Tumor-associated Myeloid-derived Suppressor Cells Research Articles

Myeloid Cells’ Evasion of Melanoma Immunity

An immune-suppressive role of myeloid-derived suppressor cells (MDSCs) in melanoma has long been speculated, whereas molecular mechanisms underlying this role are not well understood. Here, Chung and colleagues show that dendritic cell-associated, heparan sulfate proteoglycans-dependent integrin ligand (DC-HIL), a cell surface immune-modulatory molecule, is highly expressed on tumor-associated MDSCs. Genetic ablation or antibody blockade of DC-HIL delays the growth of transplantable B16 melanoma in syngeneic mice, which is accompanied by enhanced antitumor T-cell activities. These findings support a role for DC-HIL in immune evasion within the melanoma microenvironment.

Complexity and challenges in defining myeloid-derived suppressor cells.

Study of myeloid cells endowed with suppressive activity is an active field of research which has particular importance in cancer, in view of the negative regulatory capacity of these cells to the host's immune response. The expansion of these cells, called myeloid-derived suppressor cells (MDSCs), has been documented in many models of tumor-bearing mice and in patients with tumors of various origin, and their presence is associated with disease progression and reduced survival. For this reason, monitoring this type of cell expansion is of clinical importance, and flow cytometry is the technique of choice for their identification. Over the years, it has been demonstrated that MDSCs comprise a group of immature myeloid cells belonging both to monocytic and granulocytic lineages, with several stages of differentiation; their occurrence depends on tumor-derived soluble factors, which guide their expansion and determine their block of differentiation. Because of their heterogeneous composition, accurate phenotyping of these cells requires a multicolor approach, so that the expansion of all MDSC subsets can be appreciated. This review article focuses on identifying MDSCs and discusses problems associated with phenotyping circulating and tumor-associated MDSCs in humans and in mouse models. This article is protected by copyright. All rights reserved.

Activation of the COX2/PGE2 axis by immune effector cells promotes the self-limiting nature of type-1 immunity in cancer tissues (VAC3P.939)

Abstract Type-1 immune responses, mediated by IFNγ and TNFα-producing CTLs, Th1, and NK cells, are essential for effective anti-tumor immunity. Despite recent advances in the induction and stabilization of these responses by cancer immunotherapies, the clinical success of these approaches remain limited. Here, we report that the activation of type-1 immunity within the human tumor environment initiates IFNγ- and TNFα-dependent counter-regulation, driven by amplification of prostaglandin E2 (PGE2) and the key regulator of PGE2 synthesis, cyclooxygenase 2 (COX2). We demonstrate that activated NK cells and CTLs induce IFNγ/TNFα-mediated over-expression of indoleamine 2,3-dioxygenase (IDO), inducible nitric oxide synthase (iNOS/NOS2), IL-10, and COX2 by tumor-associated myeloid-derived suppressor cells (MDSCs). Importantly, this self-limiting suppressive feedback driven by type-1 immunity could be eliminated not only by neutralization of IFNγ and TNFα, the factors critically required for the anti-tumor activity of immune effector cells, but also by COX2 blockade, which counteracted the IFNγ/TNFα-driven enhancement of all other suppressive factors, amplifying the therapeutic potential of intratumoral type-1 immunity. Our data demonstrate an intrinsic mechanism underlying the self-limiting character of type-1 immunity within the human tumor environment, and provide rationale for targeting the COX2/PGE2 axis to enhance desirable type-1 responses for cancer immunotherapy.

Silibinin inhibits accumulation of myeloid-derived suppressor cells and tumor growth of murine breast cancer.

Myeloid-derived suppressor cells (MDSC)s increase in blood and accumulate in the tumor microenvironment of tumor-bearing animals, contributing to immune suppression in cancer. Silibinin, a natural flavonoid from the seeds of milk thistle, has been developed as an anti-inflammatory agent and supportive care agent to reduce the toxicity of cancer chemotherapy. The goals of this study were to evaluate the effect of silibinin on MDSCs in tumor-bearing mice and antitumor activity of silibinin in a mouse model of breast cancer. 4T1 luciferase-transfected mammary carcinoma cells were injected into in the mammary fat pad female BALB/c mice, and female CB17-Prkdc Scid/J mice. Silibinin treatment started on day 4 or day 14 after tumor inoculation continued every other day. Tumor growth was monitored by bioluminescent imaging (BLI) measuring total photon flux. Flow cytometry measured total leukocytes, CD11b+ Gr-1+ MDSC, and T cells in the blood and tumors of tumor-bearing mice. The effects of silibinin on 4T1 cell viability in vitro were measured by BLI. Treatment with silibinin increased overall survival in mice harboring tumors derived from the 4T1-luciferase breast cancer cell line, and reduced tumor volumes and numbers of CD11b+Gr-1+ MDSCs in the blood and tumor, and increased the content of T cells in the tumor microenvironment. Silibinin failed to inhibit tumor growth in immunocompromised severe combined immunodeficiency mice, supporting the hypothesis that anticancer effect of silibinin is immune-mediated. The antitumor activity of silibinin requires an intact host immune system and is associated with decreased accumulation of blood and tumor-associated MDSCs.

PGE2-Driven Induction and Maintenance of Cancer-Associated Myeloid-Derived Suppressor Cells

Myeloid-derived suppressor cells (MDSCs) are critical mediators of tumor-associated immune suppression, with their numbers and activity strongly increased in most human cancers and animal models. MDSCs suppress anti-tumor immunity through multiple mechanisms, including the manipulation of arginine and tryptophan metabolism by such factors as arginase (Arg), inducible nitric oxide synthase (iNOS/NOS2), and indoleamine-2,3-dioxygenase (IDO). Prostaglandin E2 (PGE2), a mediator of chronic inflammation and tumor progression, has emerged as a key molecule in MDSC biology. PGE2 promotes MDSC development and their induction by additional factors, directly suppresses T cell immune responses and participates in the induction of other MDSC-associated suppressive factors, including Arg, iNOS and IDO. It further promotes MDSC recruitment to tumor environments through the local induction of CXCL12/SDF-1 and the induction and stabilization of the CXCL12 receptor, CXCR4, on tumor-associated MDSCs. The establishment of a positive feedback loop between PGE2 and cyclooxygenase 2 (COX-2), the key regulator of PGE2 synthesis, stabilizes the MDSC phenotype and is required for their suppressive function. The central role of PGE2 in MDSC biology provides for a feasible target for counteracting MDSC-mediated immune suppression in cancer.

Abstract 3647: Characterization of the relationship between mature neutrophils and tumor associated granulocytic myeloid-derived suppressor cells

Abstract Myeloid-derived suppressor cells (MDSC) is a group of cells with potent immune suppressive activity that accumulate in many pathologic conditions. In cancer, most of MDSC are represented by cells with granulocytic phenotype, CD11b+Ly6ClowLy6G+, and morphology and are thus called granulocytic MDSC (G-MDSC). This phenotype and morphology are also shared by mature neutrophils (Neu) in naive mice. The relationship, if any, between G-MDSC and Neu remains unclear. In this study, we compared tumor associated G-MDSC and mature Neu. G-MDSC were freshly isolated from spleen of tumor-bearing mice. Because there are no known specific markers that differentiate between Neu and G-MDSC in tumor being mice or Neu and immature myeloid cells in naïve mice; Neu were isolated from naive mice by mobilizing them to the peritoneum (pNeu). We found that in contrast to pNeu, a substantial proportion of G-MDSC express the M-CSF receptor, CD115, CXCR4 and the CD244 molecule, also known as 2B4. pNeu have high amounts of lysosomes, endosomes and proteasomes, which was validated using immunofluorescence microscopy to measure the expression of associated proteins, LAMP2, EEA1, PSMA5, respectively. G-MDSC had significantly lower expression of LAMP2, EEA1, PSMA5. Also, G-MDSC, compared to pNeu, had significantly lower phagocytic activity, as measured by the uptake of latex beads in vitro. On the other hand, G-MDSC, but not pNeu, suppressed T-cell IFN-γ production upon antigen-specific stimulation. Interestingly, pNeu had higher arginase activity and nitric oxide production than G-MDSC, while G-MDSC had a significantly higher increase in reactive oxygen species levels upon PMA stimulation in vitro. This may explain the differences in suppressive activity. G-MDSC survived 3 days in culture, in the presence of GM-CSF. Within 24 hours in culture with GM-CSF, they became phenotypically and functionally similar to pNeu, with increased phagocytic capacity and LAMP2 expression and decreased expression of CD115, CXCR4, and CD244. These data suggest that, in cancer, G-MDSC may represent pathologically activated precursors of neutrophils. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3647. doi:10.1158/1538-7445.AM2011-3647

MiR‐223 suppresses differentiation of tumor‐induced CD11b+Gr1+myeloid‐derived suppressor cells from bone marrow cells

Tumor-associated factors are related to increased accumulation of CD11b(+) Gr1(+) myeloid-derived suppressor cells (MDSCs). However, the exact mechanism of how genetic factors control the expansion of MDSCs in tumor-bearing hosts remains elusive. Herein, we found that tumor-associated MDSCs and their subsets, mononuclear MDSCs and polymorphonuclear MDSCs, have decreased expression of miR-223 when compared to CD11b(+) Gr1(+) cells from the spleen of disease-free mice. With the differentiation of CD11b(+) Gr1(+) MDSCs from bone marrow cells (BMCs) upon exposure to tumor-associated factors, the expression of both pri-miR-223 and mature miR-223 was downregulated, indicating that the expression of miR-223 could be regulated by tumor-associated factors. Interestingly, miR-223 remarkably inhibits differentiation of BMCs into CD11b(+) Gr1(+) MDSCs in the presence of tumor-associated factors by targeting myocyte enhancer factor 2C (MEF2C). Using reconstituted s.c. tumor models, miR-223 also suppresses accumulation of CD11b(+) Gr1(+) MDSCs, whereas its targeting molecule MEF2C increases the number of MDSCs. Tumor growth is slower in mice infused by miR223-engineered BMCs than in mice infused with control transfected BMCs. As miR-223 and its target molecule MEF2C are highly conserved between mice and humans, the modulation of miR-223 in tumor-induced CD11b(+) Gr1(+) MDSCs may exert an important role in controlling the increased accumulation of CD11b(+) Gr1(+) MDSCs in patients with tumor.

The inflammasome component NLRP3 impairs antitumor vaccine by enhancing the accumulation of tumor-associated myeloid-derived suppressor cells.

The inflammasome is a proteolysis complex that generates the active forms of the proinflammatory cytokines interleukin (IL)-1β and IL-18. Inflammasome activation is mediated by NLR proteins that respond to microbial and nonmicrobial stimuli. Among NLRs, NLRP3 senses the widest array of stimuli and enhances adaptive immunity. However, its role in antitumor immunity is unknown. Therefore, we evaluated the function of the NLRP3 inflammasome in the immune response using dendritic cell vaccination against the poorly immunogenic melanoma cell line B16-F10. Vaccination of Nlrp3(-/-) mice led to a relative 4-fold improvement in survival relative to control animals. Immunity depended on CD8(+) T cells and exhibited immune specificity and memory. Increased vaccine efficacy in Nlrp3(-/-) hosts did not reflect differences in dendritic cells but rather differences in myeloid-derived suppressor cells (MDSC). Although Nlrp3 was expressed in MDSCs, the absence of Nlrp3 did not alter either their functional capacity to inhibit T cells or their presence in peripheral lymphoid tissues. Instead, the absence of Nlrp3 caused a 5-fold reduction in the number of tumor-associated MDSCs found in host mice. Adoptive transfer experiments also showed that Nlrp3(-/-) MDSCs were less efficient in reaching the tumor site. Depleting MDSCs with an anti-Gr-1 antibody increased the survival of tumor-bearing wild-type mice but not Nlrp3(-/-) mice. We concluded that Nlrp3 was critical for accumulation of MDSCs in tumors and for inhibition of antitumor T-cell immunity after dendritic cell vaccination. Our findings establish an unexpected role for Nlrp3 in impeding antitumor immune responses, suggesting novel approaches to improve the response to antitumor vaccines by limiting Nlrp3 signaling.

Abstract LB-148: Depletion of myeloid derived suppressor cells in pancreatric cancer results in decreased tumor growth

Abstract Introduction: Pancreatic adenocarcinoma (PA) is a highly aggressive and chemo-resistant malignancy with a median survival of only 6 months. Myeloid derived suppressor cells (MDSC) have been implicated in the immune suppression and growth of several malignancies. MDSC are highly prevalent in the tumor microenvironment, and represent a potential therapeutic target. Here, we assess the tumor-promoting ability of MDSC in a model of PA and explore MDSC targeting strategies using ZA, a drug known to inhibit MDSC. Methods: C57BL/6 mice were inoculated in the right flank with 100,000 or 50,000 of the syngeneic PA line, Pan02. Tumor volume was determined by standard calculation using caliper measurements. Mice were treated with Gemcitabine 20mg/kg Qwk IP and/or ZA 1ug QOD SQ once tumors were palpable. Analysis for MDSC accumulation was determined in the tumor, spleen and bone marrow of mice at sacrifice using flow cytometry for standard markers of MDSC (CD45, CD11b, Gr1, Ly6G, and Ly6C). MDSC prevalence is expressed as a percentage of total leukocytes (CD45+). Statistical analysis was performed using non-parametric student's t-test. Results: MDSC are up regulated in the spleen and bone marrow of mice with flank injected Pan02 compared to non-tumor bearing mice. MDSC accumulate over time and increase in number until 4 weeks post-inoculation (CD45 + 11b+Gr1+, Bone Marrow: 26.06% 42.04%, spleen 1.07% 7.18%, p=0.0021). Treatment of tumor bearing mice with ZA decreases tumor associated MDSC (CD45+11b+Gr1+: 50.36 29.38; CD45+11b+Ly6G+: 49.45 30.47) and tumor volume (2.45 1.71 p=0.034). ZA also works in concert with Gemcitabine to decrease MDSC (Gr1 +: 50.36 39.38; Ly6G+: 49.45 37.28;) and tumor volume (2.45 1.02, p=0.0011). Conclusion: MDSC play an integral role in the development of PA. Depletion of MDSC by ZA results in significantly smaller tumors and can be added to the standard chemotherapeutic regimen (Gemcitabine) successfully. The addition of ZA to Gemcitabine represents an attractive and innovative regimen to improve the treatment of this deadly malignancy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-148.

5-Fluorouracil Selectively Kills Tumor-Associated Myeloid-Derived Suppressor Cells Resulting in Enhanced T Cell–Dependent Antitumor Immunity

Myeloid-derived suppressor cells (MDSC) accumulate in the spleen and tumor bed during tumor growth. They contribute to the immune tolerance of cancer notably by inhibiting the function of CD8(+) T cells. Thus, their elimination may hamper tumor growth by enhancing antitumor T-cell functions. We have previously reported that some anticancer agents relied on T cell-dependent anticancer responses to achieve maximal efficacy. However, the effect of anticancer agents on MDSC has remained largely unexplored. In this study, we observed that gemcitabine and 5-fluorouracil (5FU) were selectively cytotoxic on MDSC. In vivo, the treatment of tumor-bearing mice with 5FU led to a major decrease in the number of MDSC in the spleens and tumor beds of animals whereas no significant effect on T cells, natural killer cells, dendritic cells, or B cells was noted. Interestingly, 5FU showed a stronger efficacy over gemcitabine to deplete MDSC and selectively induced MDSC apoptotic cell death in vitro and in vivo. The elimination of MDSC by 5FU increased IFN-gamma production by tumor-specific CD8(+) T cells infiltrating the tumor and promoted T cell-dependent antitumor responses in vivo. Altogether, these findings suggest that the antitumor effect of 5FU is mediated, at least in part, by its selective cytotoxic action on MDSC.

The Inflammasome Component Nlrp3 Impairs Antitumor Vaccine by Enhancing the Accumulation of Tumor-Associated Myeloid-Derived Suppressor Cells

The Inflammasome Component Nlrp3 Impairs Antitumor Vaccine by Enhancing the Accumulation of Tumor-Associated Myeloid-Derived Suppressor Cells

Mechanism of All-TransRetinoic Acid Effect on Tumor-Associated Myeloid-Derived Suppressor Cells

Myeloid-derived suppressor cells (MDSC) play an important role in tumor escape by suppressing T-cell responses. MDSC represent a group of cells of myeloid lineage at different stages of differentiation. Increased arginase activity and production of reactive oxygen species (ROS) are among the main functional characteristics of these cells. Recent studies have shown that all-trans retinoic acid (ATRA) had a potent activity in eliminating MDSC in cancer patients and in tumor-bearing mice. ATRA differentiates these cells into mature myeloid cells. However, the mechanism of this effect is unclear. Here, we have shown that ATRA dramatically and specifically up-regulated gene expression and protein level of glutathione synthase (GSS) in MDSC. This resulted in accumulation of glutathione (GSH) in these cells, observed in both mice and cancer patients. Blockade of GSH synthesis cancelled the effect of ATRA on MDSC. Accumulation of GSH in these cells using N-acetyl-L-cysteine mimicked the effect of ATRA on MDSC differentiation. Analysis of potential mechanisms of ATRA effect on GSS revealed that ATRA regulates its expression not by directly binding to the promoter but primarily via activation of extracellular signal-regulated kinase 1/2. Thus, ATRA induced differentiation of MDSC primarily via neutralization of high ROS production in these cells. This novel mechanism involves specific up-regulation of GSS and accumulation of GSH and could be used in developing and monitoring therapeutic application of ATRA.