Cleavage and cell adhesion properties of human epithelial cell adhesion molecule (HEPCAM).

Human epithelial cell adhesion molecule (HEPCAM) is a tumor-associated antigen frequently expressed in carcinomas, which promotes proliferation after regulated intramembrane proteolysis. Here, we describe extracellular shedding of HEPCAM at two α-sites through a disintegrin and metalloprotease (ADAM) and at one ß-site through BACE1. Transmembrane cleavage by γ-secretase occurs at three γ-sites to generate extracellular Aß-like fragments and at two ϵ-sites to release human EPCAM intracellular domain HEPICD, which is efficiently degraded by the proteasome. Mapping of cleavage sites onto three-dimensional structures of HEPEX cis-dimer predicted conditional availability of α- and ß-sites. Endocytosis of HEPCAM warrants acidification in cytoplasmic vesicles to dissociate protein cis-dimers required for cleavage by BACE1 at low pH values. Intramembrane cleavage sites are accessible and not part of the structurally important transmembrane helix dimer crossing region. Surprisingly, neither chemical inhibition of cleavage nor cellular knock-out of HEPCAM using CRISPR-Cas9 technology impacted the adhesion of carcinoma cell lines. Hence, a direct function of HEPCAM as an adhesion molecule in carcinoma cells is not supported and appears to be questionable.

Reducing tumor growth and angiogenesis using a triple therapy measured with Contrast-enhanced ultrasound (CEUS).

BACKGROUND: To evaluate the in vivo response by detecting the anti-angiogenic and invasion-inhibiting effects of a triple-combination-therapy in an experimental-small-animal-squamous-cell-carcinoma-model using the "flash-replenishment" (FR) method to assess tissue hemodynamics via contrast-enhanced-ultrasound (CEUS). METHODS: Human hypopharynx-carcinoma-cells were subcutaneously injected into the left flank of 22-female-athymic-nude-rats. After seven days of subcutaneous tumor growth, FR-measurements were performed on each rat. Treatment-group and control-group were treated every day for a period of one week, with the treatment-group receiving solvents containing a triple therapy of Upamostat®, Celecoxib® and Ilomastat® and the control-group solvents only. On day seven, follow-up measurements were performed using the same measurement protocol to assess the effects of the triple therapy. VueBox® was used to quantify the kinetic parameters and additional immunohistochemistry analyses were performed for comparison with and validation of the CEUS results against established methods (Proliferation/Ki-67, vascularization/CD31, apoptosis/caspase3). RESULTS: Compared to the control-group, the treatment-group that received the triple-therapy resulted in a reduction of tumor growth by 48.6% in size. Likewise, the immunohistochemistry results showed significant decreases in tumor proliferation and vascularization in the treatment-group in comparison to the control-group of 26%(p ≤ 0.05) and 32.2%(p ≤ 0.05) respectively. Correspondingly, between the baseline and follow-up measurements, the therapy-group was associated with a significant(p ≤ 0.01) decrease in the relative-Blood-Volume(rBV) in both the whole tumor(wt) and hypervascular tumor(ht) areas (p ≤ 0.01), while the control-group was associated with a significant (p ≤ 0.01) increase of the rBV in the wt area and a non-significant increase (p ≤ 0.16) in the ht area. The mean-transit-time (mTT) of the wt and the ht areas showed a significant increase (p ≤ 0.01) in the follow-up measurements in the therapy group. CONCLUSION: The triple-therapy is feasible and effective in reducing both tumor growth and vascularization. In particular, compared with the placebo-group, the triple-therapy-group resulted in a reduction in tumor growth of 48.6% in size when assessed by CEUS and a significant reduction in the number of vessels in the tumor of 32% as assessed by immunohistochemistry. As the immunohistochemistry supports the CEUS findings, CEUS using the "flash replenishment"(FR) method appears to provide a useful assessment of the anti-angiogenic and invasion-inhibiting effects of a triple combination therapy.

DCE-MRI biomarkers for monitoring an anti-angiogenic triple combination therapy in experimental hypopharynx carcinoma xenografts with immunohistochemical validation.

BACKGROUND: Novel anti-angiogenic treatments are increasingly complementing established cancer therapy strategies in head and neck tumors. Contrast-enhanced magnetic resonance imaging (MRI) can be applied for early and non-invasive therapy monitoring by non-invasive quantitative assessment of tumor microcirculation as in vivo imaging biomarkers of therapy response. PURPOSE: To monitor the anti-angiogenic effects of a novel combination therapy on experimental head and neck squamous cell carcinomas (HNSCC) with dynamic contrast-enhanced (DCE)-MRI. MATERIAL AND METHODS: Athymic rats (n = 18) with subcutaneous HNSCC xenografts were investigated by DCE-MRI before and after 7 days of a daily triple therapy regimen combining the COX-II-inhibitor celecoxib, the matrix-metalloproteinase-inhibitor GM6001, and the uPA-inhibitor upamostat. Quantitative measurements of tumor blood flow (tBF), tumor blood volume (tBV), and permeability-surface area product (PS) were calculated and validated by immunohistochemistry. RESULTS: Mean tBF and tBV in triple-therapy animals decreased significantly from day 0 to day 7 (tBF, 41.0 ± 14.2 to 20.4 ± 5.7 mL/100 mL/min; P < 0.01; tBV, 17.7 ± 3.9 to 7.5 ± 3.3%; P < 0.01). No significant effects on PS were observed in either group (P > 0.05). Immunohistochemical analysis showed a significantly lower tumor vascularity in the therapy group than in the control group (CD31), significantly fewer Ki-67+ proliferating tumor cells and significantly more Capase-3+ apoptotic tumor cells (P < 0.05). Significant (P < 0.05) correlations were observed between tBF/tBV and CD31 (tBF, r = 0.84; tBV, r = 0.70), tBV and Ki-67 (r = 0.62), as well as tBF and caspase-3 (r = -0.64). CONCLUSION: DCE-MRI may be a suitable tool for the non-invasive monitoring of the anti-vascular effects of this innovative triple therapy regimen with potential for clinical translation.

CXC chemokine receptor 4 is essential for maintenance of renal cell carcinoma-initiating cells and predicts metastasis.

In many solid tumors, cancer stem cells (CSC) represent a population with tumor-initiating, self-renewal, and differentiation potential, which can be identified by surface protein markers. No generally applicable markers are yet known for renal cell carcinoma (RCC). Two RCC cell lines (RCC-26, RCC-53) were found to differ widely in their capacity to form spheres in vitro and to establish tumors in mice, potentially reflecting differences in CSC content. A subpopulation expressing the CXC chemokine receptor 4 (CXCR4) was present only in the more tumorigenic cell line RCC-53. When grown as spheres, most of the RCC-53 cells were CXCR4-positive, expressed stem cell-associated transcription factor genes at elevated levels, and were more resistant toward the tyrosine kinase inhibitors sunitinib, sorafenib, and pazopanib. Sorted CXCR4-positive cells exhibited greater capacity for sphere formation and tumor growth-inducing potential in vivo than CXCR4-negative cells. Significantly, higher CXCR4 mRNA levels in primary RCC tumors from patients with localized but not disseminated disease predicted shorter survival. Downregulation of CXCR4 expression by small interfering RNA (siRNA) or pharmacological inhibition by AMD3100 compromised tumor sphere formation, viability of CXCR4-positive cells, and increased their responsiveness toward tyrosine kinase inhibitors. In conclusion, CXCR4 identifies a subpopulation of tumor-initiating cells in RCC cell lines and plays a role in their maintenance. The relative insensitivity of such cells to tyrosine kinase inhibitors might contribute to the development of therapy resistance in RCC patients. Future therapies therefore could combine blockade of the CXCR4 signaling pathway with standard therapies for more effective treatments of metastatic RCC.

B cells lacking the tumor suppressor TNFAIP3/A20 display impaired differentiation and hyperactivation and cause inflammation and autoimmunity in aged mice.

The ubiquitin-editing enzyme A20/TNFAIP3 is essential for controlling signals inducing the activation of nuclear factor-κB transcription factors. Polymorphisms and mutations in the TNFAIP3 gene are linked to various human autoimmune conditions, and inactivation of A20 is a frequent event in human B-cell lymphomas characterized by constitutive nuclear factor-κB activity. Through B cell-specific ablation in the mouse, we show here that A20 is required for the normal differentiation of the marginal zone B and B1 cell subsets. However, loss of A20 in B cells lowers their activation threshold and enhances proliferation and survival in a gene-dose-dependent fashion. Through the expression of proinflammatory cytokines, most notably interleukin-6, A20-deficient B cells trigger a progressive inflammatory reaction in naive mice characterized by the expansion of myeloid cells, effector-type T cells, and regulatory T cells. This culminates in old mice in an autoimmune syndrome characterized by splenomegaly, plasma cell hyperplasia, and the presence of class-switched, tissue-specific autoantibodies.

CD19-independent instruction of murine marginal zone B-cell development by constitutive Notch2 signaling.

B cell-specific gene ablation of Notch2 results in the loss of the marginal zone (MZ) B-cell lineage. To analyze the effects of constitutive Notch2 signaling in B cells, we have generated a transgenic mouse strain that allows the conditional expression of a constitutively active, intracellular form of Notch2 (Notch2IC). Expression of Notch2IC at the earliest developmental stages of the B-cell lineage completely abolished B-cell generation and led to the development of ectopic T cells in the bone marrow (BM), showing that Notch2IC is acting redundantly with Notch1IC in driving ectopic T-cell differentiation. In B cells clearly committed to the B-cell lineage induction of Notch2IC drove all cells toward the MZ B-cell compartment at the expense of follicular B cells. Notch2IC-expressing B cells reflected the phenotype of wild-type MZ B cells for their localization in the MZ, the expression of characteristic surface markers, their enhanced proliferation after stimulation, and increased basal activity of Akt, Erk, and Jnk. Notch2IC-driven MZ B-cell generation in the spleen was achieved even in the absence of CD19. Our results implicate that a constitutive Notch2 signal in transitional type 1 B cells is sufficient to drive MZ B-cell differentiation.

CD133 induces tumour-initiating properties in HEK293 cells.

The pentaspan protein CD133 (Prominin-1) is part of the signature of tumour-initiating cells for various cancer entities. The aim of the present study was to investigate the impact of ectopic CD133 expression on tumourigenic properties of otherwise CD133-negative, non-tumourigenic cells in vitro and in vivo. CD133 was stably transfected into human embryonic kidney 293 (HEK293) which was then sorted for the expression of CD133. The effects of CD133 on cell proliferation were assessed upon standard cell counting of sorted cells at various time points. Severe combined immunodeficient (SCID) mice (n = 30) were injected with HEK293 CD133(high) and CD133(low) transfectants (5 × 10(3), 1 × 10(5), or 5 × 10(6) cells per injection). The expression of CD133, Ki67, CD44s, CD44v6, and EpCAM was analysed upon immunohistochemical staining of cryosections with specific antibodies. In vitro, ectopic expression of CD133 did influence neither cell proliferation nor cell cycle distribution of otherwise CD133-negative HEK293 cells. However, CD133(high) cells generated tumours in vivo in SCID mice with at least 1,000-fold increased frequency compared to CD133(low) cells. Tumour load was also significantly increased in CD133(high) cells as compared to those tumours formed by high numbers of CD133(low) cells. Immunohistochemistry stainings disclosed no changes in Ki67, CD44s, CD44v6, or EpCAM once tumours were formed by either cell type. CD133 induces tumour-initiating properties in HEK293 cells in vivo and is potentially involved in the regulation of tumourigenicity. Future research will aim at the elucidation of molecular mechanisms of CD133-induced tumourigenicity.

Eosinophils and mast cells: a comparison of nasal mucosa histology and cytology to markers in nasal discharge in patients with chronic sino-nasal diseases.

Allergic rhinitis (AR), nasal polyps (NP) as well as chronic rhinosinusitis (CRS) are all known to be associated with eosinophilic infiltration and elevated numbers of mast cells (MC) within the mucosa. Both cell types and their markers eosinophilic cationic protein (ECP) and tryptase are utilized in the diagnosis and management of chronic sino-nasal diseases. Mucosal cytology samples were gathered by cytobrush, histological samples were obtained from the inferior turbinate. In both sample sets, the number of eosinophils and MC was determined. Their corresponding markers ECP and tryptase were quantified from nasal discharge. Patients were grouped with reference to their main diagnosis: AR (n = 34), NP (n = 25), CRS (n = 27) and controls (n = 34). Eosinophil counts from cytobrush and ECP levels were significantly elevated in NP compared to all other groups-31- and 13-fold over control, respectively. However, histologic review did not reveal any difference in eosinophil count among groups. Tryptase was significantly elevated threefold in AR versus CRS and controls. No correlation to cytological and histological MC counts could be found. ECP levels in nasal discharge as well as eosinophil counts can provide useful information with regard to the diagnosis. Likewise, tryptase concentrations can do. The presented data show that the measurement of markers in nasal discharge is superior in differentiating among diagnosis groups. Given that the collection of nasal secretions is more comfortable for patients than the more invasive techniques, we recommend first line ECP and tryptase testing performed on nasal secretions.

MMP7 is a target of the tumour-associated antigen EpCAM.

Epithelial cell adhesion molecule (EpCAM) is a single-transmembrane protein, which is involved in numerous cellular processes including cell adhesion, proliferation, maintenance of stemness of embryonic cells and progenitors, migration and invasion. Activation of signal transduction by EpCAM is warranted by regulated intramembrane proteolysis and nuclear translocation of the intracellular domain EpICD. Here, we describe matrix metalloproteinase 7 (MMP7) as a target gene of EpCAM signalling viaEpICD nuclear translocation. EpCAM and MMP7 expression pattern and levels positively correlated in vitro and in vivo, and were strongly elevated in primary carcinomas of the head and neck area. Hence, MMP7 is a novel target of EpCAM signalling.

Solitary chemosensory cells in the respiratory and vomeronasal epithelium of the human nose: a pilot study.

BACKGROUND: Recently, solitary chemosensory cells have been described in the respiratory and vomeronasal epithelium of the rodent nose. Expressing G-protein coupled receptors for sweet, umami and bitter taste transduction, these cells are thought to mediate trigeminal reflexes upon stimulation with chemical irritants. The present study analyzes human nasal mucosa for the presence of solitary chemosensory cells. METHODOLOGY: In human tissue samples from respiratory mucosa and the vomeronasal organ, gene expression of taste receptors families was studied in five patients using the Affymetrix Human Gene 1.0 ST Array and immunohistochemistry with specific antibodies. RESULTS: Immunohistochemistry revealed that solitary chemosensory cells expressing G-protein coupled receptors for sweet, umami and bitter taste transduction are present in the human nose. cDNA microarray analysis congruently showed that cells expressing bitter taste receptors accumulate in the vomeronasal organ compared to the respiratory epithelium. CONCLUSIONS: Solitary chemosensory cells expressing taste receptors are also present in the human nose. Since they are thought to mediate trigeminal reflexes, their role in the pathogenesis of nasal hyperreagibility should be elucidated in further studies.

EpCAM is involved in maintenance of the murine embryonic stem cell phenotype.

Epithelial cell adhesion molecule EpCAM is a transmembrane glycoprotein that is expressed on subsets of normal epithelia, numerous stem- and progenitor-type cells, and most carcinomas and highly overexpressed on cancer-initiating cells. The role of EpCAM in early development, particularly in stem-like cells, has remained unclear. Here, we show that the maintenance of self-renewal in murine embryonic stem (ES) cells depends on the high-level expression of EpCAM. Cultivation of ES cells under differentiation conditions in the absence of leukemia inhibitory factor (LIF) caused down-regulation of EpCAM along with decreased expression of cellular myelocytomatosis oncogene (c-Myc), Sex-determining region Y-Box 2, Octamer 3/4 (Oct3/4), and Stat3. As a consequence ES cells were morphologically differentiated and ceased to proliferate. RNA interference-mediated inhibition of EpCAM expression under self-renewal conditions resulted in quantitatively decreased proliferation, decreased Oct3/4, SSEA-1, and c-Myc expression, and diminished alkaline phosphatase activity. Conversely, exogenous expression of EpCAM partially compensated for the requirement of ES cells for LIF to retain a stem cell phenotype. Thus, murine EpCAM is a transmembrane protein, which is essential but by itself is not sufficient for maintenance of the ES cell phenotype.

Multimodal therapy for synergic inhibition of tumour cell invasion and tumour-induced angiogenesis.

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.

Initial activation of EpCAM cleavage via cell-to-cell contact.

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).

Keratin 8 expression in head and neck epithelia.

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.

Spatiotemporal patterning of EpCAM is important for murine embryonic endo- and mesodermal differentiation.

Epithelial cell adhesion molecule EpCAM is expressed in pluripotent embryonic stem cells (ESC) in vitro, but is repressed in differentiated cells, except epithelia and carcinomas. Molecular functions of EpCAM, possibly imposing such repression, were primarily studied in malignant cells and might not apply to non-pathologic differentiation. Here, we comprehensively describe timing and rationale for EpCAM regulation in early murine gastrulation and ESC differentiation using single cell RNA-sequencing datasets, in vivo and in vitro models including CRISPR-Cas9-engineered ESC-mutants. We demonstrate expression of EpCAM in inner cell mass, epiblast, primitive/visceral endoderm, and strict repression in the most primitive, nascent Flk1+ mesoderm progenitors at E7.0. Selective expression of EpCAM was confirmed at mid-gestation and perinatal stages. The rationale for strict patterning was studied in ESC differentiation. Gain/loss-of-function demonstrated supportive functions of EpCAM in achieving full pluripotency and guided endodermal differentiation, but repressive functions in mesodermal differentiation as exemplified with cardiomyocyte formation. We further identified embryonic Ras (ERas) as novel EpCAM interactor of EpCAM and an EpCAM/ERas/AKT axis that is instrumental in differentiation regulation. Hence, spatiotemporal patterning of EpCAM at the onset of gastrulation, resulting in early segregation of interdependent EpCAM+ endodermal and EpCAM-/vimentin+ mesodermal clusters represents a novel regulatory feature during ESC differentiation.

High Expression of EpCAM and Sox2 is a Positive Prognosticator of Clinical Outcome for Head and Neck Carcinoma.

Locally advanced head and neck squamous cell carcinomas (HNSCC) have limited prognosis due to frequent treatment failure. Currently, TNM-classification and human papillomavirus (HPV) infection are the sole clinical prognosticators of outcome. Tumor heterogeneity and stemness based on epithelial-mesenchymal-transition reportedly associate with therapy resistance. The capacity of epithelial marker EpCAM (EpEX), stemness regulator Sox2 and mesenchymal marker vimentin to predict clinical outcome of HSNCC patients was assessed upon immunohistochemistry staining in two cohorts of HNSCC patients treated with surgery and adjuvant radio (chemo) therapy (n = 94) and primary radio (chemo) therapy (n = 94), respectively. Prognostic values with respect to overall, disease-free and disease-specific survival were assessed in uni- and multivariate cox proportional hazard models to generate integrated risk scores. EpEX, Sox2 and vimentin displayed substantial inter- and intratumoral heterogeneity. EpEXhigh and Sox2high predicted improved clinical outcome in the discovery cohort and in the HPV-negative sub-cohort. EpEXhigh and Sox2high were confirmed as prognosticators of clinical outcome in the validation cohort treated with definitive radio(chemo)therapy. Importantly, EpEXhigh identified patients with improved survival within the HPV-negative subgroup of the validation cohort. Hence, Sox2high and particularly EpEXhigh have potential as tools to predict clinical performance of HNSCC patients, foremost HPV-negative cases, in the frame of molecular-guided treatment decision-making.

Biodistribution and radioimmunotherapy of SCCHN in xenotransplantated SCID mice with a 131I-labelled anti-EpCAM monoclonal antibody.

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.

Confocal laser endomicroscopy in head and neck malignancies using FITC-labelled EpCAM- and EGF-R-antibodies in cell lines and tumor biopsies.

Intraoperative detection of residual malignant cells at tumor margins following excision of primary tumors could help improving surgery and thus patients' outcome. The feasibility of the tumor antigens epidermal growth factor receptor (EGF-R) and epithelial cell adhesion molecule (EpCAM) for antibody-dependent confocal laser scanning endomicroscopy (CLE)-mediated visualization of malignant cells was addressed. Both tumor antigens are highly and frequently expressed in the majority of carcinomas, including head and neck squamous cell carcinomas (HNSCC), and represent prognostic and therapeutic tumor target molecules. FITC-conjugated EGF-R- and EpCAM-specific antibodies served as molecular tools for the detection of antigen-positive cells using the CLE technology. Specificity of both antibodies and their ability to discriminate tumor from non-tumor cells were assessed in vitro with human fibroblasts and PCI-1 HNSCC cell lines, and ex vivo on primary HNSCC samples (n = 11) and healthy mucosa (n = 5). Antigen specificity of the used EpCAM-specific antibody was superior to that of the EGF-R-specific antibody both in vitro and ex vivo (100% vs. 31.25%), and allowed visualization of cellular structures in CLE measurements. These results hold promise for possible future applications in humans.

Identification and functional analysis of tumor-infiltrating plasmacytoid dendritic cells in head and neck cancer.

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.