The role of myeloid-derived suppressor cells in gastrointestinal cancer

1 INTRODUCTION

Gastrointestinal (GI) cancer accounts for a third of all new diagnoses and a similar proportion of cancer-associated mortality worldwide, and its incidence and mortality rate are on the rise [1, 2]. A number of malignancies fall under the wide umbrella of GI cancer, including esophageal cancer (EC), gastric cancer (GC), colorectal cancer (CRC), hepatocellular carcinoma (HCC), and pancreatic cancer (PC) [3, 4]. Current research shows that the estimated number of increased GI cancer cases (not include pancreatic cancer) in both sexes and ages were 4,296,333 (23.8%) and the number of deaths was estimated at 2,953,693 (30.9%) in 2018, which promotes GI cancer to be the largest public health concern in general [1]. According to the World Health Organization (WHO), the number of deaths from cancer will increase by nearly 80% by 2030 worldwide, with most occurring in low- and middle-income countries [5]. A broad range of therapeutic strategies has been developed to treat these diseases, which include surgery, chemotherapy, radiotherapy, and immunotherapy. While these approaches have improved outcomes for patients with GI cancer, the overall survival rate of these patients remains relatively poor [6, 7]. This is at least in part due to the tendency of GI cancer to resist chemotherapy and escape immune surveillance via the activation and expansion of the populations of immunosuppressive cells, such as myeloid-derived suppressor cells (MDSCs), regulatory T-cell (Treg) as well as tumor-associated macrophages (TAMs), leading to immunosuppression and an increased propensity for metastasis [8-10].

MDSCs are a heterogeneous population of immature monocytes and granulocytes that have been observed in a number of murine tumor models, where they are identified by their expression of the CD11b and Gr-1 markers [11]. In humans, similar populations of immature myeloid cells (IMCs) were initially described in head and neck squamous cell carcinoma (HNSCC) [12], and their presence has since been well-documented in cancers of the esophagus, stomach, pancreas, colon, and skin [13-15]. MDSCs accumulate in tumor sites (both primary and metastatic), where they inhibit T-cell-mediated tumor clearance, and have also been found to suppress natural killer (NK) cell populations [11, 16, 17]. Furthermore, MDSCs appear to confer some level of protection to circulating tumor cells and to be involved in the formation of pre-metastatic niches (PMNs), which encourage the invasion of cancer cells [18]. Their involvement in this process has been known for decades; in the 1970s, the “seed and soil” hypothesis was conceived by Paget [19], describing the link between tumor metastasis and MDSCs. More recently, in 2005, PMNs were defined for the first time as the specific cell microenvironment that favors the colonization of metastatic cancer cells in distant organs [20].

In GI cancer patients, MDSCs levels are closely associated with both therapeutic efficacy and overall clinical outcome [21]. For example, Limagne et al. [22] reported that accumulation of MDSCs in metastatic CRC patients was correlated with the efficacy of a 5-fluorouracil (5-FU), oxaliplatin, and bevacizumab treatment regimen. Markowitz et al. [23] studied the correlation between MDSC frequency in peripheral blood mononuclear cells (PBMCs) and disease progression among 16 chemotherapy-treated PC patients and found that a higher chance of progression was observed in the patients with increased frequency of MDSCs in peripheral blood (P = 0.0013). A meta-analysis showed that the combined hazard ratio for overall survival of GI cancer patients in the association between MDSC level and prognosis was 1.26 (95% confidence interval [CI] = 1.10-1.44, P = 0.0003) [21]. Karakasheva et al. [24] reported that CD38⁺ monocytic MDSC (M-MDSC) expansion could be associated with a subset of advanced CRC patients. Additionally, a recent study showed that peripheral circulating MDSCs were significantly correlated with the prognosis of ESCC patients [25]. Since substantial progress has been made in recent years towards understanding the function of these cells, as well as their genomic and biochemical characteristics [26-28], further investigation into their role in GI cancer is still required. In this review, we summarize the known functions and effects of MDSCs in GI cancer, as well as detail the involvement of MDSCs in immunosuppression, metastasis, and prognosis. The aim of this work was to present the development of novel treatments targeting MDSCs in GI cancer in the hope of improving outcomes for patients with this condition.

2 THE ORIGIN AND PHENOTYPE OF MDSCS IN CANCERS

MDSCs are a heterogeneous population of cells that include both myeloid progenitors and IMCs. Under healthy conditions, IMCs are formed in the bone marrow and rapidly differentiate into mature macrophages, dendritic cells (DCs), or granulocytes [11]. However, IMCs are often prevented from fully differentiating into mature cells under certain pathological conditions, including cancer [12, 11], infection [29], sepsis [30], trauma [31], and some autoimmune disorders [32], leading to an expansion of this population.

In tumor bearing mice, two main MDSC subtypes have been identified, termed granulocytic MDSCs (G-MDSC) and monocytic MDSCs (M-MDSC), respectively [33]. G-MDSC is defined as CD11b⁺Ly6G⁺Ly6C^low/− cells, while M-MDSC is defined as CD11b⁺Ly6G^low/−Ly6C^high cells. Both of these cell subsets are expanded in murine tumor models and have primarily been documented in the bone marrow, peripheral blood, spleen, liver and lung tissues, and a range of tumor tissues [34].

In humans, MDSC populations are even more complex. In 1995, MDSCs bearing the cell surface marker CD34 was first reported in HNSCC patients [12]. Further researches defined the criteria for the phenotypic characterization of these cells by flow cytometry [35, 36]. Among the human PBMCs, M-MDSC is defined as CD14⁺ while G-MDSC is CD15⁺, and both these subsets are HLA-DR⁻CD33⁺. A further subset of HLA-DR⁻CD33⁺ lineage-negative (Lin⁻) early MDSCs (e-MDSCs) has since been proposed, which lack the expression of mature cell lineage markers such as CD3, CD14, CD15, CD19 and CD56. This subset contains mixed groups of MDSCs and is comprised of more immature progenitors [37]. In healthy individuals, IMCs with the phenotype described above comprise ∼0.5% of PBMCs [11]. Importantly, the evidence indicates that these subpopulations may have different functions in cancer [38], infections [39], and autoimmune diseases [32], with both G-MDSC and M-MDSC populations being expanded in those cases. Youn et al. [40] reported that the expansion of G-MDSC population was greater than that of M-MDSC subset in the spleen of 10 different tumor models of lung, breast, colon, melanoma, and sarcoma in three different strains of mice. They also found that the two subpopulations used different mechanisms to suppress T-cell function: G-MDSC mainly generated reactive oxygen species (ROS), whereas M-MDSC mainly produced nitric oxide (NO). As MDSCs are a group of myeloid cells comprised of precursors of macrophages, granulocytes, and DCs at earlier stages of differentiation, these two subsets of MDSCs remained in different differentiation directions. Previously, it has been reported that 20% of G-MDSCs differentiated to F4/80⁺ macrophages compared with more than 60% of cells differentiated from M-MDSCs in an in vitro culture system with granulocyte macrophage colony-stimulating factor (GM-CSF). Additionally, 10% of G-MDSC differentiated into CD11c⁺ DCs, whereas more than 50% of cells differentiated from M-MDSC were CD11c⁺ cells [40]. Therefore, M-MDSCs have been found to be restricted to mature macrophages and DCs in vitro more than G-MDSCs, and various tumor-derived factors (TDFs) produced by different tumor cells define the expansion of MDSC subsets.

From the perspective of GI cancer, multiple reports have shown involvement of MDSCs in tumor progression. For instance, Ding et al. [41] have demonstrated that bone marrow-derived Schlafen4⁺(SLFN4⁺) MDSCs could migrate to the stomach during Helicobacter infection. Later, they found that high MiR-130b expression in SLFN4⁺ MDSCs was responsible for neoplastic stimulation before GC. Mechanistically, miR-130b-mediated inhibition of cylindromatosis (Cyld) leads to release/activation of NF-κB, which in turn promotes MDSC immunosuppression via up-regulating the expression of arginase I (ARG1) and nitric oxide synthase 2 (NOS2). Suppression of miR-130b impaired MDSCs function and restored CD8⁺ cytotoxic T-cell infiltration of the stomach. Moreover, elevated levels of miR-130b were found in the serum of both Helicobacter-infected mice and gastric cancer patients that correlated with the respective metaplastic changes in the stomach, showing a strong correlation with pre-neoplastic changes prior to GC [42]. CD38 is a transmembrane glycoprotein and belongs to the ADP-ribosyl cyclase family, possessing both ectoenzyme and receptor functions. It was first reported by Karakasheva et al. [24] that CD38^high MDSCs are more immature than MDSCs lacking CD38 expression, possess a greater capacity to suppress activated T cells, and promote tumor growth to a greater degree, with an increased inducible nitric oxide synthase (iNOS) production in EC. Later, Karakasheva et al. [43] also proved that CD38⁺ M-MDSC expansion characterizes a subset of advanced CRC patients, and targeting M-MDSCs with an anti-CD38 monoclonal antibody would bring a benefit to the metastatic CRC patients. In GC patients, the nature of most MDSCs has been recognized as granulocyte-like cells or monocyte-like cells, which have been reported in many studies [44, 45]. Recently, a novel subset of CD45⁺CD33^lowCD11b^dim MDSCs in GC patients was identified by Zhuang's group [46]. Compared to healthy individuals, CD45⁺CD33^lowCD11b^dim MDSCs morphologically resembled neutrophils with the expression of CD66b, showing a more immunosuppressive function. Serum IL-6 and IL-8 derived from GC patients could activate granulocytic MDSCs to express ARG1 through the PI3K/AKT signalling pathway. Evidence showed that the percentage of CD45⁺CD33^lowCD11b^dim MDSCs in the peripheral blood and the levels of IL-6, IL-8, and ARG1 in the serum were positively correlated with GC progression and negatively correlated with overall patient survival.

Nevertheless, it remains unclear whether these special subsets of MDSCs would lead to a resistance to anti-cancer therapy, such as chemoresistance, as current treatments for GI cancer patients retain little effect. From a personal opinion, apart from the reported agents or clinical trials, other cytokines, chemokine ligands/receptors, genes and molecules, especially those first reported in GI cancer that affect the number and function of MDSCs, could be potential therapeutic targets and promising drugs for GI cancer.

3 THE UNDERLYING MECHANISMS OF MDSC-MEDIATED IMMUNOSUPPRESSION IN GI CANCER

Suppression of the immune system is a primary function of MDSCs in cancer and occurs both through direct targeting and release of soluble mediators that regulate immune response [11]. The main factors involved in MDSC-mediated immune suppression includes ARG1, iNOS, NADPH oxidase 2 (NOX-2), ROS, transforming growth factor β (TGFβ), programmed death-ligand 1 (PD-L1), and indoleamine 2,3-dioxygenase (IDO), which specifically target T cells and other immune cells [34] (Figure 1).

4 THE INVOLVEMENT OF MDSCS IN THE MALIGNANT PROGRESSION OF GI CANCER

MDSC-mediated tumor metastasis is a complex, multi-stage process characterized by the secretion of chemokines, cytokines, and enzymes, all of which contribute to tumor cell proliferation, invasion, survival, and chemoattraction [33]. MDSCs favor tumor cell invasion and metastasis through remodeling of the basal membrane via the secretion of matrix metalloproteinases (MMPs), which opens a route for tumor cell migration and invasion [95]. This process may also involve macrophage-1 antigen (MAC-1)/ligand intercellular adhesion molecule-1 (ICAM-1) interactions [96, 97] and TGFβ1 [98]. In addition, MDSCs protect circulating tumor cells and promote their extravasation [99], serving a critical role in the formation of circulating tumor clusters (CTCs). These CTCs are strongly associated with immune escape. Furthermore, MDSCs also support tumor cell extravasation and invasion through the release of neutrophil extracellular traps (NETs) [100] and promote tumor cell survival via the ROS-nuclear erythroid 2-related factor 2 (NRF2)/antioxidant responsive element (ARE) axis and Notch signaling pathway [101, 102]. With CTCs being favored in this way by MDSCs, patients with GI cancer are more likely to experience rapid metastasis and poor prognosis [103, 104].

In addition, MDSCs have been found to promote the generation of PMNs in different cancer patients, including GI cancer. Prior to the arrival of tumor cells to a secondary site, MDSCs infiltrate the healthy tissue and induce their transformation to future metastatic sites. The accumulation, expansion, and recruitment of MDSCs in distant organs may be induced by a range of cytokines and chemokines, which are derived from the distant organs, tumor cells, and cancer-associated fibroblasts (CAFs). In the tumor microenvironment, several tumor-derived suppressor factors (TDSFs) have been found to influence the accumulation and expansion of myeloid precursors in PMNs, including G-CSF, GM-CSF, IL-6, and Flt3-ligand [105, 106]. Meanwhile, chemokines such as CCL2, which is produced by both tumor cells and target organs, drive MDSCs into normal tissues to form PMNs [107]. Expression of CAFs, such as fibroblast activation protein (FAP) and alpha smooth muscle actin (α-SMA), further promote the recruitment and sustainment of MDSCs in PMNs via the STAT3-CCL2 signaling pathway [108].

TAMs have also been found to impact the formation of liver PMNs in a murine colon tumor model by releasing CXCL1, which attracts CXCR2-expressing MDSCs [109]. Other chemokines, such as monocyte chemoattractant protein 1 (MCP-1) [110], CCL5 [111], CCL12 [112], CCL9 [113], CCL15 [114], and CXCL17 [115], have also been reported to promote the transport of MDSCs to PMNs, although the source of these cytokines in PMNs remains unclear. Furthermore, MDSCs initiate and sustain the development of PMNs by autocrine signaling: secreting a variety of regulatory molecules including PDGF [115], vascular endothelial growth factor A (VEGFA) [116], matrix metalloproteinase 9 (MMP9) [117], bombina variegate peptide 8 (Bv8) [118], and S100 calcium-binding protein A8/A9 (S100A8/A9) [119]. For example, S100A9-deficient mice showed reduced accumulation of MDSCs in PMNs during colonic metastasis [120]. Notably, MDSCs have also been found to differentiate into fibrocytes and osteoclasts in tumor mice, which may further enhance tumor growth and metastasis in cancer patients [121, 122]. In the clinic, recent investigations have documented a correlation between MDSC frequency in GI cancer patients and progression, which is summarized in Table 1.

5 MDSCS AS A POTENTIAL PROGNOSTIC MARKER IN GI CANCER

Several clinical studies have suggested a strong relationship between prognosis and an increased frequency of MDSCs in the peripheral blood or tumor tissue of patients with GI cancer. For example, in HCC patients, a high MDSC frequency is correlated with the early recurrence after resection [123]. For CRC patients, a number of studies have demonstrated that the frequency of circulating MDSCs is correlated with reduced overall survival both before and after chemotherapy and radiotherapy, and this correlation was of clinical relevance [124]. A study including both healthy donors and ESCC patients showed that MDSC levels (CD11b⁺CD14⁺HLA^–DR^–) in the PBMCs of patients with stage IIIb or IV tumors were increased compared with those of patients with stage II or IIIa disease [125]. Also, the authors reported that 14 of the 23 patients (60.9%) in the low MDSC frequency group were responders, while only 1 of 12 patients (8.3%) in the high MDSC frequency group responded to the chemotherapy [125]. A further clinical report showed that increased peripheral blood MDSCs level was correlated with a 22% increased risk of death, based on data from 60 patients with EC, 25 patients with GC, and 54 healthy controls [13]. Another study also showed the correlation among MDSC frequency, cancer stage, and survival rates of GC patients [52]. Current studies concerning the relationship between the frequency of MDSCs and cancer progression, as well as prognosis, are summarized in Table 1. These findings suggest that the frequency of MDSCs could be used as a prognostic marker in patients with GI cancer and may also be a useful tool when selecting individual treatment regimens.

6 MDSC-TARGETED STRATEGIES FOR TREATING GI CANCER

As described above, many studies have demonstrated that the existence of MDSCs in the tumor microenvironment remained an obstacle for the efficacy of cancer treatment. Fortunately, to date, many therapeutic approaches against GI cancer by targeting MDSCs, including small molecules, vitamins, conjugates, nucleotide, and immunotherapy, have been developed. These therapies work by affecting the accumulation, recruitment, differentiation, and immunosuppression of MDSCs (Table 2).

7 CURRENT CLINICAL TRIALS IN GI CANCER TARGETING MDSCS

Based on targeting MDSCs, several therapies in GI cancer have been undergone or completed clinical trials. Here, we reviewed the website of clinicaltrials.gov and selectively summarized the therapeutic approaches involving MDSCs by different treatment regiments for GI cancer in Table 4.

For instance, a completed phase II clinical trial (NCT02090101) involved 32 participants to evaluate the influence of Anakinra on the vascularization of liver metastases by targeting VEGFA in metastatic CRC patients. Similarly, six participants were enrolled into a phase I clinical trial of VXM01 (NCT02718430), which is targeting vascular endothelial growth factor receptor 2 (VEGFR2). This completed trial was set up to examine safety, efficacy, and immune biomarkers in patients with metastatic CRC. Both the two clinical trials employed MDSCs as a secondary outcome measure. Unfortunately, neither result of these two trials were posted on the website. Another completed phase II clinical trial (NCT01507103) enrolled 124 participants to determine the impact of tecemotide (L-BLP25) administration on the mucinous glycoprotein 1 (MUC1)-specific immune response in subjects with newly diagnosed rectal cancer who are eligible for neoadjuvant therapy. In this clinical trial, the population of MDSCs (CD33⁺CD14^–) and other immune cells such as NK cells, macrophages, and DCs were set as secondary outcome measures. The treatment showed a total risk of 25.64%, but the population change of MDSCs after treatment was not found from their posted results. The limitation of this study was that not all efficacy data were analyzed as no acceptable ELISpot assay is available, which led to the discontinuation of the development of tecemotide (L-BLP25) in September 2014. Currently, there are not enough completed clinical trials that specifically target MDSCs in GI cancer, evidencing that more work should be done around this field.

8 CONCLUSIONS AND FUTURE PROSPECTS

As described in this review, MDSCs modulate several different aspects of tumor growth and metastasis, including immunosuppression, local invasion, and PMN formation. This broad range of functions suggests that these cells could be used as targets to develop both innovative liquid biopsy-based cancer diagnostics and novel anti-cancer therapeutic approaches. In future, researches investigating the regulatory functions of MDSCs in GI cancer should perhaps focus on their interactions with stromal cells from the tumor microenvironment, such as CAFs, because their role in the MDSC lifecycle remains to be fully elucidated. Furthermore, one of the most promising treatment approaches appears to be the promotion of MDSC differentiation into mature myeloid cells, as these lose their immunosuppressive functions. High-throughput technologies will be of particular use as these allow the full characterization of the genetic, epigenetic, and metabolic pathways underlying the cross-talk between MDSCs and cancer cells, as well as identifying relevant molecules. This will likely clarify some unsolved aspects of the interaction between MDSCs and GI cancer cells, laying the groundwork for more effective therapeutic strategies in the future. Finally, unlike in malignancies such as lung, renal, and skin cancers, the efficacy of immunotherapeutic agents on GI cancer has, on the whole, been much less remarkable and do not apply to the majority [166]. This could be explained by the permeated MDSCs in the tumor microenvironment, which eliminated the response of activated T cells generated by PD1/PD-L1 blockade. Therefore, the possibility of combining checkpoint-based immunotherapy with approaches targeting MDSCs may also represent a promising avenue in the development of more personalized treatments for GI cancer.

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