T Lymphocytes Restrain Spontaneous Metastases in …Microenvironment and Immunology T Lymphocytes...

Microenvironment and Immunology

T Lymphocytes Restrain Spontaneous Metastases inPermanent Dormancy

Irene Romero1, Cristina Garrido3, Ignacio Algarra4, Antonia Collado2, Federico Garrido1,3, andAngel M. Garcia-Lora1

AbstractTumor dormancy is a clinical phenomenon related to immune equilibrium during cancer immunoediting.

The mechanisms involved in dormant metastases are poorly understood due to the lack of preclinical models.Here, we present a nontransgenic mouse model in which spontaneous metastases remain in permanent immuno-mediated dormancy with no additional antitumor treatment. After the injection of a GR9-B11mouse fibrosarcomaclone into syngeneic BALB/c mice, all animals remained free of spontaneous metastases at the experimentalendpoints (3–8 months) but also as long as 24 months after tumor cell injection. Strikingly, when tumor-bearingmice were immunodepleted of T lymphocytes or asialo GM1-positive cells, the restraint on dormant disseminatedmetastatic cells was relieved and lung metastases progressed. Immunostimulation was documented at bothlocal and systemic levels, with results supporting the evidence that the immune system was able to restrainspontaneous metastases in permanent dormancy. Notably, the GR9-B11 tumor clone did not express MHCclass I molecules on the cell surface, yet all metastases in immunodepleted mice were MHC class I–positive. Thismodel system may be valuable for more in-depth analyses of metastatic dormancy, offering new opportunitiesfor immunotherapeutic management of metastatic disease. Cancer Res; 74(7); 1958–68. �2014 AACR.

IntroductionThe initiation and progression of cancer in an immuno-

competent host involve numerous interactions between tumorcells and the immune system. The immune response exercisesselective pressure against tumor cells, eliminating the moreimmunogenic phenotypes. This constant interaction betweenthe immune system and cancer cells may ultimately result inthe selection of less immunogenic "cancer-escape" variantsthat are able to survive and progress in the host (1, 2). Thediverse escape mechanisms developed by cancer cells to evadethe immune response (3–6) include the loss of surface expres-sion of MHC class I molecules (7–9). This loss may make thetumor cells invisible to T lymphocytes, allowing them to enteran "immunoblindness" stage (10).

It is feasible that some cancer cells neither progress nor aredestroyed by immune system during this selective process,remaining in a dormant stage and reaching equilibrium withthe host tumor microenvironment (11–13). Cancer dormancy

has been observed in humans (14, 15), and several experimentalstudies have reported an immunomediated control of primarytumor cells in dormancy (16–20). There is considerable evi-dence of metastasis relapse in human cancer patients afterlong periods of remission, when disseminated metastatic cellscan persist for years or even decades as minimal residualdisease (21, 22). Other clinical examples related to cancerimmune control include tumors that arise after immunosup-pressive treatments (23, 24) and cases of transplanted organscarrying an undetectable tumor that grows after immunosup-pressive treatment of the patient (25–27). These clinical phe-nomena support the existence of a state of equilibriumbetween the host and the cancer cells. The fact that immu-nosuppression can disturb this equilibrium and activate dor-mant cancer cells strongly suggests the existence of an immu-nomediated state of dormancy in these cases.

The mechanisms involved in cancer dormancy remainlargely unknown, due to difficulties in isolating dormanthuman metastatic cells and constructing preclinical modelsof dormant metastases. Here, we presented a novel nontrans-genic preclinical mouse model of permanent immunome-diated metastatic dormancy. We have used an extensivelystudied fibrosarcoma mouse model (GR9) developed in ourlaboratory composed of several tumor clones with differentMHC class I expression patterns and spontaneous metastaticcapacities (28). Thus, the metastatic capacity is elevated in theclones with high MHC class I expression and reduced in thosewith lowMHC class I expression (29).We show that anMHC-I–negative GR9-B11 tumor cell clone did not generate sponta-neous lung metastasis in immunocompetent mice, which

Authors' Affiliations: 1Dept. Analisis Clinicos e Inmunologia, UGC Labor-atorio Clínico; 2Unidad de Investigaci�on, Hospital Universitario Virgen de lasNieves, Granada; 3Departamento de Bioquímica, BiologíaMolecular e Inmu-nología III, Universidad de Granada, Granada; and 4Departamento de Cien-cias de la Salud, Universidad de Ja�en, Ja�en, Spain

Corresponding Author: Angel M. Garcia-Lora, UGC Laboratorio Clinico,Hospital Universitario Virgen de las Nieves, Av. de las Fuerzas Armadas 2,18014, Granada, Spain. Phone: 349-580-20269; Fax: 349-580-20069;E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-13-2084

�2014 American Association for Cancer Research.

CancerResearch

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remained free of metastasis until their euthanasia at the end ofthe assays (3–24 months). Strikingly, immunodepletion of T orasialo GM1-positive cells in the mice awoke the disseminatedmetastatic cells from dormancy, generating lung metastases.

Materials and MethodsCell lines and IFN-g treatmentGR9 cell line is derived from a mouse fibrosarcoma induced

by methylcholanthrene in BALB/c mice and has been exten-sively characterized in our laboratory. It is composed of cellclones with distinct H-2 class I expression patterns and met-astatic capacities (29). Spontaneous metastasis assays wereperformed with different GR9 cell clones, and one of these, theGR9-B11 clone, was selected for this study. GR9-B11 and GR9-A7 are clones obtained by a limited dilution method from theGR9 cell line. GR9-B11 and GR9-A7 cell lines were recloned bypicking up individual cells under phase-contrast microscopy.All cell lines were characterized by PCR assay using shorttandem repeat and they were also regularly tested for MHC-Igenotype and surface expression. Cell lines were maintainedin Dulbecco's Modified Eagle Medium (Sigma-Aldrich) sup-plemented with 10% FBS (Life Technologies), 2 mmol/L glu-tamine (Sigma-Aldrich), and antibiotics. In some experiments,cell lines were treated with 100 U/mL IFN-g for 48 hours(Sigma-Aldrich).

MiceEight-week-old male BALB/c and athymic nu/nu BALB/c

(Charles River Laboratories) mice were used in the experi-ments. The breeding and care of animals were undertaken incompliance with European Community Directive 86/609/CEEand Spanish law (Real Decreto 1201/2005) for the use oflaboratory animals. Housing and all experimental proceduresinvolving animals were performed according to protocolsapproved by the hospital Animal Care Committee and incompliance with the animal welfare guidelines of the NationalCommittee for Animal Experiments.

Spontaneous metastasis assayDifferent cell doses (50� 105, 25� 105, 12.5� 105, and 6.25�

105) of GR9-B11 were subcutaneously injected into the footpadof groups of syngeneic immunocompetent and nude BALB/cmice. The growth of local tumors was recorded 3 times perweek in all animals, measuring the largest diameter of eachtumor with electronic calipers. Tumors were excised when thelargest diameter reached 10 mm. The model resembles met-astatic development in humans after surgical removal of theprimary tumor.Micewere anesthetizedwith 0.04mLdiazepam(Valium, Roche) and 0.1mL ketamine (Ketolar) before removalof the primary tumors with sterilized instruments, usingelectrocautery to minimize bleeding and closing the woundswith surgical clips and adhesive. After the surgery, each animalwas housed alone until recovery from anesthesia. At the end ofthe assays, animals were anesthetized and euthanized bycervical dislocation. A complete necropsy was performed, andthe number of spontaneous metastases was counted. Localtumors and macroscopically visible metastatic nodules wereexcised, disaggregated, and adapted to tissue culture. Then, the

lungs were fixed in Bouin solution (Sigma-Aldrich) and themicrometastases were counted.

Preparation of splenic and lung leukocytesSpleens and lungs were excised and gently homogenized in a

stomacher in cold PBS (Sigma-Aldrich). A tissue fragment wasremoved and a sterile falcon cell strainer (BD Biosciences) wasused to create a single-cell suspension. Red blood cells werelysed with ACK lysing buffer (Gibco) for 5 minutes and thenwashed twice inPBS. Viable cells were counted andused for theantibody staining reaction.

Flow cytometry analysis of immune cell subsetsFor direct immunofluorescence, the following labeled anti-

bodies (Miltenyi-Biotech) were used: CD3e-APC, CD4-FITC,CD8-PE, CD25-PE, FoxP3-APC, CD19-FITC, CD49b-FITC,anti-MHC class II-APC, anti-CD11c-PE, and anti-CD11b-FITC.Isotype-matched nonimmune mouse IgGs conjugated withFITC, PE, or APC served as controls. FcR Blocking reagentwas used to block unwanted binding of antibodies to mousecells expressing Fc receptors. Immunofluorescence was doneaccording to themanufacturer's instructions (Miltenyi Biotec),using FoxP3 staining buffer to obtain optimal FoxP3 immu-nofluorescent staining. Cells were analyzed on a FACSCantocytometer (BD Biosciences). Each sample consisted of a min-imum of 5 � 104 cells and was analyzed with CellQuest-Prosoftware.

MHC class I surface expressionMHC class I surface expression was analyzed by indirect

immunofluorescence using FACS (FACScan; Becton Dickin-son) according to a standard protocol. In brief, 5 � 105 cellswere washed twice with PBS and incubated for 30 minutes at4�C with the primary antibodies anti-H-2 Kd (K9-18), anti-H-2Dd (34-5-8), and anti H-2 Ld (28.14.8 and 30.5.7), all obtainedfrom the American Type Culture Collection. The secondaryfluorescein isothiocyanate (FITC)-conjugated antibody (anti-mouse FITC IgG/Fab, Sigma-Aldrich) was used in 1:120 dilu-tion for 30 minutes at 4�C in the dark. Isotype-matchednonimmune mouse IgG and cells labeled with the fluoresce-in-conjugated antibody alone served as controls. A minimumof 1� 104 cells were analyzed with CellQuest-Pro software. Allcell lines were studied in baseline conditions and after IFN-gtreatment.

Real-time RT-PCR analysisAn mRNA isolation kit (Miltenyi-Biotech) was used to

extract mRNA from tumor cell lines. First-strand cDNA wassynthesized with 100 ng of mRNA using a High CapacityReverse Transcription Kit (Applied Biosystems) in a totalvolume of 20 mL. These cDNAs were diluted to a final volumeof 100mL. Real-time quantitative PCRanalyseswere carried outin the 7500 Fast System (Applied Biosystems), performing PCRreactions in quadruplicate and expressing the values obtainedas means � SD. Quantitative PCR was performed with thePower SYBR Green Master Mix (Applied Biosystems); theprimers and amplicon size for each gene were previouslyreported (30). GADPH and b-actin genes were used as

Spontaneous Metastases in Immune-Mediated Dormancy

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housekeeping genes. PCR conditions were 40 cycles of 15seconds of denaturation at 95�C and 60 seconds at 60�C.

Immunodepletion protocols in spontaneous metastasisassay

A total of 12.5 � 105 GR9-B11 cells were injected into thefootpad of syngeneic BALB/c mice. The growth of the tumorswas measured 3 times per week. At around 20 to 22 days, thetumors reached 10mmandwere excised as described above. At151 days, one group of mice was euthanized and the remainingmice were randomly divided among six groups. Four of thesegroups received immunodepletion treatments with monoclo-nal antibodies. The following protocols were used: (i) 100 mganti-CD4 monoclonal antibody (mAb; clone YTS191)þ 100 mganti-CD8 mAb (clone YTS 169), (ii) 100 mg anti-CD4 mAb, (iii)100 mg anti-CD8 mAb, and (iv) 50 mg anti-asialo GM1 (Wakochemicals USA Inc.). A fifth group was treated with 100 mgcontrol immunoglobulin (Polyclonal rat IgG, Sigma-Aldrich),while the remaining group received no treatment (untreatedmice). The agents were administered twice a week for 90 daysfrom day 152 after the tumor cell injection. Depletion of thecorresponding immune subpopulations in the mice was con-firmed by flow cytometry. Mice from each group were eutha-nized at the end of the treatments (day 242 post-cell injection).A complete necropsy was performed, isolating and analyzingthe lungs as described above.

Statistical analysisData were expressed as means� SD. The Student t test was

used to compare mean values. A significance level of P < 0.05was assumed for all statistical tests. SPSS 16.0.2 (IBM)was usedfor the data analyses. All statistical tests were two-sided.

ResultsH-2 class I phenotype ofGR9-B11fibrosarcoma clone cellline and local primary tumors

GR9 is a methylcholanthrene-induced fibrosarcoma gener-ated in BALB/c mice and is composed of several tumor cloneswith different H-2 class I surface expressions (28). GR9-B11fibrosarcoma cell line was derived from the GR9 tumor bylimited dilution cloning. The GR9-B11 clone was recloned bypicking up individual cells under phase-contrast microscopy.Under baseline conditions, this tumor clone cell line has anegative expression of H-2 Kd, Dd, and Ld molecules (Fig. 1A).IFN-g treatment upregulated all the three molecules (Fig. 1A).Possible mechanisms underlying the loss of surface MHC-Iexpression of GR9-B11 were investigated by analyzing thetranscriptional gene expression of antigen processing machin-ery (APM), H-2 class I heavy chains, and b2-microglobulingenes. The GR9-B11 fibrosarcoma clone was compared with apositive MHC-I GR9-A7 clone cell line, normalizing the datato the expressions of GAPDH and b-actin housekeepinggenes. Figure 1B depicts the results, using the values forGR9-A7 cells as reference (assigned a relative value of 1).GR9-B11 showed a downregulation of H-2 Ld, calreticulin,LMP2, TAP-1, and tapasin (Fig. 1B). These results demonstratethat the molecular mechanism underlying the loss of MHC-Isurface expression involves the coordinated transcriptional

downregulation of several APM components andMHC-I heavychains.

In tumor-initiating capacity (TIC) assays, four cell doses (50.0� 105, 25.0 � 105, 12.5 � 105, and 6.25 � 105 cells) were locallyinjected into the footpad in groups of 10mice. The local tumorswere removed from the animals when the largest tumor diam-eter reached 10 mm. The tumors were then adapted to tissueculture to analyze their H-2 class I surface expression incomparisonwith that of the GR9-B11 clone. The surface expres-sion of H-2 class I molecules in vitro was higher in the localtumor cell lines than in the original clone. The tumors showedpositive surface expression of H-2 Kd and Dd molecules andnegative for H-2 Ld molecule in baseline conditions (Fig. 1C).TheH-2 Kd andDdmolecules were strongly upregulated after invitro treatment with IFN-g and showed an even higher expres-sion than what was observed on the original tumor clone.However, two populations were observed with different pat-terns of expressionofH-2 Ldmolecule: in onepopulation, H-2 Ld

molecule was clearly upregulated, whereas in the other, H-2 Ld

molecule was negative (Fig. 1C). These results were observed inall local tumors analyzed regardless of the cell dose injected.

Spontaneous metastasis assays from GR9-B11 clone inimmunocompetent and nu/nu BALB/c mice

To determine the in vivometastatic capacity of the GR9-B11tumor clone, themice weremonitored weekly for spontaneousmetastatic spread after removal of primary tumors. The miceshowed no signs of disease and were eventually euthanized at90 days after the tumor excision. The autopsy revealed that allmice were metastasis-free, regardless of the cell dose used. Wethen repeated these assays and found that the mice remainedmetastasis-free at 24 months after primary tumor removal,when the mice were euthanized.

Two possible explanations for these findings were thenexplored: the disseminated metastatic cells might not becapable of migrating or invading, or GR9-B11 metastatic cellsmight be eliminated by the immune system. These possibilitieswere examined by performing spontaneous metastasis assayswith GR9-B11 cells in immunodeficient nu/nu BALB/c mice,injecting 6.25� 105 cells into two groups of 10 nude mice. Thelocal growth rate of tumors in these mice was similar to that inimmunocompetentmice, reaching a largest diameter of 10mmin 32 versus 29 days, respectively. The local tumors wereremoved when this diameter was reached and were adaptedto tissue culture. Analysis of their H-2 class I surface expressionrevealed the same H-2 class I phenotype as expressed by theprimary tumors generated in immunocompetent mice (Fig.1C). The mice were monitored for the appearance of sponta-neous metastases and were euthanized when signs of diseasewere observed. Unexpectedly, lung metastases (range, 1–8)were found in 80% of these mice (Fig. 2A). The immunocom-petent mice served as controls in this assay, and all remainedmetastasis-free (Fig. 2A). These results indicate that GR9-B11cells have intrinsic migratory and invasive capacities. Allmacroscopically visible metastatic nodules were adapted totissue culture, and H-2 class I phenotype analysis revealed thatall of the metastases had the same H-2 class I phenotype,characterized by the high baseline expression of H-2 Kd and Dd

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Cancer Res; 74(7) April 1, 2014 Cancer Research1960




molecules, which were induced by IFN-g treatment, and theabsence of surface expression of H-2 Ld molecule underbaseline conditions and after IFN-g treatment (Fig. 2B). Thisphenotype represents a new tumor variant that is not presentin the original clone but is observed in the primary tumors.To summarize, the GR9-B11 fibrosarcoma clone generated

spontaneous metastases in T-cell–immunodeficient nu/nuBALB/c mice, but not in immunocompetent BALB/c mice,and all metastases evidenced total loss of H-2 Ld moleculesurface expression.The immunogenicity of the metastases derived from nude

mice was evaluated by injecting these metastatic cell lines intothe footpad of immunocompetent BALB/cmice, administering

different cell doses (12.5 � 105 and 6.25 � 105 cells) in twogroups of 5 mice each. The metastatic cells demonstrated anelevated immunogenicity, being rejected in 67% of the miceinjected with the higher cell dose and in 40% of those injectedwith the lower dose. Moreover, these mice did not developspontaneous metastases.

Changes in immune cell subpopulations promoted byGR9-B11 tumor cells

We carried out assays to evaluate individual immune cellsubpopulations at local and systemic level in GR9-B11 tumor-bearing immunocompetent mice. Two groups of 30 mice wereeuthanized on days 25 and 50 after removal of the local tumors,

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Figure 1. H-2 class I surface expression onGR9-B11 clone and in local primarytumors. A, H-2 class I expression of theGR9-B11 fibrosarcoma cell line underbaseline conditions and after treatmentwith IFN-g (100U/mL) for 48 h: H-2 K (grayline), H-2 D (dotted line), and H-2 L (blackline). GR9-B11 is H-2–negative and allthree molecules are induced after IFN-gtreatment. B, transcription levels of H-2class I heavy chains, b2m, and severalAPM components detected by real-timeRT-PCR. Expression level of the genes ofinterest was determined with respect tolevels of b-actin and GAPDHhousekeeping genes. Data for GR9-A7are set to 1. Values, mean � SD of threeindependent experiments performed inquadruplicate; �,P

and spleen leukocyte populations were analyzed by flowcytometry (Fig. 3 and Table 1). Tumor-bearing mice showedstatistically significant changes in the lymphocyte subpopula-tions on days 25 (25 d) and 50 (50 d) in comparison with thenontumor-injected animals (NT; P < 0.05), with an increase inCD3þ (44.4 and 53.7 vs. 33.1%, respectively), CD3þCD4þ (33.3and 40.4 vs. 26.1%), and CD3þCD8þ (11.1 and 13.0 vs. 6.9%)lymphocytes, a slight increase inNKTcells (1.0 and 1.4 vs. 0.4%),and an increase in dendritic cells (8.7 and 8.0 vs. 3.0%) andmacrophages (8.6 and 7.1 vs. 3.8%; Fig. 3B and Table 1).

We also analyzed the changes in lymphocyte subpopulationsin the lungs of mice on day 25 and 50 after removal of the localtumor (Fig. 4A and Table 1), finding a major rise in thepercentage of CD3þ lymphocytes, which had increased to64% on both days, versus 51.9% in nontumor-injected mice(Fig. 4B); this expansion corresponded to increases in T-helperlymphocytes (46.2% and 50.5% vs. 40.6%, respectively) and T-cytotoxic lymphocytes (17.4 and 12.1 vs. 9.7%, respectively; Fig.4B and Table 1). We highlight that the percentage of CD8þ Tlymphocytes had increased by 180% on day 25.

T or asialo GM1-positive cells maintain dormantspontaneous metastases in a state of equilibrium

According to the above experiments, the GR9-B11 cloneproduced spontaneous pulmonary metastatic nodules in nudemice but not in immunocompetent mice, which remainedmetastasis-free and developed an immune response.We there-

fore hypothesized that disseminated metastatic cells would beeliminated by the immune system in immunocompetent miceor, alternatively, would be kept in a dormant state. Thesepossibilities were tested in a new experiment (Fig. 5A), inwhich 12.5 � 105 GR9-B11 cells were injected into the footpadin seven groups of immunocompetent BALB/c mice. The localtumors were removed at 20 to 22 days, and one of the groupswas euthanized at 151 days; the necropsy revealed that nometastases were present in any mice in this group. From day152, the mice in another four groups were treated weekly withanti-CD4 þ anti-CD8 mAbs, anti-CD4 mAb, anti-CD8 mAb, orimmunoglobulins (control Ig group; Fig. 5A). A sixth group ofmice was treated with the anti-asialo GM1 antibody (Fig. 5A).After 3 months of treatment, the mice were euthanized on day242 (Fig. 5A) and the depletion of each subpopulation wasconfirmed by flow cytometry. In the mice treated with controlimmunoglobulin, no metastatic nodules were detected in thenecropsy (Table 2). In the groups treated with anti-CD4þ anti-CD8 mAbs or with anti-CD8 mAb alone, 100% of the micedeveloped spontaneous pulmonary metastases. Similar resultswere found in the group treatedwith anti-asialo GM1 antibody,in which 87% of the mice developed metastases (Table 2).However, in the group in which CD4þ T cells alone weredepleted, only 23% of the mice developed metastases. Thenumber of metastases per mouse differed among the groups(Table 2): it ranged from 4 to 62 micrometastases in the micetreated with anti-CD4 þ anti-CD8 mAbs and from 3 to 17 in

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Figure 2. Spontaneous metastasis assaysin immunocompetent and nu/nu BALB/cmice. A, the graph depicts the mice withmetastases and the number of lungmetastases per mouse. A dot representsthe number of lungmetastases identified ineach mouse. These results werereproducible in another two independentexperiments. B, H-2 class I phenotype ofspontaneous pulmonary metastases innudemice. All metastases showed a singlephenotype under baseline conditions andafter IFN-g treatment, with positiveexpression of K and Dmolecules alone. H-2K (gray line), H-2D (dotted line), andH-2 L(black line). A representative example isdepicted.

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Figure 3. Changes in splenic leukocyte populations (lymphocytes,macrophage, anddendritic cells). Thedifferent leukocyte populationswere analyzed at days25 (25 d) and 50 (50 d) after local tumor removal in comparison with nontumor-injected mice (NT). A, a representative experiment showing B (CD3�CD19þ)and T (CD3þCD4þ and CD3þCD8þ) lymphocyte subpopulations, macrophages (MHC-IIþCD11bþ), and dendritic cells (MHC-IIþCD11cþ). B, thegraphs depict the percentages (mean � SD) of CD3þ/CD19þ, CD4þ/CD8þ, and CD11bþ/CD11cþ cells. �, P < 0.05.






50% of the group with CD8þ T lymphocyte–depleted mice butreached 100 micrometastases in the other 50% of this group; itranged from 2 to 35 micrometastases in 62% of asialo GM1-depleted mice but reached more than 100 micrometastases in

25% of this group; it ranged from 1 to 2 micrometastases in thegroup treated with anti-CD4mAb. Macrometastases were onlydetected in the mice treated with anti-CD4 mAb (13%) or anti-asialo-GM1 (30%) in a range of 1 to 2 per mouse. Mice from a

Table 1. Changes in splenic and lung leukocyte populations

Splenic leukocyte populations

CD3þCD4þ CD3� CD3� CD3þ MHCIIþc MHCIIþc

CD3þc CD4þc CD8þc CD25þFoxP3þa CD19þc CD49bþc CD49bþ CD11bþb CD11cþb

NT 33.1 � 4.3 26.1 � 3.1 6.9 � 1.8 4.4 � 0.8 61.0 � 4.2 4.6 � 1.0 0.4 � 0.3 3.8 � 0.1 3.0 � 0.225d 44.4 � 6.8 33.3 � 5.1 11.1 � 1.9 4.3 � 1.0 48.9 � 6.8 5.4 � 1.7 1.0 � 0.2 8.6 � 1.2 8.7 � 0.750d 53.7 � 5.5 40.4 � 4.1 13.0 � 2.9 3.6 � 1.7 41.0 � 4.7 3.7 � 1.0 1.4 � 0.6 7.1 � 1.4 8.0 � 1.6

Lung lymphocyte populations

CD3þCD4þ CD3� CD3� CD3þ

CD3þc CD4þc CD8þc CD25þFoxP3þa CD19þc CD49bþ CD49bþ

NT 51.9 � 6.3 40.6 � 5.5 9.7 � 1.5 1.9 � 0.7 35.2 � 5.9 12.2 � 0.9 1.8 � 0.825d 64.5 � 8.2 46.2 � 7.0 17.4 � 1.4 3.3 � 0.8 24.2 � 7.9 11.1 � 4.4 1.5 � 1.050d 64.1 � 5.2 50.5 � 6.9 12.1 � 2.2 3.7 � 0.6 26.2 � 2.1 9.6 � 4.0 1.6 � 0.3NOTE: Data are expressed as mean � SD of 30 mice of each group.aPercentage among CD4þ cells.bPercentage among MHC-IIþ cells.cP < 0.05 compared with NT group.

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-A

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19F

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-A

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19F

ITC

-A

100

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10

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1010

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3

101 102 103 104

Figure 4. Lung lymphocytes werequantified at different time points. Atdays 25 (25d) and 50 (50d) after removingthe local tumor, the lymphocytesubpopulations were measured andcompared with nontumor-injected mice(NT). A, a representative experimentshows B (CD3�CD19þ) and T(CD3þCD4þ and CD3þCD8þ)lymphocyte subpopulations. B, thegraphs depict the (mean � SD)percentages of CD3þ/CD19þ andCD4þ/CD8þ lymphocytes. �, P < 0.05.

Romero et al.





seventh group that received no treatment were maintained upto 24 months after local tumor removal and remained metas-tasis-free (Table 2).To summarize, we found a higher number of mice with

metastases and higher number of metastases in the lungs inCD8- or asialo GM1-depleted mice than in CD4-depleted mice.

The metastases remained in dormancy for 5 months andawoke when T or asialo GM1-positive cells were depleted.

After necropsy, all macroscopically visible spontaneouspulmonary metastases from immunodepleted mouse groupswere adapted to tissue culture and H-2 class I surface expres-sion was analyzed. All metastases were characterized by apositive expression of Kd and Ddmolecules and a total absenceof Ld molecule expression under baseline conditions, while allthree molecules were induced after IFN-g treatment (Fig. 5B).

DiscussionThis study describes for the first time a novel nontransgenic

mouse tumor model of permanent immunomediated meta-static dormancy. In this model, spontaneous metastases arecompletely controlled and maintained in a dormant state bythe wild-type mice immune system, with no application of anyanticancer treatment. The local primary tumors grew rapidly,and the mice remained metastasis-free after tumor removal atthe end of the assays (3–8 months) and for more than 24months. Interestingly, immunodepletion of host T or asialoGM1-positive cells promoted the awakening of dormant dis-seminated spontaneous metastatic cells, which invaded thelungs of the mice. We highlight that the spontaneous metas-tases in our model are dormant, remaining in latency through-out the life of the animals; furthermore, this dormant meta-static state is maintained by the murine immune systemthrough T and asialo GM1-positive cells, with no previousimmunization or treatment of the hosts. Thismetastatic tumor

Table 2. Number of metastases generated incontrol and depletion groups

Range

GroupsMetastasisincidence

Micro-PMs

Macro-PMs

Untreated 0/30 0 0Control Ig 0/30 0 0Anti-CD4þCD8 30/30 4–62 0Anti-CD8 30/30 3–17 (50%) 0

>100 (50%) 0

Anti–asialo-GM1 26/30 (87%) 2–35 (62%)�

1 (30%)

0 (32%)>100 (25%) 0

Anti-CD4 7/30 (23%) 1–2 (23%) 2 (13%)0 (10%)

Abbreviations: micro-PMs, pulmonary micrometastases;macro-PMs, pulmonary macrometastases.

Negative

A

B

H-2 D H-2 LH-2 K

Basal IFN-γ

100

0 060

8040

2010

012

0

2040

6080

100

FL1-Height FL1-Height

Cou

nts

Cou

nts

101 102 103 104 100 101 102 103 104

Figure 5. Spontaneous metastasis assays in immunocompetent and immunodepleted BALB/c mice. A, time schedules of the immunodepletion protocols inspontaneous metastasis assays. The mice were injected with GR9-B11 cells on day 0. The local tumors were removed between days 20 and 22. Oneof the groupswas euthanized at 151days. Five groups ofmicewere treated twice aweek for 90 daysby intraperitoneal injection ofmAbs (anti-CD4þ anti-CD8or anti-CD4 or anti-CD8 or anti-asialo GM1) or Igs (control Ig), beginning on day 152, whereas another group remained untreated. All micewere euthanized onday 242, except the group (vii) control. B, H-2 class I surface expression ofmetastases awoken fromdormancy. H-2K (gray line), H-2 D (dotted line), andH-2 L(black line). Allmacrometastases showedpositive expressionofH-2KandDmolecules alone under baseline conditions, and all threemoleculeswere inducedafter IFN-g treatment. A representative example of three independent experiments is depicted.






murine model faithfully resembles the progression of humancancers in which a long latency period with minimal residualdisease can follow the primary tumor resection, with themetastatic cells clinically manifesting years or even decadeslater (31–33). TheGR9-B11metastatic tumormodel is a uniqueand reproducible experimental system for detailed analysis ofthe phenomenon of immunomediated metastatic dormancy.

Various authors recently demonstrated that the immuneresponse can delay cancer progression. Koebel and colleaguesstudied aMCA-induced tumormousemodel and reported thatthe immune response kept occult primary tumors in a state ofequilibrium and that the cancer progressed after the jointdepletion of CD4/CD8 cells and the depletion of IFN-g or IL-12(34). Unlike the present study, they investigated dormantpreneoplastic or neoplastic tumor cells but not metastatictumor cells. Another group found that the IFN-g produced bylung NK cells played a major role against the development ofpulmonary metastases (35), although these were experimentaland not spontaneousmetastases and theywere not dormant. Itwas also reported that cancer progression was accelerated bydepletion of CD8þ T cells in a RET.ADD melanoma transgenicmice model, although the nondepleted mice also eventuallydeveloped cancer (36). In our assays, the mice remainedmetastasis-free until euthanized.

In the GR9 fibrosarcoma mouse model, MHC-I expressionon different tumor clones has been indirectly correlated within vivo TIC (28). In the present study, tumor cells grewrapidly immediately after the injection of GR9-B11, possiblyescaping from the immune system due to their altered MHC-I phenotype with low antigen presentation capacity. Aninverse behavior was reported in spontaneous metastasisassays, which showed a higher metastatic capacity in theMHC-positive GR9 tumor clones than in the MHC-negativeclones. A very high spontaneous metastatic capacity waspreviously reported for an MHC-I–positive clone of the GR9tumor model, GR9-A7, whose metastases were also MHC-I–positive (37). However, unlike findings for the GR9-B11clone, GR9-A7 tumor growth and metastatic disseminationproduced an immunosuppressive effect in the hosts. Resto-ration of the host immune response by immunotherapytreatments completely eradicated the spontaneous metas-tases and the animals remained metastasis-free (37). In thepresent study, immune stimulation was observed during theperiod of GR9-B11 primary tumor growth and disseminationof spontaneous metastatic cells, which was characterized byan increase in immune cells, especially T lymphocytes. Inboth cases, the spread of metastases was dependent on theimmune effect promoted by the primary tumor cells. Ourgroup also previously reported that GR9-B9, another tumorclone of GR9 primary fibrosarcoma, produced a largernumber of spontaneous pulmonary metastases in nu/nuBALB/c mice than in immunocompetent BALB/c mice(38, 39). These results and the present findings suggest thatimmunosurveillance plays a key role against metastaticprogression and that GR9-B11 tumor cells promote animmune response able to completely control disseminatedmetastatic cells and maintain them in a state of dormancy.This proposition is further supported by the finding that

spontaneous metastases were generated in nu/nu andimmunodepleted BALB/c mice injected with GR9-B11 tumorcells.

Baseline MHC-I surface expression was higher on allspontaneous metastases originated from the GR9-B11 tumorclone in immunodeficient and immunodepleted mice incomparison with GR9-B11 tumor cells. A similar phenome-non was previously reported for the GR9-B9 tumor clone, onwhich MHC-I expression is absent, finding that all metastasesin immunodeficient mice were MHC-I–positive under base-line conditions (38, 39). Hence, the MHC-I alterations werereversible (i.e., "soft lesions") in both clones (39, 40). Theseresults strongly suggest that MHC-I loss is not a requirementfor escape in the absence of a T-cell–mediated immuneresponse. Recent investigations in chemoresistance humanmodels of hormone-refractory prostate cancer (HRPC) iden-tified an HLA-I–negative cell subpopulation of the bulkpopulation of primary and metastatic prostate cancer tissue(41). These HLA-I–negative tumor cells exhibited resistanceto chemotherapy and their number correlated with the stageof the disease and its recurrence. In agreement with thepresent data, these HLA-I–negative tumor cells displayed ahigher TIC in immunodeficient murine hosts in vivo, with theprimary tumors again reproducing the initial HLA-I pheno-typic heterogeneity of the original tumor (41). In this context,relapse after adoptive cell transfer therapies in patients withmelanoma has been related to the reversible downregulationof antigen expression (42). In brief, MHC-I–positive andMHC-I–negative cells may exist in a dynamic and inter-changeable state of equilibrium that adapts in response tosignals from the microenvironment.

The depletion of T or asialo GM1-positive cells was sufficientto awaken disseminated spontaneous metastatic cells fromtheir dormant state. These results demonstrate the capacity ofT or asialo GM1-positive cells in wild-type mice to maintainspontaneousmicrometastases permanently occult in a state ofequilibrium. In BALB/cmice, NK cells and some otherminoritysubpopulations express asialo GM1. We hypothesize that NKcells did not exert a direct cytotoxic effect in our model,because no increase in NK cells was observed during thesystemic or local immune response generated by GR9-B11tumor cells. Furthermore, the depletion of CD8þ T lympho-cytes produced spontaneous metastases in 100% of immuno-competentmice, andGR9-B11–bearing nudemice developed aconsiderable number of spontaneous metastases, despite thelarge amount of NK cells in these hosts. It was previouslyreported that NK cells may facilitate the development of anantitumor protective CTL response independent of CD4þ Tlymphocytes (43–45). Another possibility is that a subpopula-tion of asialo GM1þ CD8þ T cells might be involved incontrolling dormant metastases in this model (46, 47). How-ever, other subpopulations of T cells should also be implicated,because the results found in CD8- versus asialo GM1-depletedtumor-bearing mice were different. In addition, we wouldhighlight that MHC-I molecules can act directly as tumorsuppressor genes, arresting cancer cell proliferation (48), andthe FHIT tumor suppressor gene is directly implicated inMHC-I cell surface expression (30). Taken together, these results

Romero et al.





suggest that the expression of MHC-I molecules on thesemetastatic cells may promote the dormant state via immuno-mediated and oncogenic suppression mechanisms. Futureinvestigations will be designed to clearly decipher the role ofMHC-I molecules in this dormant state and the molecularmechanisms that may be involved.In summary, we present a novel murine metastatic tumor

model in which the host immune response per se can fullycontrol spontaneous pulmonary metastases, maintainingthem in a state of dormancy. Dormant micrometastases wereawoken after the immunodepletion of T or asialo GM1-positivecells, revealing the major role of these immune cells in main-taining the metastases in a dormant state. This tumor modelresembles the metastatic dormancy observed in some humancancer patients. This preclinicalmetastatic tumormodel offersthe possibility of in-depth investigation of the intrinsic char-acteristics of the premetastatic niche (49) and of the mechan-ismsunderlyingmetastatic dormancy (50), offering newoppor-tunities for immunotherapeutic management of metastaticdisease. These data may help us understand how cancer mightbecome a chronic disease that persists in nonfatal form in aclinically healthy individual.

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

Authors' ContributionsConception and design: I. Romero, F. Garrido, A.M. Garcia-LoraDevelopment of methodology: I. Romero, C. Garrido, I. Algarra, A. Collado,A.M. Garcia-LoraAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): I. Romero, C. Garrido, I. Algarra, A. ColladoAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): I. Romero, I. Algarra, F. Garrido, A.M. Garcia-LoraWriting, review, and/or revision of the manuscript: I. Romero, F. Garrido,A.M. Garcia-LoraStudy supervision: F. Garrido, A.M. Garcia-Lora

AcknowledgmentsThe authors thank I. Linares, A.B. Rodriguez, and E. Arias for technical advice

and R. Davies for editorial assistance.

Grant SupportThis work was supported by grants cofinanced by FEDER funds (EU) from

the Instituto de Salud Carlos III (CP03/0111, PI12/02031, PI 08/1265, PI11/01022, RETIC RD 06/020 and RD09/0076/00165), Junta de Andalucía(Group CTS-143, and CTS-695, CTS-3952, CVI-4740 grants), and EuropeanCommunity (LSHC-CT-2004-503306, OJ 2004/c158, 18234). A.M. Garcia Lora.was supported by Miguel Servet Contract CP03/0111 and Contract I3 fromFPS and ISCIII, I. Romero by Rio-Hortega Contract CM12/00033 from ISCIII,and C. Garrido by FPU–MEC 1631.

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received July 24, 2013; revised January 7, 2014; accepted January 28, 2014;published OnlineFirst February 14, 2014.

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2014;74:1958-1968. Published OnlineFirst February 14, 2014.Cancer Res Irene Romero, Cristina Garrido, Ignacio Algarra, et al. DormancyT Lymphocytes Restrain Spontaneous Metastases in Permanent

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