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Abstract Background Squamous cell carcinoma of the head and neck (SCCHN) are highly invasive tumours with frequent local and distant recurrence. Metastasis formation requires degradation of the extracellular matrix, which is fulfilled by membrane-associated proteases such as the urokinase plasminogen activator (uPA). WX-UK1 is a competitive active site inhibitor of the protease function of uPA that impairs on the capacity of tumour cells to invade in vitro. Methods In the present study, effects of combinations of WX-UK1 with matrix metalloprotease inhibitors (MMP, galardin®) and cyclooxygenase-2 (COX-2, celecoxib®) inhibitors on tumour cell proliferation, invasion, and angiogenesis induction were evaluated. Matrigel invasion chambers and a spheroid co-cultivation model with human fibroblast served to determine the invasive potential of both FaDu (SCCHN) and HeLa (cervical carcinoma) cells, each treated with combinations of Celecoxib®, Galardin®, and WX-UK1. Results Blocking of single protease systems resulted in a significant 50% reduction of tumour cell invasion using WX-UK1, while the triple combination was even more effective with 80% reduction of invasion. Additionally, a sprouting assay with HUVEC was used to test the anti-angiogenetic potential of the triple combination, resulting in a 40% decrease in the sprouting rate. Conclusions A combined approach targeting different families of proteases and cyclooxygenases represents a promising adjuvant therapy.

Abstract Background Epithelial cell adhesion molecule EpCAM is a transmembrane glycoprotein, which is frequently over-expressed in simple epithelia, progenitors, embryonic and tissue stem cells, carcinoma and cancer-initiating cells. Besides functioning as a homophilic adhesion protein, EpCAM is an oncogenic receptor that requires regulated intramembrane proteolysis for activation of its signal transduction capacity. Upon cleavage, the extracellular domain EpEX is released as a soluble ligand while the intracellular domain EpICD translocates into the cytoplasm and eventually into the nucleus in combination with four-and-a-half LIM domains protein 2 (FHL2) and β-catenin, and drives cell proliferation. Methods EpCAM cleavage, induction of the target genes, and transmission of proliferation signals were investigated under varying density conditions using confocal laser scanning microscopy, immunoblotting, cell counting, and conditional cell systems. Results EpCAM cleavage, induction of the target genes, and transmission of proliferation signals were dependent on adequate cell-to-cell contact. If cell-to-cell contact was prohibited EpCAM did not provide growth advantages. If cells were allowed to undergo contact to each other, EpCAM transmitted proliferation signals based on signal transduction-related cleavage processes. Accordingly, the pre-cleaved version EpICD was not dependent on cell-to-cell contact in order to induce c-myc and cell proliferation, but necessitated nuclear translocation. For the case of contact-inhibited cells, although cleavage of EpCAM occurred, nuclear translocation of EpICD was reduced, as were EpCAM effects. Conclusion Activation of EpCAM's cleavage and oncogenic capacity is dependent on cellular interaction (juxtacrine) to provide for initial signals of regulated intramembrane proteolysis, which then support signalling via soluble EpEX (paracrine).

Abstract Background The intermediate filament forming protein keratin 8 (K8) is a tumour-associated antigen, which was shown to be over-expressed in a variety of malignancies. Here, we present a study of K8 expression in squamous epithelia of the head and neck area, including normal mucosa, hyperplastic and dysplastic leukoplakia, carcinomas of different sub-localisations, and lymph node metastases. Methods K8 expression was assessed upon immunohistochemistry with specific antibodies in cryosections of primary tumours of the head and neck area. Results K8 expression was characteristic of transformed tissue and marked early stages of disease, i.e. dysplastic oral leukoplakia, but not normal or hyperplastic epithelium. With the exception of carcinomas of the larynx and the tongue, K8 expression also strictly differentiated carcinomas from normal epithelium of the same origin. Furthermore, K8high was characteristic of cells, which had detached from the sites of primary tumours and had been invading the surrounding tissue at the time point of surgery. Conclusion K8 is an excellent marker for head and neck malignancies, which allows for early detection as well as for visualisation of potentially disseminating tumour cells in vivo.

Background: Squamous cell carcinoma of the head and neck (SCCHN) are highly invasive tumours with frequent local and distant recurrence. Metastasis formation requires degradation of the extracellular matrix, which is fulfilled by membrane-associated proteases such as the urokinase plasminogen activator (uPA). WX-UK1 is a competitive active site inhibitor of the protease function of uPA that impairs on the capacity of tumour cells to invade in vitro. Methods: In the present study, effects of combinations of WX-UK1 with matrix metalloprotease inhibitors (MMP, galardin®) and cyclooxygenase-2 (COX-2, celecoxib®) inhibitors on tumour cell proliferation, invasion, and angiogenesis induction were evaluated. Matrigel invasion chambers and a spheroid co-cultivation model with human fibroblast served to determine the invasive potential of both FaDu (SCCHN) and HeLa (cervical carcinoma) cells, each treated with combinations of Celecoxib®, Galardin®, and WX-UK1. Results: Blocking of single protease systems resulted in a significant 50% reduction of tumour cell invasion using WX-UK1, while the triple combination was even more effective with 80% reduction of invasion. Additionally, a sprouting assay with HUVEC was used to test the anti-angiogenetic potential of the triple combination, resulting in a 40% decrease in the sprouting rate. Conclusions: A combined approach targeting different families of proteases and cyclooxygenases represents a promising adjuvant therapy. [ABSTRACT FROM AUTHOR]

Tumors express specific antigens, which are targets of a humoral immune response. Such specific tumor-associated antigens are of increasing importance for diagnosis and immunotherapy. They allow the detection and targeting of transformed cells in vivo. Here, we describe a new technique for the isolation and identification of tumor antigens that constitute humoral immune responses. The method is an a1utoantibody-m1ediated i1d1entification of a1ntigens (AMIDA) derived from tumor tissue lysates with autologous antibodies, followed by a two-dimensional gel electrophoresis and mass spectrometry analysis. We applied AMIDA to head and neck carcinoma patients and identified 35 antigens, which can be classified in three subgroups: i.) known tumor-associated antigens including SEREX-Ag, ii.) autoimmune disease-associated antigens, and iii.) proteins of unknown function. The expression profile and the validation of a subset of antigens using specific antibodies, the real-time PCR and BioPlex technologies, will be discussed in more detail. Thus, we demonstrate here the potential of AMIDA as a promising tool for the detection of tumor antigens in their native context, i.e. in tumor cells, and describe new protocols for the validation of such proteins. [ABSTRACT FROM AUTHOR]

Background: A new promising approach to improve the outcome of head and neck squamous cell carcinoma (HNSCC) is the application of radio-labeled antibodies directed against tumor-associated antigens. Cytokeratin 8 (CK8), an intermediate filament forming protein, is shown to be de novo expressed in dysplastic lesions as well as in HNSCC. Therefore like the epithelial cell adhesion molecule CK8 seems to be a suitable anchor molecule for targeted radioimmunotherapy (RIT). The aim of this study was to investigate the biodistribution of a radio-labeled Cytokeratin 8-specific monoclonal antibody (mAb) in a SCID (severe combined immunodeficiency disease) mouse model., Materials and Methods: The mAb against CK8 was labeled with (131)I and biodistribution was tested in established HNSCC xenografts in SCID mice. The biodistribution of the mAb in the tumor and different organs was determined with a gamma counter and was calculated as % injected dose/gram tissue., Results: Initially, after systemic administration of (131)I-anti CK8 monoclonal antibody high activity was seen in all the organs. Over time the general activity decreased, whereas activity accumulated in the tumor. This activity decayed compared to the other tissues with a two- to threefold prolonged radioactive half-life., Conclusion: Specific antibody-antigen-binding is probably responsible for the prolonged radioactive half-life in the tumor and the resulting cumulative activity due to enrichment of the (131)I-anti CK8 mAb, so that Cytokeratin 8 seems to be a suitable anchor molecule for radioimmunotherapy in HNSCC.

Background: Epithelial cell adhesion molecule EpCAM is a transmembrane glycoprotein, which is frequently over-expressed in simple epithelia, progenitors, embryonic and tissue stem cells, carcinoma and cancer-initiating cells. Besides functioning as a homophilic adhesion protein, EpCAM is an oncogenic receptor that requires regulated intramembrane proteolysis for activation of its signal transduction capacity. Upon cleavage, the extracellular domain EpEX is released as a soluble ligand while the intracellular domain EpICD translocates into the cytoplasm and eventually into the nucleus in combination with four-and-a-half LIM domains protein 2 (FHL2) and beta-catenin, and drives cell proliferation., Methods: EpCAM cleavage, induction of the target genes, and transmission of proliferation signals were investigated under varying density conditions using confocal laser scanning microscopy, immunoblotting, cell counting, and conditional cell systems., Results: EpCAM cleavage, induction of the target genes, and transmission of proliferation signals were dependent on adequate cell-to-cell contact. If cell-to-cell contact was prohibited EpCAM did not provide growth advantages. If cells were allowed to undergo contact to each other, EpCAM transmitted proliferation signals based on signal transduction-related cleavage processes. Accordingly, the pre-cleaved version EpICD was not dependent on cell-to-cell contact in order to induce c-myc and cell proliferation, but necessitated nuclear translocation. For the case of contact-inhibited cells, although cleavage of EpCAM occurred, nuclear translocation of EpICD was reduced, as were EpCAM effects., Conclusion: Activation of EpCAM's cleavage and oncogenic capacity is dependent on cellular interaction (juxtacrine) to provide for initial signals of regulated intramembrane proteolysis, which then support signalling via soluble EpEX (paracrine).

Background: The mortality from squamous cell carcinoma of the head and neck (SCCHN) remains high and almost unchanged throughout the last decades. Therefore, new therapeutic strategies are urgently needed. One promising approach is the application of radio-labeled antibodies directed against tumor-associated antigens. EpCAM is a transmembrane protein, which is overexpressed on almost all SCCHN, making it a suitable anchor molecule for targeted radioimmunotherapy (RIT). The aim of this study was to establish an animal model to investigate the biodistribution and the therapeutic effect of a radio-labeled EpCAM-specific monoclonal antibody (mAb)., Materials and Methods: The mAb C215 was labeled with 131I and tested for its antitumor effect against established SCCHN xenografts in SCID mice. Initially, the biodistribution of the mAb in the tumor and different organs was determined with a gamma counter and was calculated as % injected dose/gram tissue. For therapeutic approaches 5, 15 or 25 MBq 131I-labeled mAb was injected as a single bolus into tumor-bearing mice. Control animals received either sodium chloride or the unlabeled mAb. The tumor growth and body weight of the animals were measured at various times after administration of the antibody., Results: Initially, high activity was seen in all organs after systemic administration of 13I-C215. Over time general activity decreased whereas an accumulation of activity was seen in the tumor. Tumor growth was delayed in the groups receiving either 15 MBq or 25 MBq 131I-C215 relative to control groups and the 5 MBq group. However, animals in the high-dose groups suffered from treatment-related toxicity, which led to body weight loss of more than 20%., Conclusion: Our data demonstrate that the EpCAM-specific radio-labeled mAb C215 is a promising tool to target SCCHN leading to significant tumor control. Further studies are necessary to increase efficacy and reduce toxicity of this new therapeutic approach.

Background: In some epithelial tumors, isoforms of CD44 are overexpressed and soluble isoforms detectable in serum samples are elevated. In squamous cell cancer of the head and neck (SCCHN) the alteration of CD44 isoforms could be associated with poor prognosis. A comprehensive study was undertaken to examine the value of CD44v6 as a tumor marker for SCCHN., Patients and Methods: Serum samples of SCCHN and healthy smokers were analyzed for soluble CD44v6 by ELISA. The expression of CD44 isoforms was determined by immunohistochemical staining of healthy and dysplastic tissue., Results: There was no significant difference between the serum levels of sCD44v6 in SCCHN and healthy smokers. Nor was there a correlation between the serum level of sCD44v6 and UICC stage, TNM stage or histological grading. In tissue of primary SCCHN, expression of CD44v6 was found as a strong, specific staining of the lower epithelial layers. Similar amounts of CD44v6-positive-labelled tumor cells were found in invasive carcinoma., Conclusion: Soluble CD44v6 is not a valuable tumor marker for SCCHN since the soluble form appears to be present in healthy smokers and does not reflect the stage of the disease.

Background: Tumor cells have developed several strategies to escape the immune system. One of these strategies consists of the secretion of immunosuppressive factors like interleukin-10 or prostaglandin E2 (PGE 2 ), which impair the immune system. We have demonstrated recently that tumor-derived PGE 2 down-regulates the expression of the integrin Mac-1 and the chemokine receptor CCR5 on primary monocytes, resulting in reduced adhesion and migration., Materials and Methods: In order to identify new target genes for tumor-derived factors in monocytes, we set up an in vitro system consisting of cDNA micro arrays and 2D gel electrophoresis., Results: We identified 25 genes that were differentially expressed upon incubation of cells in conditioned tumor cell supernatants as compared to cells incubated in cell culture medium. We describe in more detail that IL-1β secretion is induced by tumor supernatants and that IL-1β overexpression is also evident in monocytes from tumor patients in vivo, where expression correlates with the tumor stage. In addition, up-regulation of the plasminogen activator inhibitor-2, PAI-2, and down-regulation of the urokinase-type plasminogen activator receptor, uPAR, resulted in a reduced capability of monocytes to degrade and invade extracellular matrices., Conclusion: In summary, we describe interesting novel targets of soluble tumor-derived factors that are probably involved in the tumor-mediated immunosuppression commonly found in cancer patients., (Copyright© 2004 International Institute of Anticaner Research (Dr. John G. Delinassios), All rights reserved.)

Background: New adjuvant immunological therapies that selectively redirect effector cells towards tumour cells are currently under development. These strategies include the use of bispecific antibodies as promising tools for the elimination of disseminated tumour cells and micrometastases. At present, bispecific molecules have demonstrated their antitumour potential in investigations in vitro using monolayer cell cultures. However, their effectiveness in vivo is less clear and expressive in vitro tumour models are in high demand., Materials and Methods: Three-dimensional multicellular tumour spheroids (MTS) are of intermediate complexity between monolayer cell cultures and solid tumours in patients and therefore represent a particularly promising in vitro system., Results: We show, here, the antitumour potential of a bispecific antibody, BiUII, in three-dimensional multicellular tumour spheroids and furthermore demonstrate that BiUII triggers peripheral blood mononuclear cells to invade MTSs and elicit the production of TNFalpha, resulting in the efficient destruction of tumour cells., Conclusion: These results demonstrate that bispecific antibodies are capable of activating immune effector cells, resulting in the elimination of three-dimensional structures of tumour cells. The therapeutic potential of these antibodies in the clinical setting merits further investigations.

The antitumor activity of IFN-alpha is well established. However, the role of the plasmacytoid dendritic cell (PDC), the major producer of IFN-alpha upon viral infection, in tumor biology is unknown. We sought to study the presence and function of PDC in a human solid tumor. Here, we demonstrate that PDCs infiltrate tumor tissue of patients with head and neck squamous cell carcinoma (HNSCC). Functional activity of PDC was examined by using CpG motif containing oligonucleotides, a defined microbial stimulus for PDCs (recognized via toll-like receptor 9). We found that HNSCC diminished the ability of PDC to produce IFN-alpha in response to CpG motif containing oligonucleotide. Tumor-induced down-regulation of toll-like receptor 9 was identified as one mechanism likely contributing to impaired PDC function within the tumor environment. In tumor-draining lymph nodes, suppression of CpG-induced IFN-alpha production was less pronounced than in single-cell suspensions of primary tumor tissue. In these lymph nodes, CpG-induced IFN-alpha production was associated with increased levels of interferon-induced protein 10 and IFN-gamma and activation of CD4 and CD8 T cells. These results show for the first time the presence of PDCs in human solid tumor tissue and that tumors suppress the capacity of PDCs to produce IFN-alpha. PDCs, which in the absence of appropriate stimulation are reported to promote regulatory CD8 T cells, may contribute to an impaired T-cell-mediated immune response in HNSCC.