Extravascular optical coherence tomography of cerebral vessel walls in vivo.

PURPOSE: Evaluation of extravascular, microscope integrated OCT (iOCT) as an in vivo imaging modality of cerebral blood vessels and as an intraoperative imaging method. METHODS: Microscope integrated optical coherence tomography of major cerebral arteries (n = 13) and superficial sylvian veins (n = 5) and one incidental cerebral vasospasm (n = 1) in (n = 10) patients. Post procedural analysis of OCT volume scans, microscopic images and videos during the time of scan as well as measurements of the diameter of vessel walls and its layers with an accuracy of 7.5 µm. RESULTS: iOCT was feasible during vascular microsurgical procedures. In all scanned arteries a clear delineation of the physiological three layered vessel wall composition could be achieved. Pathological arteriosclerotic alterations of cerebral artery walls could precisely be demonstrated. Major superficial cortical veins conversely presented a mono layered composition. First in vivo measurements of vascular mean diameters were possible. Cerebral artery walls showed a diameter of 296 µm, tunica externa 78 µm, media 134 µm and interna 84 µm. CONCLUSION: For the first time the microstructural composition of cerebral blood vessels could be illustrated in vivo. Due to an outstanding spatial resolution a clear definition of physiological and pathological characteristics was possible. Therefore, microscope integrated optical coherence tomography holds promise for basic research in the field of cerebrovascular arteriosclerotic diseases and for intraoperative guidance during microvascular surgery.

Overcoming Barriers in Neurosurgical Education: A Novel Approach to Practical Ventriculostomy Simulation.

BACKGROUND: In the high-risk, high-stakes specialty of neurosurgery, traditional teaching methods often fail to provide young residents with the proficiency needed to perform complex procedures in stressful situations, with direct effects on patient outcomes. Physical simulators provide the freedom of focused, hands-on training in a more controlled environment. However, the adoption of simulators in neurosurgical training remains a challenge because of high acquisition costs, complex production processes, and lack of realism. OBJECTIVE: To introduce an easily reproducible, cost-effective simulator for external ventricular drain placements through various ventriculostomy approaches with life-like tactile brain characteristics based on real patients' data. METHODS: Whole brain and skull reconstruction from patient's computed tomography and MRI data were achieved using freeware and a desktop 3-dimensional printer. Subsequently, a negative brain silicone mold was created. Based on neurosurgical expertise and rheological measurements of brain tissue, gelatin in various concentrations was tested to cast tactilely realistic brain simulants. A sample group of 16 neurosurgeons and medical students tested and evaluated the simulator in respect to realism, haptics, and general usage, scored on a 5-point Likert scale. RESULTS: We saw a rapid and significant improvement of accuracy among novice medical students. All participants deemed the simulator as highly realistic, effective, and superior to conventional training methods. CONCLUSION: We were able to demonstrate that building and implementing a high-fidelity simulator for one of the most important neurosurgical procedures as an effective educational and training tool is achievable in a timely manner and without extensive investments.

Canonical WNT pathway inhibition reduces ATP synthesis rates in glioblastoma stem cells.

BACKGROUND: The conserved stem cell signaling network canonical Wingless (WNT) plays important roles in development and disease. Aberrant activation of this pathway has been linked to tumor progression and resistance to therapy. Industry and academia have substantially invested in developing substances, which can efficiently and specifically block the WNT signaling pathway. However, a clear clinical proof of the efficacy of this approach is still missing. Studies on the metabolomics dysregulation of cancer cells have led to innovations in oncological diagnostics. In addition, modulation of cancer cell metabolome is at the base of promising clinical oncology trials currently underway. While onco-protein activation can have profound metabolic outcomes, the involvement of stem cell signals, such as the WNT pathway, in tumor cell metabolomics is yet insufficiently characterized. MATERIAL AND METHODS: We determined live cell metabolism and bioenergetics in pathophysiological relevant, WNT-dependent glioblastoma stem cell (GSC) models. We quantified those parameters in cells with canonical WNT activity and in isogenic cells where WNT activity had been inhibited by short hairpin RNA against ß-catenin. Furthermore, we applied computational analysis of RNA sequencing to verify our functional findings in independent GSCs cohorts. RESULTS: The investigated collection of disease models allows the separation in tumors with low, moderate and high base line metabolic activity. Suppression of canonical WNT signaling led to significant reduction of total, mitochondrial, and glycolytic ATP production rates. Elevated canonical WNT transcription signature in GSCs positively correlated with transcription levels of mitochondrial ATP synthesis, whereas non-canonical WNT gene expression signature did not. CONCLUSION: The applied disease modeling technology allows the recapitulation of inter-tumoral heterogeneous metabolic properties of glioblastoma. Our data show for the first time that inhibition of canonical WNT signaling in alive GSCs functionally correlates with energy inhibition and glucose homeostasis. As this correlation occurs in GSCs from different transcriptional or epigenetic transcriptional subtypes, our results suggest that developing therapies directed against glycolysis/ATP-synthesis may be a promising strategy to overcome therapy resistance due to inter-tumoral heterogeneity and offers starting point to impair downstream signal WNT.

The prognostic significance of clinicopathological features in meningiomas: Microscopic brain invasion can predict patient outcome in otherwise benign meningiomas.

AIMS: Brain invasion (BI) was firstly defined as a single criterion of atypia in otherwise benign meningiomas in the revised fourth edition of 2016 WHO classification of brain tumours after being previously inconsistently addressed. However, recent studies have raised doubts about the prognostic significance of BI in otherwise benign meningiomas. In our study, we investigate the reproducibility of such a prognostic effect. METHODS: We identified two cohorts one consisting of 483 patients with meningioma WHO grade I (M°I) or atypical meningioma WHO grade II (M°II) from Hannover Medical School and the other including atypical meningiomas defined according to the classical WHO criteria (M°IIb) from the University Hospital Heidelberg. Follow-up data with a median observation time of 38.2 months were available from 308 cases. These included 243 M°I and 65 M°II patients with the latter group consisting of 25 patients with otherwise benign meningiomas with BI (M°IIa) and 40 with M°IIb. RESULTS: A significant difference of progression-free interval (PFI) was found between patients with M°I and M°II, M°I and M°IIa and those with M°I and M°IIb of both cohorts and each separately. However, PFI of M°IIa and M°IIb patients showed no significant difference. In the multivariate regression analysis adjusted for M°I/M°IIa versus M°IIb, sex, age, extent of resection and tumour location, BI exhibited the strongest risk of relapse (Hazard ratio: 4.95) serving as an independent predictor of PFI (p = 0.002). CONCLUSIONS: Our results clearly support the definition of BI as a single criterion of atypia in WHO classification of 2016.

Age-dependent changes of collagen alpha-2(IV) expression in the extracellular matrix of brain arteriovenous malformations.

OBJECTIVES: Brain arteriovenous malformations (bAVM) are rare vascular lesions. Recent observations challenge the congenital nature of these lesions. The underlying cellular and molecular mechanisms associated with dynamic changes of bAVM still remain unclear. The objective of this study was to explore the potential role of COL4A2 (Collagen alpha-2(IV)) in the pathophysiology of bAVM. PATIENTS AND METHODS: Expression and localization of COL4A2 were analyzed on tissue microarrays from bAVM patients (n = 60) by immunohistochemistry. Correlations between COL4A2 levels and clinical parameters were examined with Pearson's test for normally- distibuted or Spearman's Rho for not normally distributed data. Comparison between different clinical parameters was performed using t-test, non-parametric Mann-Whitney U test or Kruskal- Wallis test. Fisher's exact test was used for categorical data. RESULTS: COL4A2 was mainly expressed beneath the endothelium of vessels in the tunica media of bAVM. This pattern of expression indicates the basement membrane of the vessel walls. High levels of COL4A2 expression correlated with the age at surgery of patients (p = 0.005; R = 0.393; Spearman's Rho). The age at surgery in young (17-25 years) and old patients (55-76 years) showed a linear correlation; a greater variance of COL4A2 expression was observed in the age group between 26-54 years. CONCLUSION: This study reports for the first time the expression of COL4A2 in bAVM and suggests a potential role of COL4A2 in bAVM pathophysiology. These findings contribute to a better understanding of the microenvironment of bAVM and may foster the development of improved therapeutic strategies for this disease.

Cannabinoids in Glioblastoma Therapy: New Applications for Old Drugs.

Glioblastoma (GBM) is the most malignant brain tumor and one of the deadliest types of solid cancer overall. Despite aggressive therapeutic approaches consisting of maximum safe surgical resection and radio-chemotherapy, more than 95% of GBM patients die within 5 years after diagnosis. Thus, there is still an urgent need to develop novel therapeutic strategies against this disease. Accumulating evidence indicates that cannabinoids have potent anti-tumor functions and might be used successfully in the treatment of GBM. This review article summarizes the latest findings on the molecular effects of cannabinoids on GBM, both in vitro and in (pre-) clinical studies in animal models and patients. The therapeutic effect of cannabinoids is based on reduction of tumor growth via inhibition of tumor proliferation and angiogenesis but also via induction of tumor cell death. Additionally, cannabinoids were shown to inhibit the invasiveness and the stem cell-like properties of GBM tumors. Recent phase II clinical trials indicated positive results regarding the survival of GBM patients upon cannabinoid treatment. Taken together these findings underline the importance of elucidating the full pharmacological effectiveness and the molecular mechanisms of the cannabinoid system in GBM pathophysiology.

Rare ADAR and RNASEH2B variants and a type I interferon signature in glioma and prostate carcinoma risk and tumorigenesis.

In search of novel germline alterations predisposing to tumors, in particular to gliomas, we studied a family with two brothers affected by anaplastic gliomas, and their father and paternal great-uncle diagnosed with prostate carcinoma. In this family, whole-exome sequencing yielded rare, simultaneously heterozygous variants in the Aicardi-Goutières syndrome (AGS) genes ADAR and RNASEH2B co-segregating with the tumor phenotype. AGS is a genetically induced inflammatory disease particularly of the brain, which has not been associated with a consistently increased cancer risk to date. By targeted sequencing, we identified novel ADAR and RNASEH2B variants, and a 3- to 17-fold frequency increase of the AGS mutations ADAR,c.577C>G;p.(P193A) and RNASEH2B,c.529G>A;p.(A177T) in the germline of familial glioma patients as well as in test and validation cohorts of glioblastomas and prostate carcinomas versus ethnicity-matched controls, whereby rare RNASEH2B variants were significantly more frequent in familial glioma patients. Tumors with ADAR or RNASEH2B variants recapitulated features of AGS, such as calcification and increased type I interferon expression. Patients carrying ADAR or RNASEH2B variants showed upregulation of interferon-stimulated gene (ISG) transcripts in peripheral blood as seen in AGS. An increased ISG expression was also induced by ADAR and RNASEH2B variants in tumor cells and was blocked by the JAK inhibitor Ruxolitinib. Our data implicate rare variants in the AGS genes ADAR and RNASEH2B and a type I interferon signature in glioma and prostate carcinoma risk and tumorigenesis, consistent with a genetic basis underlying inflammation-driven malignant transformation in glioma and prostate carcinoma development.

Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase 2 Expression in Brain Arteriovenous Malformations and its Association with Brain Arteriovenous Malformation Size.

BACKGROUND AND OBJECTIVE: Brain arteriovenous malformations (bAVM) are severe conditions that can cause severe neurologic deficits and mortality. The underlying cellular and molecular mechanisms associated with bAVM growth and rupture remain unclear. The objective of this study was to explore the potential role of PLOD2 (procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2) in the pathophysiology of bAVM. METHODS: Expression and localization of PLOD2 were analyzed on tissue microarrays from patients with bAVM (n = 60) by immunohistochemistry. Correlations between PLOD2 levels and clinical parameters were examined with a Pearson test or Spearman rank correlation coefficient. Comparison between different clinical parameters was performed using a t test or nonparametric Mann-Whitney U test. A Fisher exact test was used for categorical data. RESULTS: PLOD2 was mainly expressed within the tunica media of the blood vessels. High levels of PLOD2 expression correlated with bAVM size (linear regression, P = 0.0083, R2=0.158). Small bAVM showed a higher frequency of hemorrhage compared with large ones (P = 0.001). Although PLOD2 was not directly associated with bAVM hemorrhage, high PLOD2-expressing bAVM had a lower frequency of hemorrhage compared with low or medium PLOD2-expressing bAVM (25% vs. 63% and 75%, respectively). CONCLUSIONS: This study reports for the first time the expression of PLOD2 in bAVM and suggests a potential role of PLOD2 in bAVM pathophysiology. These findings contribute to an better understanding of the microenvironment of bAVM and may foster the development of improved therapeutic strategies for this disease.

Adenosine metabolism of human mesenchymal stromal cells isolated from patients with head and neck squamous cell carcinoma.

BACKGROUND: Mesenchymal stromal cells (MSC) are a major component of the tumor microenvironment in patients with head and neck squamous cell carcinoma (HNSCC). MSC display innate and regulatory immunologic functions, very similar to many hematopoietic 'classical' immune cells. Conversion of ATP to immunosuppressive adenosine is an immunosuppressive mechanism utilized by other hematopoietic immune cells. The present study explores the adenosine metabolism of tumor derived MSC in comparison to autologous MSC from non-malignant tissue. METHODS: From HNSCC patients (n=10), paired MSC were generated from tumor tissue (tMSC) and autologous healthy control tissue (cMSC). Differentiation properties and phenotype (CD105, CD73, CD39, CD90, CD26, CD29) were compared by flow cytometry. Production of immunosuppressive adenosine (ADO) by functionally active ectonucleotidases, CD39 and CD73, was determined by luminescence and mass spectrometry. Suppressive activity of ADO was tested in CFSE proliferation assays of isolated T-cells. Plasticity of cMSC was explored after incubation with tumor-cell conditioned media. RESULTS: Differentiation into osteogenic, chondrogenic and adipogenic directions was comparable in tMSC and cMSC. Expression of ectonucleotidases, CD39 and CD73, was decreased in tMSC as compared to corresponding cMSC, which correlated with decreased ATP metabolism in mass spectrometry. Proliferation of CD4+ T-cells was significantly suppressed by exogenous ADO. Tumor-conditioned medium was unable to down-regulate ADO production in cMSC. CONCLUSION: We identified MSC of the oropharyngeal mucosa as an important producer of exogenous ADO. In patients with HNSCC, reduced expression of ADO may contribute to excessive inflammation and tumor growth.

Brain arteriovenous malformations: implications of CEACAM1-positive inflammatory cells and sex on hemorrhage.

Brain arteriovenous malformations (bAVMs) are severe conditions which, upon rupture, cause debilitating neurological deficits and even death. The exact cellular and molecular mechanisms associated with bAVM rupture are currently unclear. The objective of this study was to explore the potential role of CEA-related cell adhesion molecule-1 (CEACAM1) in bAVM pathophysiology. Expression and localization of CEACAM1 were assessed immunohistochemically in tissue microarrays from bAVM patients (n = 60). The association of CEACAM1 with clinical parameters was analyzed with Spearman's rank correlation coefficient and chi-square test. The predictive value of CEACAM1 was tested using logistic regression analysis. CEACAM1 was highly expressed in tissue-infiltrating neutrophil granulocytes. High levels of CEACAM1-positive cells were associated with bAVM rupture (hemorrhage), but not with arteriovenous malformation (AVM) size, preoperative embolization, or seizure. This association was significant (p = 0.029, chi-square) in male but not in female patients, and high CEACAM1-positive immune infiltration showed predictive significance for hemorrhage in male bAVM patients only (OR = 6.50, 95 % CI 1.09-38.63, p = 0.040). Within the ruptured bAVM group, patients with a short hemorrhage to surgery (HTS) time interval had higher levels of CEACAM1 immune infiltration than patients with long HTS. This decrease in the levels of CEACAM1 immune infiltration between the HTS short and HTS long groups was, however, significant only in female patients (p = 0.022, chi-square). Our findings substantiate the role of inflammation in the pathophysiology of bAVM and suggest the presence of sexual dimorphism in this disease.

Sex-specific clinicopathological significance of novel (Frizzled-7) and established (MGMT, IDH1) biomarkers in glioblastoma.

BACKGROUND: The Wnt receptor Frizzled-7 (FZD7) promotes tumor progression and can be currently targeted by monoclonal antibody therapy. Here, we determined the prognostic value of FZD7 for the overall survival of glioblastoma (GBM) patients, both as individual marker and taken in combination with the previously-described markers MGMT and IDH1. Additionally, we tested whether these markers (alone or in combination) exhibited sex-specific differences. RESULTS: High levels of FZD7 (FZD7high) associated with shorter survival in GBM patients; however, FZD7high was a significant predictor of poor survival only in male patients. Mutation of IDH1 significantly associated with longer survival in male but not female patients. Methylated MGMT promoter significantly associated with longer survival only in female patients. Combination of FZD7 with MGMT enhanced the prognostic accuracy and abrogated the sex differences observed upon single marker analysis. Combination of FZD7 with IDH1 was a significant predictor of survival in male GBM patients only. MATERIALS AND METHODS: Three independent cohorts of patients with primary GBM (n=120, n=108 and n=105, respectively) were included in this study. FZD7 and IDH1 were assessed by immunohistochemistry in tissue microarrays. MGMT promoter methylation was determined by methylation-specific polymerase chain reaction. Survival analysis was performed by Kaplan-Meier estimate, log-rank test and Cox proportional hazard regression. CONCLUSIONS: Our study identifies novel individual and combination markers with prognostic and, possibly, therapeutic relevance in GBM. Furthermore, our findings substantiate the importance of sexual dimorphism in this type of cancer.

The bidirectional tumor--mesenchymal stromal cell interaction promotes the progression of head and neck cancer.

INTRODUCTION: Mesenchymal stromal cells (MSC) are an integral cellular component of the tumor microenvironment. Nevertheless, very little is known about MSC originating from human malignant tissue and modulation of these cells by tumor-derived factors. The aim of this study was to isolate and characterize MSC from head and neck squamous cell carcinoma (HNSCC) and to investigate their interaction with tumor cells. METHODS: MSC were isolated from tumor tissues of HNSCC patients during routine oncological surgery. Immunophenotyping, immunofluorescence and in vitro differentiation were performed to determine whether the isolated cells met the consensus criteria for MSC. The cytokine profile of tumor-derived MSC was determined by enzyme-linked immunosorbent assay (ELISA). Activation of MSC by tumor-conditioned media was assessed by measuring cytokine release and expression of CD54. The impact of MSC on tumor growth in vivo was analyzed in a HNSCC xenograft model. RESULTS: Cells isolated from HNSCC tissue met the consensus criteria for MSC. Tumor-derived MSC constitutively produced high amounts of interleukin (IL)-6, IL-8 and stromal cell-derived factor (SDF)-1α. HNSCC-derived factors activated MSC and enhanced secretion of IL-8 and expression of CD54. Furthermore, MSC provided stromal support for human HNSCC cell lines in vivo and enhanced their growth in a murine xenograft model. CONCLUSIONS: This is the first study to isolate and characterize MSC from malignant tissues of patients with HNSCC. We observed cross-talk of stromal cells and tumor cells resulting in enhanced growth of HNSCC in vivo.

Stimulation of mesenchymal stromal cells (MSCs) via TLR3 reveals a novel mechanism of autocrine priming.

Mesenchymal stem/stromal cells (MSCs) are emerging as important regulators of innate and adaptive immunity. In this context, both proinflammatory and anti-inflammatory effects have been described for MSCs. The mechanisms mediating this functional plasticity are poorly characterized at present. Here, we investigated the inflammatory responses of MSCs isolated from human nasal mucosa (nmMSCs) upon challenge with different Toll-like receptor (TLR) ligands. We found that TLR3 ligands induced the strongest release of both proinflammatory cytokines [interleukin (IL)-6 and IL-8] and type I interferon by nmMSCs compared with other TLR ligands. Notably, TLR3 ligands triggered a biphasic cytokine response, with an early peak of type I interferon at 4 h poststimulation and a late release of proinflammatory cytokines at 24 h poststimulation. While the early interferon response was subject to direct stimulation, the proinflammatory response was regulated by factors released during the early cytokine response, which subsequently enhanced sensitivity to TLR3 ligation and amplified the production of IL-6 and IL-8 but not that of interferon. Taken together, our findings indicate that TLR3 ligands polarize the inflammatory phenotype of MSCs in a time-dependent manner. Thus, our study proposes a novel model that helps to explain the strikingly dichotomous functionality of MSCs in inflammation and immunoregulation.

The role of tumour FoxP3 as prognostic marker in different subtypes of head and neck cancer.

Expression of the forkhead transcription factor (FoxP3)--an established marker of regulatory T cells--has been found in other cell types as well, including tumour cells. Recent studies indicated that high tumour FoxP3 expression might be associated with a poor outcome of patients with several types of solid cancers. Here, we investigated the role of FoxP3 expressed by the tumour cells in the prognosis of larynx and oro-hypopharynx squamous cell carcinoma (LSCC and OHSCC)--two major subtypes of head and neck cancer. To this end, we analysed by immunohistochemistry the expression of tumour FoxP3 in tissues from 83 LSCC and 89 OHSCC patients in relation to overall survival. In multivariate analysis we found that high tumour FoxP3 expression significantly associated with poor survival in OHSCC but not in LSCC patients. In further studies, we combined the prognostic value of FoxP3 with selected markers of inflammation (cyclooxygenase-2; COX2) or with markers of enhanced tumour migration/invasion (AHNAK and CORTACTIN). Interestingly, we found that the combination of FoxP3 and AHNAK (in LSCC) or FoxP3 and CORTACTIN (in OHSCC) had significantly stronger prognostic values than either marker analysed individually. Combination of FoxP3 and COX2 enhanced the prognostic accuracy only in OHSCC. Thus, our study identifies novel individual and combination markers that might have enhanced and distinct prognostic relevance in different subtypes of head and neck cancer.

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.

AHNAK and inflammatory markers predict poor survival in laryngeal carcinoma.

AHNAK/Desmoyokin is a giant protein which has been recently linked to reorganization of the actin cytoskeleton, cellular migration and invasion. Here, we investigated the role of AHNAK in the pathophysiology of larynx carcinoma-one of the major subtypes of head and neck cancer. To this end, we analysed AHNAK expression in tumor tissues from 83 larynx carcinoma patients in relation to overall survival. We found that tumoral AHNAK overexpression significantly associated with poor survival of these patients both in univariate and multivariate analysis. In further studies, we combined the prognostic value of AHNAK with selected markers of inflammation, such as macrophage migration inhibitory factor (MIF) and tumor-infiltrating neutrophils (CD66b-positive cells). Both MIF and neutrophils have been linked to enhanced tumoral migration and poor clinical outcome in patients with orohypopharynx carcinoma-another major subtype of head and neck cancer. Interestingly, we found that synchronous high levels of AHNAK and MIF or AHNAK and neutrophils, respectively, were stronger predictors of poor survival than AHNAK alone. Synchronous high levels of all three markers were the strongest predictors of poor survival in our patient cohort. Taken together, our findings propose novel strategies for an accurate prognosis in larynx carcinoma and suggest potential mechanisms of inflammation-mediated tumor progression.

Neutrophils Activate Tumoral CORTACTIN to Enhance Progression of Orohypopharynx Carcinoma.

CORTACTIN is an actin-binding protein critically involved in cellular migration and invasion. Here, we investigated the role of CORTACTIN in the pathophysiology of orohypopharynx carcinoma - one of the major subtypes of head and neck cancer. To this end, we analyzed CORTACTIN expression in tumor tissues from 89 orohypopharynx carcinoma patients in relation to clinical parameters. We found that high tumoral CORTACTIN expression associated with poor survival, higher T-stage, and higher lymph node metastasis (N-stage) in these patients. Next, we combined the prognostic values of tumoral and stromal cell biological parameters in our patient cohort. We determined the potential interaction of tumoral CORTACTIN with tumor-infiltrating neutrophils, which have been previously linked to poor clinical outcome in orohypopharynx carcinoma patients with advanced disease. Interestingly, we found that patients with both high tumoral CORTACTIN expression and high neutrophilic infiltration had significantly worse clinical outcome than all other patients in our cohort. These findings suggest that tumoral CORTACTIN and tumor-infiltrating neutrophils might be functionally linked during progression of orohypopharynx carcinoma. In vitro, we showed that neutrophils released soluble factors which phosphorylated CORTACTIN in the tumor cells and promoted their migration. Furthermore, we demonstrated that strong CORTACTIN phosphorylation significantly correlated with strong neutrophilic infiltration in tumor tissues from orohypopharynx carcinoma patients. Taken together, our findings unravel a novel mechanism of tumor-stroma interaction, which might be relevant for a more accurate prognosis and improved therapeutic strategies in this tumor entity.

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.

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.

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.