Myeloid-derived suppressor cells in cancer: recent

Immunology and Cell Biology (2013) 91, 493–502 & 2013 Australasian Society for Immunology Inc. All rights reserved 0818-9641/13 www.nature.com/icb

REVIEW

Myeloid-derived suppressor cells in cancer: recent progress and prospects Yazan S Khaled1,2,3,4,5, Basil J Ammori1,2,3,4 and Eyad Elkord1,2,6 Immunosuppressive cells, mainly myeloid-derived suppressor cells (MDSCs) and T regulatory cells, downregulate antitumour immunity and cancer immunotherapy. MDSCs are a heterogeneous group of immature myeloid cells that negatively regulate the immune responses during tumour progression, inflammation and infection. Whilst there have been extensive laboratory investigations aimed at characterising the MDSC subsets in cancer, there remains a significant gap in our understanding of their phenotypical and functional heterogeneity. In this article, we review data concerning the phenotypical and functional role of MDSCs in cancers. Importantly, we analyse the value of MDSCs as a prognostic factor in various clinical settings and the possible therapeutic approaches towards elimination of their immunosuppressive activity and enhancement of beneficial antitumour immune responses. MDSCs promote tumour immune evasion by inhibiting T-cell responses, as well as by supporting tumour progression. Accumulation of MDSCs is associated with the progression of human cancers, and their elimination was shown to improve anti-tumour immune responses. Phenotypical characterisation of MDSCs has been poorly investigated in many human cancers and lacks comprehensive clinicopathological correlation data. Although the need for effective therapeutic agents to eliminate the MDSC suppressive effect is immense, their role has been examined only in a few clinical settings. Immunology and Cell Biology (2013) 91, 493–502; doi:10.1038/icb.2013.29; published online 25 June 2013 Keywords: myeloid-derived suppressor cells; cancer; expansion; clinical significance; therapeutic target

Tumour-induced immunosuppression is now recognised as a key element in enabling tumours to escape immune-mediated destruction.1 Despite the limited understanding of tumour immune biology and the mechanisms of induction, expansion and proliferation of immunosuppressive cells within the tumour microenvironment, advances in this field helped to identify new cancer immunotherapeutic targets. The current approaches to immunotherapy aim to enhance the immune system’s capability for surveillance of the host in order to destroy abnormal/cancerous cells and prevent cancer development. However, the diverse regulatory pathways associated with complex cellular interactions and feedback loops within the immune system have been major obstacles that have limited the clinical application and effectiveness of cancer immunotherapeutic approaches. It is now evident that immune responses in cancer are negatively regulated by immunosuppressive cells, mainly T regulatory cells (Tregs) and myeloid-derived suppressor cells (MDSCs). They are largely responsible for inhibiting host T-cell activity against tumourassociated antigens and consequently impair the effectiveness of anticancer immunotherapeutic approaches.2 Therefore, approaches which aim to reduce the deleterious effects of these immunosuppressive cells may increase the success of various immunotherapeutic modalities in cancer patients.3 However, a better understanding of

the local tumour microenvironment and the exact mechanisms of induction and/or expansion of immunosuppressive cells in circulation and the tumour milieu should provide opportunities for testing novel treatments that target such cells and alter the balance in favour of more effective antitumour immune responses. This could also be vital for the design of more effective immunotherapeutic strategies. In this review, we describe the biology of MDSCs, their phenotype and function in cancer and their mechanisms of action, as well as their clinical value in cancer prognosis and potential strategies for targeting their immunosuppressive activity. THE ORIGIN AND DEFINITION OF MDSCS Myeloid cells represent the most abundant type of haematopoietic cells in the immune system and have a huge diversity of physiological and pathological functions.4 In cancer and other pathological conditions such as trauma and sepsis, the pathway of normal physiological differentiation is partially blocked to generate pathological myeloid cells and an expansion of cells with immunosuppressive functions, hence the term myeloid-derived suppressive cells.5 Therefore, MDSCs are best defined as a heterogeneous population of activated immature myeloid cells that are characterised by a morphological mixture of granulocytic and

1Institutes of Cancer and Cardiovascular Sciences, University of Manchester, Manchester, UK; 2Biomedical Research Centre, School of Environment and Life Sciences, University of Salford, Manchester, UK; 3Department of Upper Gastrointestinal Surgery, Salford Royal Foundation Trust, Manchester, UK; 4Department of Hepatobiliary Surgery, North Manchester General Hospital, Manchester, UK; 5Section of Translational Anaesthetic and Surgical Sciences, Leeds Institute of Molecular Medicine, Leeds, UK and 6College of Medicine and Health Sciences, United Arab Emirates University, Al Ain, UAE Correspondence: Dr E Elkord, United Arab Emirates University, PO Box 17666, Al Ain, UAE or University of Salford, Peel Building, Manchester M5 4WT, UK. E-mail: [email protected] or [email protected] Received 13 April 2013; revised 26 May 2013; accepted 27 May 2013; published online 25 June 2013

MDSCs in cancer YS Khaled et al 494

monocytic cells but lack the expression of cell-surface markers associated with fully differentiated monocytes, macrophages or dendritic cells (DCs).6 However, the heterogeneity of MDSCs can also be defined according to their expression of cell-surface markers as shown below. Mice MDSCs MDSCs are defined in mice by the characteristic co-expression of myeloid lineage differentiation antigens Gr-1 and CD11b (also known as Ly6C/G and a M-integrin, respectively).7–9 Granulocytic MDSCs have a CD11b þ Ly6G þ Ly6Clow phenotype, whereas MDSCs with a monocytic morphology have a CD11b þ Ly6G Ly6Chigh phenotype.6,10–13 The exact roles of both MDSC subpopulations in pathological conditions are not fully understood; however, emerging evidence suggests that they have different functions in cancer and autoimmune diseases.10,14 In their analysis of 10 different experimental tumour models, Youn et al.6 reported that both MDSC subsets were expanded and, in keeping with other studies, noted greater expansion of the granulocytic MDSC subpopulation compared with the monocytic subset. However, their findings suggested that the level of MDSC expansion was not indicative of their suppressive features but, rather, was representative of the functional state within the tumour environment.6 More recent studies that aimed to characterise the nature of the granulocytic MDSCs in a murine tumour model showed that, although tumour granulocytic MDSCs shared common phenotype cell markers with normal mature neutrophils, they were functionally different.13,15 Therefore, the role of these surface markers remains poorly investigated and their identification is likely to help establish a

better phenotypical characterisation of MDSC subsets in human cancers. Human MDSCs The equivalent MDSCs in humans is defined as the CD14 CD11b þ CD33 þ CD15 þ phenotype or cells that express the CD33 marker but lack the expression of markers of mature myeloid and lymphoid cells and the major histocompatibility complex (MHC) class II molecule HLA-DR.16–18 This human MDSC phenotype accounts for B0.5% of peripheral blood mononuclear cells in healthy individuals with a 10fold higher level in circulation in cancer patients, such as those with renal cell carcinoma and colorectal carcinoma.3,16,17,19 The identification and isolation of human MDSC subsets have been somehow difficult because of the heterogeneous characteristics of these immature cells. However, further identification of the characteristic features of MDSC subsets in different types of human cancers prompted some researchers to redefine MDSCs according to a combination of a new set of markers, such as high levels of CD66b and low levels of CD62L and CD16.20–22 Table 1 outlines the different MDSC subsets identified in different human cancers, as well as their characteristics. These data suggest a significant diversity in the MDSC subsets in different human cancers. Gros et al.23 defined the morphology of different MDSC subsets such as CD14 þ monocytes/ macrophages, CD14 CD15 immature myeloid cells and CD14 CD15hi neutrophils/eosinophils. However, it remains unknown whether this diversity in MDSC subsets is due to different mechanisms of induction and/or expansion in various cancers or due to the different surface markers used by the investigators. Many studies that characterised the different MDSC

Table 1 Phenotype of MDSC subsets in human cancers MDSC subset

Phenotype

Cancer type

Source

Reference

LIN HLA-DR CD33 þ CD11b þ

Breast Colon

Peripheral blood

19

Unclassified 19

Lung Renal

101

Pancreatic Oesophageal

81

19,81

CD14 þ /arginase þ

Gastric Multiple myeloma

CD11b þ CD14 CD15

Melanoma

C11b þ CD14 CD15 þ CD66b þ VEGFR1 þ CD15 þ þ IL-4R þ CD124

91

19,81

95

Peripheral blood and tumour tissue

23

Renal

Peripheral blood

22

Colon Melanoma

Peripheral blood

102

CD11b þ CD14 CD15 þ CD33 þ CD11b þ CD14 CD15int

Lung Melanoma

Peripheral blood Peripheral blood and tumour tissue

103

CD14 þ CD11b þ HLA-DRlow/

Melanoma Prostate

Peripheral blood

102

Granulocytic

23

Monocytic 104 105

HCC Head and neck

95

CD14 þ IL-4R þ CD124

Colon Melanoma

Peripheral blood

CD14 þ

Melanoma

Peripheral blood and tumour tissue

102

Abbreviations: HCC, hepatocellular carcinoma; int, intermediate; MDSC, Myeloid-derived suppressor cell; VEGFR1, vascular endothelial growth factor receptor 1.

Immunology and Cell Biology

102

23


subsets have examined only the peripheral blood samples, and not the tumour microenvironment, of cancer patients to establish a clinical correlation with cancer progression. The tumour microenvironment is a rich source of tumour-associated cytokines and immunosuppressive cells and should be examined, when possible, to identify the phenotypes and functions of human MDSCs and correlate these to the clinical progression of cancers. EXPANSION AND ACTIVATION OF MDSCS IN CANCER Accumulating evidence from tumour-bearing mice and human cancers indicates that the induction and expansion of MDSCs in the tumour microenvironment is mediated by a combined effect of multiple factors including cytokines, growth factors and pro-inflammatory mediators. The list of such factors is expanding with ongoing research, but the relevant factors implicated in human cancers can be divided into two groups:24 ‘MDSC expansion promoting factors’ and ‘MDSC activating factors’. A list of these factors and their signalling pathways is shown in Table 2. MDSC expansion is facilitated by triggering a cascade through the signalling molecules that regulate cell survival, proliferation, differentiation and apoptosis, which are known as family members of Janus tyrosine kinase and signal transducer and activator of transcription 3 (STAT3).25 STAT3 is the main transcription factor involved in MDSC expansion.25 A most recent study in a melanoma murine model found that the inhibition of tumour-expressed inducible nitric oxide synthase (iNOS) with a small-molecule inhibitor (L-NIL) was associated with an inhibition of accumulation of STAT3/reactive oxygen species (ROS)-expressing MDSCs and an elimination of their suppressive function within the tumour microenvironment.26 In addition, normalised serum levels of vascular endothelial growth factor (VEGF) were associated with downregulation of the activated STAT3 and ROS production in MDSC and with reversed tumour-mediated immunosuppression.26 In another supporting study, Qu et al.27 reported that inhibition of STAT3 eliminated the suppressive activity of MDSCs in mice. Recent findings indicate that pathways downstream of STAT3 may also regulate MDSC expansion by inducing the expression of S100A8 and S100A9 proteins; these are members of the S100 calcium-binding protein family that are released in response to cell damage, infection or inflammation and are specifically involved in the accumulation and function of granulocytic MDSCs.28 Myeloid progenitor cells that express S100A8 and S100A9 were found to infiltrate areas of dysplasia

and adenoma in both human colorectal carcinoma tissue and colon tumour-bearing mice.29 In addition, these proteins are involved in the production of ROS in MDSCs and in tumour-bearing murine models; the absence of S100A9 was associated with an inhibition of MDSC accumulation in the spleen.28 Although S100A8 and S100A9 induce their effect on MDSCs through the activation of the nuclear factor-kB pathway,28 this exact mechanism is still not fully understood but seems to have an important role in regulating MDSC expansion and may provide an interesting link between immune suppression and cancer progression. MicroRNA (miRNA) molecules have attracted considerable attention as crucial members of a complex regulatory network that interact to mediate the induction and expansion of MDSCs. Although miR155, miR-223 and miR-146a may be associated with the formation of a cancer microenvironment,30,31 miR-146a has a negative regulatory role in the development of myeloid cells. Zhao et al.32 reported that the deletion of miR-146a in mice results in a myeloproliferative disorder with an enormous accumulation of MDSCs in peripheral lymphoid organs. Nevertheless, our understanding of the exact role and function of these molecules and their mechanism of action is limited, and, given the preliminary data accumulated from previous studies, miRNAs seem to have the potential to increase MDSC accumulation and/or expansion in human cancers. Accumulating evidence suggests that the immunosuppressive activity of MDSCs is dependent not only on the expansion of promoting factors but also on activating factors that exert their effect through multiple signalling pathways including STAT6, STAT1 and nuclear factor-kB.33–35 STAT1 is the major transcription factor that is activated by interferon gamma (IFNg)-mediated signalling and is responsible for the upregulation of arginase (ARG1) and iNOS expression in MDSCs within the tumour microenvironment.24,36 MDSCs from STAT1-knockout mice could not upregulate ARG1 and iNOS expression and subsequently had no inhibitory effect on T cells.24,36 Interleukin-4 (IL-4) induces ARG1 expression, whereas IFNg induces iNOS expression in myeloid suppressor cells isolated from tumour-bearing mice.34 Although some studies supported the role of the IL-4 receptor a–STAT6 pathway in reducing the immune surveillance in tumour-bearing mice, such as those with sarcoma, through blockage of ARG1 production,35 others reported that IL4Ra-deficient mice with breast cancer retained a high level of activated MDSCs after surgical resection.37 These contradicting

Table 2 Factors implicated in the expansion and/or activation of MDSCs in cancer MDSC expansion/activation process Expansion

Factors

Production source of factors

Signalling pathways

Cellular effect

COX-2106

Tumour cells

JAK protein and STAT3

Myelopoiesis stimulation inhibiting the

Prostaglandins55,106 SCF107

differentiation of MMC

M-CSF88,108 IL-6109 GM-CSF110 VEGF111 Activation

IFNg112 IL-1340 IL-434 TGF-b35,41

Activated T cells and tumour stromal cells

STAT6, STAT1 and NF-kB

MDSC activation Upregulation of iNOS and arginase

Abbreviations: COX-2, cyclooxygenase-2; GM-CSF, granulocyte/macrophage colony-stimulating factor; IL, interleukin; IFNg, interferon gamma; iNOS, inducible nitric oxide synthase; JAK, Janus kinase protein family members; M-CSF, macrophage colony-stimulating factor; MDSC, myeloid-derived suppressor cell; MMC, mature myeloid cells; NF-kB, nuclear factor-kB; SCF, stem-cell factor; STAT3, signal transducer and activator of transcription 3; TGF-b, transforming growth factor-b; VEGF, vascular endothelial growth factor.



studies may indicate that the IL-4 receptor a–STAT6 pathway may not be involved in immunosuppression in all tumour models and that the effect of this pathway is tightly controlled by cross talks between other immune cells and tumour-associated factors present within the tumour microenvironment. MECHANISMS OF MDSC IMMUNOSUPPRESSION MDSCs mediate their tumour-induced immunosuppression through several potential mechanisms. It has been shown that MDSCs mediate their suppression of T-lymphocytes in cancer through direct contact and/or through a combination of multiple major mediators such as iNOS, ARG1, cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2), transforming growth factor (TGF-b), IL-10 and Tregs.34,38–45 Here we present a summary of the mechanisms of action of these mediators as supported by recent work. ARG1, iNOS and ROS ARG1 and iNOS are two different but related enzymes that are expressed highly in MDSCs and utilise L-arginine to produce urea and NO, respectively.46,47 High levels of ARG1 expression by MDSCs can accelerate the depletion of L-arginine in the tumour microenvironment, which subsequently inhibits T-cell proliferation by causing low expression of T-cell receptors and thus suppression of the cell cycle in T cells.48 Moreover, Srivastava et al.45 have shown that MDSCs inhibit T cells through an uptake of cysteine, an essential amino acid for T-cell activation, with its consequent depletion from the microenvironment. These findings indicate that this metabolic mechanism of T-cell suppression is not unique to L-arginine and that further research is required to identify new enzymes and/or products within the tumour microenvironment that the MDSCs utilise to exert their negative immune regulatory effect. Blockage of the metabolic mechanism of MDSCs can serve as a new therapeutic target for tackling T-cell inhibition in cancers. Recently, it was found that NO significantly inhibits E-selectin expression on human endothelial cells.49 The in vitro treatment of squamous cell carcinoma tissue with iNOS inhibitor N(o)-nitro-L-arginine(L-NNA) leads to the induction of E-selectin expression.49 These findings indicate that MDSCs are critical producers of NO and that inhibition of NO production leads to restoration of E-selectin expression and potentially to an increase in T-lymphocyte recruitment into the squamous cell carcinoma tumour milieu. ROS production has become evident as a major regulator of the suppressive activity of the granulocytic MDSCs in both murine models and human cancers.50–52 In three different studies, inhibition of ROS production in vitro was associated with complete elimination of the suppressive activities of the MDSCs isolated from mice and human cancers.9,18,50 In addition, the combination of NO and ROS was associated with the production of peroxynitrite, which causes dysfunction of several proteins in target cells and nitration of the T-cell receptor, which in turn lead to suppression of CD8 þ T-cell responses.2,53 Recently, ROS was shown to induce both ERBB2 and ERBB3 receptors (members of the epidermal growth factor receptor family) in ovarian cancer, both in vitro and in vivo, via downregulation of miR-199a and miR-125b expression.54 Although the authors did not investigate the source of ROS production and only referred to it as ‘endogenous ROS’,58 these findings suggest that ROS production is associated with tumour growth and pathogenesis. However, this study has provided vital data on the mechanism by which ROS induced tumour growth.58 It is important to investigate whether the downregulation of miR-199a and miR-125b expression is associated with MDSC accumulation in other human cancers. Immunology and Cell Biology

PGE2 and COX-2 Whereas murine MDSCs express all four PGE receptors, PGE2 was the main receptor found in human MDSCs.30,55 PGE2 leads to upregulation of ARG1 and accordingly regulates MDSC-related T-cell immune suppression.30,55,56 Obermajer et al.57 found that PGE2 also promotes MDSC recruitment to the tumour microenvironment through the induction of stromal cell-derived factor-1(CXCL12/ SDF-1) chemokine and the induction and stabilisation of its related receptors CXCL12 (CXC chemokine ligand 12) and CXCR4 on the surface of tumour-associated MDSCs. The positive feedback loop between PGE2 and COX-2 for MDSC-related immune suppression provides an ideal target for cancer immunotherapy. Veltman et al.58 demonstrated that COX-2 inhibition with dietary celecoxib treatment improved immunotherapy and prevented the local and systemic expansion of all MDSC subtypes in a mesothelioma murine model. Therefore, simultaneous blockage of the PGE2 and COX-2 loop, alone or with L-arginine metabolic products (urea and NO) that are produced by ARG1 and iNOS, provides a potential target for countering MDSC-related T-cell suppression. Transforming growth factor-b TGF-b is an immunosuppressive cytokine that has been firmly associated with MDSC function and with the regulation of tumour induction and expansion.59 In a recent study for characterisation of MDSC subsets in patients with squamous cell carcinoma of the head and neck, the CD14 þ HLA-DR MDSC subset was noted to be the highest in number and produced higher levels of TGF-b compared with other subsets.60 The addition of anti-TGF-b monoclonal antibody in combination with other antibodies (anti-CD86 monoclonal antibody and anti-PD-L1 monoclonal antibody) partially restored T-cell proliferation and IFN-g production.60 This evidence indicates that MDSCs are likely to be a major source for TGF-b production, and their immunosuppressive effect is mediated by various molecules including TGF-b. In two other studies it was found that TGF-b production promoted tumour cell invasion and metastasis.61,62 In breast cancer cells, Yang et al. found that the deletion of TGF-b receptor gene type II resulted in infiltration of MDSCs into the invasive front of tumour tissues with synergistic production of further large quantities of TGF-b that led to the promotion of tumour invasion and metastasis.41 T regulatory cells Although it is not clear whether Tregs have a role in the expansion of MDSCs, emerging evidence indicates that MDSCs are involved in Treg cell differentiation through the production of several cytokines and/or through direct cell–cell interactions. In this regard, Huang et al.43 found that Treg induction by MDSCs was dependent on the presence of IFNg, IL-10 cytokines and antigen-associated activation of tumour-specific T cells but was independent of the NO mechanism in tumour-bearing mice. These data indicate that MDSCs can evade the immune surveillance by directly inhibiting T-cell responses and inducing anergic and suppressive Tregs. Recently, tumourinfiltrating MDSCs in a murine model were found to express high levels of chemokines comprising the CCR5 ligands CCL3, CCL4 and CCL5. Injection of CCL4 and CCL5 into tumour tissue resulted in a remarkable increase in tumour-infiltrating Tregs, whereas ablation of CCR5 caused a profound decrease in Tregs.63 CCL5 was found to be preferentially expressed on CD4 þ Foxp3 þ Tregs in human pancreatic cancer,64 and collectively this evidence demonstrates that MDSCs recruit higher numbers of Tregs into the tumour microenvironment by secreting several chemokines. In contrast, granulocytic MDSCs


from tumour-bearing mice unexpectedly impaired TGF-b-mediated generation of CD4 þ CD25 þ FoxP3 þ -induced Tregs and impeded the proliferation of natural Tregs without affecting FoxP3 expression.65 In addition, there is a discrepancy in evidence supporting the cell–cell interaction between MDSCs and Tregs. In a murine model of lymphoma, MDSCs induced Treg expansion through the ARG1 pathway and through presentation of tumour-associated antigens.66 In contrast, others reported that the number of Tregs was high throughout tumour growth and did not correlate with MDSC-related expansion; this indicated that the induction of Treg cells was not MDSC mediated.10 MDSCs were found to mediate the induction of Th17 response in a manner independent of MDSC—T-cell contact but via MDSC-dependent cytokine secretion.67 Further, CD40 on MDSCs is required for T-cell suppression and Treg accumulation in tumour-bearing mice;68 the blockade of CD40–CD40L interaction between MDSCs and Tregs may provide new insight into a strategy to ablate tumour immune suppression and enhance antitumour immunity. MDSCS IN CANCER The impact of MDSCs in cancer could be described as a two-staged effect; the first is an abnormal myelopoiesis and recruitment of MDSCs into the tumour tissue and the second is active MDSC cytokine production and cell–cell interactions within the environment and further progression of cancer.69–73 Although it is not clear whether these early recruited MDSCs have the immunosuppressive features of tumour-infiltrating MDSCs, evidence from a limited number of studies has shown that these myeloid cells share both the phenotype and suppressive characteristics of MDSCs.69–71 It is worth mentioning that this stage of MDSC recruitment is tightly regulated by several soluble molecules, as well as by the upregulation of STAT3 and other signalling pathways. In a most recent update on this subject, the TNF signalling pathway was found to drive MDSC accumulation into the tumour tissue to evade the immune system in a tumour-bearing murine model.74 MDSCs promote tumour immune evasion by limiting T-cell responses and infiltration into the tumour microenvironment.72 However, what remains unresolved is whether MDSCs mediate antigen-specific or nonspecific suppression of T-cell responses in the tumour environment. Although several in vitro studies have demonstrated that MDSC-mediated suppression of T cells is antigen nonspecific in nature,75,76 there is no supporting evidence from in vivo studies. MDSCs can take up soluble antigens such as tumourassociated antigens and present them to T cells in order to mediate an antigen-specific suppression.2,10 In another supporting in vitro study, the block of MHC class I molecules on the surface of MDSCs completely abrogated their inhibitory effects on T cells.77 CD8 þ T-cell suppression is also mediated strictly by MHC class I molecules as shown in an in vivo cancer model.33 Corzo et al.53 reported that MDSCs express MHC class I and lack MHC class II molecules; this supports the theory that MDSCs inhibit T-cell responses in an MHC class I-restricted manner. These data also indicate that the MDSC-mediated suppression at the tumour site is different from that which occurs in peripheral lymphoid tissues. MDSCs isolated from tumour tissue promoted tumour growth in vitro more remarkably than did the respective MDSCs isolated from the spleen when injected into control mice.69,78 Although both cells shared the same morphology and phenotype, interestingly tumour MDSCs expressed high levels of NO and ARG1 and suppressed both antigen-specific and nonspecific T cells in comparison with splenic MDSCs that only mediated a nonspecific

T-cell suppression through a ROS-mediated pathway.69,78 The exact mechanisms that regulate and affect the functions of MDSCs in tumour and peripheral tissues remain unclear. It is likely that the generation of a pro-tumour microenvironment that provides mitogenic factors has substantial influence on the expansion and function of MDSCs and that their elimination could open new horizons in cancer immunotherapy. In addition to early recruitment into the tumour milieu and promotion of immune escape, MDSCs have been shown to have a vital role in tumour angiogenesis and metastasis. They promote cancer cell dissemination by inducing factors that lead to a proangiogenic state within the tumour microenvironment. In a murine model of melanoma, it was reported that MDSCs promote cancer cell dissemination by inducing epithelial–mesenchymal transition and that CXCL5 was the main chemokine attracting MDSCs to the tumour site.73 MDSCs have also been associated with cancer angiogenesis by producing matrix metalloproteinases (MMP) and enhancing VEGF bioavailability.79 In vivo induction of TIMP-2 (MMP inhibitor) in human lung adenocarcinoma A-549 led to a remarkable decrease in MDSC infiltration into tumours as well as to suppression of angiogenesis and tumour growth.80 Clinical significance of MDSCs in human cancers The lack of definite cell markers of human MDSCs created a major obstacle in establishing a precise definition of their phenotypes and consequently limited the number of studies that examined the clinical significance of MDSCs in cancers. We identified the largest six recent studies that examined the diagnostic and prognostic significance of MDSCs in human cancer.19,23,81–84 The patients’ demographic details and study outcomes for these studies are shown in Tables 3 and 4, respectively. Whereas all studies measured the levels of MDSCs in circulation, only three studies identified their levels in tumour tissue.23,83,84 Sun et al.83 found that the proportion of MDSCs in colorectal carcinoma tissue was correlated with nodal metastases, distant metastases and tumour stage, which suggested the involvement of MDSCs in cancer development. However, the authors did not perform any functional assays and did not examine the suppressive features of MDSCs in peripheral blood or tumour tissue. Gros et al.23 characterised the nature and suppressive function of melanoma-infiltrating MDSCs in addition to that of circulating MDSCs. Unexpectedly, the melanoma-infiltrating MDSCs displayed an impaired ability to inhibit in vitro T-cell proliferation in comparison with circulating MDSCs. This suggested that the tumour-infiltrating MDSCs exhibited less immune suppressive effect than that reported in other studies.85 In addition, and in contrast to other studies,19,81–83 Gros et al. found no significant difference in the percentage and absolute number of 14 different MDSC subsets in the peripheral blood of metastatic melanoma patients when compared with controls.23 These findings highlight the complex immune regulatory role of tumour-specific/derived factors within specific tumour microenvironments and the fact that MDSCs are not universally present in all tumours or tend to decrease in numbers in advanced stages of cancer. The reports on clinical correlation between circulating/infiltrating MDSC levels and tumour stage and survival are contradicting, as shown in Table 4. There are, however, a number of potential factors that might explain this controversy. These reports include a heterogeneous population of diverse cancer types such as breast, lung, melanoma, sarcoma and gastrointestinal cancers. In addition, the focus of these reports and that of many other human studies was only directed towards the characterisation of some MDSC subsets in Immunology and Cell Biology


Table 3 Clinical studies of MDSCs in human cancer: patient demographics Author (Ref No)

No. of patients

No. of controls

106

21

Diaz-Montero et al.19

Cancer type

AJCC clinical cancer stage

Breast (n ¼ 50)

I/II (n ¼ 31)

Oesophageal (n ¼ 5) Colon n (n ¼ 10)

III (n ¼ 19) IV (n ¼ 56)

Sample processing method Fresh whole blood

Pancreatic (n ¼ 15) Non-small cell lung (n ¼ 3) Melanoma (n ¼ 4) Sarcoma (n ¼ 5) Gastric (n ¼ 2) Others (n ¼ 12) Gabitass et al.81

131

54

Pancreas (n ¼ 46) Oesophageal (n ¼ 60)

41

44

Colorectal (n ¼ 22) Pancreas (n ¼ 8)

Cryopreserved PBMC

Not assessable (n ¼ 3)

Gastric (n ¼ 25) Duffy et al.82

II/III (n ¼ 44) IV (n ¼ 84) IV (n ¼ 41)

Fresh whole blood, fresh PBMC and cryopreserved PBMC

I (n ¼ 6) II (n ¼ 12)

Fresh PBMC and tumour tissue

Hepatocellular (n ¼ 9) Gastric (n ¼ 2) Sun et al.83

49

40

Colorectal (n ¼ 49)

III (n ¼ 11) IV (n ¼ 20) Zhang et al.84

64

32

Colorectal (n ¼ 64)

I (n ¼ 6) II (n ¼ 5)

Fresh whole blood and tumour tissue

III (n ¼ 15) IV (n ¼ 38) Gros et al.23

32

10

Melanoma (n ¼ 32)

Metastatic (n ¼ 32)

Fresh whole blood and tumour tissue

Abbreviations: AJCC, American joint committee on cancer; No, number; PBMC, peripheral blood mononuclear cells.

Table 4 Clinical studies of MDSCs in human cancer: diagnostic power and correlation with clinical pathology Author (Ref

MDSC level in peripheral blood:

No)

MDSC subset identified

cancer vs normal

Diaz-Montero

Lin /low HLA-DR ,

2.85 vs 1.26%

et al.19

CD33 þ CD11b þ

MDSC value in tumour

P value

ND

Po0.0001

Correlation with clinical features MDSC percentage and absolute numbers correlate significantly with cancer stage: I/II cancer vs IV cancer: (1.96 vs 3.77%; Po0.0001) (124.1 vs 260.04 cells ml 1; Po0.0001). III cancer vs IV cancer: (2.46 vs 3.77%; P ¼ 0.014) (163.7 vs 260.04 cells ml 1; P ¼ 0.031).

Gabitass et al.81

Lin /low HLA-DR , CD33 þ CD11b þ

(2.1, 1.3,1.5) vs 0.8%

ND

Po0.001

Duffy et al.82

CD14 þ HLA-DR /low

FPBMC 5.5 vs 1.2%, CPBMC

ND

Po0.001

CD15 þ CD14

10.03 vs 4.16% whole blood 2.5 vs 0.8%

ND

Po0.003

Sun et al.83

CD33 þ HLA-DR

whole blood 59.7 vs 46.9% PBMC: 1.89 vs 0.54%

2.99%

No association between cancer stage and MDSC levels, (Po0.53) but significantly associated with survival (Po0.001) CD14 þ HLA-DR-/low MDSC are consistently elevated in the peripheral blood of patients with advanced GI cancer

Po0.05, Po0.05 MDSC in peripheral blood correlated with distant metastasis, Po0.023 MDSC correlated with cancer stage and pro-

Zhang et al.84

Lin /low HLA-DR ,

Whole blood 3.554%; vs 0.818%

4.69%

CD33 þ CD11b þ Gros et al.23

CD11b þ CD14 þ CD11b þ CD14 CD15 þ

Po0.0001

gression, Po0.028 MDSC in peripheral blood correlated with clinical cancer stage and tumour metastasis but not primary tumour size

12.6±1.1 vs 13.8±1.2% 59.2±3.4 vs 52.6±4.2%

49.8±5.3% 28.9±1.7

Po0.48, Po0.17 No significant difference in the levels of perPo0.17, Po0.202 ipheral MDSC in melanoma vs healthy individuals

Abbreviations: CPBMC, cryopreserved peripheral blood mononuclear cells; FPBMC, fresh peripheral blood mononuclear cells; MDSC, myeloid-derived suppressor cell; ND, not determined; PBMC, peripheral blood mononuclear cells.



diverse cancer types, which likely leads to an inconclusive characterisation of the suppressive features of MDSCs. The processing methods used for MDSC characterisation have also varied among studies. The analysis of MDSCs using fresh PBMCs or, more frequently, cryopreserved PBMCs offers the advantages of transport and batch analysis, which is essential for multicentre studies. However, in a recent study, the frequencies of Lin HLA-DR CD15 þ and Lin HLA-DR CD33 þ subsets from healthy individuals and cancer patients were significantly reduced and all other subsets lost their in vitro suppressive activities after cryopreservation.86 Therefore, the study of human MDSCs from fresh, rather than cryopreserved, PBMCs appears to be a better method, and measuring their levels in whole blood is more appropriate for complete characterisation of all potential subsets. The prognostic or predictive significance of MDSCs in human cancers has not been resolved completely, and is supported by data from a few clinical studies, some of which lack information on the clinical implications of circulating MDSCs. A study that measured the ratio of DC to MDSC (Lin HLA-DR CD33 þ ) in patients with advanced kidney cancer (n ¼ 20) or melanoma (n ¼ 16) found high pretreatment DC-to-MDSC ratios in patients with more favourable outcome,87 which suggest that baseline levels of circulating MDSCs may predict the effectiveness of IL-2 therapy. Potential strategies to target MDSCs in human cancers In recent years, there has been a remarkable increase in the wealth of information on tumour-associated immunosuppression and the mechanisms by which immune evasion is established. It is well recognised nowadays that MDSC-mediated immunosuppression not only contributes to tumour progression but is also one of the major obstacles that limit the effectiveness of cancer treatment. There are potential therapeutic strategies that can target MDSCs and these are being explored currently. Although many of the therapeutic targets have shown promising results in mice bearing tumours, the need for such effective agents in human cancers is immense and only a few have been tested in the clinical setting that we will discuss. The promotion of MDSC differentiation into mature cells has proven to be an effective strategy for eliminating their immunosuppressive activities. All-trans retinoic acid (ATRA) at therapeutic levels was shown to induce MDSC differentiation into DCs and macrophages, leading to a subsequent reduction in the number of MDSCs in cancer patients and mice.17,24,88 Nefedova et al.89 suggested that an upregulation of glutathione synthesis and a reduction in ROS levels were the main mechanisms involved in ATRA-mediated MDSC differentiation. In a recent randomised clinical trial involving patients with extensive-stage small cell lung cancer (n ¼ 41), systemic depletion of MDSCs using ATRA in combination with cancer vaccination led to a statistically significant improvement in the immune response to vaccination (41.7%, P ¼ 0.012) in comparison with the control group (zero) and the vaccination-only group (20%, P ¼ 0.02).90 The early data from this trial strongly suggest that therapeutic depletion of MDSCs can be used clinically to enhance the effect of cancer immunotherapy. In another clinical study, ATRA administration in patients with metastatic renal cell carcinoma markedly reduced the number of MDSCs (Lin HLA-DR CD33 þ ), increased the DC-to-MDSC ratio in the peripheral blood and improved T-cell immune response.3 ATRA was administered in an escalating pattern at doses of 50, 100 and 150 mg m 2 per day divided into three daily doses for 7 days, and the effect of ATRA was observed only when it reached the therapeutic target of 4150 ng ml 1.3 In another study involving patients with advanced renal cell carcinoma stage III/IV, ATRA therapy was associated with a reversed

immunosuppressive effect of MDSCs and improved T-cell function by promoting MDSC differentiation into antigen-presenting DCs.91 Recently, MDSCs were investigated as vectors for tumour-specific oncolytic viral therapy (vesicular stomatitis virus) to treat hepatic colorectal metastatic cancer.92 As MDSCs were superior to other immune cell types with respect to the migration to tumours, vesicular stomatitis virus delivery to the tumour tissue was potentiated by binding to MDSCs and was associated with longer-term survival of tumour-bearing mice compared with systemic administration of wildtype vesicular stomatitis virus alone. Interestingly, tumour cell death was further extended by promoting MDSC differentiation into a mature phenotype.92 Therefore, this approach can be used as a vehicle to deliver a more targeted oncolytic viral therapy as well as for promoting the differentiation of MDSCs and abrogating their suppressive ability. Neutralising the effect of tumour-derived factors in order to inhibit MDSC expansion has also been a target for immunotherapy. It is proposed that pharmacological elimination of VEGF receptor-positive MDSCs (CD11b þ VEGFR1 þ ) may have a significant impact on the therapeutic efficacy of cancer vaccines. A significant decrease in the total number of CD11b þ VEGFR1 þ MDSCs was observed in metastatic renal cell carcinoma patients treated with VEGF-specific blocking antibody (avastin).93 Recently, a therapeutic strategy based on IL-4 receptor a-signalling blockade was designed to deplete tumour-associated MDSCs and tumour-associated macrophages in mice. RNA aptamer, which blocks murine and human IL4Ra, has been generated with the potential to target a cellular mechanism of tumour immune escape that is mediated by MDSCs and tumourassociated macrophages.94 Inhibition of MDSC function by inhibiting the signalling pathways that regulate the production of their suppressive factors is another promising approach. Sildenafil, a phosphodiesterase-5 inhibitor, was able to decrease the production of both ARG1 and iNOS of tumourassociated MDSCs and restored in vitro T-cell proliferation in multiple myeloma and head and neck cancer patients.95 However, it is not clear whether this favourable effect will be observed clinically in cancer patients. Nitroaspirin inhibits the production of ROS, ARG1 and iNOS and thus eliminates the suppressive functions of MDSC.96 Recently, cimetidine (histamine type-2 receptor antagonist) was shown to suppress lung tumour growth in a murine model by inducing Fas and FasL expression in MDSCs and by regulating the caspase-dependent apoptosis pathway.97 This was associated with reduced CD11b þ Gr-1 þ MDSC accumulation in vivo and reversed MDSC-mediated T-cell suppression, as well as with improved IFN-g production in vitro.97 The effect of chemotherapeutic agents on MDSCs has been investigated in only three clinical studies on human cancers. In 17 early-stage breast cancer patients receiving doxorubicin– cyclophosphamide chemotherapy every 14 days, MDSCs (Lin HLA-DR CD33 þ CD11b þ ) were found to be significantly reduced in number in the peripheral blood because of cytotoxic elimination.19 Similarly, in a cohort study of patients with melanoma (n ¼ 77, I-IV), taxane-based chemotherapy administration was shown to reduce the number of circulating MDSCs (HLA-DR CD14 þ ).98 Gabitass et al.99 have also reported a significant decrease in the number of circulating MDSCs in patients with pancreatic (n ¼ 16) and oesophagogastric (n ¼ 23) cancer post-treatment with gemcitabine-based chemotherapy (Po0.0001) . Of clinical interest, the decrease in MDSC numbers was also observed in patients at advanced stages. Nevertheless, the cytotoxic elimination of MDSCs is not target specific and can affect other immune cells. In a recent in vitro study, gemcitabine and Immunology and Cell Biology


5-fluorouracil induced direct activation of the pyrin domaincontaining 3 protein (NLRP3)-dependent caspase-1 activation complex (termed the inflammasome) in MDSCs, which led to secretion of IL-1b, production of IL-17 and thus to the promotion of tumour growth.100 In addition, gemcitabine and 5-fluorouracil demonstrated higher antitumour effects in NLRP3 mice or wildtype mice treated with the IL-1 receptor antagonist (IL-1Ra).100 The in vivo relevance of these findings indicates that 5-fluorouracil treatment should be combined with IL-1 inhibition. CONCLUSIONS Different approaches have been explored to harness the potency of the immune system to target cancer. To date, these have been essentially focused on enhancing the immunogenicity of the tumour or on the induction and expansion of immune effectors to potentially target and eradicate the tumour. However, efforts to actively stimulate the immune system against tumours in patients have been largely disappointing despite substantial evidence that peripheral immune responses against tumour antigens can be generated. This could be due to the local tumour environment, which strongly suppresses the antitumour immune responses; therefore, there is a need for strategies that could help overcome the mechanisms of immunosuppression. Recent evidence suggests a significant role for MDSCs in immune evasion. Both monocytic and granulocytic MDSC subsets were detected and characterised in different human cancers and, since the last decade, human MDSCs have been better described. However, there is still ambiguity regarding the exact definition of MDSCs in human cancers because of the diversity of cells analysed. Accumulation of MDSCs has been associated with the progression of cancer, with some evidence that their elimination could enhance cancer immunotherapy. Determining whether MDSCs mediate an antigen-specific T-cell suppression, as well as understanding the role and function of tumour-derived MDSCs in cancer pathogenesis as against peripheral subsets, needs further investigations. Therefore, there is an immense need to perform comprehensive clinicopathological correlations between tumour progression and MDSC phenotypes and levels in tumour tissue and peripheral blood. The existing evidence, although limited, indicates that MDSC levels are inversely correlated with cancer prognosis and can aid as a predictive/prognostic marker for cancer immunotherapy. However, there is lack of evidence regarding these observations, and further prospective large clinical studies are important to validate the clinical value of MDSC levels as a potential marker for cancer stage correlation and therapeutic response. We suggest that further studies should examine not only circulating MDSCs but also benign tissue with a view to comparing it with malignant tissue. Laboratory studies involving murine models have helped to evolve a better understanding of the mechanisms of induction, expansion and trafficking of MDSCs into the tumour microenvironment. However, the data from murine models have limited clinical applications because of the different phenotypical and functional features of MDSCs in human cancers. Tumour-secreted molecules exist at significantly high concentrations within the tumour microenvironment compared with other peripheral sources, and hence the cancer tissue is an important source for identification of potential factors. In addition, tumour-derived factors may dictate the induction, expansion and trafficking of MDSCs into the tumour microenvironment. The list of these factors is expanding and their selective blockade will ultimately increase our understanding of the mechanism of MDSC expansion and also help in shaping their immune suppressive role. One of the priorities in the Immunology and Cell Biology

field of MDSC research is ascertainment of whether eliminating these cells in cancer patients is likely to have clinical benefits. Therefore, the data presented on MDSC therapeutic targeting should be incorporated into the design of future clinical trials to improve immunebased therapeutic strategies and pharmacologic modulation of MDSCs in cancers. The development of novel therapeutic agents that eliminate the activity of MDSCs in human cancers should accelerate our understanding of their biological role within the tumour microenvironment. CONFLICT OF INTEREST The authors declare no conflict of interest.

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