The pros and cons of chemokines in tumor immunology

Review

The pros and cons of chemokines in tumor immunology Antonella Viola1, Adelaida Sarukhan2*, Vincenzo Bronte3 and Barbara Molon4 1

Istituto Clinico Humanitas IRCCS and Department of Translational Medicine, University of Milan, Via Manzoni 113, 20089 Rozzano, Milan, Italy 2 Istituto Clinico Humanitas IRCCS and INSERM, 101 rue Tolbiac, 75013 Paris, France 3 Department of Pathology, Verona University Hospital, Immunology Section, 37134, Verona, Italy 4 Istituto Oncologico Veneto, Via Gattamelata 64, 35128 Padua, Italy

Innate and adaptive immune cells can intervene during tumor progression at different stages including initiation, angiogenesis, local spreading and distant metastasis formation. The net effect can be favorable or detrimental to tumor development, depending on the composition and activation status of the immune infiltrate. Chemokines can determine the distribution of immune cells in the tumor microenvironment and also affect stroma composition. Here we consider how a complex network of chemokines plays a key role in dictating the fate of a tumor. Although the field is in its infancy, we also highlight how targeting chemokines offers a tool to modulate the tumor environment with the aim of enhancing immune-mediated rejection of cancer. Chemokines and the tumor microenvironment Tumor progression is a multistep process based on cumulative cellular alterations. However, it is clear that this process does not exclusively depend on cancer cells, and it is also strongly influenced by the tumor microenvironment itself, which is composed of normal cells and their secreted factors. Among the soluble factors that shape the tumor microenvironment, chemokines (Box 1) have a multi-faceted role. On the one hand, they are responsible for recruiting immune cells that drive and orchestrate the antitumor immune response. On the other hand, chemokines can sustain tumor survival, progression and metastasis. Recent studies have highlighted the complexity of the chemokine actions in the tumor microenvironment showing, for example, that CCL2 may promote tumor metastasis [1] as well as immune-mediated tumor rejection [2] and that intratumoral, post-translational modifications of chemokines may add an additional layer of regulation [2]. As we gain a deeper understanding of the complex biology of the tumor microenvironment, chemokines and their receptors may represent attractive pharmacologic targets for therapeutic intervention in cancer patients. In this review we discuss how chemokines may affect antitumor immunity and modify the tumor microenvironment. In addition, we describe the therapeutic strategies that are currently Corresponding author: Bronte, V. ([email protected]). Keywords: chemokines; tumor; tumor immunity; tumor microenvironment. * Current address: Cellular and Molecular Immunology, Vrije Universiteit Brussels, Brussels, Belgium.

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under investigation in the laboratory and clinic to target the chemokine system in cancer. Chemokines and tumor control The idea that the immune system might recognize and destroy tumor cells was conceived several decades ago. However, the concept of immune surveillance remained controversial until the discovery of the importance of interferon (IFN)-g in promoting rejection of transplanted tumor cells [3]. This, together with improved mouse models of immunodeficiency, led to a reassessment of the role of immunity in tumor control. Mice deficient for the IFN-g receptor, the STAT1 transcription factor (required for IFNg receptor signaling) or adaptive immunity (RAG2 / mice lacking T, NKT and B cells) were all more susceptible to carcinogen-induced and spontaneous primary tumor formation [3]. These data demonstrate a role for innate and adaptive immunity in restraining tumor initiation and progression. In this regard, the amount and type of chemokines expressed by tumor and stromal cells determine the composition and extent of leukocyte infiltration and, ultimately, tumor regression or progression. Several chemokines of the CXC family, such as CXCL9, CXCL10 and CXCL11, are induced by type I (IFN-a and IFN-b) and type II (IFN-g) IFNs [4] and have been shown to inhibit angiogenesis by directly blocking proliferation [5] and migration [6] of CXCR3-expressing endothelial cells. Furthermore, they limit tumor progression by recruiting innate and adaptive immune cells that mediate antitumor immunity. Thus, NK cell accumulation in tumors depends on the presence of IFN-g and CXCL10 [7], and tumor-infiltrating lymphocytes (TILs) with high levels of CXCR3 are attracted by CXCL9 expressed by stromal cells in gastric carcinoma [8]. Importantly, tumor infiltration by NK cells, Th1 CD4+ T cells and CD8+ T cells creates a positive feedback loop within the tumor microenvironment because the released IFN-g enhances the production of CXC chemokines by tumor cells, which further limits neoangiogenesis (a concept coined as ‘immunoangiostasis’) [9]. Recruitment of activated T and NKT cells can also be driven by other, non-angiostatic chemokines such as the structurally-related chemokines CXCL16 and fractalkine/ CX3CL1. CXCL16 can be induced by irradiation [10] and

1471-4906/$ – see front matter ß 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.it.2012.05.007 Trends in Immunology, October 2012, Vol. 33, No. 10

Review Box 1. Chemokines and their receptors at a glance Chemokines  Are small cytokines with selective chemoattractant properties  Coordinate homeostatic circulation of leukocytes as well as leukocyte movement to sites of inflammation or injury  Dysregulated expression of chemokines and their receptors is involved in the development of many human diseases, including cancer  Approximately 50 chemokines (and 20 receptors) are identified to date.  They are divided into four families on the basis of the pattern of the first two of four cysteine residues of the ligand: the large CC family, the CXC family, with a single amino acid residue between the first two cysteines, the CX3C family (with only one member, CX3CL1) and the XC family (consisting of two highly related chemokines, both binding to the XCR1 receptor) Chemokine receptors  Are seven-transmembrane-spanning proteins coupled to heterotrimeric G protein, i.e., G-protein-coupled receptors (GPCRs).  Signal mainly via dissociation of Gi, activation of phosphatidylinositol 3-kinase (PI3K) and the small Rho GTPases.  The majority of chemokine receptor responses are inhibited by treatment with pertussis toxin (PTx).  However, depending on their coupling to distinct G proteins, chemokine receptors may initiate distinct signal transduction pathways and exert several biological functions.

its expression in the tumor is associated with better prognosis in renal cell cancers [11], while expression of CX3CL1 is linked to better prognosis in neuroblastoma [12] and colorectal carcinoma [13]. The proinflammatory chemokines CCL3, CCL4 and CCL5, recognized by CCR5, also play an important role in recruitment of immune cells with antitumor activity. In Ewing sarcoma, the chemokines CXCL9, CXCL10 (expressed by both tumor and stromal cells) and CCL5 (expressed by stromal cells) correlated positively with the numbers of infiltrating CD8+ T lymphocytes and negatively with tumor progression [14]. Furthermore, CCR5 expression on CD4+ and CD8+ T cells is required for increasing antitumor responses in a CCL5-dependent manner, and CCR5 activation in CD4+ T cells leads to CD40L upregulation, promotion of antigen presenting cell maturation, and enhanced CD8+ T cell crosspriming [15]. In a mouse model of ovarian cancer, high concentrations of CCL5 secreted by tumor-primed CD4+ T cells were shown to recruit CCR5+ dendritic cells (DCs) to tumor locations and to activate them through the CD40/CD40L pathway [16]. Indeed, DC infiltration of tumors is often a good prognostic marker [17] and has been associated with CCL20 production by several types of tumors [18,19] and to CCL17 in early stages of pancreatic cancer [20]. CCL17 also attracts CD4+ and CD8+ T cells, and in particular skin-homing T cells [21], and when locally delivered to established colon carcinoma lesions promoted immunemediated tumor rejection [22]. Additionally, chemokines and cytokines such as CCL19, CCL21, CXCL13, CCL17, CCL22 and IL-16 contribute to the de novo formation of tertiary lymphoid structures (TLS), described recently in lung cancers and associated with patient long-term survival [23]. DCs within TLS interact with both CD4+ T cells of memory phenotype and Th1-like CD8+ T cells [23]. Thus, TLS may represent activation sites for tumor-specific T cells entering through

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the high endothelial venules present in these lymphoid aggregates. Furthermore, B cells also associate closely with DC-T clusters [23,24] and may promote the formation of TLS by either membrane bound molecules (CD27/CD70 interaction), or by secreting lymphotoxin and chemokines (CCL22 and CCL4) that attract and stimulate T cells and DCs [25]. In addition to modulating angiogenesis and guiding immune cell recruitment to either tumor sites or TLS, chemokines might directly influence the functional program of chemoattracted cells. For example, CCR5 and CXCR4 act as costimulators on T cells [26] while CXCL10 and CCL21 induce Th1 polarization [27,28]. Evidence is accumulating that chemokines secreted by senescent cells, such as IL-8 or GROa, trigger an innate immune response that is responsible for clearing senescent lesions, resulting in control of benign and preneoplastic lesions [29]. Loss of function mutations in the receptor for these chemokines, CXCR2, was found in lung human adenocarcinomas suggesting that tissue senescence inducing signals might be necessary for tumor immune surveillance [29]. Chemokines and tumor progression Transformed cells can directly hijack chemokines by acquiring expression of chemokine receptors, such as CXCR4, with the aim of colonizing distant sites, as recently reviewed [30]. However, tumors can also modify the local chemokine microenvironment as a strategy to promote their growth (Figure 1). This is for example the case of chemokines with angiogenic activity, such as CXCL1 and CXCL8, which are pivotal to generate a microenvironment with an adequate blood supply to the growing tumor masses [31,32]. In addition, tumors may also secrete chemokines that recruit immunosuppressive cells, among which CD4+CD25+FoxP3+ regulatory cells (Tregs), F4/80+ tumor-associated macrophages (TAMs) and CD11b+Gr-1+ myeloid-derived suppressor cells (MDSCs) are the best characterized. The accumulation of this suppressive infiltrate defeats antitumor immunity and sustains the tolerogenic microenvironment that fosters tumor progression. In ovarian cancer, hypoxia inducible factor 1a (HIF1a) upregulates the intratumoral production of CCL28, which attracts Tregs that in turn establish local immunosuppression, release vascular endothelial growth factor A (VEGFA) and promote an increase in intratumoral microvasculature density [33]. Treg confinement at tumor sites fosters tumor-induced immune evasion [34], especially if this is associated with a decrease in tumor-infiltrating CD8+ T lymphocytes [34]. In ovarian cancers, Treg recruitment was additionally attributed to CCL22 that is secreted by both cancer cells and TAMs [35], whilst in Lewis Lung Carcinoma, the secretion of the same chemokine within the tumor tissues depends on MDSCs and is dynamically regulated by NK cells [36]. Recently, while investigating the mechanism by which TAMs favour Treg entry into the tumor mass, CCL20 was found to regulate CCR6+ Treg trafficking within colerectal cancer tissues [37]. Furthermore CCL1, CCL17 and CCL22 – under a range of inflammatory conditions – regulate the recruitment of CCR4 and CCR8 expressing Tregs [38]. 497

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MDSCs TILs CCL22 CCL2

CCL2

NK

VEGF-α

CXCL9 CXCL12 CXCL5

Endothelial cells

CCL2 CXCL1,CXCL2,CXCL3 CXCL5,CXCL6,CXCL7 CXCL8,CXCL12

IFN-γ IFN-γ

CXCL10

CXC3L1

Stromal cells CXCL9 CXCL10 CXCL11

Treg

tumor CCL5

CCL1,CCL17,CCL20 CCL22,CCL28

CXCL9 CXCL10

CCL20

CCL21 TAM

CXCL9 CXCL10

TILs

CCL20

CXCL16 CX3CL1

CXCL12 CCL21 CCL2

NK T Tumor-primed Act T

CCL19,CCL21 CCL17,CCL22 CXCK13,IL-6

CD4

CCL5

CCL21

CCL2 Bv8

CCL5 CXCL9 CXCL10 CXCL11

Granulocytes

MDSCs Treg

DC CCL4 CCL22

monocyte TAM

CD4 CD8

B Immunostimulating Tertiary lymphoid structure

Tolerogenic Tertiary lymphoid structure

Metastatic niche TRENDS in Immunology

Figure 1. Chemokine and cellular networks in cancer progression. The figure highlights chemokines directly released by primary tumors, which act on cells of innate and adaptive immunity to either support (right part, red) or control (left part, green) tumor development and metastatic spreading (bottom right; the pro-metastatic monocytes/ TAMs and granulocytes are shown in red while the antimetastatic granulocytes in green). The tumor-infiltrating leukocytes and lymphocytes sustain an additional wave of cytokine/chemokine release that can amplify or control immune-mediated effects. Here, only a few secondary loops are reported, such as: the interplay between stromal cells and lymphocytes; MDSCs, monocytes, TAMs and endothelial cells; different cell subsets in either immuno-stimulating or tolerogenic tertiary lymphoid structures. Abbreviations: MDSCs, myeloid-derived suppressor cells; TAMs, tumor-associated macrophages; TILs, tumor-infiltrating lymphocytes; ActT, activated T lymphocytes; DC, dendritic cells; Treg, regulatory T lymphocytes.

Oncogenic viruses encode a number of chemoattractants. By engaging CCR3, CCR8 and CCR4 receptors, respectively, the virokines vMIP-I, vMIP-II and vMIP-III (encoded by the Kaposi Sarcoma-associated herpes virus 8, KS-HHV8) divert host immune responses through the preferential recruitment of Tregs and Th2 cells [38,39], thus tuning down the intensity of the inflammatory response [40]. HHV-8 genome encodes a viral G -protein coupled receptor (vGPCR) that is constitutively active and interacts with both ELR and ELR+ CXC chemokines 498

[41,42]. The vGPCR expression in endothelial cells triggers autonomuous cell proliferation and recapitulates the formation of KS-like lesions, exerting an instructing oncogene activity [41,42]. Compelling genetic evidence affirms the unequivocal role of inflammatory chemokines in tumorigenesis [43,44]. CCL2 is found in the majority of solid cancers, including gliomas, ovarian, cervical, bladder, prostate, and breast tumors and its role in tumor progression is both prominent and multifaceted. CCL2 is one of the main

Review determinants of TAM content within tumor tissues, where it tailors macrophage differentiation toward the protumorigenic M2-phenotype [45,46]. In prostate cancer, CCL2 concurred to the generation of a fertile microenvironment for bone marrow metastasis [47]. Furthermore, high levels of CCL2 correlated with TAM accumulation, poor prognosis and lymph node metastasis in human breast and colorectal cancer patients [48,49]. A pivotal role for the CCL2 CCR2 axis in the migration of myeloid cells to the tumor has been confirmed in different human tumors and mouse tumor models [50]. MDSC accumulation within tumors favors immune evasion and facilitates cancer progression [51]. In metastatic breast cancer models, CCL2 secreted by both tumor cells and stroma drove the accumulation of inflammatory monocytes (CD11b+CD115+Gr1+Ly6C+CCR2+) to primary lesions as well as the subsequent pulmonary recruitment of metastasis-associated macrophages [1]. CCR2-expressing MDSCs accumulate in melanomas where they hamper CD8+ T cell entry to the tumor site, thus limiting the efficacy of cancer immunotherapy [52]. CCL2 remarkably influences the mobilization and the accumulation of the different MDSC subsets as CCR2 deficiency caused a significant loss of monocytic CD11b+Gr-1intLy-6Chi monocytic MDSCs, resulting in the predominance of the CD11b+Gr-1hiLy-6Cint granulocytic MDSCs within the tumor primary lesion [53]. CCR2-dependent signalling also triggers the egress of inflammatory monocytes from the bone marrow during infections; Ccr2 / mice had fewer circulating Ly6Chi monocytes in both homeostatic and pathological conditions and showed an enhanced susceptibility to infection by many pathogens [54]. Other chemokine and chemokine receptors known to support MDSC migration to tumor tissues are CXCL12CXCR4 and CXCL5CXCR2 [55]. CCL21 is released by many tumors, and as mentioned above, induces lymphoid tissue neogenesis and tumor regression. In melanoma cells transduced with CCL21, however, the neo lymphoid-like structure is replenished by Treg and MDSCs, which creates a tolerogenic environment. It is not clear how the same chemokine can give rise to lymphoid structures that can either sustain or inhibit antitumor adaptive immunity but it is likely that other tumorreleased factors can contribute to shape the environment. For example, TGFb1 can polarize intratumoral granulocytes towards an N2 phenotype with inhibitory activity on CD8+ T lymphocytes [56]. Among other factors released by tumors (Box 2), the oxysterol ligands of the liver X receptor (LXR) can negatively affect CCR7 expression in DCs, which then become ineffective in migrating to lymph nodes and priming tumor-specific T lymphocytes. LXR ligand-inactivating enzyme sulfotransferase 2B1b decreased LXR ligand production by the tumor cells and restored antitumor immune responses [57]. Recent results link tumor-released factors with contradictory roles for granulocytes in the regulation of lung metastatic niche. By overexpressing G-CSF, some tumors expand and mobilize Ly6G+Ly6C+ granulocytes that facilitate the formation of a pre-metastatic niche for the subsequent homing of circulating cancer cells [58,59]. This process is dependent on Bv8 (also known as prokineticin-2), which

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Box 2. Other tumor-associated chemotactic factors Tumors may release other chemoattractant molecules that do not belong to the chemokine family. These molecules play a role in attracting suppressive immune cells and/or in facilitating tumor cell migration and metastasis.  CSF-1: enhancement of TAM recruitment and function [99]  Calcium binding proteins S100A8 and S100A9: MDSC recruitment and activation; favor metastasis to lungs [100]  Complement byproduct C5a: granulocytic MDSC recruitment and enhancement of ROS and RNS production by monocytic MDSCs [101]  Eicosanoids PGE2 and LTB4: recruitment of DCs, macrophages, neutrophils, Th17. Treg cells; induction of tumor cell migration [102]  Cholesterol-derived oxysterols: induction of tumor cell migration [103]  VEGF: enhances tumor cell migration and metastasis [104]

induces angiogenesis, mobilizes myeloid cells from bone marrow, and attracts tumor cells to lung metastatic niches through the engagement of the prokineticin receptor-1 [58,59]. However, the pro-metastatic role of granulocytes is questioned by findings from a mammary carcinoma model, in which G-CSF release attracts granulocytes in the lung before any metastatic colonization [60]. In this case, granulocytes killed tumor cells by releasing hydrogen peroxide and thus exerted a net antimetastatic activity. In this process, CCL2 was crucial not only for chemoattraction but also for the activation of the antitumor activity in granulocytes [60]. Intratumoral production of CXCL13 might result in attraction of B lymphocytes, which may exert immunosuppressive properties [61] and promote tumor progression through diverse mechanisms like IgG production and activation of Fcg receptors on resident and recruited myeloid cells [6], or secretion of TNF-a [62] and lymphotoxin [63]. Cancers can cause inflammation by de novo production of inflammatory mediators and chemokines, as well as their receptors, as consequence of oncogenic changes [64]. RET tyrosine kinase rearrangement in thymocytes leads to expression of CCL2 and CCL20, which are known to attract monocytes and DCs, as well as chemokines that are implicated in angiogenesis, such as CXCL8 [65]. In some situations the same oncogene can control the tumor environment in a positive or negative way through effects on chemokine activity. For example, the Ras oncogene enhances the production of CXCL-8 required for the initiation of tumor-associated inflammation and neovascularization in a xenograft tumor model [66]. On the other hand, abnormal EGFR-Ras signalling pathway in keratinocytes suppresses the release of CCL27 with a consequent reduction in T lymphocyte recruitment, together with a relative increase in myeloid cells and enhanced tumor growth [67]. New concepts and therapeutic strategies Impaired homing of tumor-specific T cells to tumor sites is a major limiting step in cancer immunotherapy. This is suggested by experiments and clinical trials involving adoptive cell transfer (ACT) of in vitro expanded, tumorspecific lymphocytes. In ACT for melanoma, half of the patients failed to respond to treatment even though the majority of the transferred, circulating CD8+ T cells showed specific antitumor activity [68]. Further studies 499

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revealed that <1% of the total transferred T cells had migrated to the tumor, and the majority of these cells localized in the lung, liver, and spleen [69]. The importance of proper T cell homing is also highlighted by the strict correlation between infiltration of the primary human tumors by memory T cells, particularly Th1 and CTLs, and the high positive prognostic value for disease-free and overall survival at all disease stages [70]. For these reasons, strategies aimed at boosting the migration of T cells to tumor sites are likely to improve the efficacy of cancer immunotherapy. On the other hand,

because of their relevance in guiding tumor metastasis, chemokines and their receptors may represent specific pharmacological targets to control metastatic spread. These findings are summarized in Table 1. Delivering chemokines Several approaches have been used to increase the expression of specific chemokines within the tumor microenvironment with the rationale of increasing recruitment of immune cells. Antitumor immunity was indeed successfully stimulated following transduction of tumor cells to

Table 1. Chemokines in tumor immunology. Chemokine CCL1

Receptor(s) Pros Recruitment of CCR8 neutrophils and monocytes [71]

CCL2

CCR2, CCR3

Infiltration of CD8+ T cells within the tumor [2]

CCL3,CCL4 CCL5

CCR5

Recruitment and maturation of DCs; enhancement of CD8+ T cell activation [15,16]

CCL17

CCR4

Recruitment of DC, CD4+ and CD8+ T cells [22]

CCL19,CCL21

CCR7

Formation of tumorassociated TLS [23]

CCL22

CCR4

CCL28 CXCL1

CCR10 CXCR1, CXCR2 CXCR2 CXCR1, CXCR2 CXCR1, CXCR2

CXCL5 CXCL7 CXCL8

CXCL9,CXCL10 CXCR3 CXCL11

CXCL12

CXCR4, CXCR7

CXCL16

CXCR6

CXC3CL1

CX3CR1

500

Recruitment and stimulation of T cells and DCs [23]

Cons Recruitment of Tregs [34,38]

Therapeutic approach Intratumoral CCL1 delivery by transduction of tumor cells [71]

Recruitment of TAMs, neutrophils subsets and MDSCs [1,53]

Pharmacological reversal of CCL2 nitration/nytrosilation of CCL2; mAbs to CCL2 and CCR2, shRNA for CCR2 deliveredby nanoparticles [96] Intratumoral CCL3/CCL5 delivery by transduction of tumor cells [72,73]

Recruitment of Tregs [38]

Adenoviral-mediated expression of CCL17 by tumor cells [82] Intratumoral delivery of Recruitment and activation of Tregs and rCCL21 or CCL21 vault MDSCs; formation of nanocapsules; transfer tolerogenic TLS; of engineered DCs metastasis promotion expressing CCL21 [35,38] [76,77,79–81,91,92] Recruitment of Treg [35,38] Intratumoral injection of recombinant adenovirus encoding CCL22 [75] Recruitment of Tregs [33] Angiogenic activity and metastasis promotion [98] Recruitment of MDSCs Angiogenic activity and metastasis promotion [98] Mediates endothelial cell Blocking of intratumor migration and CXCL8 expression by neovascularization [31,32] shRNA [95]

Exprimental or clinical outcome Stimulation of lymphocyteindependent antitumor activity and tumor-specific immunity in myeloma mouse tumor model Increased tumor infiltration and regression in different mouse tumor models; synergy with standard chemotherapy in prostate cancer mouse models; control of metastasis in breast cancer mouse models Ongoing clinical trials Increased numbers of NK, CD4+, and CD8+ T cells at the tumor site in mouse thymoma; generation of long-lasting antitumor immunity in adenocarcinoma mouse model Recruitment of activated immune effector cells to tumor in mouse colon carcinoma Recruitment of T and DCs Tumor eradication of mouse lung carcinoma Ongoing clinical trials Induction of antitumor immunity in mouse lung carcinoma

Inhibition of human tumor xenograft growth in rats

Recruitment of CD8+ T, NK, and NKT cells; block in tumor cell proliferation [5,6,9] Recruitment of endothelial progenitors and MDSCs; metastasis promotion [30] Recruitment of activated T cells and NKTs [10–12] Recruitment of activated T cells, NK cells, and monocytes [12,13]

Control of metastasis in breast, Neutralizing antibodies to CXCL12; blocking prostate, lung, colorectal, peptides and siRNAs for gastric cancer, glioblastoma CXCR4 [89]

Review produce CCL1 [71], CCL5 [72], CCL3 [73], CXCL1 [74] or CCL22 [75] and the effect was attributable, at least partially, to the recruitment and activation of innate and adaptive cells to the tumor site. These studies provided proof-of-concept experiments that paved the way to novel immunotherapeutic approaches. In this regard, the most advanced project involves the chemokine CCL21. After the initial finding that intratumoral administration of recombinant CCL21 reduced tumor burden in a mouse lung cancer model [76] more translational approaches were exploited. To improve vaccination effectiveness, B16 melanoma lysatepulsed DCs were engineered to produce CCL21 [77] and injected into growing tumors resulting in TLS formation within the tumor microenvironment and recruitment and priming of T cells. This approach also led to tumor eradication in mouse lung cancer models [78,79], an outcome not observed with either the chemokine or DC alone. These results found their way into the clinic with ongoing Phase I clinical trials, based on transfer of CCL21transduced DCs, in melanoma patients at the Moffitt Cancer Center (FL, USA) [80] and in advanced nonsmall-cell lung cancer (NSCLC) at the University of California Los Angeles (CA, USA) [80]. Furthermore, a novel CCL21-vault nanocapsule for intratumoral delivery of CCL21 [81] was shown to induce recruitment of antitumor effectors, inhibit lung cancer tumor growth and reduce the frequencies of immune suppressive cells in a mouse model of orthotopic lung cancer. Transducing chemokine receptors An alternative strategy to selectively increase the number of tumor-specific CTLs inside the tumor microenvironment is based on the transduction of CTLs to express chemokine receptors specific for tumor-produced chemokines. Melanoma tumors, for example, consistently show high levels of monocyte/macrophage infiltrates, which are known to migrate in a CXCR2-dependent fashion toward CXCL1 and CXCL8 gradients present in the tumor. Thus, introduction of the CXCR2 gene into tumor-specific T cells can enhance their localization to tumors and improve antitumor immune responses [33]. In a Hodgkin’s tumor model that is known to produce CCL17, over-expression of CCR4, the receptor for CCL17, by T cells equipped with a chimeric antigen receptor that targets CD30 improved homing and antitumor activity in vivo [82]. Successful proof-of-concept studies have been also performed expressing the chemokine receptor CCR2 in T cells carrying chimeric receptors [83,84]. Targeting post-translational chemokine modifications Although one obvious problem for T cell homing in solid tumors is represented by the anarchic vasculature of cancers [85,86], other factors may limit T cell motility within the tumor. Several studies have shown that T cells do not freely travel within a tumor; instead, they remain trapped in the stroma surrounding the cancer cells [2,70,87,88]. Recently, it was demonstrated that intratumoral production of reactive nitrogen species (RNS) induces nitration/ nitrosylation of CCL2 in different human and mouse cancers. As a result, modified CCL2 could no longer attract tumor-specific CTLs, but could still recruit myeloid cells to

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the tumor. Notably, this novel mechanism of tumor escape can be pharmacologically targeted. In vivo administration of a novel drug (AT38) that blocks intratumor RNS production, induced massive T cell infiltration within the tumor. Moreover, AT38 enabled transferred CTLs to reject solid tumors, even at low T cell transfer doses, which are otherwise ineffective [2]. Thus, RNS-inhibiting molecules represent a bona fide new class of adjuvants specific for the immunotherapy of cancer. Blocking chemokines or chemokine receptors Blocking chemokines or their receptors may represent a valuable strategy to limit tumor progression by inhibiting metastasis spreading or by interfering with the tumor microenvironment. Indeed, the most advanced studies have focused on homeostatic chemokines in an attempt to inhibit organ-specific metastasis [30]. CXCR4 is the most widely expressed chemokine receptor in tumor cells and is responsible for metastasis to the most common destinations such as the lung, liver and bone marrow. As described in a recent review [30], targeting of the CXCR4CXCL12 axis is considered a crucial target for therapeutic intervention, and preclinical studies demonstrated that anti-CXCL12 agents can significantly delay primary tumor growth when the treatment is provided in a preventive setting, although it has minor antitumor effects on most established tumors [89]. CXCR4-blocking strategies are currently being evaluated in clinical trials in patients suffering from ovarian cancer, osteogenic sarcoma and acute myeloid leukaemia (ClinicalTrials. gov identifier: NCT01120457). CCR7 drives immune cells into lymph nodes, and for this reason it has a major role in the development of nodal metastasis. This is particularly important in the case of leukaemias and lymphomas, which very often express CCR7 because of their lymphoid origin [90]. In addition, the CCL19-CCR7 axis seems to have a crucial role in the recruitment of leukemic T cells to the CNS [91]. A nonmetastatic B16 melanoma cell line developed in vivo gross metastasis to lymph nodes when expressing CCR7, and the effect was abrogated by the use of neutralizing anti-CCL21 antibodies [92]. On the same line, an elegant study showed that CCR7 expression in breast cancer cells shifts metastasis from the lung to lymph nodes [93]. Altogether, these studies indicate that expression of a single chemokine receptor can define the metastatic destination of a tumor cell and the clinical and therapeutical consequences of this concept are, of course, extremely important. Other approaches aim at interfering with the tumor microenvironment. For example, CXCL8 was exploited as potential target for anti-angiogenic strategies in breast and colorectal cancers [94,95]. Administration of monoclonal antibodies against CCL2 reduced recruitment of inflammatory monocytes to lungs and inhibited the spreading of breast cancer to lung niche, resulting in survival prolongation of tumor-bearing mice [1]. AntiCCL2 antibodies can also synergize with standard chemotherapy, as shown by the tumor regression induced when they were administered together with docetaxel in mice with prostate cancers [96] and are currently being evaluated in humans in prostate and ovarian cancer [97]. 501

Review Concluding remarks In the context of several human pathologies, such as inflammatory and autoimmune diseases, chemokines and their receptors are considered interesting druggable targets, and recent discoveries suggest that targeting the chemokine system may be relevant in cancer, too. The role of chemokines in cancer biology is complex because these molecules may have both pro-tumoral and antitumoral effects, with some of them playing both roles depending on undefined factors regarding tumor biology, chemokine concentration, and post-translational modifications. This, together with the redundancy typical of the chemokine system, may discourage researchers from trying chemokine-based therapies for cancer. However, evidence is accumulating that targeting one specific chemokine – either inhibiting its signaling to block tumor metastasis or, in contrast, enhancing its local concentration to favor antitumor immunity – may dramatically inhibit tumor progression. Thus, we believe that useful therapeutic approaches will be found as we gain a deeper knowledge of the complex interaction between chemokines and cancer.

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Acknowledgments This work was supported by grants from the Ministry of Health (Progetto Giovani Ricercatori and Ricerca Finalizzata) and Ministry of Education (PRIN); the Istituto Superiore Sanita` -Alleanza Contro il Cancro (project no. ACC8); Inserm, France, and Fondazione Cassa di Risparmio delle Provincie Lombarde (grant 5808/2007). We thank Alberto Mantovani for critical reading of the manuscript.

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