adverse immunoregulatory effects of 5fu and cpt11 ...medical center ein kerem (jerusalem, israel)...

Microenvironment and Immunology

Adverse Immunoregulatory Effects of 5FU and CPT11Chemotherapy on Myeloid-Derived Suppressor Cells andColorectal Cancer Outcomes

Julia Kanterman1, Moshe Sade-Feldman1, Moshe Biton1, Eliran Ish-Shalom1, Audrey Lasry1,Aviya Goldshtein1, Ayala Hubert2, and Michal Baniyash1

AbstractColorectal cancer is associated with chronic inflammation and immunosuppression mediated by myeloid-

derived suppressor cells (MDSC). Although chemotherapy reduces tumor burden at early stages, it tends to havelimited effect on a progressive disease, possibly due to adverse effects on the immune system in dictating diseaseoutcome. Here, we show that patients with advanced colorectal cancer display enhanced MDSC levels andreduced CD247 expression and that some conventional colorectal cancer chemotherapy supports the immu-nosuppressive tumor microenvironment. A FOLFOX combined therapy reduced immunosuppression, whereas aFOLFIRI combined therapy enhanced immunosuppression. Mechanistic studies in a colorectal cancer mousemodel revealed that FOLFIRI-like therapy including the drugs CPT11 and 5-fluorouracil (5FU) damaged hostimmunocompetence in a manner that limits treatment outcomes. CPT11 blocked MDSC apoptosis and myeloidcell differentiation, increasing MDSC immunosuppressive features and mouse mortality. In contrast, 5FUpromoted immune recovery and tumor regression. Thus, CPT11 exhibited detrimental immunoregulatoryeffects that offset 5FU benefits when administered in combination. Our results highlight the importance ofdeveloping therapeutic regimens that can target both the immune system and tumor towards improvedpersonalized treatments for colorectal cancer. Cancer Res; 74(21); 6022–35. �2014 AACR.

IntroductionColorectal cancer and certain other tumors are characterized

by chronic inflammation–induced immunosuppression medi-ated by proinflammatory cells and mediators (1–4), whichsubvert the outcome of anticancer therapy. Myeloid-derivedsuppressor cells (MDSC) are the main cell population causingimmunosuppression in numerous cancers including colorectalcancer (3, 5–8). MDSCs are immaturemyeloid cells expanded inthe course of chronic inflammation, co-expressingGr1þCD11bþ

in mice and CD11bþCD14�CD33þ, LIN�HLA-DR�CD33þ, orCD14þCD11bþ in humans (6, 9).

Chemotherapeutic drugs commonly used to treat cancer,including colorectal cancer, affect not only the tumor but alsothe immune system, having a crucial impact on antitumor

responses and disease outcome (5, 10). Although chemothera-pies combat the tumors and lead to their regression, the effectson the tumor microenvironment and the immune system arenot clearly understood. Colorectal cancer is usually treatedwith multiagent regimens, and in some cases, different drugsthat act via diverse mechanisms are combined as they mayhave superior efficacy and effectiveness when administeredjointly (11). The most common protocols for colorectal cancerapproved by the FDA are combined chemotherapies FOLFIRI[folinic acid, 5-fluorouracil (5FU), and CPT11] or FOLFOX(folinic acid, 5FU, and oxaliplatin). Studies comparing betweenthese regimens indicated that in some cases FOLFOX is super-ior as it leads to higher overall survival rates (12, 13). However,other studies have demonstrated equal efficacy for thesetreatments (14).

Even though chemotherapy for stage IV colorectal cancerleads to tumor regression, in most cases, the survival time islimited. We speculated that various drugs may differentlyalter the immune status of patients with colorectal cancer,thus affecting their therapeutic effectiveness. Monitoring theimmune status of patients with stage IV colorectal cancer,before and following FOLFOX or FOLFIRI treatments,revealed that before therapy the patients displayed a sup-pressed immune status as indicated by the elevated MDSClevels and downregulated CD247, which is a key moleculethat "senses" immune functionality and regulates T-cell andnatural killer (NK) cell immune responses (15). Duringchemotherapeutic treatments, while FOLFOX reduced

1The Lautenberg Center for General and Tumor Immunology, Israel-Canada Medical Research Institute, Faculty of Medicine, The HebrewUniversity, Jerusalem, Israel. 2Sharett Institute of Oncology, HadassahUniversity Medical Center Ein Kerem, Jerusalem, Israel.

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

Corresponding Author: Michal Baniyash, The Lautenberg Center forGeneral and Tumor Immunology, Israel-Canada Medical Research Insti-tute, Faculty of Medicine, The Hebrew University, POB 12272, Jerusalem91120, Israel. Phone: 972-2-6757461; Fax: 972-2-6430834; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-14-0657

�2014 American Association for Cancer Research.

CancerResearch

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Published OnlineFirst September 10, 2014; DOI: 10.1158/0008-5472.CAN-14-0657

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accumulation of circulating MDSCs that was accompaniedby upregulated CD247 expression, FOLFIRI displayed oppo-site effects, enhancing the suppressive environment.To gain better understanding of 5FU and CPT11 adverse

effects on host immunity, we used a mouse colorectal cancermodel thatmimics the human disease (1). Herein we show thatsimilar to the patients, colorectal cancer mice display animmunosuppressive status. In assessing CPT11 and 5FUmonotherapies, we discovered that CPT11, but not 5FU,increases immunosuppression by inducingMDSC insensitivityto apoptosis, arresting their differentiation and retaining theirsuppressive features. Moreover, 5FUþ CPT11 combined treat-ment displays harmful effects, resulting in a dysfunctionalimmune response associated with cancer progression andshort survival, showing that CPT11 antagonizes the anticanceractivity of 5FU by exerting its detrimental immunoregulatoryeffects. Our data suggest a significant impact of a givenchemotherapeutic protocol on both the tumor and its immu-nosuppressive environment.

Patients and MethodsPatientsPeripheral blood samples were collected from 23 patients

with stage IV metastatic colorectal cancer before and every2 months in the course of chemotherapy treatments. Allpatients who were diagnosed with metastatic colorectalcancer underwent surgery and were not previously treatedwith chemotherapy. Twenty healthy donors were used ascontrols. The samples were taken according to the Helsinkiapproval and analyzed for the indicated immune biomar-kers in a "blinded test," not knowing the therapy specifica-tion. Following analyses completion, the specific treatmentregiments and clinical parameters were acquired frommedical records of the patients under the care of Dr. AyalaHubert at the Oncology department, Hadassah UniversityMedical Center Ein Kerem (Jerusalem, Israel) and correla-tion tests were performed.

MiceFemale C57BL/6 and BALB/c mice (aged 6–8 weeks) were

purchased from Harlan and were grown at the Hebrew Uni-versity specific pathogen-free facility. All experiments weredone in accordance with preapproved institutional protocols.

ReagentsAzoxymethane and dextran sulfate sodium (DSS) were

purchased from Sigma-Aldrich and MP Biochemicals Inc.,respectively.

In vivomouse models for colorectal cancer and a tumor-free chronic inflammationMice were injected intraperitoneally with 10 mg/kg body

weight of AOM dissolved in physiological saline twice in 2weeks' intervals. Two weeks later, 2% DSS was given in thedrinking water over 7 days, following by 14 days of regularwater (16). This cycle was repeated twice. Animals weresacrificed and analyzed 3 weeks after the last treatment.

To induce a pathology-free chronic inflammation, we used apreviously described protocol subjecting mice to heat-killedMycobacterium tuberculosis (BCG) treatment (17).

Chemotherapeutic drugsIn vivo efficacy of chemotherapeutic FDA-approved drugs on

the immune status was determined under: (i) colorectal cancerconditions: a day after the second DSS administration, che-motherapy treatment was applied intraperitoneally twice aweek in a 3-day interval for 3 weeks and (ii) chronic inflam-matory tumor-free conditions: a day after the second BCGinjection, chemotherapy treatment was applied intraperitone-ally twice a week. The chemotherapies were scaled accordingto FDA-approved dosages: 5FU and CPT11 50 mg/kg each.

The chemotherapeutic drugs' ex vivo effects were tested onMDSCs isolated from inflamed mice, using a magnetic columnseparation system (Miltenyi Biotec), as previously described(18). Two cycles of purifications, one with Gr1 antibodies(Biolegend) and a second with CD11b antibodies (Biolegend),were performed. The purity of the cell populations was morethan 95%. After purification, cells were grown in tissue culture,treated for 24 hours with 5FU and CPT11 at 1.25, 2.5, and 5mmol/L concentrations for cleaved caspase-3 detection assayandwith 5FU, CPT11, and 5FUþCPT11 (2.5mmol/L each drug)for nitric oxide (NO�) and reactive oxygen species (ROS)production assay.

In vivo depletion of MDSCsFor theMDSC depletion assay, at the same day of the second

DSS administration, CPT11-treated colorectal cancer micewere administered intraperitoneally every 3 days with 0.5 mgof anti-Gr1 mAb (RB6-8C5).

Carboxyfluorescein diacetate succinimidyl ester stainingand ex vivo T-cell proliferation assay

Splenocytes or purified T cells isolated by a magneticcolumn separation system (Miltenyi Biotec) were labeled with5 mmol/L carboxyfluorescein diacetate succinimidyl ester(CFSE; Invitrogen) and subjected to T-cell receptor–mediatedactivation as previously described (17).

Ex vivo myeloid cell differentiationMDSCs were isolated from colorectal cancer and control

(normal) mice and cultured in the presence or absence of 10ng/mL granulocyte macrophage colony-stimulating factor(GM-CSF; PeproTech) for 3 days. In some samples, 5FU andCPT11 were added to the cells, with or without GM-CSF,followed by phenotyping using flow cytometry.

Flow cytometric analysisIsolated mouse splenocytes and peripheral blood lympho-

cytes (PBL)were subjected to cell surface staining as previouslydescribed (17), using the following antibodies (Biolegend):fluorescein isothiocyanate (FITC)-labeled anti-Gr1 and anti-CD11c; phycoerythrin (PE)-labeled anti-F4/80, anti-CD3e, andanti-mNKp46; and biotinylated anti-CD11b detected withstreptavidin-Cy5. Intracellular staining for CD247 was per-formed as previously described (17) by using FITC-labeled

Adverse Effects of Chemotherapies on MDSCs in Colorectal Cancer

www.aacrjournals.org Cancer Res; 74(21) November 1, 2014 6023




anti-CD247 or biotinylated anti-CD247 (lone H146), the latterdetected with streptavidin-Cy5. Foxp3 staining was performedaccording to themanufacturer's instructions (Miltenyi Biotec).Cleaved caspase-3 staining was performed using primaryrabbit anti-cleaved caspase-3 (Cell Signaling Technology,Asp175) and secondary FITC-horse anti-rabbit antibody(Thermo Scientific). For intracellular NO� and ROS detection,diaminofluoresciein-2 diacetate (DAF-2DA) reagent (NOS 200-1; Cell Technology) and aminophenyl fluorescein (APF; 4011;Cell Technology) were used, respectively, and determined byflow cytometric analysis.

For human whole blood cell phenotyping, intracellular stain-ing of CD247 cells was performedbyfirst fixing the cells with 1%paraformaldehyde followed by washes and permeabilized with0.1% saponin. Allophycocyanin-labeled anti-CD11b and anti-CD3, PE-labeled anti-CD33, FITC-labeled anti-HLA-DR, andanti-CD247 were used, all purchased from BD Pharmingenand used according to the manufacturer's protocol. Aftersurface staining, cells were treated eBioscience-Step Fix/Lysesolution according to the manufacturer's instructions. Allsamples were analyzed using FACS Calibur with Cell Questsoftware (BD).

Cell isolation from the colonThe preparation of single-cell suspensions from colons was

performed using a modified version of a previously describedprotocol (19). Briefly, isolated colons were washed with HBSS5% FBS (Invitrogen), digested, minced, incubated for 15 min-utes at 37�C, and epithelial cell suspension was washed withRPMI. For lamina propria cells, the retained tissue was trans-ferred to collagenase/DNAse (Roche Diagnostic Corporation)solution, incubated for 1 hour at 37�C, filtrated, and washedwith RPMI.

Quantitative PCR analysisTotal RNA was recovered from colon cells, splenocytes, or

isolated MDSCs and subjected to real-time PCR analysis aspreviously described (17). The sequences of the oligonucleo-tides used are listed in Supplementary Table S1.

Western blot analysisCells isolated from the spleen or colon were analyzed by

Western blotting for the expression of various proteinsas previously described (17). The antibodies used for immu-noblotting were: anti-S100A9, anti-S100A8, and anti-a-tubu-lin. Specific antibodies were detected by anti-rabbit oranti-goat antibodies conjugated to horseradish peroxidase(HRP; Jackson ImmunoResearch), followed by enhancedchemiluminescence and exposure at blotting reader (Bio-Rad software).

Histopathology and immunohistochemistryParaffin-embedded colon tissue sections were prepared

from colorectal cancer, colorectal cancer–5FU, or colorectalcancer–CPT11–treated and control untreated mice andstained with hematoxylin and eosin solution. For immunohis-tochemistry, after antigen retrieval, sections were incubated at4�Cwith primary antibodies: anti-b-catenin (BD) and anti-Gr-1

(Biolegend). For immunohistochemical staining, universalimmunoperoxidase polymer for mouse tissues (414311F; His-tofine) was used, based on anHRP-labeled polymer conjugatedto anti-rat. After incubation for 30 minutes, slide staining wascompleted by 3- to 5-minute incubation with DAB þ Chro-mogen (Lab Vision), followed by counterstaining with hema-toxylin. As a control, samples were stained with each antibodyand reagent individually.

Statistical analysisStatistical analyses were performed using GraphPad Prism

5.04. Averaged values are presented as the mean� SEM.Whencomparing two groups, statistical significance was determinedusing 2-tailed Student t test. Whenmore than two groups wereinvestigated, anANOVAwas performed. Survival analyseswereassessed using the Fisher exact test.

For the human experiments, paired t test was used tocompare samples from the same patients before and afterFOLFOX or FOLFIRI treatment. Control and colorectal cancergroups were investigated by ANOVA.

ResultsFOLFOX and FOLFIRI therapies of patients withcolorectal cancer display opposite effects onCD11bþCD33þHLA-DR� myeloid cells and immunestatus

Progression of colorectal cancer involves the development ofa chronic inflammatory and immunosuppressive milieu. Wetherefore proposed that some chemotherapies may have lim-ited beneficial effects due to their harmful impact on thepatients' immune status. To verify this possibility, we firstassessed the immune status of 23 patients with stage IVcolorectal cancer before a given chemotherapy in comparisonto 20 healthy donors. The percentage of CD11bþCD33þHLA-DR�MDSCs in the patients' peripheral blood was significantlyhigher (12.65%� 1.35%, P < 0.01) than in healthy donors (5.35%� 1.05%; Fig. 1A). Moreover, we found a significant increasedproduction of both NO� and ROS inMDSCs, as compared withhealthy donors (Fig. 1B), showing their immunosuppressivefeatures in the blood of patientswith colorectal cancer.We alsofound an inverse correlation between the percentage of cir-culating MDSCs and CD247 expression in the patients withcolorectal cancer (Fig. 1C), suggesting an impaired immunestatus associated with the disease. To examine the impact ofFOLFOX and FOLFIRI on immune parameters of patients withstage IV colorectal cancer, we followed the effects of thesedrugs on the kinetics of MDSC levels and the association withCD247 expression. All patients were treated with chemother-apy for at least 6 months. PBL analysis revealed decreasedlevels of circulatingMDSCs following FOLFOX treatments (Fig.1D, left), which were associated with a tendency of upregulatedCD247 expression (Fig. 1D, right). In contrast, during thecourse of FOLFIRI treatment of patientswith colorectal cancer,theMDSC percentage continuously increased, correlating withCD247 downregulation (Fig. 1E). These results suggest anadverse impact of the different chemotherapies on the immunestatus of patients with colorectal cancer.

Kanterman et al.

Cancer Res; 74(21) November 1, 2014 Cancer Research6024




CPT11 but not 5FU treatment increases MDSCaccumulation at the tumor site and supports colorectalcancer growthThe harmful effects of FOLFIRI treatment on the immune

status of patients with colorectal cancer and the reportedbeneficial effects of 5FU, which induces MDSC apoptosis andtumor regression in mice (20), suggest that CPT11 might

antagonize the anticancer activity of 5FU by exerting its det-rimental immunoregulatory effects. To further investigatewhether indeed 5FU and CPT11 display an adverse effect onthe immune system, we used a mouse-inducible colorectalcancer model based on AOM/DSS treatments (Fig. 2A). Inthis model, developing colorectal cancer is accompanied bychronic inflammationand immunosuppression, reminiscentof

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Figure 1. FOLFOX and FOLFIRItherapies display opposite effectson CD11bþCD33þHLA-DR�

myeloid cells and CD247expression in patients withmetastatic colorectal cancer.A, CD33þHLA-DR� MDSCs weredetected in PBLs of healthy donorsand patients with colorectalcancer, gating on CD11bþ cellsby flow cytometric analysis.Representative dot plots of MDSClevels in healthy donors andpatients with colorectal cancer arepresented (left), as well as theMDSC percentages of 20 healthydonors and 23 patients (right).B, NO� (left) and ROS (right) levelsproduced by MDSCs from healthydonors and patientswith colorectalcancer are presented as meanfluorescence intensity (MFI), gatingon CD11bþCD33þHLA-DR� cells.C, PBLs from the healthy donorsand patients with colorectal cancerdescribed inAandBwere analyzedby flow cytometry for CD247expression presented as MFI,gating on CD3þ cells. D and E, thepercentage of circulating MDSCs(left) and the expression of CD247in T cells (right) from patients withcolorectal cancer before (control)and after FOLFOX (patients 1–6; D)or FOLFIRI (patients 7–10; E)treatments. Dashed lines representthe normal mean values ofpercentage of MDSCs and CD247levels that were measured in gatedCD3þ cells and are presented asthe expression in the experimentalgroup relative to the mean ofexpression in healthy donors (as100%). ��, P < 0.01; ��, P < 0.001.






spontaneous human colorectal cancer (21). Kinetic analysis ofMDSC during colorectal cancer development revealed theirgradual accumulation in the blood along with disease progres-sion (Fig. 2B). When MDSC level became stable and adenomaswere evident, mice were treated with either CPT11 or 5FU for 3weeks. CPT11 monotherapy did not prevent tumorigenesis asno apparent differences in tumor loads within the colon were

observed when compared with untreated colorectal cancermice (Fig. 2C). This stood in sharp contrast to the dramaticeffect of 5FU toward a decreased tumor load and recovery ofcolon architecture (Fig. 2C). Moreover, immunohistochemicalanalysis showedamassiveb-cateninaccumulation in thenucleiof tumorcells both incolons fromuntreatedandCPT11-treatedcolorectal cancer mice (Supplementary Fig. S1), suggesting

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Figure 2. Differential effects of5FU and CPT11 monotherapieson the tumor and colonmicroenvironments in colorectalcancer mice. A, schematicrepresentation of themousemodelfor colorectal cancer. B, kineticstudy of Gr1þCD11bþ (MDSC)accumulation. Blood sampleswerecollected from mice duringcolorectal cancer developmentand progression at the indicatedtime points and tested for MDSCaccumulationby flowcytometry. C,histopathology of colonic lateneoplasms developed in AOM/DSS-treated mice was determinedby hematoxylin and eosin staining.Original magnifications, �200 andinsets �40. D, immunostaining forMDSCs in colons (top) and tumors(bottom). Original magnifications,�200. E, MDSC accumulationwithin the colons was evaluated byflow cytometric analysis. Graphsrepresent the absolute number ofMDSCs within the lamina propria(left) and the epithelium (right).F, the lamina propria (left) andepithelium (right) fractions isolatedfrom the colons of colorectalcancermicewereanalyzed forNO�

production by flow cytometricanalysis gating on the MDSCpopulation. Graphs representproduction levels, as shown bymean fluorescence intensity (MFI).All in vivo experiments involved sixmice per group and were repeatedthree times, yielding similar results.Graphs (means of triplicates �SEM, n ¼ 6) are representativeof a typical experiment ofthree performed. �, P < 0.05;��, P < 0.001; ns, nonsignificant.

Kanterman et al.





tumor progression (16). Histologic analyses of colons fromCPT11-treated colorectal cancer mice demonstrated not onlya loss of entire crypts and surface epithelial layer but also amassive leukocyte infiltration into themucosa (Fig. 2C). Impor-tantly, immunohistochemical probing of MDSCs within thecolon revealed elevated levels in untreated and in CPT11-treated but not in 5FU-treated colorectal cancer mice (Fig.2D, top). The same correlation between MDSC accumulationand thegiven treatmentwas alsoobservedwhen testing tumorsin colons (Fig. 2D, bottom). Analysis of cells generated from thecolon laminapropria and epitheliumdepicted increasedMDSCnumbers in the CPT11 treated as compared with untreatedcolorectal cancer mice (Fig. 2E). A significantly reducedMDSCinfiltration was observed in both the lamina propria andepithelium following 5FU treatment (Fig. 2E and Supplemen-tary Fig. S2A). Furthermore, a significant reduction of NO�

production was found in lamina propria and epithelial MDSCsfrom5FU-treatedcolorectal cancermice (Fig. 2F),whereasafterCPT11 treatment, the NO� levels remained elevated as com-pared with the colorectal cancer untreated mice. These resultsstrengthen the harmful effects of CPT11 supporting MDSCaccumulation and suppressive features at the tumor site.

CPT11 treatment increases systemicimmunosuppression and counteracts 5FU beneficialeffectsWe next investigated whether CPT11 and 5FU also dif-

ferently affect the systemic immunosuppressive state.Untreated, CPT11-, and 5FU þ CPT11–treated colorectalcancer mice display stronger inflammatory response asindicated by the enlarged spleen size as compared with5FU-treated colorectal cancer or control mice (Fig. 3A) andby the significantly decreased MDSC numbers in spleens of5FU-treated colorectal cancer mice as compared with thoseof the untreated colorectal cancer mice or those treated withCPT11 or 5FU þ CPT11 (Fig. 3B). Interestingly, none of themonotherapies altered the high percentage of regulatory Tcells (CD4þFoxp3þTregs) observed in colorectal cancer mice(Supplementary Fig. S3), showing that mainly MDSCs areaffected by 5FU and CPT11.Moreover, we found that while MDSCs from 5FU-treated

colorectal cancer mice displayed a significantly reduced NO�

and ROS production, MDSCs from CPT11- or 5FU þ CPT11–treated colorectal cancer mice displayed elevated levels, ascompared with untreated colorectal cancer mice (Fig. 3C). Exvivo studies showed that 5FU administration does not alterNO� or ROS production in purified cultured MDSCs, but theaddition of CPT11 or 5FUþ CPT11 to the medium resulted intheir elevation (Supplementary Fig. S4A), suggesting a directeffect of the drugs on MDSC suppressive features. Both mono-cytic (CD11bþ Ly6Chigh Ly6G�) and granulocytic (CD11bþ

Ly6Clow Ly6Gþ) cell populations showed increased NO� (Sup-plementary Fig. S4B, left) and ROS (Supplementary Fig. S4B,right) production upon CPT11 or 5FU þ CPT11 treatment.However, the monocytic population displayed a more pro-nounced NO� production, whereas granulocytic populationshowed more ROS production (Supplementary Fig. S4A andS4B). Thus, CPT11 supports the immunosuppressive environ-

ment when applied alone or in a combination with 5FU,affecting the whole MDSC population.

We next evaluated the effect of the given chemotherapies onthe immune status and tested the function of the whole T-cellpopulation (Fig. 3D) and CD8þ T cells (Supplementary Fig.S4C) from all experimental groups along with the expressionlevels of CD247 in CD3þ (Fig. 3E) and in CD8þ T cells(Supplementary Fig. S4D). The results revealed a decreasedT-cell proliferative ability following both CPT11 and 5FU þCPT11 treatments. In contrast, 5FU treatment of colorectalcancer mice did not affect T-cell proliferation, as comparedwith untreated colorectal cancer mice. Moreover, T-cell func-tion was correlated with CD247 expression levels; low CD247levels were obtained in T cells from colorectal cancer miceuntreated and treated with CPT11 or a 5FU þ CPT11 combi-nation, whereas elevated levels were obtained upon 5FUtreatment. Similar results were found in T cells isolated fromthe lamina propria and epithelium, inversely correlatingwith the local generated immunosuppressive environment(Supplementary Fig. S2B). Thus, CPT11 and 5FU adverselyaffect not only the tumor but also the immune system, pointingat the dominating harmful effects of CPT11 when combinedwith 5FU.

CPT11 harmful effects on the host's immune function aremediated via MDSCs

The observed MDSC elevation and increased tumor load inCPT11-treated colorectal cancer mice (Figs. 2–4) suggest animpact of CPT11 on MDSC-induced cancer progression. Wehypothesized that MDSC depletion could reduce CPT11 harm-ful effect on the immune status, thus enhancing the antitumoreffect. Indeed, in vivo MDSC depletion in CPT11-treated colo-rectal cancer mice (Supplementary Fig. S5) as indicated by thenegligible MDSC levels as compared with CPT11 and 5FU þCPT11–treated colorectal cancer mice (Fig. 4A) led to analmost complete regression of the tumors (Fig. 4B). Moreover,histopathologic analysis revealed a differentiated adenocarci-noma in the colons of colorectal cancer mice, CPT11-, and 5FUþ CPT11–treated colorectal cancer mice, whereas onlyfew aberrant crypt foci and tumors were detected in colonsof 5FU-treated colorectal cancer mice and CPT11-treatedMDSC-depleted colorectal cancer mice (Fig. 4C). This patternindicates a beneficial effect of 5FU that attenuates colorectalcancer progression with a harmful contribution of CPT11,supporting immunosuppression and tumor progression viaits effects onMDSCs. The daily recorded vitality and survival ofthemice show a rapid deterioration, with increased death ratesin colorectal cancer mice treated with CPT11 or 5FUþ CPT11,as compared with the 5FU, or CPT11-treated MDSC-depletedcolorectal cancer mice, or even to untreated colorectal cancermice (Fig. 4D).

MDSCs are insensitive to apoptosis under CPT11treatment but become susceptible after 5FU treatment

We next aimed to explore the mechanisms responsible forthe opposite effects of 5FU andCPT11 onMDSC accumulation.These drugs could differently affect MDSC levels by changingtheir sensitivity to apoptosis, as previously reported for 5FU






(20). Indeed, a significant increased cleaved caspase-3 expres-sion, an indicator for apoptosis, was observed within splenicMDSCs from 5FU-treated colorectal cancer mice, similar tothat detected in MDSCs from control mice (Fig. 5A). Incontrast, following CPT11 or 5FUþ CPT11 treatment, MDSCsdisplayed decreased cleaved caspase-3 levels, as in splenicMDSCs of untreated colorectal cancer mice (Fig. 5A). More-over, ex vivo studies revealed that 5FU but not CPT11 leads tocleaved caspase-3 upregulation in purified cultured MDSCs ina dose-dependent manner (Fig. 5B and C). Interestingly, 5FU-induced apoptosis was evident only in nondifferentiatedMDSCs (Fig. 5D) whereas dendritic cells (Fig. 5E) and macro-phages (Fig. 5F) were insensitive, suggesting an exclusive effectof 5FUon the immaturemyeloid cell population.We also foundthat 5FU controls both monocytic and granulocytic purified

cultured MDSC populations, with the monocytic populationbeing more sensitive, as indicated by the enhanced cleavedcaspase-3 expression upon 5FU addition or when combinedwith CPT11 (Supplementary Fig. S6A). We also show that thedrugs did not affect the apoptotic state of other immunecells as T (CD3þ) and B (B220þ) lymphocytes (Supplemen-tary Fig. S6B). These results underscore the direct apoptoticeffects of 5FU on immature MDSCs as opposed to theapoptosis insensitivity of MDSCs to the CPT11 or CPT11þ 5FU treatments.

5FU and CPT11 directly affect myeloid cell maturationand suppressive activity

To investigated whether 5FU and CPT11 also affect MDSCmaturation, we first assessed expression of S100A8/9

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27.61%

Normal CRC CRC+CRC+CRC+5FU CPT11 5FU+

CPT11

*

NS

E


CRC+5FU+

CPT11

CD

247

expr

essi

on (

MF

I)NS

**

T-c

ell p

rolif

erat

ion

(%)

40

30

20

10

0

40

30

20

10

0

80

60

40

20

0

80

60

40

20

0

60

40

20

0

150

100

50

0

Figure 3. A combined 5FUþCPT11therapy abrogates recovery fromimmunosuppression duringcolorectal cancer progression.Colorectal cancer mice weresubjected to 5FU, CPT11, or a 5FUþ CPT11 combination starting atweek 8 or left untreated. Threeweeks after the second DSStreatment, mice were sacrificedand spleens were analyzed. A,representative spleens of thedifferent experimental groups arepresented. B, MDSC accumulationwasmeasured in the spleen by flowcytometric analysis and thepercentage and absolute numbersare presented. C, splenocytesisolated from normal, colorectalcancer, and 5FU-, CPT11-, and5FU þ CPT11–treated colorectalcancermicewereanalyzed forNO�

and ROS production by flowcytometric analysis, gating onMDSCs. Graphs represent meanfluorescence intensity (MFI). D, T-cell proliferative response wasassessed by monitoring celldivisions of gated CFSE-labeledThy1.2þ (CD90þ) T cells upon TCR-CD28–mediated activation.Representative histograms ofproliferative activity are shown(left), and the percentage ofproliferating cells was calculatedand compared with steady-statelevels of nonactivated cells in eachgroup (right). E, splenocytes fromthe experimental groups wereanalyzed for CD247 expressionlevels indicated by MFI, gating onCD3þ cells. All in vivo experimentsinvolved six mice per group andwere repeated three times, yieldingsimilar results. Graphs (means oftriplicates � SEM, n ¼ 6) arerepresentative of a typicalexperiment of three performed.�, P < 0.05; ��, P < 0.01; ��,P < 0.001; ns, nonsignificant.

Kanterman et al.





proinflammatory proteins, which are induced in the course oftumorigenesis and chronic inflammation and playing a role incontrolling MDSC accumulation and retention in their imma-ture suppressive state (7, 17, 22). While 5FU treatment ofcolorectal cancer mice induced a significant decrease in

S100A8/9 mRNA and protein levels in the spleen as comparedwith control mice, increased S100A8/9 levels were observedfollowing CPT11 treatment (Fig. 6A andB). Similar results wereobtained when assessing the colon (Supplementary Fig. S7A),suggesting that 5FU supports MDSC transition from an

Gr1

+ C

D11

b+ c

ells

(%

)

Normal

60

40

20

0CRC CRC+

5FUCRC+CPT11

CRC+5FU+

CPT11

CRC+CPT11+anti-Gr1

*****

**

A

D

CRC+CPT11

NormalCRCCRC+5FU

CRC+5FU+CPT11CRC+CPT11+anti-Gr1S

urvi

val (

%)

Days

CRC CRC+5FU

CRC+CPT11

CRC+5FU+

CPT11

CRC+CPT11+anti-Gr1

Normal

1 cm

B

CRC+5FU+CPT11 CRC+CPT11+anti-Gr1

Normal CRC

CRC+5FU CRC+CPT11

200 µm

C

200 µm 200 µm

200 µm200 µm

200 µm

100

80

60

40

20

040 50 60 70 80

Figure 4. CPT11 harmful effects supporting tumor progression are mediated via MDSCs. Colorectal cancer mice were subjected to 5FU, CPT11, or a 5FUþCPT11 combination starting at week 8 or left untreated. At the same day of the second DSS administration, CPT11-treated colorectal cancer mice wererandomly separated into two groups; one group continued with CPT11 treatment, whereas the second group was treated with anti-Gr1 mAb for MDSCdepletion in addition toCPT11 treatment. Threeweeks after the secondDSS treatment,micewere sacrificedandPBLs (A) andcolons (B andC)were analyzed.A, MDSC accumulation was measured in PBLs by flow cytometric analysis. Graph represents the percentage of MDSCs in each experimental group.Representative colon structures are presented in B, and histopathology analyses of colons in colorectal cancer mice were determined by hematoxylin andeosin staining in C. Original presentedmagnification,�100. D, Kaplan–Meier curve (n¼ 20) of colorectal cancer, 5FU-treated, CPT11-treated, 5FUþCPT11–treated, orMDSC-depleted CPT11-treated colorectal cancermice. All in vivo experiments involved sixmice per group andwere repeated three times, yieldingsimilar results. Graphs (means of triplicates � SEM, n ¼ 6) are representative of a typical experiment of three performed. ��, P < 0.01; ��, P < 0.001.






immature suppressive stage toward differentiated nonsup-pressive myeloid phenotype (17). Indeed, 5FU treatment ofcolorectal cancer mice resulted in a significant shift towarddifferentiated dendritic cells and macrophages (Fig. 6C) andto matured antigen-presenting cells, shown by the inducedCD80 and MHCII expression (Fig. 6D). In contrast, CPT11treatment blocked myeloid cell differentiation in vivo (Fig.6C and D).

When testing the ex vivo direct effects of the drugs on GM-CSF–mediated colorectal cancer–derived MDSC differenti-ation, we detected that, similar to the in vivo effects, CPT11prevented cell differentiation after both 48 hours (data notshown) and 72 hours (Fig. 6E) as compared with MDSCsincubated with GM-CSF only. In contrast, 5FU enabledMDSC differentiation to dendritic cells and macrophages(Fig. 6F). Interestingly, ex vivo CPT11-mediated MDSC dif-ferentiation blockade was associated with increased mRNAlevels of the proinflammatory mediators TNFa (Supplemen-tary Fig. S7B) S100A9 (Supplementary Fig. S7C), whereasthe 5FU-induced MDSC differentiation correlated withdecreased levels of these factors. Thus, 5FU directly affectsthe differentiation pathway of MDSCs, whereas CPT11 exhi-

bits a differentiation blockade capacity when added to GM-CSF–treated MDSCs.

5FU and CPT11 opposing effects on MDSCs are tumor-independent

To examine whether the immunoregulatory effects of 5FUand CPT11 are tumor-dependent, we used a mouse model forchronic inflammation and associated immunosuppression(15), described in the Materials and Methods and Fig. 7A. The5FU beneficial and CPT11 harmful effects were similar to thoseobserved in the colorectal cancer model. 5FU significantlyreduced MDSC levels (Fig. 7B and C and Supplementary Fig.S8A) and NO� and ROS production (Fig. 7D). Treatment with5FU also elevated cleaved caspase-3 levels (Fig. 7E), as com-pared with inflamed-untreated mice. In contrast, CPT11 or5FUþ CPT11 treatments induced opposite effects (Fig. 7B–E).No changes in CD4þFoxp3þ Tregs percentage were detected(Supplementary Fig. S8B), confirming that these chemothera-pies specifically affect MDSCs.

We next assessed whether the 5FU and CPT11 oppositeeffects onMDSCs have different impacts on the host's immunecompetence. Assessment of the drugs' effects on total T-cell

A

1.25 505FU µmol/L

2.5

***

Cle

aved

cas

pase

-3 (

MF

I)

1.25 50CPT11 µmol/L

2.5Cle

aved

cas

pase

-3 (

MF

I)

CBC

leav

ed c

aspa

se-3

(M

FI)

1.25 50µmol/L

2.5

*** ***

Cle

aved

cas

pase

-3 (

MF

I)

1.25 50µmol/L

2.5

F

1.25 50µmol/L

2.5Cle

aved

cas

pase

-3 (

MF

I)

ED

Cle

aved

cas

pase

-3 (

MF

I)

Normal

30

20

10

0

30

20

10

0

15

10

5

0

20

15

10

5

0

50

40

30

20

10

0CRC CRC+ CRC+ CRC+

5FU CPT115FU+ CPT11

*

NS

CPT115FU

CPT115FU

CPT115FU

50

40

30

20

10

0

Figure 5. 5FU and CPT11 directly and differently affect MDSC sensitivity to apoptosis. A, splenicMDSCs from each groupwere analyzed for the expression ofactivated (cleaved) caspase-3 by flow cytometric analysis, gating on MDSCs. To assess the direct effect of 5FU (B) and CPT11 (C) on cleaved caspase-3expression, primary MDSCs isolated from spleens of colorectal cancer mice (n ¼ 6) were ex vivo incubated with various doses of the drugs for 3 days andsubjected to flow cytometric analysis. To assess which cells are affected by the chemotherapeutic drugs, MDSCs isolated from spleens of colorectal cancermice were cultured ex vivo with 10 ng/mL of GM-CSF in the absence or presence of scaled doses (0, 1.25, 2.5, and 5 mmol/L) of 5FU or CPT11 for 3 days.Cleavedcaspase-3 levelswere thenevaluatedon theprimaryMDSCs (D), differentiatedCD11cþCD11bþdendritic cells (E), andF4/80þCD11bþmacrophages(F). All experiments involved six mice per group and were repeated three times, yielding similar results. Graphs (means of triplicates � SEM, n ¼ 6) arerepresentative of a typical experiment of three performed. �, P < 0.05; ��, P < 0.001; ns, nonsignificant.

Kanterman et al.





B

CD

11b+

CD

11c+

cel

ls (

%)

NS

**

Normal CRC CRC+5FU

CRC+CPT11

C

**

Normal CRC CRC+5FU

CRC+CPT11

***NS

CD

11c+

MH

CII+

CD

80+

cells

(%

)

F4/

80+ M

HC

II+ C

D80

+

cells

(%

)

Normal CRC CRC+5FU

CRC+CPT11

***NS

2.5

D

A

mR

NA

(R

Q)

CRC

CRC+

CPT1

1

CRC+

5FU

CRC

CRC+

CPT1

1

CRC+

5FU

*

*

*

*S100A8S100A9

αTub

CRC CRC+5FU

CRC+CPT11

S100A8

S100A9

CD

11b+

F4/

80+ c

ells

(%

)

NS

*

Normal CRC CRC+5FU

CRC+CPT11

E

ControlGM-CSF

1.250

CD

11b+

CD

11c+

cel

ls (

%)

CPT11 μmol/L

5 10 2.5

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CD

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)

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CD

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%)F

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250 5 10

5FU μmol/L

CD

11b+

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ells

(%

)

2.5

1.250 5 10 2.

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250 5 10

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NS

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20

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20

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20

15

10

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20

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50

40

30

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10

0

50

40

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10

0

50

40

30

20

10

0

Figure 6. 5FU and CPT11 directlyaffect myeloid cell differentiation,maturation, and suppressiveactivity. S100A8/9 mRNA (A) andprotein (B) levels were evaluated inMDSCs isolated from the spleenof colorectal cancer mice orcolorectal cancer mice treated with5FU or CPT11 (n ¼ 4). a-Tubulinlevels served as a control. C and D,the differentiation and maturationof myeloid cells within the spleensof the different experimentalgroups were evaluated by testingthe levels of CD11cþCD11bþ

dendritic cells and F4/80þCD11bþ

macrophages (C), and the CD80and MHCII expression (D),respectively. E and F, MDSCsisolated from spleens of colorectalcancer mice were ex vivo culturedwith 10 ng/mL GM-CSF in theabsence or presence of scaleddoses (0, 1.25, 2.5, 5, and 10mmol/L) of CPT11 (E) or 5FU (F) for3 days. The phenotype ofdifferentiated dendritic cells (left)and macrophages (right) was thenevaluated. All in vivo experimentsinvolved six mice per group andwere repeated three times, yieldingsimilar results. Graphs (means oftriplicates � SEM, n ¼ 6) arerepresentative of a typicalexperiment of three independentperformed. Ex vivo experimentsinvolved four mice per group andwere repeated three times, yieldingsimilar results. Graphs (means oftriplicates � SEM, n ¼ 4) arerepresentative of a typicalexperiment of three performed.Data shown are mean � SEM.�, P < 0.05; ��, P < 0.01;��, P < 0.001. (2-way ANOVA).ns, nonsignificant.






A

Gr1

+ C

D11

b+ c

ells

(%

)

***

B

C

−

NS0−2 0−6

Harvest

−7

BCG injection

1st

Days

−14

Chemotherapy treatment

+2

2nd 3rd

−−−

−−

−

++++

+

+

++

Inflamed5FU

CPT11

Normal Inflamed Inflamed+5FU

Inflamed+CPT11

Inflamed+5FU+CPT11

19.53%46.34% %60.95%23.65%10.4

CD

11b

Gr1

Gr1

+ C

D11

b+ c

ells

(%

)

−−−−

−−

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++++

+

+

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CPT11

**

**

D

RO

S p

rodu

ctio

n (M

FI)

***

NS

−−−−

−−

−

++++

+

+

++

Inflamed5FU

CPT11

NO

− pro

duct

ion

(MF

I)p

()

**

NS

−−−−

−−

−

++++

+

+

++

Inflamed5FU

CPT11

Cle

aved

cas

pase

-3 (

MF

I)

***

NS

−−−−

−−

−

++++

+

+

++

Inflamed5FU

CPT11

E

18.36%

3.13%Non-

activated

Cel

l cou

nt

Inflamed+5FU+

CPT11

Inflamed

63.02%

CFSE

Normal

41.89%

19.41%

18.02%

Inflamed+5FU

Inflamed+CPT11

T-ce

ll pr

olife

ratio

n (%

)

−−−−

−−

−

++++

+

+

++

Inflamed5FU

CPT11

**

NS**

*

**

NS

−−−−

−−

−

++++

+

+

++

Inflamed5FU

CPT11

Spe

cific

allo

gene

ic c

ell c

lear

ance

(%

)

F

80

60

40

20

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80

60

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80

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0

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80

60

40

20

0

Figure 7. 5FU and CPT11 opposite effects on the chronic inflammatory environment are tumor independent. A, mouse model for chronic inflammation wasestablishedby three repeated injectionsof heat-killedBCGbacteria. Aday after the secondBCG injection,micewere treated twiceaweekwith 5FU,CPT11, ora 5FU þ CPT11 combination. PBLs (B) and spleens (C) from normal, inflamed, inflamed 5FU-treated, CPT11-treated, or 5FU þ CPT11–treated mice wereanalyzed for MDSC accumulation by flow cytometric analysis. Representative dot plots of MDSCs (C, left) and the percentage within the spleen (C, right) andPBLs (B) are shown. D, splenocytes were analyzed for NO� (left) and ROS (right) production by flow cytometric analysis, gating on the MDSC population.Graphs represent production levels as shown bymean fluorescence intensity (MFI). E, the expression of cleaved caspase-3 was analyzed by flow cytometry,gating on MDSC populations. F, splenocytes were labeled with CFSE and activated with anti-CD3 and anti-CD28 antibodies or left nonactivated. Theproliferative response was assessed by monitoring cell divisions of gated CFSE-labeled Thy1.2þ (CD90þ) T cells. Representative histograms of proliferativeactivity are shown (left), and the percentage of proliferating cells was calculated and compared with steady-state levels of nonactivated cells in eachgroup (right). G, NK cell–mediated clearance of CFSE-labeled allogeneic (CFSElow) and syngeneic (CFSEhigh) splenocytes was evaluated by monitoring theratio between CFSElow/CFSEhigh in the spleen (top) and PBLs (bottom) in each experimental group. Graphs (means of triplicates � SEM, n ¼ 5) arerepresentative of a typical experiment of three independent performed. �, P < 0.05; ��, P < 0.01; ��, P < 0.001; ns, nonsignificant.


Kanterman et al.




activity and specifically CD8þ T cells revealed a significantrecovery of CD247 expression in the spleen and PBLs as well asT-cell proliferation following 5FU but not CPT11 or 5FU þCPT11 treatments (Fig. 7F and Supplementary Fig. S8C andS8D). Similar effects were also detected when analyzing NKcells; downregulated CD247 expression detected in NK cellsfrom chronically inflamed mice was almost completely recov-ered upon 5FU but not CPT11 treatment (Supplementary Fig.S8E). Because NK cell activity is mediated via natural cytotox-icity receptors (NCR), which associate with and depend onCD247, we assessed NK cell in vivo function by monitoring theclearance of adoptively transferred allogeneic cells. A completerecovery of NK cell activity both in the spleen and PBLs wasdetected following 5FU treatment (Fig. 7G), along with asignificant decreased clearance of allogeneic cells after CPT11or 5FU þ CPT11 treatments. These results indicate that 5FUandCPT11 opposite effects onMDSCs are tumor-independent,displaying a significant impact on effector T-cell and NK cellresponsiveness under chronic inflammatory conditions.

DiscussionColorectal cancer appears in most cases as adenocarcinoma

that develops from the lining of the large intestine (colon) andrectum and is supported and progressed by chronic intestinalinflammation as in patients with inflammatory bowel disease(23–25).Despite the clinical progress indetectionand treatment,colorectal cancer remains one of the major causes of cancer-related death. In inflammation-driven tumors, an immunosup-pressive microenvironment, which is characterized by MDSCaccumulation within the tumor and periphery (1, 8, 17, 26, 27),poses a serious obstacle in cancer chemotherapy, attenuatingthe capacity of conventional drugs to evoke a robust antitumorimmunity.In the present study, we highlight novel mechanisms

underlying the action of the commonly used 5FU and CPT11chemotherapies, showing their effect not only on the tumorbut also on its immunosuppressive environment. Our initialstudies with patients with stage IV colorectal cancer showthat before treatment the patients displayed an immuno-suppressive status indicated by elevated MDSC levels andthe downregulated CD247 expression, which is critical forT-cell and NK cell activities. Moreover, we demonstrate thatwhile FOLFOX treatment of patients with colorectal cancerled to a decrease in MDSC levels and a gradual upregulationof CD247 expression, FOLFIRI had opposite and harmfuleffects. Such adverse effects between drugs could have asignificant impact on the overall antitumor response anddisease outcome. Our results were obtained using a limitednumber of patients as an initial proof of concept. Random-ized clinical studies using larger patient cohort should beperformed comparing these therapies to validate our initialobservations.Recent data demonstrated that 5FU treatment leads to a

selective MDSC apoptosis and tumor regression in mice (20).Hence, the harmful effects of FOLFIRI on the immune statusof patients with colorectal cancer suggested that CPT11might have detrimental immunoregulatory effects that offset

the anticancerous impact of 5FU when given together. Thiswas confirmed by testing the effects of 5FU and CPT11 on acolorectal cancer mouse model, showing that colorectalcancer mice display immunosuppressive features, similarto patients with colorectal cancer manifested by elevatedMDSC levels, impaired T-cell function associated withdownregulated CD247 expression. A comparison betweenthe effects of 5FU and CPT11 mono or combined 5FU þCPT11 therapies revealed that the overall effect of the CPT11in both therapies was harmful; yielding a strong immuno-suppression mediated via MDSCs and associated with arapid disease progression and decreased survival as com-pared with the beneficial effects of 5FU alone. These resultssuggest that CPT11 reinforces the immunosuppressive envi-ronment in colorectal cancer mice and patients and dom-inates the beneficial effects of 5FU in the FOLFIRI regimen,thus leading to an overall harmful effect.

Detailed analysis of the mechanisms underlying CPT11and 5FU adverse effects and the affected pathways revealedthat in colorectal cancer mice, 5FU reduced MDSC levels,both by inducing their apoptotic death and by enforcingmyeloid cell differentiation to mature macrophages anddendritic cells. The former drug effect is associated withelevated levels of cleaved caspase-3 and the latter involves adecreased expression of the proinflammatory S100A8/9 pro-teins, known to induce MDSC differentiation arrest(17, 22, 28). In contrast, treatment with CPT11 had oppositeeffects on colorectal cancer–associated MDSCs. CPT11 alsodirectly affects MDSCs by boosting secretion of proinflam-matory cytokines such as TNFa, which is a key MDSCregulator, inducing their differentiation arrest via S100A8/9and increasing their suppressive activity (17). Thus, in untreat-ed and CPT11-treated colorectal cancer mice, but not in 5FU-treated colorectal cancer mice, multiple changes occur in thetumor micro- and macroenvironments that directly enhancetumor growth and support indirectly its progression by inhi-biting antitumor immunity.

Immunosuppressive MDSCs that accumulate during colo-rectal cancer and upon treatment with CPT11 or a 5FU þCPT11 combination produce elevated levels of NO� and ROSthat could induce DNA damage and formation of colonicadenomas (29). In addition, S100A8/9 produced upon CPT11treatment could interact with receptors for advanced-glyca-tion end products (RAGE) and carboxylated glycans expressedon colorectal cancer cells, thereby promoting activation ofMAPK and NF-kB signaling pathways. These, in turn, upregu-late a cohort of proteins that promote leukocyte recruitment,angiogenesis, tumormigration, and establishment of premeta-static niches in distal organs (28). Moreover, the generatedproinflammatory environment can affect Wnt/b-catenin sig-naling as reflected herein by nuclear accumulation ofb-cateninin tumors of untreated and CPT11-treated colorectal cancermice. The attenuated immunosuppression during 5FU treat-ment improves the therapeutic outcome as it enables thegeneration of anticancer immunity and superior tumorregression.

Our findings regarding the immunosuppression relief by5FU are corroborated by a recent report demonstrating that






5FU does not induce ROS activation in MDSCs. In this study,however, the authors also demonstrated that 5FU induces adirect activation of the NLRP3 inflammasome through therelease of cathepsin B from lysosomes, leading to secretion ofIL1b, production of IL17, and tumor growth. NLRP3 activationin the tumor microenvironment could diminish antitumorimmunity by facilitating migration of MDSCs to the tumorsite (30). Hence, multiple regulatory pathways of MDSC func-tion are targeted by 5FU and the drug susceptibility of both thetumor and its environment will dictate the therapy outcome;5FU could accelerate IL1b secretion and inflammasome acti-vation (5) as well as attenuate Treg activity and promoteautoreactive T-cell expansion (31). Herein, we show that eventhough Tregs are elevated in both inflamed and colorectalcancer mice, upon 5FU treatment, MDSCs and their suppres-sive activity are significantly decreased. These effects weresufficient to improve T-cell and NK cell antitumor activities,indicating that in the absence of MDSCs, other factors do notnecessarily worsen host immune function.

Taken together, we demonstrate that conventional mea-surements of chemotherapeutic effects on the tumor areinsufficient to evaluate their curative effectiveness. Rather, itismandatory to assess the drug effects on the immune functionas well, as the combined impact on both the tumor andimmune system will dictate the disease outcome. We wish tostress the significance ofMDSC-mediated immunosuppressiveenvironment and its sensitivity to chemotherapy in determin-ing themost appropriate therapeutic regimen.While our studyalludes to patients with advanced disease, it may bear evengreater relevance to patients with colorectal cancer at earlierstages when monitored before and following treatment. Wealso suggest that by monitoring the host's immune functionusing unique biomarkers as CD247 and MDSCs, the efficacy ofa given treatment could be evaluated andmodified accordinglyif required. Once MDSC-mediated immunosuppression isdetected, modalities leading to MDSC elimination, differenti-ation, or neutralization should be considered as auxiliarytherapy (32) in addition to the antitumor chemotherapy or

to the currently developing anti-colorectal cancer immune–based treatments as specific tumor-infiltrating lymphocytes oranti-CTLA4 and/or anti-PD1 antibodies (33, 34) that willnecessitate a functional immune system to gain efficienttreatment efficacies. Thus, a selection of appropriate antitu-mor combined therapy should lead to an improved design offuture cancer personalized treatments.

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

Authors' ContributionsConception and design: J. Kanterman, M. Sade-Feldman, M. Biton, E. Ish-Shalom, M. BaniyashDevelopment of methodology: J. Kanterman, M. Sade-FeldmanAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J. Kanterman, M. Sade-Feldman, A. HubertAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J. Kanterman, M. Sade-Feldman, M. Biton, E. Ish-Shalom, A. Goldshtein, A. LasryWriting, review, and/or revision of the manuscript: J. Kanterman, M. Sade-Feldman, M. Biton, M. BaniyashStudy supervision: M. Baniyash

AcknowledgmentsThe authors gratefully acknowledge the support of the Society of Research

Associates of the Lautenberg Center, the Concern Foundation of Los Angeles,and the Harold B. Abramson Chair in Immunology. They also thank Z. Chevroni-Lutzky (Cytotoxic Department, Hadassah Medical Organization, Jerusalem,Israel) for providing 5FU and CPT11 chemotherapeutics for treatments;E. Pikarsky (Pathology Department, Hadassah Medical Organization) for ana-lyzing histopathological samples; E. Sasson (Oncology Department, HadassahMedical Organization) for collecting blood samples of patients with colorectalcancer; and E. Yafe-Nof, Y. Klieger, and L. Wang for reviewing this article.

Grant SupportThis study was supported by the Israel Science Foundation (ISF), the Israeli

Ministry of Health, the Joint German-Israeli Research Program (DKFZ), the IsraelCancer Research Fund (ICRF), the United States-Israel Binational ScienceFoundation (BSF), and by the Joseph and Matilda Melnick Funds.

The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received March 6, 2014; revised August 20, 2014; accepted August 26, 2014;published OnlineFirst September 10, 2014.

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2014;74:6022-6035. Published OnlineFirst September 10, 2014.Cancer Res Julia Kanterman, Moshe Sade-Feldman, Moshe Biton, et al. Cancer Outcomes

ColorectalChemotherapy on Myeloid-Derived Suppressor Cells and Adverse Immunoregulatory Effects of 5FU and CPT11

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