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Seminars in Cancer Biology 22 (2012) 319– 326

Contents lists available at SciVerse ScienceDirect

Seminars in Cancer Biology

jou rn al h om epa g e: www.elsev ier .com/ locate /semcancer

eview

elanoma-induced immunosuppression and its neutralization

iktor Umansky ∗, Alexandra Sevkokin Cancer Unit, German Cancer Research Center and University Hospital Mannheim, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany

r t i c l e i n f o

eywords:elanoma

hronic inflammationumor microenvironmentmmunosuppression

yeloid-derived suppressor cells

a b s t r a c t

Malignant melanoma is characterized by a rapid progression, metastasis to distant organs, and resis-tance to chemo- and radiotherapy. Well-defined immunogenic capacities of melanoma cells shouldallow a successful application of different immunotherapeutic strategies. However, the overall resultsof immunotherapeutic clinical studies are not satisfactory. These paradoxical observations are supposedto be due to the profound immunosuppression mediated by different mechanisms dealing with alter-ations in tumor and surrounding stroma cells. Melanoma microenvironment has been characterized bya remarkable accumulation of highly immunosuppressive regulatory leucocytes, in particular, myeloid-derived suppressor cells (MDSCs). Their migration, retention and high activity in the tumor lesions havebeen demonstrated to be induced by chronic inflammatory conditions developing in the tumor microen-

vironment and characterized by the long-term secretion of various inflammatory mediators (cytokines,chemokines, growth factors, reactive oxygen and nitrogen species, prostaglandins etc.) leading to furthercancer progression. Here, we discuss the role of chronic inflammation in the recruitment and activationof MDSCs in melanoma lesions as well as therapeutic approaches of MDSC targeting to overcome tumorimmunosuppressive microenvironment induced by chronic inflammation and enhance the efficiency of

pies.

melanoma immunothera

. Introduction

Malignant melanoma is a potentially fatal form of skin can-er characterized by a rapid progression, metastasis to regionalymph nodes and distant organs as well as by a limited efficiencyf currently applied therapeutics such as chemo- and radiother-py. Its incidence and death rates have been increasing in theSA and Europe faster than any those of other cancers [1,2]. Since

his increase is mostly seen in relatively young adults, the num-er of life years lost per melanoma death is higher than thator most other solid tumors [1,2]. Numerous evidences suggesthat melanoma is an immunogenic tumor. For example, an infil-ration of human melanoma lesions with lymphocytes has beenhown to correlate with the better clinical outcome [3]. Further-ore, primary melanoma can display a spontaneous histological

egression in some patients [4]. In addition, large numbers ofelanoma-associated antigens have been identified as well as

he development of spontaneous T cell reactions and antibodyroduction against various antigens (such as Melan-A, gp100 orY-ESO-1) has been reported in patients with advanced melanoma

5,6]. This well-documented immunogenicity of malignant skinelanoma has made this disease a preferred target for investigating

ifferent immunotherapeutic strategies based on the melanoma

∗ Corresponding author. Tel.: +49 621 383 3773.E-mail address: V.Umansky@dkfz-heidelberg.de (V. Umansky).

044-579X/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.oi:10.1016/j.semcancer.2012.02.003

© 2012 Elsevier Ltd. All rights reserved.

antigen-specific and -nonspecific immunostimulation or adoptivetransfer of melanoma-specific activated T cells [7,8]. However,despite some positive reports, the overall results of immunothera-peutic clinical studies are not satisfactory.

These paradoxical results of clinical trials in malignantmelanoma seem to be due to the intensive immunosuppressionmediated by different mechanisms dealing with structural andfunctional changes both in tumor and stroma cells. These mech-anisms were reported to include on the side of melanoma cells(i) the absence of co-stimulatory molecules [9], (ii) a down reg-ulation in the expression of tumor associated antigens [10], (iii)MHC class I molecules [11], and (iv) ligands for natural killer cellreceptors [12], as well as (v) an intensive secretion of immuno-suppressive factors such as vascular endothelial growth factor(VEGF), transforming growth factor (TGF)-�, interleukin (IL)-10,nitric oxide (NO) or prostaglandins [9,13]. The contribution ofstroma cells in the observed immune escape has been charac-terized by a strong recruitment, expansion and accumulation ofimmunosuppressive cells in the tumor microenvironment suchas CD4+CD25+Foxp3+ regulatory T cells [9,14], myeloid-derivedsuppressor cells (MDSCs) [9,15], tumor-associated macrophages(TAMs) [9,16], Tie2-expressing monocytes (TEMs) [17], N2 polar-ized subset of neutrophils [18,19], and regulatory/tolerogenic

dendritic cells (DCs) [20].

This complex immunosuppressive network is supposed to bestimulated by chronic inflammatory conditions developing in thetumor microenvironment (Fig. 1). Indeed, chronic inflammation

320 V. Umansky, A. Sevko / Seminars in Cancer Biology 22 (2012) 319– 326

Fig. 1. Soluble mediators of chronic inflammation secreted by the melanoma and host cells that include various cytokines (like IL-1�, IL-6, IFN-�, TNF-� etc.), chemokines (suchas CCL2, CCL3, CXCL8 etc.), and growth factors (e.g., TGF-�, VEGF GM-CSF etc.) can induce the migration in tumor lesions and activation of many immunosuppressive leucocytessuch as myeloid-derived suppressor cells (MDSC), regulatory T cells (Treg), M2 polarized macrophages (TAM), regulatory dendritic cells (regDC), and Th2 lymphocytes.A n spec nd CDa

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ctivated MDSC, producing high amounts of nitric oxide (NO) and reactive oxygeontribute to the inhibition of antitumor responses mediated by effector CD4 (Th1) and apoptosis.

as been demonstrated to correlate with the tumor onset androgression [21–26]. An inflammatory microenvironment ensuesuring tumor growth due to the long-term secretion of various

nflammatory mediators (cytokines, chemokines, growth factors,eactive oxygen and nitrogen species, prostaglandins) by the tumornd/or stroma cells [15,21,26,27]. These mediators were foundo support tumor development by stimulating protumor muta-ions, resistance to apoptotic cell death, and neovascularization24,27]. Moreover, many of these chronic inflammatory factorsere demonstrated to induce the migration, expansion and acti-

ation of various immunosuppressive lymphoid and myeloid cells,n particular MDSCs, into the tumor site [9,15,26–28]. In thiseview, we will first focus on the importance of chronic inflam-atory mediators in the recruitment and activation of MDSCs inalignant melanoma lesions and then discuss various therapeutic

pproaches to overcome tumor immunosuppressive microenvi-onment induced by chronic inflammation.

. Melanoma and chronic inflammation

Chronic infection and inflammation have long been consid-red to be strongly linked with cancer progression; however, onlyuring the last decade has this complex association commencedo be deciphered [22,24,29]. Although human malignant skin

elanoma is not generally associated with apparent inflammation,everal recent publications highlighted the critical importance of

chronic inflammatory microenvironment, especially the role ofarticular cytokines and chemokines for melanoma initiation and

rogression [30]. It has been reported that patients with advancedetastatic melanoma display a Th2 pattern of immune homeo-

tasis (resembling a state of chronic inflammation) as reflectedy an accumulation of cytokines IL-4, IL-5, IL-10 and IL-13 as

cies (ROS) as well as expressing elevated levels of arginase (ARG)-1 considerably8T cells by inducing a down-regulation of the TCR �-chain, an arginine deprivation,

well as chemokines CCL5 (RANTES), CCL11 (Eotaxin) and CXCL10(IP-10) in patients’ plasma [31]. In contrast, patients with com-pletely resected melanoma or healthy donors were characterizedby Th1 biased immune homeostasis. This reprogramming of sys-temic immunity towards the dominance of Th2 cytokines could bemediated by tumor-derived VEGF leading to tumor progression andmetastasis [31]. Furthermore, such Th2 bias has been also demon-strated in patients with renal cell carcinoma [32]. Interestingly,both malignancies were characterized by increased levels of VEGFproduction [33,34].

Melanoma cells have been demonstrated to produce a varietyof chemokines including CCL2, CCL5, CXCL1, CXCL2, CXCL3, CXCL5,CXCL6, CXCL7, CXCL8, and CXCL10 [35]. Although chemokineswere first described with respect to the leucocyte migration, itbecame very soon clear that chemokines are produced by many celltypes. Chemokines secreted by melanoma cells have been found toinduce tumor growth, angiogenesis, metastasis and alterations incellular components of the tumor microenvironment [30,36,37].Furthermore, melanoma cells were able to influence fibroblastsand macrophages in the tumor stroma to produce a number ofchemokines such as CCL2, CXCL8 and CXCL12 as well as growthfactors like VEGF, TGF-� or tumor necrosis factor (TNF)-� andcytokines (IL-1, IL-6 or IL-8). On the other side, stromal cells canfurther stimulate the chemokine production by tumor cells cre-ating thereby autocrine and paracrine loops of the tumor growthstimulation [38–40].

Importantly, chemokine expression was found to be steadilyincreased in the process of melanoma progression mediated by

the activation of the nuclear factor (NF)-�B family of transcriptionfactors, whereas blocking NF-�B signaling could strongly decreasethe endogenous chemokine concentration and suppress melanomaprogression in different mouse models [35,41]. Moreover, the

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dentification of several other transcription factors such as AP-1nd signal transducer and activator of transcription 3 (STAT3) thatogether with the NF-�B can induce the production of chemokinesnd other proinflammatory mediators like TNF-�, IL-1, IL-6, IL-, cyclooxygenase-2 (COX-2), matrix metalloproteases, VEGF haverovided the molecular basis for the link between chronic inflam-ation and cancer. This connection is indicated by (i) the induction

f tumor development under chronic inflammatory conditions andii) the formation of chronic inflammatory microenvironment inumor lesions, which strongly stimulates tumor growth and metas-asis [21,22,24–27]. Furthermore, recent publications indicatedhat a number of oncogenes are able not only to promote uncon-rolled cell proliferation and stimulate their resistance to apoptosisut also to activate a cascade of proinflammatory molecules. Inarticular, components of the RAS-RAF signaling pathway haveeen demonstrated to induce the NF-�B activation, which resulted

n the enhanced production of proinflammatory cytokines andhemokines [42–44]. This can lead to the never ending chronicnflammatory process as reflected by the constant release of theseytokines, chemokines and growth factors, recruitment and activa-ion of myeloid immunosuppressive cells like MDSCs and TAMs asell as by the stimulation of signal transduction pathways facili-

ating melanoma growth and metastasis [26,27,45,16].

. Mouse melanoma model for studying chronicnflammation

The study of chronic inflammatory mediators produced atifferent phases of malignant skin melanoma initiation and pro-ression needs an establishment of reliable, clinically relevantouse melanoma models. Most conventional animal melanomaodels are based on the transplantation of tumor cells (e.g., B16), inhich the disease progression and tumor-stroma cell interactionso not correspond to the clinical situation. In contrast to trans-lantation models, recently described transgenic and knock-outouse melanoma models closely resemble human skin malignantelanoma with regard to etiology, genetic alterations, histopathol-

gy and clinical development [46–48]. In one of these models,ouse melanocytes express the human ret transgene controlled

y the mouse metallothionein I promoter-enhancer [46]. A con-tant activation of Ret kinase, a member of the receptor tyrosineinase family [49] resulted in the stimulation of other down-tream kinases like mitogen-activated protein kinase and c-Juns well as matrix metalloproteinases leading to the spontaneouskin melanoma development [46]. After a short latency (20–70ays of age), ret transgenic animals developed skin tumors onhe head (nose, ears, eyes), neck, back, or tail with metastases inymph nodes, lungs, liver, brain, and the bone marrow [46,50]. This

etastatic profile shows a high similarity to metastasis observedalignant melanoma patients [51]. Immunohistologic analysis of

rimary tumors and metastases revealed the morphology of malig-ant melanoma supported by a strong expression of a number ofelanoma associated antigens such as S100, tyrosinase, tyrosi-

ase related protein (TRP)-1, TRP-2 and gp100. Transgenic miceithout macroscopic tumors were able to develop both anti-

en non-specific and antigen-specific T cell-mediated immuneesponses after appropriate stimulation in vitro and in vivo, whichid not significantly vary from those responses in non-transgenic

ittermates.To address the involvement of chronic inflammatory mediators

n melanoma progression, we investigated the production of rel-

vant cytokines, chemokines and growth factors in primary skinelanomas and metastatic lymph nodes as well as in the cell line

hat was established from primary melanomas isolated from retransgenic mice (Ret melanoma cells). Analysis at the mRNA level

cer Biology 22 (2012) 319– 326 321

revealed that these cells strongly express IL-6, VEGF, and TGF-�1 [52]. Moreover, significant levels of VEGF and TGF-�1 at theprotein level were demonstrated by ELISA in the supernatants col-lected from cultured Ret melanoma cells. Investigating primaryskin melanomas, we detected considerable amounts of IL-6, VEGF,and TGF-�1 both at the mRNA and protein levels [52]. It is impor-tant to note that the concentration of VEGF in the melanoma lysatesmeasured by the Bio-Plex technique was found to be distinctlyelevated in tumors with the larger weight (taken as an indicatorof melanoma progression). A similar VEGF accumulation has beenreported in patients with advanced metastatic melanoma [53]. Inaddition, concentrations of VEGF and IL-6 were also considerablyhigher in the serum from melanoma-bearing animals than in non-transgenic littermates [52].

Analyzing other chronic inflammatory mediators, we found anassociation of increased amounts of IL-1� and GM-CSF both inskin melanomas and metastatic lymph nodes with acceleratedmelanoma growth [54]. IL-1� was reported to be a major upstreamcytokine, which mediated inflammation by inducing a cascade ofproinflammatory molecules promoting thereby tumor invasive-ness and progression [55,56]. The positive correlation has been alsoobserved between elevating concentrations of TNF-� in melanomalesions and tumor progression in ret transgenic mice. Although thisfactor has been originally described as inducing tumor cell death,recent numerous publications documented its different procanceractivities, including an induction of the release of proinflamma-tory cytokines such as CCL2 (MCP-1) and CXCL8 (IL-8) by varioustumor cells [57–59]. Indeed, increased levels of Ccl-2 have beendetected in melanoma samples removed from transgenic mice[60]. In addition, in these mice, primary cutaneous melanomas andmetastatic lymph nodes were able to accumulate high amounts ofIFN-� [54]. This cytokine is known to be released by activated Tcells and is considered as one of the principal mediators of anti-tumor T cell-dependent immune responses [61,62]. However, itmay also have protumor effects under certain circumstances deal-ing, in particular, with the sustained antigenic stimulation of T cellsand the development of chronic inflammation [63,64]. Under theseconditions, IFN-� has been reported to stimulate nitric oxide pro-duction by MDSC that represent an important mechanism of theirimmunosuppressive activity [65,66]. Moreover, IFN-� as well asall above-mentioned inflammatory mediators have been demon-strated to be critical for driving MDSC expansion, migration intotumor lesions and for keeping their suppressive phenotype in thetumor microenvironment, lymphatic organs and the peripheralblood [15,26–29,67].

4. MDSC in the inflammatory melanomamicroenvironment

MDSCs have been described as an extremely heterogeneouspopulation of immature myeloid cells representing precursors ofgranulocytes, macrophages, and DCs [15,28,67–69]. Whereas inmice, they have been reported to be positive for Gr1 and CD11bmarkers, the situation with human MDSC is much more com-plicated due to the absence of human analog of Gr1 and thenecessity to use other markers like CD15 [68]. In mice, it hasbeen demonstrated that MDSCs could be divided on granulo-cytic CD11b+Ly6G+Ly6Clow and monocytic CD11b+Ly6G+/−Ly6Chigh

subpopulations, which may differ in their immunosuppressivepathways [15,68]. A number of signaling pathways triggered byvarious proinflammatory mediators have been demonstrated to

be responsible for the MDSC expansion and activation. Amongthem are the STAT family of factors (STAT3, STAT6 and STAT1),NF-�B as well as S100 calcium-binding proteins A8 (S100A8) andS100A9 that induce a strong activation of inducible NO synthase

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iNOS) and arginase (ARG)-1, the up-regulated production of TGF-, and the expression of cyclin D1, MYC and survivin [15,69–71].rincipal mechanisms of MDSC-mediated immunosuppression inumor-bearing mice are linked to the inhibition of antitumor T-ell responses through the enhanced activity of ARG-1, iNOS andADPH oxidase complex. This results in the depletion of l-arginine,n amino acid essential for the protein synthesis by T lymphocytes72] as well as in the increased production of NO, peroxynitrite andeactive oxygen species (ROS) [15,69,72,73].

NO has been shown to mediate T cell apoptotic death and tonhibit signaling pathways regulating the synthesis and release ofytokine that are crucial for T cell antitumor functions [74,75]. Moreecently, it has been demonstrated a capability of NO to induce theitration of T cell receptors (TCR) on tumor infiltrating lympho-ytes (TIL) impairing thereby their recognition and killing of targetumor cells [76]. Moreover, intratumoral production of NO andeactive nitrogen species has been found to induce CCL2 chemokineitration hindering intratumoral T cell infiltration and anti-tumoreactivity [77]. In addition, accumulated in the tumor microenvi-onment NO can stimulate the development of chemoresistance inumor cells thorough an inactivation of the caspase cascade [78].ther recently described mechanisms, by which MDSCs can down-

egulate T cell activities include (i) the sequestration of cystinelocking the delivery of cysteine, another amino acid critical for

lymphocyte functions [79], and (ii) the reduction of T cell migra-ion to lymph nodes via the down-regulation of l-selectin, a T celloming marker, leading to the alteration of T cell priming [80].

We have investigated numbers and activities of Gr1+CD11b+

DSCs in melanoma lesions (skin tumors and lymph node metas-ases) as well as in the spleen and bone marrow [54]. It haseen demonstrated that a remarkable accumulation of MDSCsmong tumor infiltrating leucocytes significantly correlated withhe increasing weight of these primary tumors. Moreover, quicklyrowing tumors showed elevated MDSC frequencies within infil-rating leucocytes. Interestingly, when testing the migration ofther important immunosuppressive cells such as Tregs to theumor site, we found their accumulation in smaller tumors followedy the decrease in Treg amounts in larger lesions [60]. Significantlylevated levels of MDSCs were detected also in metastatic lymphodes as well as in the spleen and bone marrow of melanoma-earing mice [54]. Similar observations on MDSCs recruitmentave been earlier demonstrated in different transplantation tumorouse models and cancer patients [15,28,68,81,82]. These data

ndicate that an observed enhanced production of numerousnflammatory mediators during melanoma progression in ret trans-enic mice may attract MDSCs into tumor lesions. Furthermore,DSC accumulation in the site of chronic inflammation has been

reviously described in the mouse chronic inflammatory model27]. One of the important consequences of the enhanced MDSCroportion in tumor-bearing hosts could be diminished numbersf mature myeloid cells like DCs [15,28]. Indeed, we have recentlybserved a significant decrease in numbers of mature conventionalCs in melanoma lesions and lymphoid organs from ret transgenicice [52].Enriched in the tumor microenvironment MDSCs are able to dis-

lay a high level of activation reflected by intensive NO production,nd ARG-1 expression associated with their strong capacity to sup-ress T cell activities in in vitro assays [15,54,69,73,83]. One of theajor mechanisms of MDSC-mediated blocking of T-cell functions

s associated with a remarkable decrease in the expression of T-celleceptor �-chain [84,85], which plays a critical role in coupling theCR-mediated antigen recognition to diverse signal transduction

athways [86]. In ret transgenic spontaneous melanoma model,

profound down-regulation of TCR �-chain expression has beenetected in T lymphocytes infiltrating primary and metastaticelanoma lesions as well as in cells localized in lymphoid organs

cer Biology 22 (2012) 319– 326

[54]. Moreover, a reduction of �-chain levels has been reported notonly in T cells from patients with different tumor types [87,88], butalso in mice with chronic inflammation [84,86], suggesting againan amazing resemblance of both pathological processes. Further-more, a direct MDSC inhibitory effect on TCR �-chain expressionhas been documented upon in vitro coculture of MDSCs isolatedfrom tumor-bearing mice or animals under chronic inflammatoryconditions with normal T lymphocytes [54,84,86]. Based on theseobservations, we suggest that measuring �-chain levels in T cellsderived from tumor-bearing host directly ex vivo without theirwithdrawal from the immunosuppressive microenvironment forin vitro cultures could provide a more accurate characteristic oftheir functional capacity. Taken together, all above mentioned find-ings suggest a strong linkage among developing tumors, chronicinflammation, and immunosuppression.

5. Neutralization of immunosuppression in melanoma

A number of treatment approaches were applied during the lastyears in order to overcome MDSC-induced immunosuppressionunder different pathological conditions, in particular in cancer andchronic inflammation [89]. These strategies were mostly focusedon the reduction of either MDSC numbers or their immunosup-pressive activities in the tumor-bearing hosts. The down-regulationof MDSC frequencies could be achieved by (i) stimulating theirdifferentiation into mature DCs and macrophages [90], (ii) block-ing their generation from earlier precursors [91], or (iii) by theirselective elimination using particular chemotherapeutic agentssuch as 5-fluorouracil or gemcitabine [92,93]. Another approach islinked to the attenuation of MDSC immunosuppressive activities.It could be achieved by interfering with the IL-13/IL-4R�/STAT6signaling pathway using an RNA aptamer that blocks an impor-tant for the MDSC function receptor IL4R� (CD124) resulting intheir elimination and the limitation of tumor progression in mice[94]. Furthermore, MDSC activity has been found to be suppressedupon the administration of COX-2 inhibitors, which block the syn-thesis of prostaglandin E2 [95,96]. In addition, an application ofnitroaspirin has been reported to reduce nitration of proteins in thetumor microenvironment and to suppress iNOS and ARG-1 activ-ities in MDSCs impairing their functions [97]. Direct inhibitors ofiNOS and ARG-1 have been also found to attenuate MDSC functions[89,98].

Recently, it has been demonstrated that phosphodiesterase(PDE)-5 inhibitors [sildenafil (Viagra) and tadalafil (Cialis)], whichare widely used for the treatment of erectile dysfunction,pulmonary hypertension and cardiac hypertrophy [99], couldunexpectedly display considerable antitumor effects in varioustransplantation mouse models by neutralizing MDSC immuno-suppressive functions that resulted in the TIL enrichment andactivation [98,100,101]. A major molecular mechanism of the MDSCinhibition was linked to the increasing intracellular concentra-tions of cyclic guanosine monophosphate (cGMP) leading to thedown-regulation of iNOS and ARG-1 activities and decreased NOsynthesis in MDSCs. In order to overcome the chronic inflamma-tory conditions associated with the MDSC activation in melanomalesions of ret transgenic mice, the PDE-5 inhibitor sildenafil wasapplied in animals with established skin tumors. Long-term drugadministration with drinking water (for 6 weeks) resulted in aremarkable prolongation of the survival of tumor-bearing mice[54]. Importantly, a significant reduction of key inflammatorycytokines, chemokines and growth factors such as IL-1�, VEGF, GM-

CSF, IL-6, Ccl2, and Ccl3 in primary skin tumors was found upon thetreatment, demonstrating the earlier unknown anti-inflammatoryeffect of sildenafil, which needs further detailed investigation [54].Moreover, sildenafil induced a significant decrease in the number

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f infiltrating metastatic lymph nodes MDSCs, which express onheir surface proinflammatory protein S100A9 important for thexpansion and retention of these cells [102,103]. All these alter-tions in the level of numerous chronic inflammatory factors in theelanoma microenvironment could be responsible for observed

iminution in MDSC frequencies [54]. In accordance with pre-ious publication on transplantable tumor mouse models [100],he biochemical mechanism of sildenafil effects in ret transgenic

elanoma-bearing mice involved the elevation of cGMP concentra-ions associated with down-regulation of NO production and ARG-1xpression in tumor infiltrating MDSCs, resulting in the inhibitionf their immunosuppressive activities [54].

Since MDSCs have been recently reported to support neovascu-arization in tumor-bearing hosts [104], one cannot exclude that thenhibition of MDSC functions could lead to the suppression of tumoreoangiogenesis. In addition, we found that antitumor effects ofildenafil were strongly associated with elevated TIL frequencies inetastatic lesions [54]. Moreover, the function of these T cells was

artially restored as reflected by an observed enhancement of TCR-chain expression in TIL. It is important to note that a systemiclimination of CD8+ T cells in sildenafil-treated mice with deplet-ng anti-CD8 monoclonal antibodies led to a complete abrogationf antitumor effects induced by this drug, highlighting the mecha-ism of the therapy dealing with the restoration of antitumor T-celleactivity [54]. Interestingly, when testing by tetramer staining, weould not detect any increase in frequencies of melanoma antigen-pecific CD8+ TIL or an elevation of �-chain expression in these cellss compared with total CD8+ tumor infiltrating lymphocytes, indi-ating that the T-cell recovery mediated by the sildenafil therapyas observed in all T cells independent of their antigen specificity.

ince the level of TCR �-chain expression in TIL have been recentlyemonstrated as a prognostic and survival biomarker in canceratients [88,105], it could be useful for the evaluation of the host

mmune status and of the efficiency of tumor immunotherapies.ince intracellular cGMP may stimulate tumor cell proliferation,e tested the PDE-5 expression also in the Ret melanoma cell

ine. However, this enzyme isoform was detected only in tumorDSCs that infiltrate primary skin melanomas but not in the Ret

ells suggesting that sildenafil was not able to stimulate the tumorell proliferation due to the accumulation of intracellular cGMP.oreover, chronic administration of sildenafil failed to induce any

mpairment of the expression of another isoform of PDE (PDE-6)resent in the mouse retina [106], indicating the absence of poten-ial side effects related to the PDE-6 inhibition in the retina. Theseata suggest that PDE-5 inhibitors could be safely applied neutral-

zing immunosuppression in melanoma microenvironment.Another strategy to overcome immunosuppressive melanoma

icroenvironment may involve the application of chemother-peutic drugs at particular doses. Conventional chemotherapyased on maximum tolerated doses (MTD) is widely used at theresent time to kill quickly proliferating tumor cells. However, thishemotherapeutic approach usually results in a severe general tox-city, and considerable local and systemic immunosuppression inumor bearing hosts that further stimulate a rapid proliferation ofhemoresistant tumor cells and fast tumor progression. Diminutionf the chemotherapeutic doses to the moderately low ones (20–33%f MTD) has been recently demonstrated not only to limit somendesirable side effects of conventional chemotherapy in canceratients but also to increase tumor cell immunogenicity by induc-

ng immunogenic cell death through efferocytosis and to releaseoluble immunogenic factors from dying tumor cells [107]. In con-rast to classical apoptosis, which is considered to be tolerogenic,

fferocytosis mediated by calreticulin and LRP/91/Rac-1 pathwayhowed its immunostimulatory potential [107,108].

A new concept of cancer chemotherapy has been recently devel-ped. In this case, cytotoxic drugs were applied at ultra-low,

cer Biology 22 (2012) 319– 326 323

non-cytotoxic and non-cytostatic doses (3–5% of MTD), and thewhole approach was designated as chemoimmunomodulation[109,110]. In contrast to conventional therapies, no suppressionof proliferation or apoptosis of tumor cells, hematopoietic cellsor immune cells in vitro were detected. However, chemoim-munomodulation has been demonstrated to induce the antitumorimmune responses by stimulating immune cell functions andenhancing the immunogenic properties of tumor cells. In particu-lar, various chemotherapeutic agents (such as vincristine, paclitaxelor methotrexate) co-cultured at ultra-low, non-cytotoxic concen-trations with human DCs were able to induce the stimulation DCmaturation as well as their ability to induce T cell proliferation andactivation [109,110]. Being administered in vivo at ultra-low, non-cytotoxic doses, all above-mentioned chemotherapeutics exerteda direct stimulatory effect on DCs as reflected by an up-regulationof components of the MHC class I antigen processing machinerythat was correlated with an increased expression of MHC classII and such co-stimulatory molecules as CD40, CD80, and CD86[111,112]. Furthermore, in transplantable tumor model (Lewis lungcarcinoma), chemoimmunomodulation with paclitaxel applied inconjunction with an intratumoral DC vaccination has been foundto suppress tumor development and to increase the frequencies ofCD4+ and CD8+ T cells infiltrating tumors [113].

When testing in ret transgenic mouse model of spontaneousmalignant melanoma, the administration of paclitaxel at ultra-low, non-cytotoxic doses into mice with established skin tumorsresulted in a significant prolongation of mouse survival and retar-dation of melanoma development (V.U., A.S., unpublished results).Similar to the impact of sildenafil in the same mouse melanomamodel, we observed a considerable inhibition of the production ofvarious chronic inflammatory factors in melanoma lesions includ-ing IL-�, IL-6, TNF-�, IFN-�, GM-CSF, and IL-10. These alterationshave been found to be linked to a considerable reduction of MDSCnumbers in primary skin tumors and their decreased ability toproduce an immunosuppressive molecule NO as compared to theuntreated group. Interestingly, the treatment of normal C57BL/6mice could also result in a significant decrease in the amount ofGr1+CD11b+ immature myeloid cells, which are considered as anMDSC counterpart in healthy mice (V.U., A.S., unpublished results).Such impairments of potential immunosuppressive cells wereshown to correlate with an increased amounts of effector CD8+ andCD4+ T-cells in lymphoid organs and to enhance an efficiency of thepeptide immunization in these animals. Direct in vitro treatment ofgenerated CD11b+Gr1+ MDSCs with paclitaxel in ultra-low concen-trations revealed the stimulation of their differentiation towardsmature DC, which was found to be independent of TLR-4 [114]. In rettransgenic melanoma model, the capability of MDSC isolated fromskin melanoma lesions upon the paclitaxel treatment to suppressthe proliferation of normal T cells has been found to be significantlylower than that of MDSC from untreated tumor-bearing mice. As aresult, the number of CD8+ T cells infiltrating melanoma lesions waselevated and the expression level of TCR �-chain in these cells wasup-regulated as compared to the untreated group. Furthermore,the depletion of CD8+ T lymphocytes in paclitaxel-treated animalsresulted in a complete abrogation of the beneficial impact of thischemotherapeutic drug indicating thereby a critical role of CD8+

T cells in the mechanism of therapeutic efficiency of paclitaxel atultra-low, non-cytotoxic doses.

6. Conclusion

Malignant skin melanoma is characterized by a complex asso-ciation of chronic inflammatory mediators and myeloid regulatorycells that play a critical role in generating a highly immunosuppres-sive microenvironment. Despite a considerable immunogenicity

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Fig. 2. Neutralization of chronic inflammatory melanoma microenvironment by thePDE-5 inhibitor sildenafil or paclitaxel in ultra-low, non-cytotoxic doses in vivoleads to the diminution of MDSC frequencies and suppressive activity (reflectedby decreased NO production and ARG-1 expression). These changes are associatedwct

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ith the restoration of antitumor activities of T-cell effector CD4 (Th1) and CD8Tells (indicated by an enhancement of TCR �-chain expression levels), which leadso a significant retardation of melanoma progression.

f melanoma cells leading to early enrichment and activation ofelanoma-specific CD8+ and CD4+ T lymphocytes, this strongly

mmunosuppressive network could eliminate these effector cellsia apoptosis or severely damage their antitumor functions. Dur-ng the last years, intensive investigations of different solubleroinflammatory factors have been performed in patients withdvanced melanoma focusing on chemokines (like CCL2, CCL5,XCL8, CXCL10 etc.), growth factors (such as GM-CSF, VEGF, andGF-�) and immunosuppressive cytokines (including IL-1�, IL-6,L-10, TNF-�, and IFN-�). However, in most studies, these factors

ere measured patients’ plasma since samples from melanomaesions are not easily available. Therefore, an extensive utilization ofreclinical mouse models that include genetic and signaling path-ays alterations typical for human melanoma seems to be of critical

mportance. These models will permit to investigate soluble proin-ammatory mediators and immunosuppressive myeloid cells notnly systemically but also at the tumor site.

Using one of such transgenic melanoma model, which resem-les human malignant melanoma, we found a striking elevation in

evels of proinflammatory cytokines, chemokines and growth fac-ors in melanoma lesions that correlated with increased amountsf highly immunosuppressive MDSCs and decreased TCR �-chainxpression in CD8+ and CD4+ TIL. Targeting MDSCs (as reflectedy the down-regulation of their numbers and suppressive func-ions) and diminishing concentrations of chronic inflammatory

ediators in the melanoma microenvironment milieu (e.g., withhe PDE-5 inhibitor sildenafil or chemoimmunomodulation withaclitaxel) enabled a partial restoration of antitumor T-cell activ-

ties leading to a dramatic retardation of spontaneous melanomarogression (Fig. 2). Therefore, it would be critically important (as

prerequisite for an effective melanoma immunotherapy) to mon-tor the initial immune status of patients including the evaluationf inflammatory cytokines, chemokines and growth factors, MDSCrequencies as well as the level of TCR �-chain expression in CD8+

nd CD4+ T lymphocytes. Based on this information, the neutraliza-ion of chronic inflammatory and immunosuppressive melanoma

icroenvironment should be performed before applying any fur-her immunotherapies.

onflict of interest

The authors declare that they have no conflict of interest.

cer Biology 22 (2012) 319– 326

Acknowledgements

This project has been funded by the DKFZ-MOST Cooperation inCancer Research (grant CA128, to V.U.), Dr. Mildred Scheel Foun-dation for Cancer Research (grant 108992, to V.U.), theInitiativeand Networking Fund of the Helmholtz Association within theHelmholtz Alliance on Immunotherapy of Cancer (to V.U.).

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