3528.Full-Text.Pdf

Published OnlineFirst March 28, 2011; DOI: 10.1158/0008-5472.CAN-10-4523

Cancer Microenvironment and Immunology Research

Unraveling Cancer Chemoimmunotherapy Mechanisms by Gene and Protein Expression Profiling of Responses to Cyclophosphamide

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

Abstract Certain 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 argued that 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 CTX on gene expression in bone marrow, spleen, and peripheral blood, and on cytokine expression in plasma and bone marrow 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 recognition receptors, inflammatory mediators, growth factors, cytokines, chemokines, and chemokine receptors. These factors are involved in sensing CTX myelotoxicity and activating repair mechanisms, which, in turn, stimulate immunoactivation events that promote efficacy. In particular, CTX induced a T-helper 17 (Th17)-related gene þ þ signature associated with an increase in Th17, Th1, and activated CD25 CD4 Foxp3 T lymphocytes and a slight recovery of regulatory T cells. By analyzing gene and protein expression kinetics and their relationship to the antitumor efficacy of different therapeutic schedules of combination, we determined that optimal timing for performing adoptive immunotherapy is approximately 1 day after CTX treatment. Together, our findings highlight factors that may propel the efficacy of chemoimmunotherapy, offering a mechanistic glimpse of the important immune modulatory effects of CTX. Cancer Res; 71(10); 3528–39. 2011 AACR.

Introduction been studied in preclinical models (4–7), as well as in clinical trials (8–11). Although immunotherapy represents a promising nontoxic Several mechanisms have been considered to explain this anticancer strategy, the different treatment modalities paradoxical phenomenon, among them great relevance was employed so far in the clinical setting resulted in limited attributed to the reduction of regulatory T-cell (Treg) number and sporadic successes, highlighting the need for the identi- and function, observed in both the mouse model (12, 13) and fication of effective strategies to enhance its clinical efficacy cancer patients (14). Additional CTX-mediated immunoacti- (1). Mounting evidence suggests that combination therapies vating effects include increased tumor infiltration by lympho- may produce synergistic antitumor responses and that certain cytes, functional activation of B and T cells, and homeostatic chemotherapeutic agents, rather than being immunosuppres- proliferation, occurring during the recovery phase that follows sive can, under defined conditions, act as strong adjuvants for chemotherapy-mediated myelolymphodepletion and that, as a either adoptive or active immunotherapy (2, 3). Currently, bystander effect, can promote antitumor immunity (5, 6, 15, cyclophosphamide (CTX) represents the gold standard immu- 16). Notably, preconditioning the host with CTX also induces þ nomodulatory chemotherapeutic drug, and the antitumor the expression of type I IFN, leading to the expansion of CD4 þ efficacy of its combination with immunotherapy has long and CD8 T cells exhibiting a memory phenotype (17). More recent data indicate that CTX can restore an activated polyfunctional helper phenotype in tumor-specific adoptively 1 þ Authors' Affiliations: Department of Cell Biology and Neurosciences, transferred CD4 T cells through an IFN-a/b-dependent Istituto Superiore di Sanita, and 2Department of Internal Medicine/Rheu- matology, La Sapienza University, Rome, Italy mechanism (18). Further studies underscore the effects of this drug not only on the adaptive but also on the innate Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). immune system. In this regard, it was shown that CTX- þ Corresponding Author: Federica Moschella, Viale Regina Elena 299, induced expansion of vaccine-specific CD8 T cells is asso- 00161, Rome, Italy. Phone: 39-064-990-2474; Fax: 39-064-990-3641. ciated with increased number and activation status of den- E-mail: [email protected] dritic cells (DC; refs. 19, 20). doi: 10.1158/0008-5472.CAN-10-4523 In a previous study, we had shown that CTX acts through 2011 American Association for Cancer Research. the production of hitherto unidentified soluble factors

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sustaining the proliferation, survival, and activity of trans- array slides, and printed with 13,443 70mer oligonucleotides ferred lymphocytes (5). Subsequently, we reported that the (Operon version 1.1; CRIBI Microarray Service, University of synergistic anticancer activity of chemotherapy and immu- Padua, Italy). The platform has been submitted to the Gene notherapy was associated with the induction of a "cytokine Expression Omnibus (GEO) database (accession GPL5098). storm," occurring primarily in the bone marrow of treated mice (16). Upregulated factors included hematopoietic Data analysis growth factors [granulocyte macrophage colony-stimulating Scanning and image file processing were carried out with factor (GM-CSF) and interleukin (IL)-1b], cytokines regulat- the GenePix 4200A instrument (Axon Instruments), and the þ ing homeostatic expansion and memory CD8 T-cell survi- obtained data were filtered with BRB ArrayTool (23) to val (IL-7, IL-15, IL-2, and IL-21) and cytokines involved in exclude spots below a minimum intensity (200), flagged the polarization toward a T-helper (Th)1 type of immune and with diameters less than 25 mm and normalized by using response (IFN-g). The present study aims at investigating Lowess Smoother. Only genes expressed in 70% of samples the mechanisms underlying the immunomodulatory activity were analyzed in subsequent statistical analyses (all done by of CTX by means of genomic and proteomic approaches. In using the log2-tranformed ratios). particular, we evaluated the effect of CTX on the gene Average linkage unsupervised hierarchical clustering, with expression of bone marrow, spleen, and peripheral blood Pearson correlation distance measure, was carried out with mononuclear cells (PBMC) and on the protein levels of 32 genes whose expression differed by at least 1.5-fold from the cytokines in plasma and bone marrow lysates of tumor- median in at least 20% of samples. Following median center- bearing mice at different times after a single chemother- ing, results were visualized with Treeview software (24). apeutic treatment. The overall results provide new insights Statistically significant (P 0.001) differentially expressed into the mechanisms by which CTX can render immune genes between posttherapy and pretreatment samples were lymphocytes capable of eradicating metastatic tumors and identified with supervised class comparison (parametric t- provide a scientific rationale for performing adoptive immu- test) and further analyzed by using Cluster and TreeView notherapy early after chemotherapy. softwares. Gene annotation and KEGG (Kyoto Encyclopedia of Genes Materials and Methods and Genomes) pathway analyses were carried out by means of DAVID (Database for Annotation, Visualization, and Inte- Animals, tumor cells, and chemoimmunotherapy grated Discovery) bioinformatic tool (25). Enriched biological Six- to 7-week-old DBA/2 mice (Harlan) were implanted processes and pathways were ranked according to EASE score, with 2 106 IFN-a/b-resistant, in vivo passaged, highly indicating the abundance of genes fitting each class in pro- metastatic 3Cl-8 Friend leukemia cells (3Cl-8 FLC; ref. 21). portion to the number of genes expected to be in each class by CTX (Sigma–Aldrich) was administered intraperitoneally chance, calculated on the global composition of the array. 4 days after tumor injection. Five hours later, some mice received the adoptive transfer of 40 106 lymphomonocytes Intracellular cytokine staining and flow cytometry derived from spleens of tumor-immunized mice, prepared as Splenocytes were stratified over Lympholyte-M Gradient in ref. 16. Vaccination was carried out with IFN-a-producing (Cederlane) for lymphocyte enrichment. For Treg and acti- þ tumor cells (IFN-a1-Cl-11; ref. 22). Mice were vaccinated with vated CD4 lymphocyte analyses, cells were stained with anti- either irradiated (100 Gy) cells or a tumor lysate obtained from CD4-FITC and anti-CD25-PE (BD Biosciences), and fixed, 3 freeze-thaw cycles. Vaccination was carried out twice (3 permeabilized, and stained with anti-Foxp3-APC according times for lysates) at a time interval of 2 weeks. The experi- to the manufacturer's instructions (eBioscience). mental protocols were approved by the Istituto Superiore di For Th1 and Th17 analyses, cells were stimulated overnight Sanita Review Board. with 100 ng/mL PMA and 1 mg/mL Ionomycin, in the presence of 1 mg/mL Brefeldin-A and 0.7 mg/mL Monensin (GolgiPlug, RNA isolation, labeling, and hybridization GolgiStop, BD-Bioscience) to prevent cytokine secretion. Fol- Total RNA was obtained by TRIZOL reagent extraction lowing staining with anti-CD4-FITC, cells were fixed and (Invitrogen) for spleens and by RNeasy (Qiagen) purification permeabilized, and intracellular staining was carried out by for bone marrow cells (femurs and tibias) and PBMC using anti-IFN-g-PE-Cy7 and anti-IL-17-APC (e-Bioscience). (obtained by gradient centrifugation with Lympholyte-Mam- Acquisition and analysis were carried out with FACSCalibur mals-Cederlane). flow cytometer (BD-Bioscience) and FlowJo software. Produc- þ Amino-allyl modified antisense RNA (aRNA) was synthe- tion of IFN-g and IL-17 was evaluated in CD4 in both sized in 1 amplification round from 2 mg total RNA by using unstimulated (data not shown) and PMA/I stimulated sam- the Amino Allyl MessageAmp II aRNA Amplification Kit ples. (Ambion), in accordance with manufacturer's instructions, and its quality was assessed with the 2100 Bioanalyzer (Agilent Multiplex proteomic analysis of cytokines, chemokines, Technologies). aRNAs were coupled to monoreactive Cy3 or and growth factors Cy5 dyes (GE Healthcare), fragmented (RNA Fragmentation bone marrow cells were obtained from femurs and tibias. Reagents, Ambion), mixed, and cohybridized overnight in After lysing erythrocytes through a 3-minute incubation in humidifying chambers at 42 C onto prehybridizated micro- 0.16 mol/L Tris-buffered NH4Cl (pH 7.2), cells were lysed in

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Bio-Plex cell lysis buffer. The mouse group I (23-plex) and IFN-a1-Cl-11 resulted in complete tumor regression in 100% of group II (9-plex) Bio-Plex cytokine assays were carried out mice with no recurrences or metastases (Fig. 1A). ACT from according to the manufacturer's instructions (Bio-Rad). Beads nonvaccinated mice had no effect either alone or in combina- were read on the Bio-Plex 200 suspension array system, and tion with CTX (data not shown). Noteworthy, when chemother- data were analyzed by using Bio-Plex Manager software. apy was followed by ACT from tumor lysate-vaccinated mice, tumor growth was delayed (more than with CTX alone), but no Results tumor regressed (Fig. 1B). These results, along with previously published data (5, 16), show that the therapeutic effectiveness of The combination of CTX and adoptive immunotherapy this chemoimmunotherapy approach depends on CTX- results in complete tumor regression mediated immunomodulatory mechanisms potentiating adop- A highly metastatic tumor model, 3Cl-8 FLC (21), was used tive immunotherapy and that also the quality of the antitumor to assess the antitumor effectiveness of the combination of a immunity induced in donor mice has an impact on the out- single CTX injection and adoptive cell transfer (ACT) of come. Irradiated tumor cells may, in fact, still produce IFN-a, lymphomonocytes from tumor-vaccinated syngeneic mice, which influences the quality of the vaccine-induced immune according to previously published protocols (5). Donor mice response (26). In addition, irradiation has been shown to induce were vaccinated with either irradiated (Fig. 1A) or lysed an immunogenic cell death leading to optimal immunization of (Fig. 1B) IFN-a–producing tumor cells (IFN-a1-Cl-11), based mice against subsequent tumor challenge (27). on the fact that these cells were shown to be effective cancer vaccines (22). Global gene expression analysis of the effects of CTX on ACT alone had no significant effect on tumor growth (Fig. 1) bone marrow, spleen, and PBMC and mouse survival (data not shown), whereas CTX alone To gain insights into the CTX-mediated effects responsible induced a transient delay in tumor growth but ultimately for the antitumor efficacy of chemoimmunotherapy, we eval- tumors developed in all mice. In contrast, the sequential treat- uated the impact of a single CTX treatment on the gene ment with CTX and ACT from mice vaccinated with irradiated expression 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. 18 The experiment was carried out in tumor-bearing mice to take 16 A into account the possible immunomodulation induced by the 14 tumor implant itself. The data discussed in this article have 12 10 been deposited in National Center for Biotechnology 8 Information's GEO and are accessible through GEO Series accession number GSE27440 (28). 6 CTR 4 To get an overall picture of the transcriptional modulation 2 by CTX and of its kinetics, data were preliminarily subjected to CTX 0 unsupervised hierarchical clustering analysis (Fig. 2A). In both 18 lymphoid organs and PBMC, this analysis segregated samples 16 B ACT into 2 principal clusters reflecting distinct gene expression 14 patterns, one including ctr and samples taken 5 days post- 12 CTX+ACT treatment and the other containing samples from day 1 and 2, 10 showing that CTX has a strong impact on gene expression, 8 inducing at early time points (1–2 days) the modulation of Mean tumor diameter (mm ± SE) 6 a large number of genes, that, however, return to baseline by 4 day 5. 2 To identify genes undergoing a statistically significant dif- 0 0 5 10 15 20 25 30 35 40 45 ferential expression, microarray data were reanalyzed by using a supervised approach (class comparison), followed by cluster- Days after tumor implant ing analysis. Class comparison was conducted gathering in one class samples from day 1 and 2 and comparing them with ctr samples, based on the fact that, on separate comparisons Figure 1. Antitumor efficacy of the combination of CTX and adoptive between day 1, day 2, and day 5 versus ctr, most genes under- immunotherapy. DBA/2 mice were implanted s.c. with 2 106 3Cl-8 FLC. After 4 days, some mice (n ¼ 5) were injected with either CTX (83 mg/kg) or going a statistically significant modulation of expression at saline (CTR) and others received the adoptive transfer of 40 106 spleen day 1 showed a similar profile at day 2 (Supplementary Fig. S1). lymphomonocytes from tumor-vaccinated donor mice (ACT). In mice In the bone marrow, 1 to 2 days after CTX administration, treated with chemoimmunotherapy, ACT was carried out 5 hours after CTX the expression of 1,123 genes was significantly (P 0.001) injection (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 the modulated with respect to ctr (580 overexpressed and 543 average tumor diameter of 5 mice per group SE. Representative downregulated; Fig. 2B and Supplementary Table S1). Most experiments out of 5 are shown. transcripts returned to baseline levels at day 5.

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Unsupervised

A BM Spleen PBMC Day 1 Day 2 ctr Day 5 Day 1/2 ctr/Day 5 ctr Day 5 Day 1/2 A3-a A3-b A4-a A4-b A1-a A1-b A2-a A2-b B3-a B3-b B2-a B2-b B4-a B4-b B1-a B1-b D1-a D1-b D3-a D3-b D4-a D4-b C3-a C3-b C4-a C4-b C1-a C1-b C2-a C2-b B1-a B1-b B2-a B2-b B3-a B3-b B5-a B5-b B4-a B4-b A1-a A1-b A2-a A2-b A5-a A5-b A3-a A3-b A4-a A4-b B4-a B4-b B2-a B2-b B5-a B5-b B3-a B3-b A1-a A1-b A5-a A5-b A3-a A3-b A2-a A2-b A4-a A4-b C2-a C2-b C1-a C1-b C3-a C3-b C4-a C4-b C5-a C5-b D1-a D1-b D2-a D2-b D3-a D3-b D5-a D5-b D4-a D4-b C1-a C1-b C2-a C2-b C5-a C5-b C3-a C3-b C4-a C4-b D1-a D1-b D5-a D5-b D3-a D3-b D2-a D2-b D4-a D4-b

Supervised

B BM Spleen PBMC ctr Day 5 Day 1/2 ctr/Day 5 Day 1/2 Day 5 ctr Day 5 Day 1 Day 2 A1-a A1-b A2-a A2-b A3-a A3-b A5-a A5-b A4-a A4-b B1-a B1-b B4-a B4-b B2-a B2-b B3-a B3-b B5-a B5-b A2-a A2-b A4-a A4-b A3-a A3-b A1-a A1-b A5-a A5-b B4-a B4-b B5-a B5-b B2-a B2-b B3-a B3-b A1-a A1-b A2-a A2-b A3-a A3-b A4-a A4-b B1-a B1-b B2-a B2-b B4-a B4-b B3-a B3-b D1-a D1-b D2-a D2-b D3-a D3-b D4-a D4-b D5-a D5-b C1-a C1-b C3-a C3-b C4-a C4-b C5-a C5-b C2-a C2-b D1-a D1-b C1-a C1-b C5-a C5-b C3-a C3-b C2-a C2-b C4-a C4-b D2-a D2-b D4-a D4-b D5-a D5-b D3-a D3-b D1-a D1-b D3-a D3-b D4-a D4-b C1-a C1-b C3-a C3-b C2-a C2-b C4-a C4-b

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 CTX treatment. After amplification, aRNAs were labeled with Cy5, mixed with Cy3-labeled "reference" aRNA (from a pool of the BM of untreated mice) and hybridized to 13.5 k oligonucleotide-based microarray. Each oligonucleotide was spotted as duplicate on the slide allowing technical replicates for each sample (indicated as -a and -b). A, unsupervised hierarchical clustering of genes displayed a 1.5-fold change from the median expression value for that gene 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 a global 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.

In case of spleen, 868 genes were significantly differentially were subjected to gene ontology–based and KEGG pathway- expressed at day 1–2 (445 overexpressed and 423 downregu- based annotation by means of the DAVID bioinformatic tool lated; Fig. 2B and Supplementary Table S2). Noteworthy, day 5 (29). Interestingly, the biological processes found to be more samples were more similar to day 1–2 samples than to ctr, significantly (P 0.05) stimulated by CTX in the bone marrow indicating that gene expression changes in the spleen are included genes regulating defense response, developmental more durable than in the bone marrow. processes, and response to external/chemical stimuli (see In PBMC, 1,084 genes were significantly modulated by CTX Table 1 and Supplementary Table S4, for a detailed list of (557 overexpressed and 527 downregulated), all of which went upregulated genes). The most significantly represented KEGG back to baseline by day 5 (Fig. 2B and Supplementary Table S3). pathways included chemokine and B-cell-receptor (BCR) signaling pathways, cytokine–cytokine receptor interaction, and Functional classification of genes differentially apoptosis (Fig. 3A and Supplementary Figs. S2 and S3). Both expressed in bone marrow by gene ontology and analyses indicated that upregulated genes comprised mostly KEGG pathway analysis immune-related genes. To characterize the observed transcriptional profiles Increased transcripts included several chemokines, that is, according to biological function and to identify functional CCL21C, CCL21B (ligands for CCR7), CXCL2 (MIP-2, chemo- and molecular pathways involved in CTX-mediated immuno- tactic for polymorphonuclear leukocytes and hematopoietic modulation, the lists of upregulated and downregulated genes stem cells, HSC), CXCL7 (proplatelet basic protein) and

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CXCL12, þ Table 1. Functional annotation charts of genes (chemoattractant for CD133 HSC). Upregulated che- CCR6 CCR9 IL8RB modulated 1–2 days after CTX administration in mokine receptors included , , and (Supple- mentary Fig. S2 and Tables S1 and S4). Of note, CTX treatment bone marrow and spleen also induced increased expression of genes involved in cyto- JAK3 GRK6 PIK3CG Genes upregulated in bone marrowa Nb Pc kine/chemokine signaling pathways ( , , , VAV2, IKBKG, and NFkB; Supplementary Fig. S3A). Accord- IL1B NOS2 Defense response 27 0.019 ingly, (IL-1b) and (nitric oxide synthase 2), impor- Pattern specification process 17 0.020 tant mediators of inflammation, were upregulated Response to external stimulus 29 0.028 (Supplementary Tables S1 and S4). Behavior 20 0.033 In addition, the receptors for granulocyte colony-stimulating CSF3R FLT1 KDR – Response to chemical stimulus 23 0.044 factor (G-CSF; ), for VEGF ( , ), and for TNF Embryonic development 25 0.045 related apoptosis-inducing ligand (TRAIL; TNFRSF10B) were upregulated at day 1–2. Noticeably, the upregulation of IL17B and of its receptor IL17R was accompanied by increased expres- a b c Genes downregulated in bone marrow N P sion of IL1B, of the receptor for TGF-b (TGFBR2), and of FOXH1 and GREM1 (TGF-b signaling pathway), which are essential for Cell division 26 1.05E-06 Th17 differentiation (30; Supplementary Tables S1 and S4). RNA processing 25 9.84E-04 Among the immune-related genes that CTX induced in the Primary metabolic process 243 0.0013 bone marrow, we also found immunoglobulin heavy chain-2 Cellular metabolic process 243 0.0014 (IGH-2) CD22 (BCR coreceptor), and BCR signaling molecules Macromolecule metabolic process 214 0.0027 (VAV2, PIK3CG, NFkB; Supplementary Fig. S3B). Moreover, Cell cycle 39 0.0076 calreticulin (CALR), whose exposure is required for the immu- Cellular component organization 103 0.0093 nogenicity of cell death, 2 alarmins (DEFB2 and DEFCR4), the and biogenesis stress response factor (HSF2), and several soluble and cell- Establishment of cellular localization 41 0.013 associated pattern recognition receptors (PRR) responsible for Establishment of protein localization 35 0.017 the recognition of apoptotic/necrotic cells, such as mannose Chromosome segregation 6 0.041 binding lectin (MBL1), FCN1 (ficolin A), PFC (complement protein properdin), and complement component C1q subu- Genes upregulated in the spleena Nb Pc nits (C1QA and C1QB), were upregulated 1 to 2 days after treatment (Supplementary Tables S1 and S4). Regulation of immune system 11 0.0028 The functional classification of downregulated genes process showed that the most significantly enriched biological classes Immune response 25 0.0082 included genes regulating cell division, RNA processing, meta- Cellular developmental process 72 0.0131 bolic processes, establishment of cellular localization, and Death 33 0.0157 chromosome segregation (Table 1). These results suggest that Leukocyte activation 15 0.0178 CTX induces the reduction of biosynthetic/metabolic pro- Macromolecule localization 33 0.0249 cesses and cell cycle presumably as a consequence of its Regulation of multicellular organismal 18 0.0271 cytotoxic activity and that, at the same time, it stimulates process the expression of a plethora of immune-related genes. Cell proliferation 27 0.0299 Cellular localization 34 0.0386 Functional classification of genes differentially Cellular component organization 83 0.0471 expressed in spleen and PBMC by gene ontology and KEGG pathway analysis As is the case with bone marrow, the most significantly Genes downregulated in the spleena Nb Pc enriched biological classes of genes upregulated in the spleen Cell cycle 43 1.81E-06 were related to the regulation of immune response, whereas Cell division 22 2.21E-06 cell cycle, chromosome segregation, and metabolic process Chromosome segregation 8 6.00E-04 classes were enriched for downregulated genes (Table 1 and Secondary metabolic process 6 0.0171 Supplementary Table S4). Also, KEGG pathway enrichment Cellular metabolic process 181 0.0406 analysis showed a great similarity between bone marrow and spleen, because enriched pathways included chemokine and aFunctional annotation charts by means of DAVID bioinfor- BCR signaling pathways, cytokine–cytokine receptor interac- matic tool (biological process, level 2). tion, and apoptosis. In addition, in the spleen lysosome and bNumber of genes in each class. TGF-b signaling pathways were also enriched (Fig. 3B and cEASE score, a modified Fisher exact P-value; only P-values Supplementary Figs. S4 and S5). 0.05 were considered. The entire list of genes present on Within immune-related transcripts, some were increased the array was used as background. also in bone marrow (CCL21C, CXCL7, CXCL12, IL1B, CD22, TGFBR2), whereas others were peculiarly overexpressed in the

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P (–log10) A 0 0.5 1 1.5 2 2.5 3 3.5 C Chemokine signaling pathway P (–log ) Axon guidance 10 0 0.5 1 1.5 2 2.5 3 3.5 Cytokine-cytokine receptor interaction Chronic myeloid leukemia Regulation of autophagy B-cell receptor signaling pathway Phenylalanine metabolism

Focal adhesion Gap junction Prostate cancer Vascular smooth muscle contraction Pathways in cancer Small cell lung cancer Apoptosis

B P (–log10) D E 0 0.5 1 1.5 2 2.5 3 3.5 BM Spleen BM Spleen B-cell receptor signaling pathway Pathways in cancer Lysosome Cytokine-cytokine receptor interaction 519 42 321 438 80 181 Chemokine signaling pathway 11 6 Pancreatic cancer 14 76 26 159 mTOR signaling pathway Focal adhesion

Small cell lung cancer 459 338 Amino sugar and nucleotide sugar metabol. Apoptosis TGF-β signaling pathway PBMC PBMC

Figure 3. Plots illustrating KEGG pathways significantly enriched within genes upregulated by CTX and Venn diagrams indicating the degree of overlap between gene expression profiles of bone marrow (BM), spleen, and PBMC. (A, B, C) The genes upmodulated 1–2 days after CTX treatment were clustered 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. The number of genes exhibiting common (or uncommon) modulation of expression in each tissue is shown. spleen, such as the C-type lectin receptors (CLR) CLEC4A2 IL1F8, IL17A). As is the case with the spleen, CTX induced (DCIR), CD209B (SIGNR-1), CLEC4N (Dectin-2), and CLEC7A the expression of CD164 (expressed by primitive HSC), of the (Dectin-1), involved in the uptake of apoptotic cells by DC, and CLR CD209B and of HIF1A (Supplementary Table S3). Only one genes involved in CLR signal-transduction pathway (SYK, BCL- biological process (establishment of cellular localization) was 10). Noteworthy, PTX3, which inhibits the capture of apoptotic enriched within downregulated genes (data not shown). More- cells by DC, was downregulated after treatment. Moreover, we over, the results of KEGG pathway analysis were not similar to observed the induction of genes associated with various to those for bone marrow or spleen (Fig. 3C). processes of DNA repair, cell death, autophagy, and drug To directly compare the correspondence of bone marrow, resistance (DDIT4, HIF1A, CLU, ATG7, HSP70–2; Supplemen- spleen, and PBMC transcriptional profiles, the overlap of the tary Tables S2 and S4). gene lists was examined in Venn diagrams. Interestingly, the Additional upregulated genes comprised AHR (regulator of spleen displayed a profile intermediate between bone mar- Treg and Th17 cell differentiation), factors regulating inflam- row and PBMC, which were the least similar to each other mation (IL-1 family members IL1B, IL1R1, IL1RAP) and cell (Fig.3DandE);thiscouldberelatedtothefactthat,inmice, proliferation (TNFSF13-APRIL, TNFSF13B-BAFF), and growth the spleen is an active hematopoietic organ and, at the same factors acting on different cell populations (FGF1, HBEGF, time, comprises fully mature cells. The most significantly IGF1, PDGFB, VEGFA; Supplementary Fig. S4 and Tables S2 enriched biological classes of genes simultaneously upregu- and S4). Noticeably, IL2RG (IL-2 receptor g chain), which is lated in bone marrow and spleen were defense response and involved in the response to homeostatic cytokines IL-2, IL-7, response to stress and to external stimuli (data not shown). IL-9, IL-15, and IL-21, was overexpressed following treatment, Altogether these results suggest that hematopoietic organs corroborating previous findings indicating that CTX increases (spleen and bone marrow) are the most responsive organs to the expression of homeostatic cytokines (16). the damage mediated by CTX and react to it by activating Although in PBMC no significant enrichment of any biolo- immune-related genes. gical process class was found within upregulated genes, we To validate the results obtained by transcriptional profiling, could identify some cytokine-encoding transcripts (IL12A, the expression of selected genes was assessed by real-time

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A B Lymphocytes CD4+/IFNγ+ CD4+/IL-17+ CD4+CD25+ CD4+CD25+ – + CD4+ cells Foxp3 Foxp3 45 6 6 40 10

5 × 35 30 4 25 3 20 2 15 10 1 Number of cells ×

Fold change versus ctr versus Fold change 5 0 0 **** *** *** * * 02 468101214 Day 4 Day 7 Day 10 Day 14 Days after CTX C

8,68 ctr 0,83 16,04 day 10 2,08 9.18 ctr 0,34 18.80 day 10 0,56 103 103 103 103

102 102 102 102

101 101 101 101

0 87,41 3,08 0 77,55 4,33 0 89,23 1.25 0 79,55 1,09 FL1-H: CD4 FL1-H: 10 10 CD4 FL1-H: 10 10 100 101 102 103 100 101 102 103 100 101 102 103 100 101 102 103

FL3-H: IFN-γ FL4-H: IL-17

Figure 4. Effect of CTX treatment on CD4þ T-cell subsets. A, representative FACS analysis (out of 2 independent experiments) showing the effect of þ þ CTX on the absolute number of IFN-g-producing (Th1), IL-17-producing (Th17), and activated (CD4 CD25 Foxp3 ) 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 cell subtype 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 per group. C, representative dot plots showing percentages of Th1 and Th17 in untreated versus CTX-treated mice (day 10).

PCR. In accordance with the results obtained by microarray, exhibited a lower rebound, evident at a later time (significant CARL and CLU transcript levels were augmented early after only at day 14; Fig. 4A). These results indicate that CTX þ treatment in bone marrow and spleen (but not in PBMC). induces an imbalance in CD4 T-cell subtypes, favoring Moreover, although the expression of IL1B was increased at effective immunity and disadvantaging tolerance, and corro- day 2 and decreased at day 5 in all tissues, CDCA8 was borate recent findings suggesting that CTX induces Th17 cells downregulated at day 2 and returned to baseline by day 5 in cancer patients (32). However, it should be noted that Tregs þ (Supplementary Fig. S7 and Supplementary Methods). are only held back with respect to other CD4 populations, because their number is anyway restored to pretreatment Effect of CTX on T-helper subpopulations level, indicating that this is a transient imbalance and not a Because Th17 cells were shown to exert a protective role permanent depletion. In Fig. 4B, the effect of CTX on lym- þ against murine cancer by providing a more significant help phocytes and CD4 T cells is shown. CTX injection induced a þ than Th1 cells for tumor-specific CD8 T cells (31), the novel transient depletion of lymphocytes followed by a rebound of observation of a Th17-related gene signature induced by CTX their number above baseline without significantly altering the þ prompted us to evaluate, in the spleen, the modulation of this proportion of CD4 T cells (Fig. 4B). T-cell subtype at different times following CTX injection. The þ þ þ modulation of Th1, Th17, Tregs (CD4 /CD25 /Foxp3 ), and CTX treatment stimulates a "storm" of þ þ þ activated CD4 T cells (CD4 /CD25 /Foxp3 ) was examined cytokines, chemokines, and growth factors simultaneously by intracellular staining followed by FACS To characterize the cytokine response to CTX at the protein analysis. Interestingly, the absolute numbers of IFN-g– and level, plasma and bone marrow lysates from tumor-bearing þ þ IL-17–producing CD4 T cells and those of activated CD4 / mice were subjected to Bio-Plex cytokine assays, which þ CD25 /Foxp3 lymphocytes underwent a transient decrease allowed the simultaneous analysis of 32 analytes. 7 days following chemotherapy and were significantly (P Three days (7 for IL-9) after chemotherapy, the levels of 0.05) augmented above the levels of untreated mice at day 10 IL-3, IL-5, IL-9, CCL2, CCL4, CCL11, G-CSF, and GM-CSF and 14 (Fig. 4A). Th1, as expected, showed the most pro- were significantly (P 0.05) increased in plasma with nounced expansion, reaching a 6-fold increase compared with respect to untreated mice, whereas IL-12, CXCL2, and þ untreated mice, and Th17 and activated CD4 T cells under- macrophage colony-stimulating factor (M-CSF) were went a 2.5 and a 3.8 increment, respectively. We did not find reduced (Fig. 5A, B, and C). þ CD4 T cells coexpressing IL-17 and IFN-g (data not shown). Regarding the bone marrow, CTX induced a transient In contrast, Treg cell numbers, although showing kinetics of depletion of cell numbers (and accordingly of their total þ depletion similar to those of the other CD4 subpopulations, protein content) at day 3, and a rebound, overcoming baseline,

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A Plasma D BM cell lysate 30 700 3 12 25 600 10 500 20 2

) 8 400 6 15 6 300 ( × 10 pg/mL 10 4 1 200

pg/number of cells 2 5 100 0 0 0 0 day 0 day 1 day 3 day 7 day 10 day 0 day 1 day 3 day 7 day 10 IL-1β 1.15 2.13* 2.71* 0.39* 1.21 IL-3 2.63 3.19 4.18* 2.52 2.61 IL-9 1.27 2.78 10.66* 0.69 0.42 IL-5 11.78 13.13 23.35* 17.32 10.53 IL-12(p40) 0.03 0.04* 0.16* 0.02 0.02 IL-9 197.62 244.31 332.80 577.23* 315.02 IL-15 0.12 0.27* 0.90* 0.19 0.11 IL- 12(p40) 424.66 461.28 311.64* 245.84* 502.17 IL-18 0.08 0.18* 0.70* 0.02* 0.02* IFN-γ 0.03 0.06 0.32* 0.03 0.01* B E 20 3 1,400 100 90 15 1,200 2

80 ) 1,000 6 70 10

800 60 ( × 10 50 1 600 5 pg/mL 40 pg/number of cells 400 30 20 0 0 200 day 0 day 1 day 3 day 7 day 10 10 CCL2 0.56 1.81* 5.78* 0.56 0.33 0 0 day 0 day 1 day 3 day 7 day 10 CCL3 1.47 2.72* 8.14* 1.03* 1.25 CCL2 248.1 304.98 419.33* 249.94 216.74 CCL4 0.49 0.82 2.22* 0.41 0.44 CCL5 4.58 7.47 14.10* 1.66 3.48 CCL4 58.1 63.93 85.25* 58.85 67.48 CXCL1 0.53 1.05* 3.27* 0.33 0.31 CCL11 542.9 633.55 1103.5* 793.81 654.01 CXCL2 0.04 0.11* 0.49* 0.02* 0.03 CXCL2 12.1 11.00 11.55 8.32* 6.28* CXCL9 2.27 3.27 12.99* 1.86 1.18 LIF 0.01 0.05* 0.18* 0.02 0.01 140 4 3,000 250 C F 120

2,500 200 100 3 ) 2,000 6 80 150 2

( × 10 60 1,500

pg/mL 100 40 1,000 1 pg/number of cells 20 50 500 0 0 day 0 day 1 day 3 day 7 day 10 0 0 PDGF-b 33.54 49.23* 117.2* 56.96* 26.05 day 0 day 1 day 3 day 7 day 10 VEGF 44.59 90.23* 5.97* 53.77 37.17 PDGF -b 2615 2105 1140* 2503 1310* FGF-2 0.33 0.93* 2.88* 0.50 0.68 M-CSF 1995 1583* 1819 1678* 1536* M-CSF 0.67 1.04* 0.66 0.63 0.79 G-CSF 96.6 151.8 204.9* 79.8 97.4 G-CSF 0.07 0.19* 0.12 0.03 0.09 GM-CSF 84.4 110.4 130.5* 101.8 104.2 GM-CSF 0.14 0.27 1.08* 0.04 0.07

Figure 5. CTX modulates the protein levels of cytokines, chemokines, and growth factors in plasma and bone marrow (BM). Before (day 0) and at the indicated time 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 of the protein levels of cytokines (A, D), chemokines (B, E), and growth factors (C, F) was analyzed by Bio-Plex cytokine assay. Data represent the mean SE of the protein levels from 3 individual mice tested in duplicate. *, P 0.05, unpaired t-test.

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30 CTR ACT CTX

10 0/6 0/6 0/6 0 Figure 6. The timing of the 30 CTX + ACT Day 0 CTX + ACT Day 1 CTX + ACT Day 2 combination of CTX and adoptive immunotherapy dictates the 20 antitumor efficacy. Mouse treatment was done as shown in Fig. 1. In mice treated with 10 chemoimmunotherapy, ACT was 5/6 4/6 1/6 carried out either 5 hours (day 0), 0 or at different times (day 1, 2, 3, 6, and 9) after chemotherapy. Data 30 CTX + ACT Day 3 CTX + ACT Day 6 CTX + ACT Day 9 are reported as the average tumor diameter of 6 mice per group Mean tumor diameter (mm ± SE) SE. The number of surviving mice 20 (out of 6) is indicated.

0/6 0/6 0/6 0 0 14 28 42 56 70 0 14 28 42 56 70 0 14 28 42 56 70

Days after tumor implant

at day 10 (Supplementary Fig. S6). Nevertheless, the analysis of The antitumor efficacy of the combined therapy cytokine expression per cell, that is, after normalizing the depends on the timing of combination concentration of each cytokine to the number of cells at each The kinetics of gene and protein expression modulation time point, showed that the amount of several cytokines (IL- showed that CTX-mediated immunomodulation is induced 1b, IL-9, IL-12, IL-15, IL-18, and IFN-g) increased significantly early after treatment and rapidly turned off. On the basis of (P 0.05) at day 3 and went back to baseline or below baseline these results, we investigated whether the timing of optimal by day 7 (Fig. 5D). Noticeably, the chemokines CCL2, CCL3, combination of CTX and ACT was congruent, and possibly CCL4, CCL5, CXCL1, CXCL2, CXCL9, and LIF and the growth dependent, on the kinetics. As shown in Fig. 6, the antitumor factors platelet-derived growth factor (PDGF)-basic, fibroblast efficacy of chemoimmunotherapy was maximal when ACT growth factor (FGF)-2, M-CSF, G-CSF, and GM-CSF followed was carried out either 5 hours or 1 day after chemotherapy (i. the same kinetics (Fig. 5E-F). Of note, in both plasma and bone e., at the time of the majority of gene expression changes), marrow, the majority of protein expression changes took place resulting in the cure of 5 out of 6 and 4 out of 6 tumor-bearing early (day 3), immediately following gene expression modula- mice, respectively, and was completely lost if donor cells were tion (day 1 and 2) and preceding the recovery of cell numbers infused 3 days after chemotherapy, indicating that transferred and, as for transcript, the protein expression modulation was cells should be infused as early as possible to enable the transient. benefit of the CTX-mediated cytokine milieu. These data not only represented the validation at the protein levels of some of the changes in gene expression Discussion assessed either by microarray analysis (i.e., IL-1b,CXCL2, PDGF, FGF) or previously by real-time PCR (IL-15, IFN-g, Different studies have addressed systematically the effects GM-CSF; ref. 16), but also further expanded this informa- of CTX on several of the multiple cells and factors driving tion pointing out the many cytokines, chemokines, and tumor-induced tolerance and antitumor immunity (reviewed growth factors regulating the recovery of bone marrow in ref. 2). However, such hypothesis-driven approaches, cells after the myelosuppressive stress exerted by CTX although deeply clarifying the contribution of each single and constituting the microenvironment capable of support- actor, do not take into account the complexity of CTX- ing HSC proliferation, differentiation, and mobilization to mediated immunomodulation. High-throughput analyses, the circulation. such as genomics and proteomics, can efficiently complement

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these type of studies, giving a comprehensive picture of a cell cross-priming (38) and Dectin-1 and Dectin-2, whose role complex situation, uncovering unknown factors and driving in the clearance of apoptotic cells has been recently under- new hypotheses. Previously, the antitumor efficacy of che- scored (39), and that, after ligand engagement, are able to moimmunotherapy was believed to mainly rely on the induc- induce DC activation and Th1/Th17 differentiation via a SYK- tion of lymphopenia by chemotherapy, which creates "room" dependent signaling pathway that leads to the activation of for the adoptive transfer of tumor-specific lymphocytes (10, NFkB (40–42). Strikingly, the upregulation of Dectins was 11), or on the selective depletion of Tregs (12, 14). Although associated with increased expression of SYK and of the CTX indeed depletes Tregs in vivo, we and others have shown adaptor BCL-10. Of note, it is possible to speculate that the that these cells can repopulate the periphery (Fig. 4; ref. 33), Th17 signature induced by CTX and the expansion of Th1 and highlighting the transient and possibly limiting nature of this Th17 cells observed in the rebound phase after chemotherapy hypothesis. We argued that the tolerogenic milieu induced by depends on the activation of this signaling pathway after tumors is multifactorial and includes Tregs as well as other "sensing" the damage of chemotherapy by PRR. In this regard, factors and that CTX conditioning can not only mitigate early after CTX injection, we observed the induction of genes tolerogenic mechanisms but also produce a multifactorial associated with DNA repair, cell death, autophagy, and drug immunogenic milieu favoring immunotherapeutic interven- resistance (DDIT4, HIF1A, CLU, ATG7, and HSP70–2; ref. 43– tions. Consistent with such a view, the present study was 45). designed to unravel the multiple factors underlying the ability In physiologic settings, the engulfment of apoptotic cells by of CTX to potentiate cancer immunotherapy. Taken together, phagocytes inhibits the release of inflammatory cytokines and microarray analyses show that CTX modulates the expression leads to T-cell tolerance. The perception of danger signals may of an unexpectedly high number of genes in lymphoid organs, have a role in tipping the balance from tolerance to inflam- leading on one hand to the downregulation of genes involved mation and effective T-cell activation (46). Accordingly, we in cell cycle and metabolic processes, which is in accordance show here that after CTX treatment several IL-1 family with the inherent toxicity of an anticancer agent, and, on the members are upregulated in the spleen and both transcript other hand, to the upmodulation of immunomodulatory and protein levels of proinflammatory cytokines (IL-1b, IL-17, genes. An important finding, stemming from both genomic IL-18, IFN-g, LIF) and chemokines (CCL2, CCL3, CCL4, CCL5; and proteomic analyses, is that CTX-mediated immunomo- ref. 47) are augmented in bone marrow. Remarkably, IL-1b and dulation is early and transient, as previously shown in a IL-18, the most important mediators of inflammation, control different tumor model (RBL-5 lymphoma), by real-time PCR innate and adaptive (including Th1-Th17) responses (48). analyses of a limited panel of cytokines (GM-CSF, IL-1 b, IL-7, Moreover, CTX increased the expression of NOS2, an enzyme IL-15, IL-2, IL-21, IL-13, and IFN-g); ref. 16). These data point responsible for the generation of free radical NO and involved out that, to be effective, chemotherapy and immunotherapy in inflammation, the expression of which has been shown to should be combined within a short time window. Remarkably, be triggered by IL-17 via the transcription factor NFkB (49). this hypothesis was confirmed by tumor growth experiments The proinflammatory action of IL-17 induces the expression of showing that the synergistic antitumor effectiveness of CTX target genes such as CXCL1, CXCL2, and defensins (50), which and ACT strictly depends on the timing of combination and is were indeed augmented in bone marrow and plasma. IL-17 is progressively lost as days pass between 1 treatment and the also an important modulator of hematopoiesis through the other (Fig. 6). induction of GM-CSF, G-CSF, and CCL2 (51), which were Recent data suggest that the antitumor efficacy of che- augmented in bone marrow lysates and plasma. Moreover, motherapy (especially anthracyclines) depends on the induc- HIF1A and its target gene VEGFA levels were increased in the tion of immunogenic apoptosis of tumor cells that stimulate spleen, which is in accordance with data showing that CTX antitumor immunity (34). Key elements dictating the immu- induces hypoxia along with HIF1A and VEGFA upregulation, nogenicity of anthracycline-mediated cell death are the sur- playing a key role in inflammation and hematopoiesis (52). face exposure of CARL and the release of HMGB1, which, in The hypothesis stemming from these data is that CTX turn, activate TLR-4 (27). Studies from our group showed that induces an immunogenic apoptosis not only of tumor cells CTX induces CARL exposure and release of DC-activating but also of host bone marrow and spleen cells and, at the same soluble factors by apoptotic tumor cells, resulting in increased time, activates inflammatory mediators, leading to the pro- þ cross-presentation of tumor antigens to CD8 T cells (35). motion of hematopoiesis and immune response. In accor- Here we show for the first time that CTX can augment, in bone dance with this hypothesis, the apoptosis of self T cells was þ marrow cells, the simultaneous expression of CARL and of 2 shown to elicit self-reactive CD8 T cells, thus inducing a PRRs (MBL1 and C1Q) involved in its recognition and in systemic immunoactivation during HIV infection (53). This apoptotic cell clearance (36). In addition, CTX augmented premise also suggests that CTX may be suitable for enhancing the transcript levels of other danger signals, that is, the the efficacy of immunotherapy in adjuvant settings, in which alarmins DEFCR4 (defensin-a-4) and DEFB2 (defensin-b-2), mounting an effective vaccine-induced antitumor immunity the first involved in macrophage and T-cell chemoattraction in the absence of tumor burden is fundamental to prevent and the second able to induce DC maturation via TLR-4 (the recurrences. In this regard, we have recently shown that same receptor of HMGB1; ref. 37). dacarbazine, an alkylating agent similar to CTX, can enhance þ Interestingly, additional DC-specific CLRs were upregulated CD8 memory T-cell responses to peptide-based vaccination þ in the spleen, namely SIGNR1 and DCIR, involved in CD8 T- in resected melanoma patients (54).

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We have previously shown that the antitumor effectiveness mediated immunomodulation. Although with the present of chemoimmunotherapy depends on adoptively transferred experimental design we could not ascertain in which cell þ CD4 T cells (16) and, among them, Th17 was proved to subtype the expression of a certain gene or protein was exhibit a stronger therapeutic efficacy than Th1 (31). Several modulated, this study describes for the first time all the þ authors reported differential effects of CTX on CD4 T cells, sensor, transducer, and effector factors involved in the host including a Th2 to Th1 shift in cytokine production (16, 55). response to CTX, opens novel hypotheses that may guide The present data reveal for the first time that CTX induces an further experimentation, and, lastly, supplies a clear-cut environment simultaneously favoring Th1, Th17, and acti- indication of the short time window in which chemotherapy þ vated CD4 T cells, suggesting that this milieu may condition and ACT should be combined to maximize chemoimmu- þ not only host cells but also transferred tumor-specific CD4 notherapy effectiveness against cancer. T cells, thus leading to improved tumor control. Taken together, our data sustain the fascinating hypoth- Disclosure of Potential Conflicts of Interest esis that CTX-mediated immunomodulation stems directly from its cytotoxicity that produces DNA damage, cell cycle No potential conflicts of interest were disclosed. arrest, and cell homeostasis perturbation not only of tumor cells but also of host highly proliferating cells, such as bone Acknowledgments marrow and splenocytes. DNA-damage responses are complex and involve "sensor" proteins perceiving danger and We are grateful to Cinzia Gasparrini, Annamaria Fattapposta, and Angela Fresolone for helpful secretarial assistance. transmitting signals to "transducer" proteins, which, in turn, convey signals to "effector" factors (56). Cells damaged or Grant Support killed by CTX within lymphoid organs would therefore expose/release danger signals that in turn, through PRR The research was partially supported by grants from the Italian Associa- signaling, would transiently activate proinflammatory path- tion for Research against Cancer (AIRC) and from the Italian Ministry of ways and create a beneficial environment aimed at repairing Health (Integrated Project on Oncology and "ISS Project for Alliance against Cancer"). the damage and able, as a bystander effect, to promote The costs of publication of this article were defrayed in part by the payment innate and adaptive immunity, including Th1 and Th17 of page charges. This article must therefore be hereby marked advertisement in responses. When an immune intervention (vaccination or accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ACT) is carried out during the recovery phase, also tumor- Received December 17, 2010; revised February 28, 2011; accepted March 12, specific immune cells can take advantage of this drug- 2011; published OnlineFirst March 28, 2011.

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www.aacrjournals.org Cancer Res; 71(10) May 15, 2011 3539

Unraveling Cancer Chemoimmunotherapy Mechanisms by Gene and Protein Expression Profiling of Responses to Cyclophosphamide

Federica Moschella, Mara Valentini, Eleonora Aricò, et al.

Cancer Res 2011;71:3528-3539. Published OnlineFirst March 28, 2011.

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