Unraveling Cancer Chemoimmunotherapy Mechanisms by Gene … · factors that may propel the efficacy of chemoimmunotherapy, offering a mechanistic glimpse of the important immune modulatory

Microenvironment and Immunology

Unraveling Cancer Chemoimmunotherapy Mechanisms byGene and Protein Expression Profiling of Responses toCyclophosphamide

Federica Moschella1, Mara Valentini1, Eleonora Aricò1, Iole Macchia1, Paola Sestili1,Maria Teresa D’Urso1, Cristiano Alessandri2, Filippo Belardelli1, and Enrico Proietti1

AbstractCertain chemotherapeutic drugs, such as cyclophosphamide (CTX), can enhance the antitumor efficacy of

immunotherapy because of their capacity to modulate innate and adaptive immunity. Indeed, it has been arguedthat this capacity may be more significant to chemotherapeutic efficacy in general than is currently appreciated.To gain insights into the core mechanisms of chemoimmunotherapy, we methodically profiled the effects of CTXon gene expression in bonemarrow, spleen, and peripheral blood, and on cytokine expression in plasma and bonemarrow of tumor-bearing mice. Gene and protein expression were modulated early and transiently by CTX,leading to upregulation of a variety of immunomodulatory factors, including danger signals, pattern recognitionreceptors, inflammatory mediators, growth factors, cytokines, chemokines, and chemokine receptors. Thesefactors are involved in sensing CTX myelotoxicity and activating repair mechanisms, which, in turn, stimulateimmunoactivation events that promote efficacy. In particular, CTX induced a T-helper 17 (Th17)-related genesignature associated with an increase in Th17, Th1, and activated CD25þCD4þFoxp3� T lymphocytes and a slightrecovery of regulatory T cells. By analyzing gene and protein expression kinetics and their relationship to theantitumor efficacy of different therapeutic schedules of combination, we determined that optimal timing forperforming adoptive immunotherapy is approximately 1 day afterCTX treatment. Together, our findings highlightfactors that may propel the efficacy of chemoimmunotherapy, offering a mechanistic glimpse of the importantimmune modulatory effects of CTX. Cancer Res; 71(10); 3528–39. �2011 AACR.

Introduction

Although immunotherapy represents a promising nontoxicanticancer strategy, the different treatment modalitiesemployed so far in the clinical setting resulted in limitedand sporadic successes, highlighting the need for the identi-fication of effective strategies to enhance its clinical efficacy(1). Mounting evidence suggests that combination therapiesmay produce synergistic antitumor responses and that certainchemotherapeutic agents, rather than being immunosuppres-sive can, under defined conditions, act as strong adjuvants foreither adoptive or active immunotherapy (2, 3). Currently,cyclophosphamide (CTX) represents the gold standard immu-nomodulatory chemotherapeutic drug, and the antitumorefficacy of its combination with immunotherapy has long

been studied in preclinical models (4–7), as well as in clinicaltrials (8–11).

Several mechanisms have been considered to explain thisparadoxical phenomenon, among them great relevance wasattributed to the reduction of regulatory T-cell (Treg) numberand function, observed in both the mouse model (12, 13) andcancer patients (14). Additional CTX-mediated immunoacti-vating effects include increased tumor infiltration by lympho-cytes, functional activation of B and T cells, and homeostaticproliferation, occurring during the recovery phase that followschemotherapy-mediated myelolymphodepletion and that, as abystander effect, can promote antitumor immunity (5, 6, 15,16). Notably, preconditioning the host with CTX also inducesthe expression of type I IFN, leading to the expansion of CD4þ

and CD8þ T cells exhibiting a memory phenotype (17). Morerecent data indicate that CTX can restore an activated poly-functional helper phenotype in tumor-specific adoptivelytransferred CD4þ T cells through an IFN-a/b-dependentmechanism (18). Further studies underscore the effects ofthis drug not only on the adaptive but also on the innateimmune system. In this regard, it was shown that CTX-induced expansion of vaccine-specific CD8þ T cells is asso-ciated with increased number and activation status of den-dritic cells (DC; refs. 19, 20).

In a previous study, we had shown that CTX acts throughthe production of hitherto unidentified soluble factors

Authors' Affiliations: 1Department of Cell Biology and Neurosciences,Istituto Superiore di Sanit�a, and 2Department of Internal Medicine/Rheu-matology, La Sapienza University, Rome, Italy

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

Corresponding Author: Federica Moschella, Viale Regina Elena 299,00161, Rome, Italy. Phone: 39-064-990-2474; Fax: 39-064-990-3641.E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-10-4523

�2011 American Association for Cancer Research.

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sustaining the proliferation, survival, and activity of trans-ferred lymphocytes (5). Subsequently, we reported that thesynergistic anticancer activity of chemotherapy and immu-notherapy was associated with the induction of a "cytokinestorm," occurring primarily in the bone marrow of treatedmice (16). Upregulated factors included hematopoieticgrowth factors [granulocyte macrophage colony-stimulatingfactor (GM-CSF) and interleukin (IL)-1b], cytokines regulat-ing homeostatic expansion and memory CD8þ T-cell survi-val (IL-7, IL-15, IL-2, and IL-21) and cytokines involved inthe polarization toward a T-helper (Th)1 type of immuneresponse (IFN-g). The present study aims at investigatingthe mechanisms underlying the immunomodulatory activityof CTX by means of genomic and proteomic approaches. Inparticular, we evaluated the effect of CTX on the geneexpression of bone marrow, spleen, and peripheral bloodmononuclear cells (PBMC) and on the protein levels of 32cytokines in plasma and bone marrow lysates of tumor-bearing mice at different times after a single chemother-apeutic treatment. The overall results provide new insightsinto the mechanisms by which CTX can render immunelymphocytes capable of eradicating metastatic tumors andprovide a scientific rationale for performing adoptive immu-notherapy early after chemotherapy.

Materials and Methods

Animals, tumor cells, and chemoimmunotherapySix- to 7-week-old DBA/2 mice (Harlan) were implanted

with 2 � 106 IFN-a/b-resistant, in vivo passaged, highlymetastatic 3Cl-8 Friend leukemia cells (3Cl-8 FLC; ref. 21).CTX (Sigma–Aldrich) was administered intraperitoneally4 days after tumor injection. Five hours later, some micereceived the adoptive transfer of 40 � 106 lymphomonocytesderived from spleens of tumor-immunized mice, prepared asin ref. 16. Vaccination was carried out with IFN-a-producingtumor cells (IFN-a1-Cl-11; ref. 22). Mice were vaccinated witheither irradiated (100 Gy) cells or a tumor lysate obtained from3 freeze-thaw cycles. Vaccination was carried out twice (3times for lysates) at a time interval of 2 weeks. The experi-mental protocols were approved by the Istituto Superiore diSanit�a Review Board.

RNA isolation, labeling, and hybridizationTotal RNA was obtained by TRIZOL reagent extraction

(Invitrogen) for spleens and by RNeasy (Qiagen) purificationfor bone marrow cells (femurs and tibias) and PBMC(obtained by gradient centrifugation with Lympholyte-Mam-mals-Cederlane).Amino-allyl modified antisense RNA (aRNA) was synthe-

sized in 1 amplification round from 2 mg total RNA by usingthe Amino Allyl MessageAmp II aRNA Amplification Kit(Ambion), in accordance with manufacturer's instructions,and its quality was assessed with the 2100 Bioanalyzer (AgilentTechnologies). aRNAs were coupled to monoreactive Cy3 orCy5 dyes (GE Healthcare), fragmented (RNA FragmentationReagents, Ambion), mixed, and cohybridized overnight inhumidifying chambers at 42�C onto prehybridizated micro-

array slides, and printed with 13,443 70mer oligonucleotides(Operon version 1.1; CRIBI Microarray Service, University ofPadua, Italy). The platform has been submitted to the GeneExpression Omnibus (GEO) database (accession GPL5098).

Data analysisScanning and image file processing were carried out with

the GenePix 4200A instrument (Axon Instruments), and theobtained data were filtered with BRB ArrayTool (23) toexclude spots below a minimum intensity (200), flaggedand with diameters less than 25 mm and normalized by usingLowess Smoother. Only genes expressed in 70% of sampleswere analyzed in subsequent statistical analyses (all done byusing the log2-tranformed ratios).

Average linkage unsupervised hierarchical clustering, withPearson correlation distance measure, was carried out withgenes whose expression differed by at least 1.5-fold from themedian in at least 20% of samples. Following median center-ing, results were visualized with Treeview software (24).

Statistically significant (P � 0.001) differentially expressedgenes between posttherapy and pretreatment samples wereidentified with supervised class comparison (parametric t-test) and further analyzed by using Cluster and TreeViewsoftwares.

Gene annotation and KEGG (Kyoto Encyclopedia of Genesand Genomes) pathway analyses were carried out by means ofDAVID (Database for Annotation, Visualization, and Inte-grated Discovery) bioinformatic tool (25). Enriched biologicalprocesses and pathways were ranked according to EASE score,indicating the abundance of genes fitting each class in pro-portion to the number of genes expected to be in each class bychance, calculated on the global composition of the array.

Intracellular cytokine staining and flow cytometrySplenocytes were stratified over Lympholyte-M Gradient

(Cederlane) for lymphocyte enrichment. For Treg and acti-vated CD4þ lymphocyte analyses, cells were stained with anti-CD4-FITC and anti-CD25-PE (BD Biosciences), and fixed,permeabilized, and stained with anti-Foxp3-APC accordingto the manufacturer's instructions (eBioscience).

For Th1 and Th17 analyses, cells were stimulated overnightwith 100 ng/mL PMA and 1 mg/mL Ionomycin, in the presenceof 1 mg/mL Brefeldin-A and 0.7 mg/mL Monensin (GolgiPlug,GolgiStop, BD-Bioscience) to prevent cytokine secretion. Fol-lowing staining with anti-CD4-FITC, cells were fixed andpermeabilized, and intracellular staining was carried out byusing anti-IFN-g-PE-Cy7 and anti-IL-17-APC (e-Bioscience).Acquisition and analysis were carried out with FACSCaliburflow cytometer (BD-Bioscience) and FlowJo software. Produc-tion of IFN-g and IL-17 was evaluated in CD4þ in bothunstimulated (data not shown) and PMA/I stimulated sam-ples.

Multiplex proteomic analysis of cytokines, chemokines,and growth factors

bone marrow cells were obtained from femurs and tibias.After lysing erythrocytes through a 3-minute incubation in0.16 mol/L Tris-buffered NH4Cl (pH 7.2), cells were lysed in

Mechanisms of Antitumor Efficacy of Chemoimmunotherapy

www.aacrjournals.org Cancer Res; 71(10) May 15, 2011 3529




Bio-Plex cell lysis buffer. The mouse group I (23-plex) andgroup II (9-plex) Bio-Plex cytokine assays were carried outaccording to the manufacturer's instructions (Bio-Rad). Beadswere read on the Bio-Plex 200 suspension array system, anddata were analyzed by using Bio-Plex Manager software.

Results

The combination of CTX and adoptive immunotherapyresults in complete tumor regression

A highly metastatic tumor model, 3Cl-8 FLC (21), was usedto assess the antitumor effectiveness of the combination of asingle CTX injection and adoptive cell transfer (ACT) oflymphomonocytes from tumor-vaccinated syngeneic mice,according to previously published protocols (5). Donor micewere vaccinated with either irradiated (Fig. 1A) or lysed(Fig. 1B) IFN-a–producing tumor cells (IFN-a1-Cl-11), basedon the fact that these cells were shown to be effective cancervaccines (22).

ACT alone had no significant effect on tumor growth (Fig. 1)and mouse survival (data not shown), whereas CTX aloneinduced a transient delay in tumor growth but ultimatelytumors developed in all mice. In contrast, the sequential treat-ment with CTX and ACT frommice vaccinated with irradiated

IFN-a1-Cl-11 resulted in complete tumor regression in 100% ofmice with no recurrences or metastases (Fig. 1A). ACT fromnonvaccinated mice had no effect either alone or in combina-tion with CTX (data not shown). Noteworthy, when chemother-apy was followed by ACT from tumor lysate-vaccinated mice,tumor growth was delayed (more than with CTX alone), but notumor regressed (Fig. 1B). These results, along with previouslypublished data (5, 16), show that the therapeutic effectiveness ofthis chemoimmunotherapy approach depends on CTX-mediated immunomodulatory mechanisms potentiating adop-tive immunotherapy and that also the quality of the antitumorimmunity induced in donor mice has an impact on the out-come. Irradiated tumor cells may, in fact, still produce IFN-a,which influences the quality of the vaccine-induced immuneresponse (26). In addition, irradiation has been shown to inducean immunogenic cell death leading to optimal immunization ofmice against subsequent tumor challenge (27).

Global gene expression analysis of the effects of CTX onbone marrow, spleen, and PBMC

To gain insights into the CTX-mediated effects responsiblefor the antitumor efficacy of chemoimmunotherapy, we eval-uated the impact of a single CTX treatment on the geneexpression of primary (bone marrow) and secondary (spleen)lymphoid organs and of PBMC. Microarray analysis, evaluat-ing the expression of 13,443 genes, was carried out before (ctr)and at different times (1, 2, and 5 days) after chemotherapy.The experiment was carried out in tumor-bearing mice to takeinto account the possible immunomodulation induced by thetumor implant itself. The data discussed in this article havebeen deposited in National Center for BiotechnologyInformation's GEO and are accessible through GEO Seriesaccession number GSE27440 (28).

To get an overall picture of the transcriptional modulationby CTX and of its kinetics, data were preliminarily subjected tounsupervised hierarchical clustering analysis (Fig. 2A). In bothlymphoid organs and PBMC, this analysis segregated samplesinto 2 principal clusters reflecting distinct gene expressionpatterns, one including ctr and samples taken 5 days post-treatment and the other containing samples from day 1 and 2,showing that CTX has a strong impact on gene expression,inducing at early time points (1–2 days) the modulation ofa large number of genes, that, however, return to baseline byday 5.

To identify genes undergoing a statistically significant dif-ferential expression, microarray data were reanalyzed by usinga supervised approach (class comparison), followed by cluster-ing analysis. Class comparison was conducted gathering in oneclass samples from day 1 and 2 and comparing them with ctrsamples, based on the fact that, on separate comparisonsbetween day 1, day 2, and day 5 versus ctr, most genes under-going a statistically significant modulation of expression atday 1 showed a similar profile at day 2 (Supplementary Fig. S1).

In the bone marrow, 1 to 2 days after CTX administration,the expression of 1,123 genes was significantly (P � 0.001)modulated with respect to ctr (580 overexpressed and 543downregulated; Fig. 2B and Supplementary Table S1). Mosttranscripts returned to baseline levels at day 5.

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Figure 1. Antitumor efficacy of the combination of CTX and adoptiveimmunotherapy. DBA/2 mice were implanted s.c. with 2 � 106 3Cl-8 FLC.After 4 days, somemice (n¼ 5) were injected with either CTX (83 mg/kg) orsaline (CTR) and others received the adoptive transfer of 40 � 106 spleenlymphomonocytes from tumor-vaccinated donor mice (ACT). In micetreated with chemoimmunotherapy, ACT was carried out 5 hours after CTXinjection (CTX þ ACT). Donor mice were vaccinated with either irradiated[100 Gy (A)] or lysed (B), IFNa1-Cl-11 tumor cells. Data are reported as theaverage tumor diameter of 5 mice per group � SE. Representativeexperiments out of 5 are shown.

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Cancer Res; 71(10) May 15, 2011 Cancer Research3530




In case of spleen, 868 genes were significantly differentiallyexpressed at day 1–2 (445 overexpressed and 423 downregu-lated; Fig. 2B and Supplementary Table S2). Noteworthy, day 5samples were more similar to day 1–2 samples than to ctr,indicating that gene expression changes in the spleen aremore durable than in the bone marrow.In PBMC, 1,084 genes were significantly modulated by CTX

(557 overexpressed and 527 downregulated), all of which wentback to baseline by day 5 (Fig. 2B and Supplementary Table S3).

Functional classification of genes differentiallyexpressed in bone marrow by gene ontology andKEGG pathway analysisTo characterize the observed transcriptional profiles

according to biological function and to identify functionaland molecular pathways involved in CTX-mediated immuno-modulation, the lists of upregulated and downregulated genes

were subjected to gene ontology–based and KEGG pathway-based annotation by means of the DAVID bioinformatic tool(29). Interestingly, the biological processes found to be moresignificantly (P� 0.05) stimulated by CTX in the bone marrowincluded genes regulating defense response, developmentalprocesses, and response to external/chemical stimuli (seeTable 1 and Supplementary Table S4, for a detailed list ofupregulated genes). The most significantly represented KEGGpathways included chemokine and B-cell-receptor (BCR) sig-naling pathways, cytokine–cytokine receptor interaction, andapoptosis (Fig. 3A and Supplementary Figs. S2 and S3). Bothanalyses indicated that upregulated genes comprised mostlyimmune-related genes.

Increased transcripts included several chemokines, that is,CCL21C, CCL21B (ligands for CCR7), CXCL2 (MIP-2, chemo-tactic for polymorphonuclear leukocytes and hematopoieticstem cells, HSC), CXCL7 (proplatelet basic protein) and

BM Spleen PBMC BDay 5 ctr/Day 5ctr Day 1/2 Day 5Day 1/2 Day 5 Day 1ctr Day 2

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Figure 2. Gene expression profiles induced by CTX in bone marrow (BM; 5 mice), spleen (5 mice), and PBMC (4 pools of 4 mice) of tumor-bearing mice.RNA was isolated either before (ctr; indicated as sample A in the dendrograms) or at day 1 (sample B), day 2 (sample C), and day 5 (sample D) after CTXtreatment. After amplification, aRNAs were labeled with Cy5, mixed with Cy3-labeled "reference" aRNA (from a pool of the BM of untreated mice) andhybridized to 13.5 k oligonucleotide-based microarray. Each oligonucleotide was spotted as duplicate on the slide allowing technical replicates foreach sample (indicated as -a and -b). A, unsupervised hierarchical clustering of genes displayed a 1.5-fold change from the median expression value for thatgene in no less than 20% of samples. B, cluster analyses of statistically significantly (P � 0.001) modulated genes. Class comparison (BRB-ArrayTools)was carried out gathering in one class samples taken 1 and 2 days following CTX treatment and comparing them to ctr samples. The result of aglobal permutation test (which indicates the probability of selecting those numbers of genes if there were no real differences between the classes) was zero.Treeview software was used for visualization of results. For each gene, expression levels above or below the median are shown in red or green, respectively.






CXCL12, (chemoattractant for CD133þHSC). Upregulated che-mokine receptors included CCR6, CCR9, and IL8RB (Supple-mentary Fig. S2 and Tables S1 and S4). Of note, CTX treatmentalso induced increased expression of genes involved in cyto-kine/chemokine signaling pathways (JAK3, GRK6, PIK3CG,VAV2, IKBKG, and NFkB; Supplementary Fig. S3A). Accord-ingly, IL1B (IL-1b) and NOS2 (nitric oxide synthase 2), impor-tant mediators of inflammation, were upregulated(Supplementary Tables S1 and S4).

In addition, the receptors for granulocyte colony-stimulatingfactor (G-CSF; CSF3R), for VEGF (FLT1, KDR), and for TNF–related apoptosis-inducing ligand (TRAIL; TNFRSF10B) wereupregulated at day 1–2. Noticeably, the upregulation of IL17Bandof its receptor IL17Rwasaccompaniedby increasedexpres-sion of IL1B, of the receptor for TGF-b (TGFBR2), and of FOXH1and GREM1 (TGF-b signaling pathway), which are essential forTh17 differentiation (30; Supplementary Tables S1 and S4).

Among the immune-related genes that CTX induced in thebone marrow, we also found immunoglobulin heavy chain-2(IGH-2) CD22 (BCR coreceptor), and BCR signaling molecules(VAV2, PIK3CG, NFkB; Supplementary Fig. S3B). Moreover,calreticulin (CALR), whose exposure is required for the immu-nogenicity of cell death, 2 alarmins (DEFB2 and DEFCR4), thestress response factor (HSF2), and several soluble and cell-associated pattern recognition receptors (PRR) responsible forthe recognition of apoptotic/necrotic cells, such as mannosebinding lectin (MBL1), FCN1 (ficolin A), PFC (complementprotein properdin), and complement component C1q subu-nits (C1QA and C1QB), were upregulated 1 to 2 days aftertreatment (Supplementary Tables S1 and S4).

The functional classification of downregulated genesshowed that the most significantly enriched biological classesincluded genes regulating cell division, RNA processing, meta-bolic processes, establishment of cellular localization, andchromosome segregation (Table 1). These results suggest thatCTX induces the reduction of biosynthetic/metabolic pro-cesses and cell cycle presumably as a consequence of itscytotoxic activity and that, at the same time, it stimulatesthe expression of a plethora of immune-related genes.

Functional classification of genes differentiallyexpressed in spleen and PBMC by gene ontologyand KEGG pathway analysis

As is the case with bone marrow, the most significantlyenriched biological classes of genes upregulated in the spleenwere related to the regulation of immune response, whereascell cycle, chromosome segregation, and metabolic processclasses were enriched for downregulated genes (Table 1 andSupplementary Table S4). Also, KEGG pathway enrichmentanalysis showed a great similarity between bone marrow andspleen, because enriched pathways included chemokine andBCR signaling pathways, cytokine–cytokine receptor interac-tion, and apoptosis. In addition, in the spleen lysosome andTGF-b signaling pathways were also enriched (Fig. 3B andSupplementary Figs. S4 and S5).

Within immune-related transcripts, some were increasedalso in bone marrow (CCL21C, CXCL7, CXCL12, IL1B, CD22,TGFBR2), whereas others were peculiarly overexpressed in the

Table 1. Functional annotation charts of genesmodulated 1–2 days after CTX administration inbone marrow and spleen

Genes upregulated in bone marrowa Nb Pc

Defense response 27 0.019Pattern specification process 17 0.020Response to external stimulus 29 0.028Behavior 20 0.033Response to chemical stimulus 23 0.044Embryonic development 25 0.045

Genes downregulated in bone marrowa Nb Pc

Cell division 26 1.05E-06RNA processing 25 9.84E-04Primary metabolic process 243 0.0013Cellular metabolic process 243 0.0014Macromolecule metabolic process 214 0.0027Cell cycle 39 0.0076Cellular component organization

and biogenesis103 0.0093

Establishment of cellular localization 41 0.013Establishment of protein localization 35 0.017Chromosome segregation 6 0.041

Genes upregulated in the spleena Nb Pc

Regulation of immune systemprocess

11 0.0028

Immune response 25 0.0082Cellular developmental process 72 0.0131Death 33 0.0157Leukocyte activation 15 0.0178Macromolecule localization 33 0.0249Regulation of multicellular organismal

process18 0.0271

Cell proliferation 27 0.0299Cellular localization 34 0.0386Cellular component organization 83 0.0471

Genes downregulated in the spleena Nb Pc

Cell cycle 43 1.81E-06Cell division 22 2.21E-06Chromosome segregation 8 6.00E-04Secondary metabolic process 6 0.0171Cellular metabolic process 181 0.0406

aFunctional annotation charts by means of DAVID bioinfor-matic tool (biological process, level 2).bNumber of genes in each class.cEASE score, amodified Fisher exactP-value; onlyP-values� 0.05 were considered. The entire list of genes present onthe array was used as background.

Moschella et al.





spleen, such as the C-type lectin receptors (CLR) CLEC4A2(DCIR), CD209B (SIGNR-1), CLEC4N (Dectin-2), and CLEC7A(Dectin-1), involved in the uptake of apoptotic cells by DC, andgenes involved in CLR signal-transduction pathway (SYK, BCL-10). Noteworthy, PTX3, which inhibits the capture of apoptoticcells by DC, was downregulated after treatment. Moreover, weobserved the induction of genes associated with variousprocesses of DNA repair, cell death, autophagy, and drugresistance (DDIT4, HIF1A, CLU, ATG7, HSP70–2; Supplemen-tary Tables S2 and S4).Additional upregulated genes comprised AHR (regulator of

Treg and Th17 cell differentiation), factors regulating inflam-mation (IL-1 family members IL1B, IL1R1, IL1RAP) and cellproliferation (TNFSF13-APRIL, TNFSF13B-BAFF), and growthfactors acting on different cell populations (FGF1, HBEGF,IGF1, PDGFB, VEGFA; Supplementary Fig. S4 and Tables S2and S4). Noticeably, IL2RG (IL-2 receptor g chain), which isinvolved in the response to homeostatic cytokines IL-2, IL-7,IL-9, IL-15, and IL-21, was overexpressed following treatment,corroborating previous findings indicating that CTX increasesthe expression of homeostatic cytokines (16).Although in PBMC no significant enrichment of any biolo-

gical process class was found within upregulated genes, wecould identify some cytokine-encoding transcripts (IL12A,

IL1F8, IL17A). As is the case with the spleen, CTX inducedthe expression of CD164 (expressed by primitive HSC), of theCLR CD209B and ofHIF1A (Supplementary Table S3). Only onebiological process (establishment of cellular localization) wasenriched within downregulated genes (data not shown). More-over, the results of KEGG pathway analysis were not similar toto those for bone marrow or spleen (Fig. 3C).

To directly compare the correspondence of bone marrow,spleen, and PBMC transcriptional profiles, the overlap of thegene lists was examined in Venn diagrams. Interestingly, thespleen displayed a profile intermediate between bone mar-row and PBMC, which were the least similar to each other(Fig. 3D and E); this could be related to the fact that, in mice,the spleen is an active hematopoietic organ and, at the sametime, comprises fully mature cells. The most significantlyenriched biological classes of genes simultaneously upregu-lated in bone marrow and spleen were defense response andresponse to stress and to external stimuli (data not shown).Altogether these results suggest that hematopoietic organs(spleen and bone marrow) are the most responsive organs tothe damage mediated by CTX and react to it by activatingimmune-related genes.

To validate the results obtained by transcriptional profiling,the expression of selected genes was assessed by real-time

Chemokine signaling pathway

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Pathways in cancer

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519 42 321 438

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Figure 3. Plots illustrating KEGG pathways significantly enriched within genes upregulated by CTX and Venn diagrams indicating the degree ofoverlap between gene expression profiles of bone marrow (BM), spleen, and PBMC. (A, B, C) The genes upmodulated 1–2 days after CTX treatment wereclustered in different KEGG pathways by using DAVID and the significantly enriched pathways (P � 0.05, EASE score) are shown for BM (A), spleen(B), and PBMC (C). The lists of genes significantly up- (D) and downregulated (E) 1–2 days following CTX treatment were used to create the Venn diagrams. Thenumber of genes exhibiting common (or uncommon) modulation of expression in each tissue is shown.






PCR. In accordance with the results obtained by microarray,CARL and CLU transcript levels were augmented early aftertreatment in bone marrow and spleen (but not in PBMC).Moreover, although the expression of IL1B was increased atday 2 and decreased at day 5 in all tissues, CDCA8 wasdownregulated at day 2 and returned to baseline by day 5(Supplementary Fig. S7 and Supplementary Methods).

Effect of CTX on T-helper subpopulationsBecause Th17 cells were shown to exert a protective role

against murine cancer by providing a more significant helpthan Th1 cells for tumor-specific CD8þ T cells (31), the novelobservation of a Th17-related gene signature induced by CTXprompted us to evaluate, in the spleen, the modulation of thisT-cell subtype at different times following CTX injection. Themodulation of Th1, Th17, Tregs (CD4þ/CD25þ/Foxp3þ), andactivated CD4þ T cells (CD4þ/CD25þ/Foxp3�) was examinedsimultaneously by intracellular staining followed by FACSanalysis. Interestingly, the absolute numbers of IFN-g– andIL-17–producing CD4þ T cells and those of activated CD4þ/CD25þ/Foxp3� lymphocytes underwent a transient decrease7 days following chemotherapy and were significantly (P �0.05) augmented above the levels of untreated mice at day 10and 14 (Fig. 4A). Th1, as expected, showed the most pro-nounced expansion, reaching a 6-fold increase compared withuntreated mice, and Th17 and activated CD4þ T cells under-went a 2.5 and a 3.8 increment, respectively. We did not findCD4þ T cells coexpressing IL-17 and IFN-g (data not shown).In contrast, Treg cell numbers, although showing kinetics ofdepletion similar to those of the other CD4þ subpopulations,

exhibited a lower rebound, evident at a later time (significantonly at day 14; Fig. 4A). These results indicate that CTXinduces an imbalance in CD4þ T-cell subtypes, favoringeffective immunity and disadvantaging tolerance, and corro-borate recent findings suggesting that CTX induces Th17 cellsin cancer patients (32). However, it should be noted that Tregsare only held back with respect to other CD4þ populations,because their number is anyway restored to pretreatmentlevel, indicating that this is a transient imbalance and not apermanent depletion. In Fig. 4B, the effect of CTX on lym-phocytes and CD4þ T cells is shown. CTX injection induced atransient depletion of lymphocytes followed by a rebound oftheir number above baseline without significantly altering theproportion of CD4þ T cells (Fig. 4B).

CTX treatment stimulates a "storm" ofcytokines, chemokines, and growth factors

To characterize the cytokine response to CTX at the proteinlevel, plasma and bone marrow lysates from tumor-bearingmice were subjected to Bio-Plex cytokine assays, whichallowed the simultaneous analysis of 32 analytes.

Three days (7 for IL-9) after chemotherapy, the levels ofIL-3, IL-5, IL-9, CCL2, CCL4, CCL11, G-CSF, and GM-CSFwere significantly (P � 0.05) increased in plasma withrespect to untreated mice, whereas IL-12, CXCL2, andmacrophage colony-stimulating factor (M-CSF) werereduced (Fig. 5A, B, and C).

Regarding the bone marrow, CTX induced a transientdepletion of cell numbers (and accordingly of their totalprotein content) at day 3, and a rebound, overcoming baseline,

A

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0,56

Figure 4. Effect of CTX treatment on CD4þ T-cell subsets. A, representative FACS analysis (out of 2 independent experiments) showing the effect ofCTX on the absolute number of IFN-g-producing (Th1), IL-17-producing (Th17), and activated (CD4þCD25þFoxp3�) T-helper cells and Treg cells(CD4þCD25þFoxp3þ) in the spleen. Analyses were conducted by gating on lymphocytes. Results are shown as fold change of absolute numbers of each cellsubtype with respect to untreated mice. Data represent the mean fold change of 3 individual mice versus 3 untreated mice. *, P � 0.05, unpaired t-test.B, Effect of CTX treatment on the number of lymphocytes and CD4þ T cells. Data represent the mean absolute number of cells � SE of 3 mice pergroup. C, representative dot plots showing percentages of Th1 and Th17 in untreated versus CTX-treated mice (day 10).

Moschella et al.





0 0

IL-3 2.63 3.19 4.18* 2.52 2.61IL-5 11.78 13.13 23.35* 17.32 10.53

IL-9 197.62 244.31 332.80 577.23* 315.02IL-12(p40) 424.66 461.28 311.64* 245.84* 502.17

day 0 day 1 day 3 day 7 day 10

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542.9 633.55 1103.5* 793.81 654.01

248.1 304.98 419.33* 249.94 216.7458.1 63.93 85.25* 58.85 67.48

12.1 11.00 11.55 8.32* 6.28*CCL11

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M-CSF 1995 1583* 1819 1678* 1536*G-CSF 96.6 151.8 204.9* 79.8 97.4

GM-CSF 84.4 110.4 130.5* 101.8 104.2


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0.03 0.04* 0.16* 0.02 0.02

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4.58 7.47 14.10* 1.66 3.480.53 1.05* 3.27* 0.33 0.31

0.01 0.05* 0.18* 0.02 0.01

0.04 0.11* 0.49* 0.02* 0.03

0.49 0.82 2.22* 0.41 0.44


CXCL9CXCL2

LIF

CCL5CXCL1

CCL4CCL3CCL2

PDGF-b 33.54 49.23* 117.2* 56.96* 26.05VEGF 44.59 90.23* 5.97* 53.77 37.17FGF-2 0.33 0.93* 2.88* 0.50 0.68M-CSF 0.67 1.04* 0.66 0.63 0.79G-CSF 0.07 0.19* 0.12 0.03 0.09GM-CSF 0.14 0.27 1.08* 0.04 0.07

day 0 day 1 day 3 day 7 day 100 0

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Figure 5.CTXmodulates the protein levels of cytokines, chemokines, and growth factors in plasma and bone marrow (BM). Before (day 0) and at the indicatedtime points after CTX, plasma and BM cells were collected from tumor-bearing mice and BM was lysed in Bio-Plex cell lysis buffer. The variation ofthe protein levels of cytokines (A, D), chemokines (B, E), and growth factors (C, F) was analyzed by Bio-Plex cytokine assay. Data represent themean � SE of the protein levels from 3 individual mice tested in duplicate. *, P � 0.05, unpaired t-test.






at day 10 (Supplementary Fig. S6). Nevertheless, the analysis ofcytokine expression per cell, that is, after normalizing theconcentration of each cytokine to the number of cells at eachtime point, showed that the amount of several cytokines (IL-1b, IL-9, IL-12, IL-15, IL-18, and IFN-g) increased significantly(P� 0.05) at day 3 and went back to baseline or below baselineby day 7 (Fig. 5D). Noticeably, the chemokines CCL2, CCL3,CCL4, CCL5, CXCL1, CXCL2, CXCL9, and LIF and the growthfactors platelet-derived growth factor (PDGF)-basic, fibroblastgrowth factor (FGF)-2, M-CSF, G-CSF, and GM-CSF followedthe same kinetics (Fig. 5E-F). Of note, in both plasma and bonemarrow, the majority of protein expression changes took placeearly (day 3), immediately following gene expression modula-tion (day 1 and 2) and preceding the recovery of cell numbersand, as for transcript, the protein expression modulation wastransient.

These data not only represented the validation at theprotein levels of some of the changes in gene expressionassessed either by microarray analysis (i.e., IL-1b, CXCL2,PDGF, FGF) or previously by real-time PCR (IL-15, IFN-g ,GM-CSF; ref. 16), but also further expanded this informa-tion pointing out the many cytokines, chemokines, andgrowth factors regulating the recovery of bone marrowcells after the myelosuppressive stress exerted by CTXand constituting the microenvironment capable of support-ing HSC proliferation, differentiation, and mobilization tothe circulation.

The antitumor efficacy of the combined therapydepends on the timing of combination

The kinetics of gene and protein expression modulationshowed that CTX-mediated immunomodulation is inducedearly after treatment and rapidly turned off. On the basis ofthese results, we investigated whether the timing of optimalcombination of CTX and ACT was congruent, and possiblydependent, on the kinetics. As shown in Fig. 6, the antitumorefficacy of chemoimmunotherapy was maximal when ACTwas carried out either 5 hours or 1 day after chemotherapy (i.e., at the time of the majority of gene expression changes),resulting in the cure of 5 out of 6 and 4 out of 6 tumor-bearingmice, respectively, and was completely lost if donor cells wereinfused 3 days after chemotherapy, indicating that transferredcells should be infused as early as possible to enable thebenefit of the CTX-mediated cytokine milieu.

Discussion

Different studies have addressed systematically the effectsof CTX on several of the multiple cells and factors drivingtumor-induced tolerance and antitumor immunity (reviewedin ref. 2). However, such hypothesis-driven approaches,although deeply clarifying the contribution of each singleactor, do not take into account the complexity of CTX-mediated immunomodulation. High-throughput analyses,such as genomics and proteomics, can efficiently complement

CTR

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Figure 6. The timing of thecombination of CTX and adoptiveimmunotherapy dictates theantitumor efficacy. Mousetreatment was done as shownin Fig. 1. In mice treated withchemoimmunotherapy, ACT wascarried out either 5 hours (day 0),or at different times (day 1, 2, 3, 6,and 9) after chemotherapy. Dataare reported as the average tumordiameter of 6 mice per group �SE. The number of surviving mice(out of 6) is indicated.

Moschella et al.





these type of studies, giving a comprehensive picture of acomplex situation, uncovering unknown factors and drivingnew hypotheses. Previously, the antitumor efficacy of che-moimmunotherapy was believed to mainly rely on the induc-tion of lymphopenia by chemotherapy, which creates "room"for the adoptive transfer of tumor-specific lymphocytes (10,11), or on the selective depletion of Tregs (12, 14). AlthoughCTX indeed depletes Tregs in vivo, we and others have shownthat these cells can repopulate the periphery (Fig. 4; ref. 33),highlighting the transient and possibly limiting nature of thishypothesis. We argued that the tolerogenic milieu induced bytumors is multifactorial and includes Tregs as well as otherfactors and that CTX conditioning can not only mitigatetolerogenic mechanisms but also produce a multifactorialimmunogenic milieu favoring immunotherapeutic interven-tions. Consistent with such a view, the present study wasdesigned to unravel the multiple factors underlying the abilityof CTX to potentiate cancer immunotherapy. Taken together,microarray analyses show that CTX modulates the expressionof an unexpectedly high number of genes in lymphoid organs,leading on one hand to the downregulation of genes involvedin cell cycle and metabolic processes, which is in accordancewith the inherent toxicity of an anticancer agent, and, on theother hand, to the upmodulation of immunomodulatorygenes. An important finding, stemming from both genomicand proteomic analyses, is that CTX-mediated immunomo-dulation is early and transient, as previously shown in adifferent tumor model (RBL-5 lymphoma), by real-time PCRanalyses of a limited panel of cytokines (GM-CSF, IL-1 b, IL-7,IL-15, IL-2, IL-21, IL-13, and IFN-g); ref. 16). These data pointout that, to be effective, chemotherapy and immunotherapyshould be combined within a short time window. Remarkably,this hypothesis was confirmed by tumor growth experimentsshowing that the synergistic antitumor effectiveness of CTXand ACT strictly depends on the timing of combination and isprogressively lost as days pass between 1 treatment and theother (Fig. 6).Recent data suggest that the antitumor efficacy of che-

motherapy (especially anthracyclines) depends on the induc-tion of immunogenic apoptosis of tumor cells that stimulateantitumor immunity (34). Key elements dictating the immu-nogenicity of anthracycline-mediated cell death are the sur-face exposure of CARL and the release of HMGB1, which, inturn, activate TLR-4 (27). Studies from our group showed thatCTX induces CARL exposure and release of DC-activatingsoluble factors by apoptotic tumor cells, resulting in increasedcross-presentation of tumor antigens to CD8þ T cells (35).Here we show for the first time that CTX can augment, in bonemarrow cells, the simultaneous expression of CARL and of 2PRRs (MBL1 and C1Q) involved in its recognition and inapoptotic cell clearance (36). In addition, CTX augmentedthe transcript levels of other danger signals, that is, thealarmins DEFCR4 (defensin-a-4) and DEFB2 (defensin-b-2),the first involved in macrophage and T-cell chemoattractionand the second able to induce DC maturation via TLR-4 (thesame receptor of HMGB1; ref. 37).Interestingly, additional DC-specific CLRs were upregulated

in the spleen, namely SIGNR1 and DCIR, involved in CD8þ T-

cell cross-priming (38) and Dectin-1 and Dectin-2, whose rolein the clearance of apoptotic cells has been recently under-scored (39), and that, after ligand engagement, are able toinduce DC activation and Th1/Th17 differentiation via a SYK-dependent signaling pathway that leads to the activation ofNFkB (40–42). Strikingly, the upregulation of Dectins wasassociated with increased expression of SYK and of theadaptor BCL-10. Of note, it is possible to speculate that theTh17 signature induced by CTX and the expansion of Th1 andTh17 cells observed in the rebound phase after chemotherapydepends on the activation of this signaling pathway after"sensing" the damage of chemotherapy by PRR. In this regard,early after CTX injection, we observed the induction of genesassociated with DNA repair, cell death, autophagy, and drugresistance (DDIT4, HIF1A, CLU, ATG7, and HSP70–2; ref. 43–45).

In physiologic settings, the engulfment of apoptotic cells byphagocytes inhibits the release of inflammatory cytokines andleads to T-cell tolerance. The perception of danger signals mayhave a role in tipping the balance from tolerance to inflam-mation and effective T-cell activation (46). Accordingly, weshow here that after CTX treatment several IL-1 familymembers are upregulated in the spleen and both transcriptand protein levels of proinflammatory cytokines (IL-1b, IL-17,IL-18, IFN-g , LIF) and chemokines (CCL2, CCL3, CCL4, CCL5;ref. 47) are augmented in bonemarrow. Remarkably, IL-1b andIL-18, the most important mediators of inflammation, controlinnate and adaptive (including Th1-Th17) responses (48).Moreover, CTX increased the expression of NOS2, an enzymeresponsible for the generation of free radical NO and involvedin inflammation, the expression of which has been shown tobe triggered by IL-17 via the transcription factor NFkB (49).The proinflammatory action of IL-17 induces the expression oftarget genes such as CXCL1, CXCL2, and defensins (50), whichwere indeed augmented in bone marrow and plasma. IL-17 isalso an important modulator of hematopoiesis through theinduction of GM-CSF, G-CSF, and CCL2 (51), which wereaugmented in bone marrow lysates and plasma. Moreover,HIF1A and its target gene VEGFA levels were increased in thespleen, which is in accordance with data showing that CTXinduces hypoxia along with HIF1A and VEGFA upregulation,playing a key role in inflammation and hematopoiesis (52).

The hypothesis stemming from these data is that CTXinduces an immunogenic apoptosis not only of tumor cellsbut also of host bone marrow and spleen cells and, at the sametime, activates inflammatory mediators, leading to the pro-motion of hematopoiesis and immune response. In accor-dance with this hypothesis, the apoptosis of self T cells wasshown to elicit self-reactive CD8þ T cells, thus inducing asystemic immunoactivation during HIV infection (53). Thispremise also suggests that CTX may be suitable for enhancingthe efficacy of immunotherapy in adjuvant settings, in whichmounting an effective vaccine-induced antitumor immunityin the absence of tumor burden is fundamental to preventrecurrences. In this regard, we have recently shown thatdacarbazine, an alkylating agent similar to CTX, can enhanceCD8þ memory T-cell responses to peptide-based vaccinationin resected melanoma patients (54).






We have previously shown that the antitumor effectivenessof chemoimmunotherapy depends on adoptively transferredCD4þ T cells (16) and, among them, Th17 was proved toexhibit a stronger therapeutic efficacy than Th1 (31). Severalauthors reported differential effects of CTX on CD4þ T cells,including a Th2 to Th1 shift in cytokine production (16, 55).The present data reveal for the first time that CTX induces anenvironment simultaneously favoring Th1, Th17, and acti-vated CD4þ T cells, suggesting that this milieu may conditionnot only host cells but also transferred tumor-specific CD4þ

T cells, thus leading to improved tumor control.Taken together, our data sustain the fascinating hypoth-

esis that CTX-mediated immunomodulation stems directlyfrom its cytotoxicity that produces DNA damage, cell cyclearrest, and cell homeostasis perturbation not only of tumorcells but also of host highly proliferating cells, such as bonemarrow and splenocytes. DNA-damage responses are com-plex and involve "sensor" proteins perceiving danger andtransmitting signals to "transducer" proteins, which, in turn,convey signals to "effector" factors (56). Cells damaged orkilled by CTX within lymphoid organs would thereforeexpose/release danger signals that in turn, through PRRsignaling, would transiently activate proinflammatory path-ways and create a beneficial environment aimed at repairingthe damage and able, as a bystander effect, to promoteinnate and adaptive immunity, including Th1 and Th17responses. When an immune intervention (vaccination orACT) is carried out during the recovery phase, also tumor-specific immune cells can take advantage of this drug-

mediated immunomodulation. Although with the presentexperimental design we could not ascertain in which cellsubtype the expression of a certain gene or protein wasmodulated, this study describes for the first time all thesensor, transducer, and effector factors involved in the hostresponse to CTX, opens novel hypotheses that may guidefurther experimentation, and, lastly, supplies a clear-cutindication of the short time window in which chemotherapyand ACT should be combined to maximize chemoimmu-notherapy effectiveness against cancer.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We are grateful to Cinzia Gasparrini, Annamaria Fattapposta, and AngelaFresolone for helpful secretarial assistance.

Grant Support

The research was partially supported by grants from the Italian Associa-tion for Research against Cancer (AIRC) and from the Italian Ministry ofHealth (Integrated Project on Oncology and "ISS Project for Alliance againstCancer").

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

Received December 17, 2010; revised February 28, 2011; accepted March 12,2011; published OnlineFirst March 28, 2011.

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2011;71:3528-3539. Published OnlineFirst March 28, 2011.Cancer Res Federica Moschella, Mara Valentini, Eleonora Aricò, et al. CyclophosphamideGene and Protein Expression Profiling of Responses to Unraveling Cancer Chemoimmunotherapy Mechanisms by

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