.Giacone, G. Grignani, F. Carnevale-Schianca, M. Aglietta, D. SangioloAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): L. GammaitoniStudy supervision: L. Gammaitoni, F. Picciotto, G. Grignani,M. Aglietta, D.Sangiolo

AcknowledgmentsThe authors thank Dr. W. Cui (IRDB, Imperial College London, London,

United Kingdom) who provided the phOCT4.EGFP1 vector and Dr. ElisaVignawhoprovided the transfer vector pRRL.sin.PPT.hPGK.EGFP.Wpre (LV-PGK.EGFP). The authors also thank Joan Leonard (Leonard Editorial Ser-vices, LLC) for the linguistic revision and editorial assistance and E. Lantelmefor sorting services.

Grant SupportThis work was supported by grants from Progetti di Ricerca Rete Onco-

logica Piemonte-Valle d’Aosta, Associazione Italiana Ricerca sul Cancro–AIRC I.G. grant no. 11515, Associazione Italiana Ricerca sul Cancro–AIRC 5� 1000, and University of Torino-Progetti di Ateneo 2011 grant RETHE-ORTO 11RKTW. The fellowships of L. Giraudo, M. Todorovic, and Y.Pignochino are sponsored byMIUR (University of Turin) and the fellowshipof G. Mesiano is sponsored by an Associazione Italiana Ricerca sul Cancro–AIRC I.G. grant no. 11515.

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 January 8, 2013; revised May 15, 2013; accepted June 10, 2013;published OnlineFirst June 21, 2013.

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Published OnlineFirst June 21, 2013.Clin Cancer Res Loretta Gammaitoni, Lidia Giraudo, Valeria Leuci, et al. Stemness FeaturesAutologous Metastatic Melanoma Including Cells with Effective Activity of Cytokine-Induced Killer Cells against

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