The Biomedical Outlook: Focus on Cancer Series (3)

MDSCs in Progression of Solid Cancers: Where We Stand

Introduction

The tumour microenvironment (TME) is characterised by a heterogeneity of cell types, including immune cells and fibroblasts which play a role in tumour progression, chiefly via paracrine signalling. One cell type that has attracted much attention in the past decade is the myeloid derived suppressor cell (MDSC). MDSCs represent a heterogeneous immature cell population of the myeloid lineage that fail differentiation into granulocytes, macrophages or dendritic cells. MDSCs are estimated to comprise around 5% of total cells in solid tumours[1] and have been found to correlate with clinical stage in various cancers. In mice, MDSCs typically co-express the markers Gr1 and CD11b while human MDSCs are commonly delineated by the CD14- CD11b+ immunophenotype[2]. The expansion of immature myeloid cells (IMCs) in bone marrow is induced by tumour-derived paracrine factors also linked to inflammation including IL-6, IL1β, PGE₂ and GM-CSF. Other TME components, including M2 macrophages and endothelial cells, release cytokines and chemokines involved in MDSC activation and recruitment[3]. This article expatiates on the role of TME MDSCs in the processes of tumour immunosuppression and angiogenesis. In the second part of this review the current progress on MDSCs in therapy will be discussed with a view to informing future research.

MDSCs in Immunosuppression

Immunosuppression is perhaps the best characterised feature of MDSCs. One of the key features of MDSCs is high expression of iNOS and arginase (ARG1) both involved in metabolism of L-arginine to L-ornithine and urea[4]. CD8+ cytotoxic T cells represent the key immune defence in cancer. L-arginine is purported to support the anti-tumour immune response mediated by T cells, in part by upregulating expression of IL-2, which promotes T cell clonal expansion, and upregulating the CD3ζ chain which is required for T cell activation following antigen challenge [5, 6]. Thus, depletion of L-arginine is associated with T-cell hyporesponsiveness both in vitro and in vivo, which may be compounded by cell surface expression of CTLA4 by MDSCs [7]. The role of reduced L-arginine bioavailability on macrophages, the other major immune cell type with anti-tumour activity, has not been fully elucidated. iNOS is abundantly expressed by macrophages which therefore depletes local l-arginine concentrations. While some studies report reduced iNOS expression and reduced macrophage viability in low L-arginine conditions[8], others report no change[9]. Further work is clearly needed in this area. The high release of reactive oxygen species (ROS) from MDSCs also impairs CD8+ T cell reponses [10], while new evidence suggests MDSC derived peroxynitrite (ONOO^-) may promote nitration of the T-cell receptor (TCR) to inhibit its recognition of tumour antigens[11]. Moreover, TGF-β, IL-10 and IDO released from transformed cells have been shown to mediate induction of regulatory T cells (Tregs), thereby contributing to an immunosuppressive milieu[12-14]. Emerging evidence suggests MDSCs also release these signalling molecules, with TGF-β and IDO expression being linked directly to immunosuppression via Tregs[15]. Thus, therapies targeting MDSCs may also limit Treg-mediated immunosuppression.

Natural killer (NK) cells are a major innate immune component which detects altered MHC-1 expression on tumour cells relative to normal cells. Mouse models in which NK cells are depleted show accelerated tumour growth despite normal CD8+ T cell populations, highlighting the pivotal anti-tumour role of NK cells[16, 17]. Striking new research by Park and colleagues reveals that adoptively transferred immature NK cells in mice undergo a phenotypic switch to MDSCs in vivo, a phenomenon dependent on IL-2 abrogation[18]. This suggests a mechanism whereby MDSC-produced arginase depletes IL-2 expression by T cells, which then enables generation of further MDSCs from infiltrating NK cells. Furthermore, NK cell activity was found to be impaired in mouse models of liver cancer via membrane-bound TGF-β1[19]. M1 macrophages have also been found to express this ligand, indicating possible synergism between these cells and MDSCs in immunosuppression [20].

MDSCs in Angiogenesis

The TME as well as transformed epithelial cells have roles to play in inducing angiogenesis. A number of pro-angiogenic factors including VEGF, MMPs 2 and 9 and the chemokines CXCL5 and CXCL2[21, 22]. A complex multidirectional cross-talk is known to occur, with epithelial cells expressing VEGF and PGE₂ which subsequently binds EP receptors on MDSCs to stimulate release of pro-angiogenic peptides, while MDSC-derived IL-10 and TGF-β augment tumour growth and ultimately angiogenesis[23]. MDSCs themselves are also a potent source of PGE₂, meaning autocrine signalling cannot be ruled out. Kujawski et al. reveal that constitutive STAT-3 activation in MDSCs is required for expression of many of the aforementioned angiogenic signalling factors[24], in harmony with other studies that show the antiangiogenic agent sunitinib is capable of promoting renal cell carcinoma regression via STAT3 inhibition[25].The potent anti-tumour activity seen following treatment of tumours with various circumin analogues designed to inhibit STAT-3 may be partially ascribed to its effects on MDSC-induced angiogenesis [26]. STAT3 dimerisation is important for the activity of pro-angiogenic mediators follow receptor ligation including VEGF , bFGF and angiopoeitin [27-29].

Early evidence from Yang and colleagues suggests MDSCs may be able to transdifferentiate into ECs, based on the observation that radiolabelled Gr1+CD11b+ cells were found incorporated into the endothelial wall over a period of time together with upregulation of V-CAM, a classical endothelial cell marker. Indeed, MDSC plasticity has been noted in other studies, with differentiation into macrophages and dendritic cells. Recently, Zhang et al. identified expansion of cells with fibrocyte-like properties in metastatic paediatric sarcoma patients[30]. Similar to other MDSCs, these cells have roles to play in immunosuppression and angiogenesis in vitro. Thus characterising the pathways and signalling cascades that lead to development of these cells may be important for identifying novel drug targets.

While anti-angiogenic therapies such as Bevacizumab and sunitinib have proven efficacy in extending survival of patients with advanced solid tumours, resistance to these therapies has been observed, with MDSCs being implicated. Finke et al observed sunitinib resistance to be associated with persistence of a neutrophil-like MDSC population dependent on ambient GM-CSF[1]. The mechanism of action for sorafanib, a multikinase inhibitor used extensively for advanced kidney and lung cancer, involves depletion of MDSCs alongside Tregs. Thus, alterations in MDSC phenotypes may be a conceivable resistance mechanism in this setting. Similarly, although it has not been directly demonstrated, it is plausible that MDSCs mediate resistance to bevacizumab both by release of pro-angiogenic factors and immunosuppression on CD8+ T cells and NK cells and thereby impair antibody-mediated cell dependent cytoxicity (ADCC). Clearly, understanding of resistance mechanisms will make existing therapies more effective. The pivotal role of MDSCs in the processes herein described is summarised in Figure 1 below:

Figure 1: Transformed epithelial cells induce MDSCs via VEGF and PGE2. Ambient MDSCs release pro-angiogenic factors that act on endothelial cells (ECs) together with those derived directly from transformed cells. Transdifferentiation of MDSCs into ECs may occur. MDSCs inhibit the tumoricidal activities of NK cells and CD8+ CTLs while inducing Tregs which further impair CD8+ CTL function. All abbreviations are defined in the main text.

MDSCs as Biomarkers and Therapeutic Targets

The prominent role of MDSCs in enabling acquisition of a number of tumour hallmarks including immune evasion, angiogenesis and metastasis has led to investigation of numerous therapeutic strategies. As the quantity and phenoytpes of MDSCs may alter the course of disease and response to therapy, there has been a drive to identify if they may be an appropriate biomarker. Cole et al. noted that MDSC correlated directly with levels with circulating tumour cells in the blood, possibly allowing both MDSCs and CTCs to be used together to stratify patients with greater accuracy (meeting abstract). Despite correlating increased MDSC load with poorer overall survival in metastatic breast cancer, MDSCs were quantified on a binary scale (i.e. cell counts higher or lower than the median). Dividing patients into distinct categories based on MDSCs as a percentage of tumour volume may be more judicious. Zhang et al. further found circulating MDSCs to be elevated in patients with colorectal cancer, while their levels also correlated to clinical stage[31]. Similar findings are seen in gastric cancer[32] and breast[33] while recent work khaled did not find the same trend in pancreatic cancer. This suggests tumour and tissue-specific roles of MDSCs and further work is needed to clarify the prognostic value of MDSCs.

Given the important role of the COX2-PGE₂ axis in MDSC induction, Rodriguez and colleagues found the COX-2 inhibitor SC-58125 effective in both reducing MDSC numbers and inhibiting Arginase 1 expression in murine lung cancer models[34]. Similar findings have emerged for glioma[35] and mesothelioma [36]. In both these studies, MDSC depletion was associated with improved CTL infiltration. While celecoxib is a widely used selective COX-2 inhibitor, its prolonged use is associated with higher risk of adverse cardiovascular events. Selective inhibitors of the terminal prostaglandin E₂ synthase mPGES1 (which lies downstream of COX-2) are emerging and may provide a safer alternative to reduce MDSC activity [37]. As PGE₂ expression has been linked to endothelial cell proliferation, migration, tubologenesis and mural cell recruitment, disruption of COX-2 may also be a useful anti-angiogenic strategy[38, 39].

Another option for circumventing immunosuppression is to inhibit iNOS to limit l-arginine metabolism. Jayeraman et al. used the small molecule L-NIL to this effect and found reductions in ROS production and STAT-3 activation while observing improved CD8+ CTL infiltration in melanoma murine models[40].

The enzyme PDE5 plays a role antagonising the effects guanalyl cyclase by degrading cGMP and upregulating ARG1 and NOS. Interest is now focussed on phosphodiesterase-5 (PDE5) inhibitors to target MDSCs. These agents are already in clinical use for conditions such as erectile dysfunction and pulmonary hypertension. Initial work by Serafini et al. found sildenafil to effectively enhance CD8+ CTL infilitration and cytotoxicitiy, while decreasing ARG1 and NOS activity[41]. However, subsequent work by Pyriochou et al. found sildenafil to drive endothelial cell proliferation and tubologenesis in vivo contingent on MEK and P38 activation[42]. These conflicting findings bring into question the value of PDE5 inhibitors. Nevertheless, evidence of their efficacy is currently being sought alongside other agents in clinical trials for multiple myeloma (NCT01374217), orophyrangeal carcinoma (NCT00843635), non-small cell lung cancer (NCT00752115) and pancreatic cancer (NCT01342224). A potential avenue for future research is to determine safety and efficacy of dual COX2 and PDE5 blockade.

An emerging concept in this field is to capitalise on the propensity of MDSCs to migrate to tumour tissue for immunotherapy. Chandra et al. developed an attenuated form of Listeria Monocytogenes which, upon injection into BALB mice, were able to survive and grow inside MDSCs[43]. Following chemotaxis of MDSCs to the TME, Listeria were able to infect surrounding primary and metastatic lesions and thereby promote an improved innate and adaptive anti-tumour immune response. It remains to be seen whether this approach is feasible in higher organisms.

Conclusions

Our understanding of the molecular mechanisms regulating the effects of MDSCs in the TME is gaining rapid momentum, enabling the investigation of novel targeted agents. The results of the clinical trials described above in particular will be eagerly anticipated. The potential of MDSCs as prognostic biomarkers in a variety of solid tumours will also require further elucidation in the drive towards personalised medicine.

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