Claudia A. Dumitru

Neutrophils and granulocytic myeloid-derived suppressor cells: immunophenotyping, cell biology and clinical relevance in human oncology

Accumulating evidence indicates that myeloid cells are critically involved in the pathophysiology of human cancers. In contrast to the well-characterized tumor-associated macrophages, the significance of granulocytes in cancer has only recently begun to emerge. A number of studies found increased numbers of neutrophil granulocytes and granulocytic myeloid-derived suppressor cells (GrMDSCs) both in the peripheral blood and in the tumor tissues of patients with different types of cancer. Most importantly, granulocytes have been linked to poor clinical outcome in cancer patients which suggests that these cells might have important tumor-promoting effects. In this review, we will address in detail the following major topics: (1) neutrophils and GrMDSCs in the peripheral blood of cancer patients—phenotype and functional changes; (2) neutrophils and GrMDSCs in the tumor tissue—potential mechanisms of tumor progression and (3) relevance of neutrophils and GrMDSCs for the clinical outcome of cancer patients. Furthermore, we will discuss the advantages and disadvantages of the current strategies used for identification and monitoring of human MDSCs. We propose a six-color immunophenotyping protocol that discriminates between monocytic MDSCs (MoMDSCs), two subsets of GrMDSCs and two subsets of immature myeloid cells in human cancer patients, thus, allowing for an improved characterization and understanding of these multifaceted cells.

Polymorphonuclear granulocytes in human head and neck cancer: enhanced inflammatory activity, modulation by cancer cells and expansion in advanced disease.

The progression of epithelial cancer is associated with an intense immunological interaction between the tumor cells and immune cells of the host. However, little is known about the interaction between tumor cells and polymorphonuclear granulocytes (PMNs) in patients with head and neck squamous cell carcinoma (HNSCC). In our study, we investigated systemic PMN‐related alterations in HNSCC, the role of tumor‐infiltrating PMNs and their modulation by the tumor microenvironment. We assessed the infiltration of HNSCC tissue by PMNs (retrospectively) and systemic PMN‐related alterations in blood values (prospectively) in HNSCC patients (n = 99 and 114, respectively) and control subjects (n = 41). PMN recruitment, apoptosis and inflammatory activity were investigated in an in vitro system of peripheral blood PMNs and a human HNSCC cell line (FaDu). HNSCC tissue exhibited considerable infiltration by PMNs, and strong infiltration was associated with poorer survival in advanced disease. PMN count, neutrophil‐to‐lymphocyte ratio and serum concentrations of CXCL8 (interleukin‐8), CCL4 (MIP‐1β) and CCL5 (RANTES) were significantly higher in the peripheral blood of HNSCC patients than in that of controls. In vitro, HNSCC‐conditioned medium inhibited apoptosis of PMNs, increased chemokinesis and chemotaxis of PMNs, induced release of lactoferrin and matrix metalloproteinase 9 by PMNs and enhanced the secretion of CCL4 by PMN. Our findings demonstrate alterations in PMN biology in HNSCC patients. In vitro, tumor‐derived factors modulate cellular functions of PMNs and increase their inflammatory activity. Thus, the interaction between HNSCC and PMNs may contribute to host‐mediated changes in the tumor microenvironment.

Modulation of neutrophil granulocytes in the tumor microenvironment: Mechanisms and consequences for tumor progression

Accumulating evidence indicates a critical role of myeloid cells in the pathophysiology of human cancers. In contrast to the well-characterized tumor-associated macrophages (TAMs), the significance of granulocytes in cancer has only recently begun to emerge. Increased numbers of neutrophil granulocytes have been observed both in the peripheral blood and in the tumor tissues of patients with different types of cancer. Importantly, these studies linked neutrophils to poor clinical outcome in cancer patients which suggests that these cells might have important tumor-promoting activities. Indeed, a number of functional in vitro and in vivo studies demonstrated that tumors stimulated neutrophils to promote angiogenesis and immunosuppression, as well as migration, invasion and metastasis of the tumor cells. Therefore, it became necessary to understand the mechanisms modulating the changes in the biology and functions of neutrophils in the context of the tumor microenvironment. In this review we will discuss several functions of neutrophils that might contribute to tumor progression. Furthermore, we will address in detail the cellular and molecular mechanisms that control modulation of neutrophils in the tumor microenvironment, such as recruitment to the tumor site (chemotaxis), prolonged survival and enhanced release of protumoral mediators.

Ceramide-induced cell death in malignant cells

Ceramide has been shown to be capable to trigger apoptosis in almost any cell, including tumor cells. Ceramide is generated by a de novo pathway or by sphingomyelinases. Sphingomyelinases hydrolyze sphingomyelin in biological membranes to release ceramide and they are named acid, neutral and alkaline sphingomyelinase depending on their maximum activity at acid, neutral and alkaline pH values, respectively. Stimuli that trigger a release of ceramide to mediate apoptosis include CD95, TNF-receptor, DR5, gamma-irradiation, cytotoxic drugs, UV-light, bacteria, viruses, some forms of developmental death, anti-CD20 and disruption of the cells contact with its matrix, to name a few. Here, we will focus on the role of acid sphingomyelinase in malignant tumors, which function in apoptosis is best characterized and documented by genetic models. We will discuss concepts that unify the biological actions of ceramide and describe the role of ceramide in important anti-tumor treatment modalities, such as gamma-irradiation and chemotherapy.

Tumor-derived macrophage migration inhibitory factor modulates the biology of head and neck cancer cells via neutrophil activation

Macrophage migration inhibitory factor (MIF) is an inflammatory cytokine that has been reported to enhance the aggressiveness and metastatic potential of tumor cells. However, the mechanisms through which MIF influences tumor development and progression are not understood. The objectives of our study were to assess the effects of tumor‐derived MIF on neutrophils in head and neck cancer (HNC) and to identify possible feedback effects on tumor cells. To this end, we used an in vitro system to model the interaction between human HNC cells and neutrophils. In addition, we analyzed expression of MIF in tissues from HNC patients in relation to neutrophilic infiltration and clinical parameters. Our results show that human HNC is infiltrated by neutrophils proportional to the levels of tumoral MIF. Strong MIF expression by the tumor is associated with higher lymph node metastasis and reduced survival in HNC patients. In vitro, MIF modulated functions of human neutrophils by inducing chemokine CXC motif receptor 2(CXCR2)‐dependent chemotaxis, enhancing neutrophil survival and promoting release of chemokine C‐C Motif Ligand 4 (CCL4) and matrix metalloprotease 9(MMP9). Further, neutrophils activated with tumor‐derived MIF enhanced migratory properties of HNC cells. In conclusion, our data indicate that the effects of tumor‐derived MIF on neutrophils represent an additional mechanism by which MIF might contribute to tumor progression.

Protumor and antitumor functions of neutrophil granulocytes

Neutrophils are primary inflammatory cells and absolutely essential to protect the host during the early phases of microbial infection. Their role in cancer is less clear. Current evidence suggests that neutrophils show high functional plasticity and can adopt protumor and antitumor activity. Protumor neutrophils are functionally related to the recently described granulocytic myeloid-derived suppressor cells. We propose a model in which homeostatic chronic recruitment and activation of neutrophils result in mainly protumor activity. In contrast, therapeutic interventions in many cases elicit acute activation, enhance direct effector functions as well as indirect regulatory functions of neutrophils with potent antitumor activity. Conversion of protumor activity of neutrophils into antitumor activity by means of appropriate stimulation or modulation may offer new possibilities in biologic therapy of cancer.

Peripheral Blood Neutrophil Granulocytes from Patients with Head and Neck Squamous Cell Carcinoma Functionally Differ from Their Counterparts in Healthy Donors

Solid tumors such as head and neck squamous cell carcinoma (HNSCC) display an intense interaction between tumoral factors and the immune system. Functional modulation of tumor-infiltrating and peripheral blood immune cells plays an important role during tumor progression. In this pilot study we compared biological functions of polymorphonuclear granulocytes (PMN) from the peripheral blood of HNSCC patients and healthy subjects. PMN were simultaneously isolated from the peripheral blood of HNSCC patients and healthy donors for functional analysis (apoptosis, production of reactive oxygen species (ROS), cytokine release and immunophenotyping). PMN from HNSCC patients showed a significantly lower inducible production of ROS (P = 0.02) and reduced spontaneous apoptosis (P = 0.008) compared with PMN from healthy donors. Under standard culture conditions, there was no significant difference regarding the release of inflammatory cytokines between PMN from HNSCC patients and PMN from healthy donors. Confirming previous observations, serum concentrations of PMN-related cytokines were significantly higher in the peripheral blood of HNSCC patients than in that of controls. Importantly, immunophenotyping revealed an increased number of immature PMN in PMN fractions isolated from HNSCC patients. Peripheral blood PMN from HNSCC patients and healthy donors show distinct functional differences. The presence of increased numbers of immature stages of PMN in HNSCC patients may partly contribute to the changes observed. After recruitment to and infiltration of the tumor, PMN may be further modulated in the local tumor microenvironment. This pilot study justifies functional analyses of myeloid cells in larger cohorts of patients with HNSCC.

Doxorubicin enhances TRAIL-induced cell death via ceramide-enriched membrane platforms

Previous studies indicated that signalling via CD95 and DR5 is greatly enhanced by the formation of ceramide-enriched membrane platforms. Here, we employed this concept to convert doses of subtherapeutic TRAIL that were unable to release ceramide and kill leukemic B-cells or ex vivo T lymphocytes, into a very effective apoptotic stimulus. Ceramide production was induced by application of sub-toxic doses of doxorubicin that resulted in an activation of the acid sphingomyelinase (ASM), release of ceramide and formation of ceramide-enriched membrane platforms. The latter served DR5 to cluster after application of very low doses of TRAIL in combination with doxorubicin. Genetic deficiency of the ASM abrogated doxorubicin-induced ceramide release, as well as clustering of DR5 and apoptosis induced by the combined treatment of doxorubicin and TRAIL. These data show that local release of ceramide potentiates very low, otherwise inactive doses of TRAIL that may represent a novel therapeutic concept to treat tumors.

A novel p38-MAPK signaling axis modulates neutrophil biology in head and neck cancer.

Neutrophils are emerging as important mediators in cancer progression. Recent studies associated neutrophils with poor clinical outcome of HNC patients and showed that HNC induces recruitment, survival, and release of proinflammatory factors by neutrophils in vitro. The molecular mechanisms through which HNC and other cancers modulate neutrophil biology are currently unknown. To explore these mechanisms, we used an in vitro system that models the interaction between human HNC cells and neutrophils or neutrophilic‐differentiated HL‐60 cells, respectively. We show that HNC‐derived factors activate p38‐MAPK in neutrophils, which partly promotes neutrophil survival, but not neutrophil recruitment and motility. Most importantly, HNC‐induced p38‐MAPK activation strongly stimulates the release of CCL4, CXCL8, and MMP9 by neutrophils. We identify CREB and interestingly, p27 phosphorylated at T198 as downstream members of the HNC‐induced p38‐MAPK signaling cascade. Using siRNA technology, we demonstrate that p27 and CREB mediate the release of CCL4 and CXCL8 and that CREB, additionally, mediates the release of MMP9. These data unravel novel molecular mechanisms involved in regulation of neutrophil proinflammatory functions. Our studies on human HNC tissues indicate that tumor‐infiltrating neutrophils might be a major source of CCL4 and particularly, MMP9 in cancer patients. Thus, our findings provide novel, mechanistic insights relevant for the pathophysiology of HNC and possibly, other types of cancer as well.

Lysosomal ceramide mediates gemcitabine-induced death of glioma cells

Acid sphingomyelinase-induced ceramide release has been shown by many studies to induce apoptosis in response to various stimuli. However, the mechanisms of acid sphingomyelinase/ceramide-mediated death signaling following treatment with chemotherapeutic drugs have not been fully elucidated thus far. The present study demonstrates that treatment of glioma cells with clinically achievable doses of gemcitabine results in acid sphingomyelinase activation, lysosomal accumulation of ceramide, cathepsin D activation, Bax insertion into the mitochondria, and cell death. Pharmacological inhibition or genetic deficiency of acid sphingomyelinase prevented these events while overexpression of the enzyme sensitized cells to gemcitabine. Likewise, inhibitors of lysosomal functions also prevent gemcitabine-induced cell death. Our data indicate a critical role of the acid sphingomyelinase/ceramide system for gemcitabine-induced signaling and suggest that lysosomal ceramide accumulation mediates cell death induced by a chemotherapeutic drug.