immune-dependent and independent antitumor activity of...

Tumor and Stem Cell Biology

Immune-Dependent and Independent Antitumor Activity ofGM-CSF Aberrantly Expressed by Mouse and HumanColorectal Tumors

Rocio G. Urdinguio1, Agustin F. Fernandez1, Angela Moncada-Pazos2, Covadonga Huidobro1,Ramon M. Rodriguez1, Cecilia Ferrero1, Pablo Martinez-Camblor4, Alvaro J. Obaya3, Teresa Bernal5,Adolfo Parra-Blanco6, Luis Rodrigo6, Maria Santacana7, Xavier Matias-Guiu7, Beatriz Soldevilla8,Gemma Dominguez8, Felix Bonilla8, Santiago Cal2, Carlos Lopez-Otin2, and Mario F. Fraga1,9

AbstractGranulocyte-macrophage colony-stimulating factor (GM-CSF/CSF2) is a cytokine produced in the hematologic

compartment that may enhance antitumor immune responses, mainly by activation of dendritic cells. Here, weshow that more than one-third of human colorectal tumors exhibit aberrant DNA demethylation of the GM-CSFpromoter and overexpress the cytokine. Mouse engraftment experiments with autologous and homologous colontumors engineered to repress the ectopic secretion of GM-CSF revealed the tumor-secreted GM-CSF to have animmune-associated antitumor effect. Unexpectedly, an immune-independent antitumor effect was observed thatdepended on the ectopic expression ofGM-CSF receptor subunits by tumors. Cancer cells expressingGM-CSF andits receptor did not develop into tumors when autografted into immunocompetent mice. Similarly, 100% of thepatients with human colon tumors that overexpressed GM-CSF and its receptor subunits survived at least 5 yearsafter diagnosis. These data suggest that expression of GM-CSF and its receptor subunits by colon tumors maybe a useful marker for prognosis as well as for patient stratification in cancer immunotherapy. Cancer Res; 73(1);395–405. �2012 AACR.

IntroductionThe immune system can identify and destroy nascent

tumor cells, in a process termed cancer immunosurveillance,which plays an important role in the defense against cancer(1, 2). The ability of the immune system to fight tumor cellshas been used in the development of cancer immunotherapytreatments based on enhancing host antitumor responses(3). The cytokine granulocyte-macrophage colony stimulat-ing factor (GM-CSF, also known as CSF2) functions as a

hematologic cell growth factor, stimulating blood stem cellsto produce granulocytes and monocytes (4, 5). In addition,GM-CSF leads to protective immunity, mainly by stimulatingthe recruitment, maturation, and function of dendriticcells (6–8), the most potent antigen-presenting cell (9). Thiseffect of the cytokine on dendritic cells leads to the activa-tion of the immune system against specific antigens (10).Therefore, one approach to cancer immunotherapy is vac-cination with tumor-irradiated cells engineered to secreteGM-CSF (6, 7, 11–13). In this context, the action of GM-CSFon dendritic cells allows the immune system to be activatedagainst the specific antigens directly provided by the tumorcells (10). This strategy has, for instance, been used invaccination with irradiated GM-CSF–secreting melanomacells resulting in enhanced host responses through improvedtumor antigen presentation by recruited dendritic cells andmacrophages (6).

GM-CSF has been shown to be ectopically secreted by celllines derived from a variety of solid tumors (14, 15) but theimmune-independent effect of tumor-secreted GM-CSF isstill poorly understood. Contradictory results from differentgroups have shown that GM-CSF could either exert anantiproliferative effect (16, 17) or promote tumor growth(18–20). Furthermore, no reproducible influence on tumorgrowth rate has been reported (21, 22). GM-CSF has alsobeen reported to be secreted by some primary tumors(23–27) but, to the best of our knowledge, there are noreports to date showing the expression of GM-CSF by

Authors' Affiliations: 1Cancer Epigenetics Laboratory, HUCA, 2Departa-mento de Bioquímica y Biología Molecular, and 4Department of FunctionalBiology, Institute of Oncology of Asturias (IUOPA), Universidad de Oviedo,Oviedo, Spain; 3Unidad de Apoyo a la Investigaci�on CAIBER, OIB, Oviedo,Spain; 5Servicio de Hematología and 6Department of Gastroenterology,Hospital Universitario Central de Asturias (HUCA), Oviedo, Spain; 7Depart-ment of Pathology and Molecular Genetics and Research Laboratory,Hospital Universitari Arnau de Vilanova, University of Lleida, IRBLLEIDA,Lleida, Spain; 8Servicio deOncologíaM�edica, Hospital Universitario Puertade Hierro Majadahonda, Facultad de Medicina, Universidad Aut�onoma deMadrid,Madrid, Spain; 9Department of Immunology andOncology,Nation-al Center for Biotechnology, CNB-CSIC, Cantoblanco, Madrid, Spain

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Mario F. Fraga, Cancer Epigenetics Laboratory(IUOPA), HUCA, C/Dr. Emilio Rodriguez Vigil s/n, Bloque Polivalente A,4� planta, 33006Oviedo, Spain. Phone: 34-985-109-475; Fax: 34-985-109-475; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-12-0806

�2012 American Association for Cancer Research.

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primary colon tumor tissues nor its possible effects on theproliferation of this tumor type. Furthermore, the molecularmechanisms involved in the ectopic expression of GM-CSF bytumor cells are still largely unknown.Here,weprovide evidencefor the possible molecular mechanism involved and describethe antitumor effect of the process in colorectal cancer.

Materials and MethodsqRT-PCR

Total RNA was extracted from mouse colon cell lines,human colon cancer cell lines, and human samples usingTRIzol Reagent (Invitrogen) following the manufacturer'sprocedure. DNA was removed with DNase I treatment withDNA-free kit (Ambion/Applied Biosystems) and cDNA wasprepared using SuperScript II Reverse Transcriptase kit(Invitrogen) following the manufacturer's recommenda-tions. We ran PCR reactions in triplicate using Sybr GreenMaster Mix (Applied Biosystems) in a 7900HT Real-TimePCR System (Applied Biosystems). We conducted relativequantification of gene expression based on standard curvetransformations of Ct values. Results from each target genewere normalized against their respective ubiquitous house-keeping gene. Primer sequences are listed in SupplementaryTable S2.

Bisulfite pyrosequencingGenomic DNA isolation was conducted according to a

standard phenol-chloroform extraction protocol. Bisulfitemodification of DNA was conducted with the EZ DNA Meth-ylation-Gold kit (Zymo Research) following themanufacturer'sinstructions. After PCR amplification of the region of interestwith specific primers, pyrosequencing was conducted usingPyroMarkQ24 reagents, vacuumprepworkstation, equipment,and software (Qiagen). Primer sequences for murine Gm-CSfand human GM-CSF are shown in Supplementary Table S2.

Human cell lines and human samplesHuman colon cancer cell lines were cultured according to

American Type Culture Collection recommendations. Celllines were authenticated using short tandem repeat profilingof an extracted DNA sample using AmpF‘STR Identifiler forhigh resolution screening and intraspecies cross-contami-nation detection by Bio-Synthesis, Inc. Briefly, cell lines weregrown at 37�C in 5% CO2 in Dulbecco's Modified Eagle'sMedia supplemented with 10% FBS. Human cells weretreated with the demethylating agent 5-aza-20-deoxycytidine(AdC; Sigma) at various concentrations (2.5 and 5 mmol/L)for 72 hours. Healthy human- and primary tumor-colonsamples were obtained from the Hospital UniversitarioPuerta de Hierro (Madrid, Spain) and the Asturias TumorBank of the Institute of Oncology (Asturias, Spain). Tissuecollection and analyses were approved by the appropriateinstitutional review boards in accordance with national andEU guidelines.

Murine cell lines and shGm-CSf stable transfectionMouse colon cancer cell lines were kindly provided by Dr.

Ignacio Melero [Centro de Investigacion M�edica Aplicada

(CIMA), Navarra, Spain] and cultured at 37�C in 5% CO2 inRPMI supplemented with 10% FBS and 50 mmol/L b-mercap-toethanol. MC38 and CT26 colon cancer cell were stablytransfected with a HuSH 29-mer shRNA construct againstMusmusculus Csf2 in pGFP-V-RS vector or a HuSH 29-mer Non-Effective Scrambled pGFP-V-RS vector (OriGene) following themanufacturer's instructions. We selected different constructsof the 4 provided by the supplier to generate 2 independentclones from each cell line with Gm-CSf interference as well as ascramble.

In vitro growth experimentsCell viability was determined by MTT assay on cells stably

transfected with scramble and shGm-CSf vectors as describedby Mosmann and colleagues (28). Ten replicates per conditionand time point were assessed. Absorbance at 595 nm wasmeasured with an automated microtitre plate reader PowerWave WS (BioTek). Cell proliferation rate was established bycell counting on consecutive days in scramble and shGm-CSfcells. Cells were seeded in triplicates in 24-well plates at aconcentration of 1� 103 per well. Cells were collected daily for5 days and viable cells, as assessed by trypan blue staining, werecounted under a microscope in a hemocytometer. Colonyformation assay was conducted as described in Franken andcolleagues (29). Cells were then seeded at different densities(50, 100, and 200 cells) and after 2 weeks, stable colonies werefixed and stained with a mixture of 6.0% Glutaraldehyde and0.5% Crystal Violet in water. After washing and being left to airdry, colonies were counted. The Plating Efficiency was calcu-lated as a mean of the Plating Efficiency of the different celldilutions.

Tumor transplantation and in vivo treatment studiesFor tumor grafting, we subcutaneously (s.c.) injected 1 to 4�

105 tumor cells into each flank of recipient mice (Nude or NU/NU, C57BL/6 and BALB/c females, 6 to 8 week old, CharlesRiver). Anti-PD1 was purchased from BioXCell and adminis-tered by intraperitoneal injection of 250 mg. Tumor volumeswere measured twice or 3 times a week with calipers andcalculated using the following formula: tumor volume¼ 0.4�(length � width2). After sacrifice, tumors were excised andweighed.

ImmunohistochemistryTissue microarray (TMA) blocks were sectioned at a thick-

ness of 3 mm, dried for 1 hour at 65� before the pretreatmentprocedure: deparaffinization, rehydration, and epitope retriev-al in the Pre-Treatment Module, PT-LINK (DAKO) at 95�C for20 minutes in �50 Tris/EDTA buffer, pH 9. The antibodyagainst CD3 (clone M-20, sc-1127, Santa Cruz Biotechnology)was used to stain the sections, after blocking endogenousperoxidase. Following incubation, the reaction was visualizedwith the EnVision Detection Kit (DAKO) using diaminobenzi-dine chromogen as a substrate. Sections were counterstainedwith hematoxylin. Appropriate negative controls were alsotested. For immunofluorescence, TMA sections were pre-treated with sodium-citrate buffer pH 6 (5 minutes, 100�C).Slides were blocked with 10% donkey serum (2 hours) and

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Cancer Res; 73(1) January 1, 2013 Cancer Research396




incubated with the antibody against CSF2RB (LS-B6993, Life-span Biosciences) and active-beta-catenin (05-665, Millipore)overnight, 4�C. After rinsing with Dulbecco's phosphate-buff-ered saline, slides were incubated with Alexa 594 (A11072, LifeTechnologies) or Alexa 488-conjugated secondary antibody(A11017, Life Technologies). DNA was stained with 40,6-dia-midino-2-phenylidole and tissue sections imaged using a dig-ital camera connected to a Leica microscope DMRXA.

RNA in situ hybridizationThe experiments of RNA in situ hybridization were con-

ducted with the QuantiGene ViewRNA ISH Tissue Assay Kit(Affymetrix) and the QuantiGene ViewRNA TYPE1 # Probe Setfor HUMAN-CSF2-colony stimulating factor 2 (granulocyte-macrophage; Affymetrix) following themanufacturer's instruc-tions. Images were obtained using a confocalmicroscope LeicaTCS-SP2-AOBS.

Cytotoxicity assaysAfter mouse grafting, peripheral lymphocytes were isolated

from blood using Lympholyte-M (Tebu-bio). The protocol forthe isolation of tumor-infiltrating lymphocytes was adaptedfrom Radoja and colleagues (30). Cytotoxicity assays wereconducted to determine cell-mediated cytotoxicity against celllines injected in the mice flanks with CytoTox 96 Non-Radio-active Cytotoxicity Assay (Promega) following the manufac-turer's instructions.

ELISACulture medium aliquots were frozen in liquid nitrogen

immediately after extraction and kept at�80�C until analysis.ELISA was conducted with LEGEND MAX Mouse GM-CSFELISA kit (BioLegend) for mouse Gm-CSf detection andHuman GM-CSF ELISAPRO kit (Mabtech) for human GM-CSFdetection, following the instructions of the manufacturers.

Statistical analysesFor statistical comparisons we used Student's t test (paired

and unpaired). We analyzed colon tumor-free survival usingKaplan–Meier log-rank test. A P value of less than 0.05 wasconsidered statistically significant.

ResultsFrequent aberrant promoter demethylation andoverexpression of GM-CSF in human colorectal cancer

To characterize GM-CSF expression in colon cancer, weanalyzed GM-CSF mRNA levels in 124 paired samples ofprimary colon tumors and their healthy counterparts, andincluded peripheral blood mononuclear cells (PBMC) as apositive control. We found that samples from healthy colonepithelium showed very low, or absent, GM-CSF expression,whereas many tumors expressed GM-CSF above PBMClevels (Fig. 1A). GM-CSF overexpression by tumors (consid-ered as more than 2-fold increase over the levels from theircorresponding healthy counterparts) was observed in 37.9%(47 of 124) of patients. We also studied 10 colon cancer celllines (CCL) and found that 6 of them (Caco2, HCT15,HCT116, HT29, RKO, and SW48) did not express GM-CSF,whereas 4 (SW480, DLD1, COLO205, and Co115) showedexpression values similar to, or above, PBMC levels (Fig. 1Aand Supplementary Fig. S1A). Our results show that GM-CSFis frequently overexpressed in primary colon tumors as wellas in colon cancer cell lines.

A large number of molecular alterations in cancer occur atthe epigenetic level (31). Cancer cells show aberrant gain orloss of DNA methylation at the promoter region of manygenes (32); alterations that are directly involved in tumor-igenesis as they can induce either repression of tumorsuppressor genes or activation of oncogenes (32, 33). Tostudy the possible aberrant epigenetic regulation of GM-CSFin colon cancer, we determined the methylation status of

Figure 1. GM-CSF overexpression and DNAmethylation levels at theGM-CSF promoter in human primary colon cancer. A, GM-CSFmRNA expression levelswere determined by quantitative real-time PCR in 124 paired samples of primary colon tumors (Tumor) and their healthy counterparts (HC, healthy colon).Results from PBMCs were included as a positive control of GM-CSF expression. Human GM-CSF mRNA levels are expressed as a ratio in relation toglyceraldehyde-3-phosphate dehydrogenase mRNA levels. B, bisulfite pyrosequencing results from 74 colon primary tumors (Tumor) and their 74paired HC tissues. A schematic representation of the studied region and themethylation values from 2 representative coupled samples are shown on the left.Black depictsmethylatedCpG andwhite indicates unmethylatedCpG. On the right, a boxplot shows themethylation results of the 74 paired human samples.A significant difference in methylation values of tumor samples compared with their healthy counterparts was found (P < 0.001, paired t test).

Antitumor Activity of GM-CSF in Colorectal Tumors

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GM-CSF promoter in 10 CCLs. This analysis showed that GM-CSF promoter was densely methylated in the 6 CCLs (Caco2,HCT15, HCT116, HT29, RKO, and SW48) that had very lowlevels of GM-CSF expression, whilst it was almost unmethy-lated in the 4 CCLs (SW480, DLD1, COLO205, and Co115)that presented high levels of GM-CSF expression (Supple-mentary Fig. S1A and S1B). With the aim of clarifyingwhether GM-CSF promoter demethylation is also a frequentin vivo event, we analyzed 74 primary colon adenocarcino-mas and their 74 paired healthy colon tissues. Healthytissues presented an average methylation level of 83.5%,whereas the mean methylation level of the tumors was61.5% (P < 0.001; Fig. 1B). We detected hypomethylation (atleast a 20% change) at the GM-CSF promoter in 38 of the 74tumors (51.4%), thereby confirming this as a common eventin vivo (Fig. 1B). To evaluate whether demethylation of theGM-CSF promoter is related to a genome-wide demethyla-tion process, something frequently observed in tumor cells(34), we analyzed the methylation levels of the LINE-1sequence by bisulfite pyrosequencing (35). A significantassociation (P < 0.001) between LINE-1 and GM-CSF hypo-methylation was found (Supplementary Fig. S2), therebyconsolidating the notion that DNA hypomethylation playsan important role in the aberrant regulation of GM-CSF incolon cancer.

Concurrent GM-CSF overexpression (Fig. 1A) and GM-CSFpromoter DNA demethylation (Fig. 1B, right) suggested thatDNA methylation plays an important role in the regulationof GM-CSF expression. To get a deeper insight into thisissue, we analyzed the consequences of in vitro demethy-lation. Firstly, we measured GM-CSF promoter methylationand GM-CSF expression levels in the HCT116 cell linecompared with a double DNMT1 and DNMT3B knock-outcell line (DKO) derived from HCT116. We observed genet-ically induced GM-CSF promoter hypomethylation andconsistent GM-CSF overexpression in the DKO cell line(Supplementary Fig. S1C), supporting the existence of arelationship between GM-CSF promoter methylation and itsexpression. Furthermore, we analyzed GM-CSF mRNA levelsin 4 colon cancer cell lines incubated with 2 differentconcentrations of the demethylating drug AdC. Treatmentwith this drug resulted in marked reactivation of GM-CSFexpression in a dose-dependent manner in cell lines dis-playing high levels of GM-CSF promoter methylation (HCT15and RKO), but not in those with low levels of methylation(Colo205 and Co115; Supplementary Fig. S1D). These datasupport the notion that aberrant GM-CSF promoter deme-thylation plays an important role in GM-CSF overexpressionin colon cancer.

To verify that GM-CSF promoter methylation and mRNAexpression levels are related to protein levels and to determinewhether ectopically overexpressed GM-CSF was secreted bycells, we measured GM-CSF protein levels in the culture mediaof 2 colon cancer cell lines (HCT116 and DLD1) by ELISA. Weconfirmed that GM-CSF protein levels are directly associatedwith mRNA levels and, inversely related to GM-CSF promotermethylation in both colon cancer cell lines (SupplementaryFig. S1E). This suggests that promoter demethylation-related

GM-CSF overexpression leads to secretion of the cytokine bycolon cancer cells.

Immune-dependent antitumor effect of GM-CSFectopically secreted by colon tumors

Given the antitumor effect of GM-CSFwhen administered asa vaccine in an immunotherapy context (6, 11), we testedwhether the overexpression of this cytokine by colon cancercells couldmimic this antitumor action. To evaluate a possibleimmune-dependent effect of GM-CSF in colon cancer, weknocked down Gm-CSf expression by stable RNA interferencein the murine Gm-CSf–expressing colon cancer cell lines CT26and MC38, which are poorly methylated at the Gm-CSf pro-moter (Supplementary Fig. S3A, S3B, S3C, and S4A). To discarda possible immune-independent effect of Gm-CSf, we analyzedthe expression of the Gm-CSf plasmatic membrane receptorsubunits Csf2ra and Csf2rb in these samemurine colon cancercell lines. MC38-derived clones expressed Csf2ra, whereasCT26-derived clones did not. All clones expressed similar lowlevels of Csf2rb (Supplementary Fig. S3D and S4B). Then, we s.c.injected CT26 cells with knocked-down Gm-CSf expression(shGm-CSf) and an isogenic scramble-transfected clone(scramble) that overexpressed Gm-CSf into a cohort of autol-ogous immune-competent BALB/c mice. As CT26 clones donot express Csf2ra (Supplementary Fig. S3D and S4B), thismodel allows the comparison of 2 colon cell lines, differing intheir Gm-CSf expression, when they encounter an immune-competent in vivo environment, excluding immune-indepen-dent effects. Abolishing Gm-CSf expression in CT26 cellsresulted in a marked increase in tumor formation in vivo (Fig.2A and Supplementary Fig. S4C). At the time the mice weresacrificed, the Gm-CSf expressing CT26 tumors had, on aver-age, half the volume and weight of their non-Gm-CSf –expres-sing counterparts (Fig. 2A and Supplementary Fig. S4C). Thisled us to investigate the possible role of the immune systemreasoning that it could be responsible for the observed anti-tumor effect of Gm-CSf (7, 36, 37). We studied T lymphocytetumor infiltration conducting immunohistochemistry oftumors resected from BALB/c mice. On average, we foundmore T infiltration in Gm-CSf expressing tumors than in theirsilenced counterparts (Fig. 2B). In addition, we measured thespecific lysis conducted by lymphocytes isolated from BALB/cmice blood and found that lymphocytes from mice carryingGm-CSf expressing tumors provoked a higher percentage ofcell lysis than those frommicewith nonexpressing tumors (Fig.2C). We further addressed this issue using tumor-infiltratinglymphocytes extracted from tumors generated by scramble oran additional shGm-CSf clone of CT26 cell line in BALB/cmice.As shown in Supplementary Fig. S4D, we obtained theminimalrequired amount of lymphocytes for cytotoxicity assays in only4 cases. While 4 cases are not sufficient for statistical analysis,we observed a tendency toward higher cytotoxicity specificlysis from lymphocytes extracted from Gm-CSf–expressingtumors (Supplementary Fig. S4E): results that we consider toprovide evidence for the role of Gm-CSf in activating theimmune system to limit tumor growth. Thus, ectopic expres-sion of this cytokine by tumors might have an antitumor effectmediated by the immune system.

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Immune-independent antitumor effect of GM-CSFectopically secreted by colon tumorsWe observed in vitro that scramble and shGm-CSf clones

from CT26 and MC38 cell lines showed variable growthpatterns (Fig. 3A and Supplementary Fig. S5). ReducedGm-CSf expression was associated with increased cell via-bility, cell growth, and colony formation ability in MC38 cellsbut its effects were not significant in CT26 cells (Fig. 3A andSupplementary Fig. S5). This observation led us to considerthat, in addition to the immune-dependent effects describedabove, Gm-CSf expression may exert an immune-indepen-dent effect on tumor growth (16–20). This possibility wassupported by the direct relationship between growth effectsand the expression of Gm-CSf receptor subunits (38): onlywhen both Gm-CSf receptor subunits were present (in

MC38) could we detect a decrease in cell growth rateattributable to Gm-CSf (Fig. 3A and Supplementary Fig.S5). This immune-independent effect was confirmed in vivoby the s.c. injection of a scramble-transfected Gm-CSf–expressing MC38 clone and a Gm-CSF–silenced MC38 cloneinto immunodeficient nude mice, thereby allowing us toavoid interference of Gm-CSf effects on the immune system.Gm-CSf expression in scramble MC38 cells resulted in asignificant reduction in tumor formation in vivo; at the timeof sacrifice, Gm-CSf expressing MC38 tumors had, on aver-age, 20% the volume and 20% the weight of those lackingGm-CSf expression (Fig. 3B). Thus, our data suggest that theimmune-independent antitumor action of tumoral Gm-CSfdepends on the ectopic expression of Gm-CSf receptors bythe tumor.

Figure 2. Immune-dependent antitumor effects of Gm-CSf on tumor growth in vivo. A, diagram of the in vivo model studied for the analysis of the immune-dependent effects ofGm-CSf showing thatBALB/cmice (n¼12)were s.c. injectedwith scramble or shGm-CSfCT26 cells in both flanks.Macroscopic tumorswere observed and regularly measured for 24 days after injection. Evolution of tumor growth measured as tumor volume and a graph with endpointtumor weights are shown. At the time the mice were sacrificed, scramble CT26 tumors (blue line and blue circles) had significantly higher volume and weightthan CT26 tumors lacking Gm-CSf expression (red line and red triangles). Photographs of 1 representative mouse from each analyzed group areincluded. B, IHC of tumors from immune-dependent experiment with BALB/c mice. Two representative images obtained from IHC are shown (anti-CD3þ

staining appears in brown). The percentage of CD3þ staining was higher in tumors originated from Gm-CSf expressing scramble CT26 cells as shownin the graph on the right. C, percentage of specific lysis carried out by lymphocyte isolated from blood from immune-dependent experiment BALB/c mice(n ¼ 7) when confronting the respective CT26 clones they had been primed for. Lymphocytes extracted from mice with scramble CT26 tumors causedhigher specific lysis that reached significance at lower effector:target ratios. Results are expressed as the mean � SEM. �, P < 0.05; ��, P < 0.01;��, P < 0.001 (Student's t test).






Synergic immune-dependent and immune-independentantitumor effect of tumoral GM-CSF

We studied the combinatory effect of Gm-CSf and itsreceptor subunits' expression using Gm-CSf–expressing andsilenced MC38 clones injected into their autologous immune-competent C57BL/6 mice. In this model, where both immune-dependent and immune-independent effects coexist, we foundthat the MC38 scramble clone was detectable only for the first10 days before disappearing completely (Fig. 4A and Supple-mentary Fig. S6). This effect did not occur on the wild-type

MC38 clone (which does not express Csf2ra; SupplementaryFig. S7), which supports the notion that the immune-indepen-dent effect is crucial for the complete suppression of tumorgrowth in our mouse model, where the combination ofimmune-dependent and immune-independent effects ofGm-CSf leads to the complete loss of tumor formationcapability.

To determine whether our findings in mice would also holdfor human clinical samples, we analyzedGM-CSF, CSF2RA, andCSF2RB expression in a set of 124 colorectal cancer patients on

Figure 3. Immune-independent antitumor effects of Gm-CSf on tumor growth in vitro and in vivo. A, characterization of in vitro growth of MC38 and CT26transfected cells. Cell viability was determined by MTT assay on scramble- and shGm-CSf–stably transfected cells. Ten replicates were assessed per timepoint. Cell proliferation rate was established by taking daily cell counts of scramble and shGm-CSf cells. Three independent replicates were conducted foreach timepoint. Colony formation assaywas conducted seeding cells at different densities (50, 100, and 200 cells). Results are represented in the bar diagramas the mean colony-formation units (CFU)� SEM of 3 independent replicates for each density. Scramble-transfected cell lines are shown in blue and shGm-CSf–transfected cell lines in red. MC38 derived clones are represented by continuous lines and CT26 derived clones by discontinuous lines. Gm-CSfexpression only caused significant differences in cell proliferation and CFU in MC38 derived cells. B, diagram of the in vivomodel studied for the analysis ofimmune-independent effects showing that nudemice (n¼12)were s.c. injectedwith scramble (on left flank, represented in blue) and shGm-CSf (on right flank,represented in red) MC38 cells. Evolution of tumor growth measured as tumor volume and a graph depicting endpoint tumor weights are also shown. TheMC38 scramble cell line produced significantly lower tumor volume andweight in nudemice. A photograph of 1 representativemouse from this experiment isincluded. Results are expressed as the mean � SEM. �, P < 0.05; ��, P < 0.01; ��, P < 0.001 (Student's t test).

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whom a long clinical follow-up was available. GM-CSF over-expression (more than 2-fold) was observed in 37.9% (47 of 124)of patients. CSF2RA and CSF2RB were overexpressed (morethan 2-fold) in 22.6% (28 of 124) and 27.2% (34 of 124) ofpatients, respectively. Demographic and clinical characteris-tics of the patients included in our study are shown inSupplementary Table S1.To test whether GM-CSF and its receptor were expressed by

primary colon cancer cells, we conducted RNA in situ hybrid-ization of GM-CSF as well as the immunofluorescence detec-tion of its receptor. As shown in Supplementary Fig. S8, weconfirmed that cells from tumor colon epithelia aberrantlyexpress thesemolecules. As expected, RNA in situhybridizationof GM-CSF was positively detected in tonsil and undetectablein muscle tissue (Supplementary Fig. S8A). GM-CSF receptorwas located in the membrane of colon tumor epithelial cells,and colocalized with beta-catenin staining (which specificallymarked epithelial cells, but not stroma cells or infiltratedlymphocytes; Supplementary Fig. S8B).Concurrent overexpression of GM-CSF, CSF2RA, and

CSF2RB, which was observed in 6.5% (8 of 124) of patients,was strongly associated with an increase in overall survivalrates (Fig. 4B). All the patients with tumors overexpressing, the3 genes survived at least 5 years from diagnosis. This effect was

not observed when comparing patients that overexpressedGM-CSF without concurrent changes in receptor subunitlevels, or in patients that overexpressed both receptor subunitswithout changes in GM-CSF expression (Supplementary Fig.S9A and S9B), which sustains the notion that ectopic expres-sion of GM-CSF and its receptor subunits by human colorectaltumors has a strong antitumor effect. Our data show that in100% of cases where the 3 genes were overexpressed overallsurvival was dramatically increased. We would thus proposethat GM-CSF in combination with its receptor subunits shouldbe considered a valuablemarker for diagnosis and prognosis inclinical studies that may prove helpful in the stratification ofpatients. Furthermore, demethylation-associated GM-CSFoverexpression accompanied by CSF2RA and CSF2RB over-expression is a possible indicator of outcome in colorectalcancer patients.

Anti-PD-1 adjuvant therapy improves the antitumoreffects of tumoral GM-CSF

Anti-PD-1 is an antibody raised against Programmed death 1(PD-1; ref. 39), that enhances T cell activity in chronic pathol-ogies such as cancer (7, 40, 41). The combination of PD-1blockade with GM-CSF–secreting tumor cell immunotherapyhas been recently shown to significantly improve antitumor

Figure 4. Combination of immune-dependent and immune-independent antitumor effects invivo. A, diagram of the in vivo modelstudied for the analysis of thecombination of immune-dependentand immune-independent effectsshowing that C57BL/6 mice (n ¼ 8)were s.c. injectedwith scramble (blueline) or shGm-CSf (red line) MC38cells in both flanks. Evolution oftumor growth measured as tumorvolume is shown. The MC38scramble clone was only detectablefor the first 10 days, disappearingcompletely after that. Photographsof 1 representative mouse from eachanalyzed group are included. Resultsare expressed as the mean � SEM.��, P < 0.001 (Student's t test). B,Kaplan–Meier study of overallsurvival from a cohort of 124 patientsin which GM-CSF, CSF2RA, andCSF2RB expression levels wereanalyzed in both healthy colontissues and colon cancer samples.Expression of the 3 genes wasindependent of patient sex (P ¼0.828, Mann–Whitney U test), andthe age of onset (P ¼ 0.957, Mann–Whitney U test). The group ofpatients with overexpression of the 3genes in the tumor sample (greenline) shows a 100%overall survival at5-year follow up (P ¼ 0.021, Kaplan–Meier log-rank test).






responses (41). To determine whether anti-PD-1 also potenti-ates the antitumor effect of Gm-CSf ectopically secreted bytumor cells, we injected Gm-CSf–expressing scramble andshGm-CSf CT26 cells into their autologous BALB/c mice andadministrated PBS or anti-PD-1 treatment. At the moment ofsacrifice, we found a higher percentage of reduced tumorgrowth in mice with Gm-CSf–expressing tumors when theywere treated with anti-PD-1 (Fig. 5). Furthermore, we found a25% remission of tumors inmice developing Gm-CSF–silencedCT26 tumorswhen theywere treatedwith anti-PD-1, whereas a50% remission or reduction of tumor growth progression wasobserved when the anti-PD-1 treatment was administered tomice developing Gm-CSf–secreting CT26 tumors (Fig. 5).

DiscussionThe cytokine GM-CSF was described because of its ability as

a colony-stimulating factor to promote the formation of gran-ulocytes and monocytes (4). However, GM-CSF also influencesdendritic cells and this property is currently being used invarious immunotherapy strategies to stimulate the antitumoraction of the immune system (12). Importantly, the first USFood and Drug Administration approval in history for atherapeutic cancer vaccine was granted to Sipuleucel-T (Pro-venge; Dendreon, Inc.) for castration-resistant prostate cancer.Sipuleucel-T is an autologous dendritic cell-based vaccine inwhich cells are loadedwith a fusion protein including GM-CSF.Reinfusion of such activated cells into the patient showed a4-month benefit relative to the control group in randomizedphase III clinical trials (12, 42). Another strategy, calledOncoVEXGM-CSF (BioVex), involves the administration ofGM-CSF through the second generation of oncolitic Herpessimplex type 1 virus encoding human GM-CSF. The virus isgenetically reprogrammed to attack cancerous cells. This

vaccine is being studied in a phase III trial for patients withadvanced melanoma because of the promising results fromprevious trials (43). In addition, the GVAX (Cell Genesys)approach in immunotherapy involves the administration ofirradiated tumor cells that have been engineered to secreteGM-CSF and, in this manner, activate dendritic cells and theimmune systemwith the antigens provided by thewhole tumorcells. A significant body of preclinical data supported itsantitumor efficacy, especially in combination with agents suchas anti-CTLA-4, and anti-PD-1 (41, 44). However, phase IIIclinical trials of GVAX in patients with prostate cancer did notproduce significant results (12). GM-CSF is a valuable tool incancer immunotherapy but there are still several unansweredquestions that need to be addressed before the complexity ofthe results from clinical trials can be explained.

Herein, we show that aberrant DNA demethylation of GM-CSF can lead to its ectopic secretion by colorectal tumors.However, the natural role of DNA methylation at the GM-CSFpromoter is still largely unknown. According to previous DNAmethylation data fromHuman InfiniumMethylation Array 27k(Illumina; ref. 45), GM-CSF promoter is methylated in humanstem cells and human healthy primary tissues but not in TCD4þ and T CD8þ lymphocytes, that is, it follows a tissue-specific pattern. Furthermore, comparing this methylationdata with available gene expression data from Amazonia"Human body index" series (46), which comprises humantranscriptome list annotations, we see that only the cells withGM-CSF promoter DNA demethylation (T CD4þ and CD8þ

lymphocytes) are able to express this cytokine after activation(Supplementary Fig. S10). Therefore, GM-CSF expression couldbe under a double regulatory mechanism with a first level ofregulation in which loss of DNA methylation would allowfurther gene expression after cell activation. Thus, GM-CSF

Figure5. Anti-PD-1 treatment of engrafted tumors in vivo. Diagramof the in vivomodel studied for the analysis of anti-PD-1 effect on tumors that differed in theirGm-CSf expression showing that BALB/cmice (n¼ 16) were s.c. injectedwith scramble or shGm-CSf CT26 cells in both flanks and treated on days 3, 6, 9, 12,and 24 with intraperitoneal injection of PBS or anti-PD-1. At the time of sacrifice, we found a higher percentage of reduction of tumor growth in micethat hadGm-CSf–expressing tumors andwere treatedwith anti-PD-1. Furthermore, therewas a 25% remission of tumors inmice treatedwith anti-PD-1whenthe tumor did not express Gm-CSf (red bars), whereas there was a 50% remission, or growth arrest, in mice treated with anti-PD-1 that carried Gm-CSf–expressing scramble cells (blue bars). Photographs of 1 representative mouse from each analyzed group are also included.

Urdinguio et al.





promoter demethylation might be important in priming thisspecific hematopoietic gene for activation/expression inresponse to external stimuli (45). This natural mechanism ofregulation could suffer aberrant changes in colorectal cancersleading to its ectopic secretion by the tumor cells. Furtherstudies are necessary to decipher the role of DNA methylationin the natural regulation of GM-CSF and the molecularmechanisms that lead to its loss in colon cancer cells.Our data indicate that tumor-secreted GM-CSF can induce

immune-dependent antitumor responses but that it also has astrong direct antitumor action when the tumors express GM-CSF receptors. Although the immune-dependent antitumoreffect of GM-CSF has been known for some time, hence its usein immunotherapy (12, 47), this is the first time that GM-CSFectopically secreted by tumor cells is shown to have immune-dependent antitumor effects. This observation might beimportant for the clinical use of GM-CSF vaccines, and furtherstudies should determine whether the effects of these vaccinesare affected by tumor-secretedGM-CSF. On the other hand, theimmune-independent effect of tumor-secreted GM-CSF is stillpoorly understood. Some studies have found it to exert anantiproliferative effect (16, 17), whereas others have shown it topromote growth (18–20) and yet others found it to have noreproducible influence on growth rate (21, 22). Two mainpoints may explain, to a certain extent, these contradictoryresults: firstly, the dose of GM-CSF usedmaywell be relevant tothe evaluation of its effects at a physiologically relevant leveland, secondly, the presence of GM-CSF receptor should also beascertained to ensure that the studied cells do in fact have themeans to signal GM-CSF presence. Our study goes further inthe evaluation of immune-independent effects of GM-CSF aswe investigate the effects in vivo and find that tumors withconcurrent expression of Gm-CSf and its receptor do not growwhen injected in their autologous immunocompetent mice.Furthermore, human colorectal tumors with concurrent over-expression lead to a very good prognosis and a 100% 5-yearoverall survival rate. These findings support the notion of thestrong role of the GM-CSF receptor in the immune-indepen-dent action of GM-CSF on tumor growth and indicate theimportance of verifying the expression of this receptor bytumor cells when selecting a strategy for the treatment ofcolorectal cancer. On the other hand, a previous report on skincancer (48) showed the tumorigenic role of GM-CSF when thiscytokine is combined with G-CSF. Together, these resultsindicate that the role of GM-CSF in cancer is complex and itstumorigenic effect might depend on the tumor context.Unexpectedly, our data show that promoter demethylation-

associated expression of GM-CSF in colon cancer cells impairstumorigenesis suggesting that not all the epigenetic alterationsoccurring in a tumor necessarily favor tumorigenesis. Thispossibility is in line with reports showing that, in many genepromoters, aberrant de novo hypermethylation is not respon-sible for gene repression as these genes are already repressed inhealthy tissues (49). Collectively, these data imply that anumber of DNA methylation alterations in cancer are notsubject to growth selection (49), which is in consonance withthe differentiation between "driver" and "passenger" modifica-tions (previously referred to as mutations, but nowadays

acknowledged to also include epigenetic alterations) in thegenome of cancer cells during the tumorogenic process (50,51). Even though driver modifications are considered causa-tive in the development of a tumor, other changes that couldbe considered passenger modifications, that is, those notdirectly related with the tumor development, may becomecrucial for tumor progression and be considered driverchanges as long as they give tumor advantage against antitu-mor therapies.

Our results on human primary tumors may have clinicalimplications, as they suggest that expression ofGM-CSF and itsreceptor subunits are indicators of good prognosis in colorec-tal cancer. In addition, tumoral expression of the GM-CSFreceptor could also be useful for patient stratification in cancerimmunotherapy (i.e., tumors expressing GM-CSF receptormaybe better candidates to receive GM-CSF vaccination). Thestrong immune-independent antitumor activity of GM-CSFreported in this study suggests that future clinical trials withGM-CSF–secreting vaccines should also take into account theexpression of the GM-CSF receptor by tumors. Moreover,the results obtained in previous Phase III clinical trials withthese vaccines (37, 52) should be reevaluated taking intoaccount the tumor expression of GM-CSF receptor subunits.In addition, our results indicate that anti-PD-1 treatment mayenhance the antitumor effect of Gm-CSf ectopically secreted bytumors. Further studies are necessary to determine whetherpatients with tumors expressingGM-CSF are better candidatesto receive anti-PD-1 or other enhancers of the clinical activityof GM-CSF (53).

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

Authors' ContributionsConception and design: R.G. Urdinguio, A.F. Fernandez, A.J. Obaya, L. Rodrigo,C. Lopez-Otin, M.F. FragaDevelopment of methodology: R.G. Urdinguio, A.F. Fernandez, A. Moncada-Pazos, C. Huidobro, R.M. Rodriguez, P. Martinez-Camblor, M. Santacana, X.Matias-Guiu, M.F. FragaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): R.G. Urdinguio, A.F. Fernandez, C. Huidobro, T.Bernal, A. Parra-Blanco, L. Rodrigo, B. Soldevilla, G. Dominguez, S. Cal, M.F. FragaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): R.G. Urdinguio, A.F. Fernandez, A. Moncada-Pazos,C. Ferrero, P. Martinez-Camblor, M. Santacana, X. Matias-Guiu, B. Soldevilla, G.Dominguez, M.F. FragaWriting, review, and/or revision of the manuscript: R.G. Urdinguio, A.F.Fernandez, A. Moncada-Pazos, L. Rodrigo, F. Bonilla, S. Cal, C. Lopez-Otin, M.F.FragaAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): R.G. Urdinguio, A.F. Fernandez, M.Santacana, X. Matias-Guiu, M.F. FragaStudy supervision: R.G. Urdinguio, A.F. Fernandez, A.J. Obaya, L. Rodrigo, C.Lopez-Otin, M.F. Fraga

AcknowledgmentsThe authors thank Dr. IgnacioMelero, Juan Dubrot, and Asis Palazon for their

advice with mouse cell lines culture and the staff of the Animal Facilities at theUniversity of Oviedo for technical and animal care assistance.

Grant SupportThis work has been financially supported by IUOPA (R.G. Urdinguio, A.F.

Fernandez, and R.M. Rodriguez); Fundacion Cientifica de la aecc (R.G. Urdin-guio); FIS (FI07/00380 to C. Huidobro); the SpanishMinistry of Health (PI061267;PS09/024549 to M.F. F Fraga); the FIS/FEDER (PI11/01728 to A.F. Fernandez);the ISCIII (CP11/00131 to A.F. Fernandez); the Spanish National Research






Council (CSIC; 200820I172 to M.F. Fraga); and the Community of Asturias(FYCIT IB09-106 to M.F. Fraga). The IUOPA is supported by the Obra SocialCajastur, Spain.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked

advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate thisfact.

Received March 2, 2012; revised September 28, 2012; accepted September 28,2012; published OnlineFirst October 29, 2012.

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2013;73:395-405. Published OnlineFirst October 29, 2012.Cancer Res Rocio G. Urdinguio, Agustin F. Fernandez, Angela Moncada-Pazos, et al. Aberrantly Expressed by Mouse and Human Colorectal TumorsImmune-Dependent and Independent Antitumor Activity of GM-CSF

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