[Neutrophils in the tumor microenvironment-foes or friends?] / Neutrophile Granulozyten im Tumormikromilieu ­ Feind oder Freund?

Neutrophils are the most abundant white blood cells in the human circulation and are usually associated with inflammation and with fighting infections. In recent years, the role of these cells during cancer progression has been a matter of increasing interest. Tumor-associated neutrophils (TANs) accumulate in cancer patients and represent an important negative prognostic marker in a broad variety of neoplasms. Accordingly, TANs represent a highly attractive therapeutic target. TAN may exhibit tumor-promoting or -inhibiting functions. Pro-tumor neutrophils support tumor angiogenesis and growth and promote metastatic dissemination of tumors via establishment of the premetastatic niche. Studies in animal models have already shown that the depletion of TANs or the inhibition of their migration bears therapeutic potential. Multiple pathways and mediators that induce pro-tumoral functions in neutrophils have been identified. In this review, we provide an up-to-date overview of the pro- and anti-tumor properties of neutrophils as well as the environmental cues that regulate these distinct functions. We also report on our own work that comprehensively investigated the role of neutrophils in head and neck cancer.

[The microbiome in head and neck tumors-initial findings and outlook]. / Das Mikrobiom bei Kopf-Hals-Tumoren ­ erste Erkenntnisse und Ausblick.

Technical progress in molecular biology has allowed for a more detailed analysis of the composition of the human microbiome in recent years. Inter- and intraindividual differences in microbiome composition have been demonstrated, which in part correlate with the occurrence of certain diseases. For some of the so-called oncomicrobes, a direct relationship between their effect on the host organism and carcinogenesis has been demonstrated, predominantly for gastrointestinal cancers. Initial results for head and neck cancer show inter- and intraindividual differences in the local microbiota of the tumor environment, with certain bacterial strains over- or underrepresented. Our results confirm these findings, e.g., by showing a relative abundance of fusobacteria in tumor tissue while streptococci were relatively reduced. Currently available results show a high degree of inter- and intraindividual variation, thus requiring larger patient cohorts for functional analyses.

[Immunotherapy in head and neck cancer]. / Immuntherapie bei Kopf-Hals-Karzinomen.

The physiological immune response to malignant cells is based on the interaction of antigen-presenting cells, such as dendritic cells and macrophages, with T and B lymphocytes. CD8(+) effector and natural killer cells are primarily responsible for tumor cell lysis. Tumor cells exploit several mechanisms to influence the body's immune system and promote development and progress of solid head and neck malignancies. Via regulatory T cells, myeloid-derived suppressor cells, tumor-associated macrophages, and cancer-associated fibroblasts, tumor cells promote development of suppressive signaling pathways that enable tumor progression. Novel immune therapeutics aim to influence these signaling pathways. Current studies are investigating agents which influence immune-stimulating or immune-suppressive cytokines, as well as drug-based Toll-like receptor activation and vaccination in head and neck cancer. Development of monoclonal antibodies allows for direct and highly specific binding of therapeutics to cell receptors - recently discovered immune checkpoint receptors are particularly intriguing targets. Monoclonal antibodies directed specifically toward T cell-stimulating receptors such as CD28 and CD134, or immunosuppressive receptors CTLA-4 and PD-1, are currently under investigation and have shown promising results.

[Immunotherapy of head and neck cancer. Current developments]. / Immuntherapie von Kopf-Hals-Karzinomen. Aktuelle Entwicklungen.

In order to improve the prognosis for patients with head and neck squamous cell cancer (HNSCC) the introduction of new therapeutic strategies is necessary. The concept of immunotherapy has been applied and improved for several years and recent studies have used tumor-specific antigens which facilitates targeted oncologic therapy. However, immunotherapy is hampered by the fact that immunosuppressive mechanisms are pronounced and relevant effector cells are suppressed, especially in patients with HNSCC. Successful immunotherapy could induce an antitumor immune response by restitution of these cell populations. Current anti-tumor immunotherapy includes unspecific immune stimulation, genetic modification of tumor and immune cells, the use of monoclonal antibodies, e.g. cetuximab, adoptive cell transfer and tumor vaccination. In the future, these biologic therapies alone or in combination with conventional therapeutic regimens could present a valuable therapeutic option for HNSCC patients.

[Trying to unravel an unresolved issue in regenerative medicine: gene expression profiling of MSCs]. / Genexpressionsmuster mesenchymaler Stammzellen: ein ungelöster Aspekt in der regenerativen Medizin.

BACKGROUND: Mesenchymal stem cells (MSCs) are adult fibroblastoid progenitor cells. Because of their immunoregulatory properties and their so-called trophic effects, MSCs play an important role in tissue regeneration, inflammation and trauma. Tissue trauma and challenge, for example during radiotherapy or infection, result in the release of so-called "danger molecules", which may be derived from dying cells or incoming pathogens. The molecular response of MSCs to this tissue stress remains largely elusive. MATERIAL AND METHODS: In this study we examined the cell biological response of MSCs derived from human parotid glands (pgMSCs) and used bacterial endotoxin as a model of tissue stress and inflammation. PgMSCs from 3 donors were isolated, expanded and tested for classical tri-lineage plus myogenic differentiation. The cell biological response to the model "stressor" endotoxin was examined by low density gene expression arrays. RESULTS: Through immunofluorescence and immunohistochemistry we were able to proof osteogenic, adipogenic, chondrogenic, and myogenic differentiation potential characteristic for stem cells. In vitro, gene expression analysis showed a characteristic modulation of MSCs after stimulation with endotoxin Lipopolysaccharide (LPS). Specifically, receptors and ligands typically involved in immune regulation, such as interleukins, TGF-ß, tumor necrosis factors (TNF), and toll-like receptors (TLR), were regulated. CONCLUSION: Our study elucidates some key functions and molecules, which are regulated in MSCs during tissue stress and inflammation. A thorough understanding of their cell biological function will aid future rationale therapeutic application of MSCs.

Dendritic cell generation and CD4+ CD25high FOXP3+ regulatory t cells in human head and neck carcinoma during radio-chemotherapy.

BACKGROUND: Regulatory T cells (Treg) and dendritic cells (DC) play an important role in tumor immunity and immune escape. However, their interplay and the effects of anti-cancer therapy on the human immune system are largely unknown. METHODS: For DC generation, CD14⊃+ monocytes were enriched by immunomagnetic selection from peripheral blood of advanced head and neck squamous cell carcinoma (HNSCC) patients and differentiated into immature DC using GM-SCF and IL-4. DC maturation was induced by addition of TNFα. The frequency of CD4⊃+CD25⊃highFOXP3⊃+ Treg in HNSCC patients was analyzed before and after radio-chemotherapy (RCT) by four-color flow cytometry. RESULTS: In HNSCC patients, the frequency of Treg (0.33 ± 0.06%) was significantly (p = 0.001) increased compared to healthy controls (0.11 ± 0.02%), whereas RCT had variable effects on the Treg frequency inducing its increase in some patients and decrease in others. After six days in culture, monocytes of all patients had differentiated into immature DC. However, DC maturation indicated by CD83 up-regulation (70.7 ± 5.5%) was successful only in a subgroup of patients and correlated well with lower frequencies of peripheral blood Treg in those patients. CONCLUSION: The frequency of regulatory T cells is elevated in HNSCC patients and may be modulated by RCT. Monocyte-derived DC in HNSCC patients show a maturation deficiency ex vivo. Those preliminary data may have an impact on multimodality clinical trials integrating cellular immune modulation in patients with advanced HNSCC.

[Innovations in biomarker development: a pathway towards individualized cancer therapy?]. / Moderne Methoden der Biomarkerentwicklung: eine Chance für die individualisierte Tumortherapie?

Despite multiple medical and scientific achievements, cancer remains a leading cause of death worldwide. Next to imaging technologies, molecular methods for early detection and for monitoring of the course of disease are of increasing interest. Thus, over the past years numerous studies have focused on the identification of biomarkers for cancer diagnosis, prognosis and response to therapy. The study of biomarkers seems to pose a high degree of complexity because many different types of molecules may, in principle, serve as potential biomarkers. In addition, these molecules can be produced either by the tumor or by the tumor-host in response to the presence of cancer. In this review the authors will address several major topics encompassed by the field of biomarker research. They will discuss the primary sources from which biomarker candidates can be 'mined' as well as the technological or methodological challenges associated with identification of biomarkers. Furthermore, the review will focus on current biomarker candidates for head and neck squamous cell carcinoma (HNSCC), with particular interest on several molecules yielding potential relevance for detection and prognosis of this type of cancer. Finally, several biomarker candidates with predictive potential for the response to therapy of HNSCC patients will be discussed, since identifying such molecules is crucial for developing individually-tailored and improved therapeutic strategies.

In vitro chemosensitivity of head and neck cancer cell lines.

BACKGROUND: Systemic treatment of head and neck squamous cell carcinoma (HNSCC) includes a variety of antineoplastic drugs. However, drug-resistance interferes with the effectiveness of chemotherapy. Preclinical testing models are needed in order to develop approaches to overcome chemoresistance. - METHODS: Ten human cell lines were obtained from HNSCC, including one with experimentally-induced cisplatin resistance. Inhibition of cell growth by seven chemotherapeutic agents (cisplatin, carboplatin, 5- fluorouracil, methotrexate, bleomycin, vincristin, and paclitaxel) was measured using metabolic MTT-uptake assay and correlated to clinically-achievable plasma concentrations. - RESULTS: All drugs inhibited cell growth in a concentration-dependent manner with an IC50 comparable to that achievable in vivo. However, response curves for methotrexate were unsatisfactory and for paclitaxel, the solubilizer cremophor EL was toxic. Cross-resistance was observed between cisplatin and carboplatin. - CONCLUSION: Chemosensitivity of HNSCC cell lines can be determined using the MTT-uptake assay. For DNA-interfering cytostatics and vinca alkaloids this is a simple and reproducible procedure. Determined in vitro chemosensitivity serves as a baseline for further experimental approaches aiming to modulate chemoresistance in HNSCC with potential clinical significance.

[Salivary gland stem cells : Can they restore radiation-induced salivary gland dysfunction?]. / Stammzellen aus Speicheldrüsen : Neue Therapieoption für die postradiogene Xerostomie?

Adult stem cells are actively investigated in the fields of regenerative medicine and tissue engineering, as they exhibit specific characteristics that make them promising candidates for cellular therapies. Depending on their tissue of origin these characteristics include long-term proliferation and the capacity to differentiate into various cell types. To date adult stem cells have been isolated from a multitude of tissues. Non-embryogenic adult tissues contain only small numbers of such stem cells and the derivation of such tissues can cause comorbidities. Therefore, there is ongoing interest in the identification and characterisation of novel cell sources for stem cell isolation and characterisation.Recently, salivary gland tissue has also been explored as a possible source of stem cells, first in animals and later in humans. Such salivary gland-derived stem cells might be useful in the treatment of radiation-induced salivary gland hypofunction, and possibly also in other diseases with loss of acinar cells, such as sequelae of radio iodine treatment or Sjögren's disease.In this paper we review the current status of salivary gland stem cell biology and application and discuss the possible role of stem cells in the development of novel therapies for salivary gland dysfunctions such as postradiogenic xerostomia.

[Diagnostic and prognostic biomarkers in head and neck squamous cell carcinoma]. / Tumormarker und Prognosefaktoren bei Plattenepithelkarzinomen der Kopf-Hals-Region.

Classical prognostic factors for squamous cell carcinoma of the head and neck (HNSCC) are based on general parameters such as tumor stage or histological grading and only allow for a rough estimation of the clinical course. However, predicting individual responses to treatment remains challenging and diverging clinical courses of same-stage HNSCC stage remain obscure. The need for a better understanding of the individual genomic or proteomic signature of HNSCC resulted in a great number of publications on novel biomarkers. Still, in most cancer centres therapy planning and risk appraisal are solely based on the classical factors with only a few exceptions such as HPV status in oropharyngeal carcinoma. Future improvements in biomarker research will probably be achieved with sets of various genomic and proteomic markers as provided by microarray technology. This review highlights the criteria for a successful biomarker candidate, gives an overview on the most important new biomarkers, and introduces the principles of genomic and proteomic biomarker chips.

[Coagulation factors as potential tumour markers in patients with head and neck carcinomas?]. / Gerinnungsfaktoren als Potenzielle Tumormarker bei Patienten mit Kopf-Hals-Karzinomen?

OBJECTIVE: Carcinomas can have influence on the coagulation system by different factors. Locally pathological changes of metabolism, neo-vascularisation, oxygenation and tissue pressure as well as locally and systemically activities of the tumor cells, are part of it. The coagulation situation in patients with head and neck carcinomata is characterized only insufficiently till now. MATERIAL AND METHODS: In a prospective pilot study 20 male patients with squamous-cell carcinomas of the head and neck area were subjected to a detailed coagulation diagnostics pre and post therapeutically and, age and sex corrected, compared with a control group (n=37). RESULTS: For the routine parameters PTT, Quick, TZ and INR no differences between the groups could be recognized. For the tumour patients a statistically significant increase arose for the acute phase proteins like factor I (fibrinogen), factor VIII, factor IX, von- Willebrand antigen and activity before therapy. Increased values were found also for plasmin, factor II, factor V and the thrombin-antithrombin-III-complex (TAT) whereas the values for antithrombin-III were degraded significantly. In the tumour patients the pre-therapeutical increased values for the activation marker TAT brought themselves back to normal after the tumour ablative therapy. CONCLUSIONS: TAT could be suitable as a potential tumour marker but also for relapse tumours. To evidence this, a study of longer duration and with a larger number of patients is necessary.

Malignant and non-malignant lung tissue areas are differentially populated by natural killer cells and regulatory T cells in non-small cell lung cancer.

Even though the lung represents a special immune compartment with the capacity of a high inflammatory response, ineffective anti-tumour immunity is common in lung-associated malignancies. We asked whether a differential composition of the immune cell infiltrate in malignant (MLTAs) and non-malignant lung tissue areas (N-MLTAs) exists and might potentially contribute to this effect. We performed a comparative analysis of immune cells residing in MLTAs and N-MLTAs of non-small cell lung cancer (NSCLC) patients. To this end, we used immunophenotyping and functional analyses on directly isolated immune cells and tissue arrays on archived paraffin-embedded specimens. A strong T cell infiltration was prominent in both tissue compartments whereas CD4(+)CD25(+)CD127(-) T regulatory cells were present in MLTAs only. Nonetheless, concurrent functional ex vivo T cell analyses revealed no significant difference between T cells of MLTA and N-MLTA, suggesting that tumour-infiltrating T cells were not functionally impaired. Interestingly, T cell infiltration was less pronounced in specimens with a high neutrophilic infiltrate. NK cell infiltration was strikingly heterogenous between MLTA and N-MLTA. While NK cells were almost absent in the malignant tissue regions, non-malignant counterparts were selectively populated by NK cells and those NK cells showed strong cytotoxic activity ex vivo. We report that malignant and non-malignant tissue areas in NSCLC are selectively infiltrated by certain immune cell types with NK cells being displaced from the tumour tissue. These phenomena have important implications for tumour immunology of NSCLC and should be considered for the development of future immunologic intervention therapies.

Growth factors and scaffold composition influence properties of tissue engineered human septal cartilage implants in a murine model.

Several surgical disciplines apply cartilage grafts for reconstructive purposes and have to overcome the scarcity of donor sites for this unique tissue. Employing the techniques of tissue engineering, cartilage might be generated in reasonable amounts for clinical purposes. Application of growth factors together with biochemical and biomechanical scaffold properties influence the process of ex vivo transplant production. The aims of this study are: 1) to investigate the influence of IGF-1 and TGFbeta-2 on tissue engineered human septal cartilage in vitro and in vivo after transplantation in nude mice; 2) to analyse the effect of the polydioxanone (PDS) content of the biodegradable Ethisorb E210 scaffold on the properties of the implanted constructs. Cells were three-dimensionally cultured on biodegradable Ethisorb E210 (PGA-PLA-copolymer fleeces with polydioxanone (PDS) adhesions), or on E210 scaffolds with a reduced polydioxanone content. Wet weight (ww), GAG-, and hydroxyprolin-content, as well as the cellularity of the neocartilage constructs were quantitatively evaluated. Additionally, the in vivo resorption of the two types of cell carriers was monitored. Addition of growth factors clearly increased the wet weight of the in vitro cultured constructs before transplantation. After transplantation, high PDS content improved the in vivo stability and macroscopic morphometric appearance of the tissue engineered specimens and led to enhanced deposition of glycosaminoglycans in transplanted constructs. Hydroxyproline content of the implants was not affected by either growth factors or PDS content. These data suggest a role for IGF-1 and TGFbeta-2 in preparative in vitro culture of chondrocytes before implantation, while PDS content of the scaffold is important for in vivo properties of the implanted material.

[Cell-based strategies for salivary gland regeneration]. / Zellbasierte Strategien für die Speicheldrüsenregeneration.

Xerostomia as a side effect of radiotherapy or due to Sjögren's disease leads to considerable impairment of the quality of life of the affected patients. Preventive treatment approaches such as intensity-modulated radiotherapy, surgical transfer of a submandibular gland to a site outside the radiation field or administration of amifostin during radiation treatment are not yet completely established in clinical practice and are not applicable for all patients. Symptomatic treatment with pilocarpin or synthetic saliva leads to an improvement of the symptoms only in some patients, and in the case of pilocarpin significant systemic anticholinergic side-effects might occur. Because large numbers of patients are affected and current treatment options are not satisfactory, it is essential to develop new treatment options. In parallel with the in vitro production of functional salivary gland constructs by means of tissue engineering techniques, attempts are currently under way to experimentally restore salivary gland function by genetic treatment approaches such as transfection of the affected salivary glands with aquaporins or pro-angiogenic factors. In addition, the in vivo application of stem cells is under investigation. In the present paper, we discuss the clinical and radiobiological background of xerostomia and highlight possible innovative future treatment options.

[Effect mechanism of intravesical BCG immunotherapy of superficial bladder cancer]. / Wirkmechanismen der intravesikalen BCG-Immuntherapie des oberflächlichen Harnblasenkarzinoms.

Immunotherapy for treatment of solid cancer mostly is an experimental treatment. In contrast, intravesical immunotherapy of superficial bladder cancer with bacille Calmette-Guérin (BCG) is clinically well established and accepted worldwide because of better results compared to topical chemotherapy. BCG is currently regarded as the most successful immunotherapy of cancer. Unfortunately the mechanism of action has not yet been fully clarified. This article gives an overview on the complex research on the mechanisms of actionhighly successful therapy.

Optimizing syngeneic orthotopic murine bladder cancer (MB49).

The syngeneic orthotopic murine bladder cancer model MB49 is hampered by unreliable tumor implantation. We optimized this model by a simple modification of the standard implantation technique in three groups of mice. Fifty thousand (group I), 20,000 (group II), or 10,000 (group III) tumor cells were implanted into cauterized bladders by transurethral instillation, and dwell time was prolonged to 3 h. Tumor take, survival, and bladder weights were determined as outcome variables. To verify whether this modification maintained its sensitivity to topical immunotherapy, an initial tumor load of 100,000 MB49 cells was given, and mice were treated intravesically with Bacillus Calmette-Guérin or phosphate-buffered saline. The prolonged dwell time of tumor cells resulted in take rates of 100% in all three groups. Survival and bladder weights were significantly correlated with the number of instilled cells. Even with the highest tumor load, Bacillus Calmette-Guérin therapy improved survival and reduced bladder weights significantly, as compared to PBS. Thus, the modified model is highly reliable and maintains its susceptibility to topical immunotherapy.

Perforin-mediated lysis of tumor cells by Mycobacterium bovis Bacillus Calmette-Guérin-activated killer cells.

Immunotherapy with Bacillus Calmette-Guérin (BCG) is clinically established in the treatment of superficial bladder cancer. In our attempt to clarify the underlying immunological mechanism, we could previously show that stimulation of PBMC with BCG leads to the generation of cytotoxic BCG-activated killer (BAK) cells. Among others, these BAK cells as well as lymphokine-activated killer (LAK) cells have been suggested as possible effector cells during BCG therapy. To understand BCG-induced activation of effector lymphocytes more precisely, we investigated the lytic pathways of human BAK cells and compared BAK cell cytotoxicity with LAK cell cytotoxicity. Perforin and Fas ligand (FasL) are the major cytolytic molecules of cytotoxic lymphocytes. Our results demonstrate that BAK and LAK cells showed an increased expression of perforin and FasL as compared with unstimulated controls. Killing of T-24 bladder tumor as well as Jurkat cells by BAK and LAK cells was predominantly mediated via perforin as demonstrated by a drastically reduced lysis in the presence of concanamycin A and EGTA/MgCl2, respectively. In contrast, lysis (radioactive release assay) and membrane disintegration (Annexin V binding) of both targets by BAK and LAK cells could not be blocked with an inhibitory anti-FasL monoclonal antibody (NOK-1). Nevertheless, T-24 and Jurkat were susceptible to killing by recombinant soluble FasL and by Chinese hamster ovary cells expressing membrane-bound FasL. We conclude that cellular mediators of BCG effector mechanisms, such as BAK and LAK cells, kill their targets via perforin and independent of the FasL pathway. Because we also found increased numbers of perforin-expressing lymphocytes in patients after BCG therapy, our findings have potential clinical relevance because BCG therapy would not be impaired by FasL resistance of target cells, which recently has been described for some tumors.

A member of the ClpB family of stress proteins is expressed during heat shock in Leishmania spp.

We have identified and isolated the Leishmania major homologue to the bacterial ClpB gene and to the yeast Hsp104 gene. ClpB in Leishmania major is a single-copy gene and encodes a low-abundance mRNA which is induced several-fold during a heat stress. We raised antibodies against the product of the recombinant gene and show that the leishmanial ClpB encodes a predominantly cytoplasmic protein of approx. 100 kDa which is detectable in Leishmania promastigotes of various species after exposure to elevated temperatures. We, therefore, term this protein Hsp100.

Chemical Stress does not Induce Heat Shock Protein Synthesis in Leishmania donovani.

The cellular heat shock response in kinetoplastid protozoa is regulated exclusively at a post-transcriptional level. The heat-inducibility of heat shock protein synthesis is retained under actinomycin C(1) which indicates an inducible translation of heat shock mRNAs. We have also assessed the ability of various chemicals known to be effective triggers of the heat shock response in higher eukaryotes to induce heat shock protein synthesis in Leishmania donovani. None of the tested chemicals elicited a stress response. We propose that the lack of transcription regulation in the kinetoplastida precludes a stress response under chemical stress.

Impact of human papilloma virus infection on the response of head and neck cancers to anti-epidermal growth factor receptor antibody therapy.

Infection with human papillomaviruses (HPVs) characterizes a distinct subset of head and neck squamous cell cancers (HNSCCs). HPV-positive HNSCC preferentially affect the oropharynx and tonsils. Localized HPV-positive HNSCCs have a favorable prognosis and treatment outcome. However, the impact of HPV in advanced or metastatic HNSCC remains to be defined. In particular, it is unclear whether HPV modulates the response to cetuximab, an antibody targeting the epidermal growth factor receptor (EGFR), which is a mainstay of treatment of advanced HNSCC. To this end, we have examined the sensitivity of HPV-positive and -negative HNSCC models to cetuximab and cytotoxic drugs in vitro and in vivo. In addition, we have stably expressed the HPV oncogenes E6 and E7 in cetuximab-sensitive cancer cell lines to specifically investigate their role in the antibody response. The endogenous HPV status or the expression of HPV oncogenes had no significant impact on cetuximab-mediated suppression of EGFR signaling and proliferation in vitro. Cetuximab effectively inhibited the growth of E6- and E7-expressing tumors grafted in NOD/SCID mice. In support, formalin-fixed, paraffin-embedded tumor samples from cetuximab-treated patients with recurrent or metastatic HNSCC were probed for p16(INK4a) expression, an established biomarker of HPV infection. Response rates (45.5% versus 45.5%) and median progression-free survival (97 versus 92 days) following cetuximab-based therapy were similar in patients with p16(INK4A)-positive and p16(INK4A)-negative tumors. In conclusion, HPV oncogenes do not modulate the anti-EGFR antibody response in HSNCC. Cetuximab treatment should be administered independently of HPV status.