The role of the epithelial cell adhesion molecule EpCAM in cancer progression remains largely unclear. High expression of EpCAM in primary tumors is often associated with more aggressive phenotypes and EpCAM is the prime epithelial antigen in use to isolate circulating tumor cells (CTCs) and characterize disseminated tumor cells (DTCs). However, reduced expression of EpCAM was associated with epithelial-to-mesenchymal transition (EMT) and reports on a lack of EpCAM on CTCs emerged. These contradictory observations might reflect a context-dependent adaption of EpCAM expression during metastatic progression. To test this, EpCAM expression was monitored in esophageal cancer at different sites of early systemic disease. Although most of the primary esophageal tumors expressed high levels of EpCAM, the majority of DTCs in bone marrow lacked EpCAM. In vitro, downregulation of EpCAM expression at the plasma membrane was observed in migrating and invading cells, and was associated with a partial loss of the epithelial phenotype and with significantly decreased proliferation. Accordingly, induction of EMT through the action of TGFβ resulted in substantial loss of EpCAM cell surface expression on esophageal cancer cells. Knock-down or natural loss of EpCAM recapitulated these effects as it reduced proliferation while enhancing migration and invasion of cancer cells. Importantly, expression of EpCAM on DTCs was significantly associated with the occurrence of lymph node metastases and with significantly decreased overall survival of esophageal cancer patients. We validated this observation by showing that high expression of EpCAM promoted tumor outgrowth after xenotransplantation of esophageal carcinoma cells. The present data disclose a dynamic expression of EpCAM throughout tumor progression, where EpCAMhigh phenotypes correlate with proliferative stages, whereas EpCAMlow/negative phenotypes associated with migration, invasion and dissemination. Thus, differing expression levels of EpCAM must be taken into consideration for therapeutic approaches and during clinical retrieval of disseminated tumor cells.

Disseminierte Tumorzellen solider Tumoren in Knochenmark und Lymphknoten wie auch der Nachweis zirkulierender Tumorzellen im Blut der Patienten dienen zunehmend als Surrogatmarker für systemische Therapieansätze. Zur Detektion epithelialer Zellen in den genannten Gewebekompartimenten wird insbesondere das Zelloberflächenprotein EpCAM (CD326) verwendet (u.a. beim FDA-anerkannten CellSearch-System) und mit Edrecolomab und Catumaxomab stehen zudem zwei zugelassene therapeutische Antikörper zur Verfügung. Da bislang keine systematischen Daten zur Prävalenz von disseminierten EpCAM-positiven Tumorzellen vorliegen, haben wir EpCAM-positive Zellen aus dem Knochenmark von Patienten mit unterschiedlichen Karzinomen des Magen-Darm-Traktes isoliert.

Die umfassende genetische und phänotypische Analyse disseminierter Tumorzellen, unter denen sich potentielle Vorläufer späterer Metastasen befinden, stellt in vivo nicht zuletzt aufgrund ihrer Seltenheit weiterhin eine große Herausforderung dar. Zum besseren Verständnis der grundlegenden Mechanismen zur Metastasierung solider Tumoren haben wir ein Tumor-Xenograft-Modell in der SCID-Maus etabliert, welches die Isolierung disseminierter Tumoreinzelzellen erlaubt.

Mithilfe eines Tissue Microarrays, welcher die Tumorproben von 98 Patienten mit einem Adenokarzinom (EAC) und von 118 Patienten mit einem Plattenepithelkarzinom des Ösophagus (ESCC) umfasst, untersuchten wir die Expression und Bedeutung des Zelladhäsionsmoleküls EpCAM im Zuge der Progression von Ösophagustumoren. Als Referenz bestimmten wir die Expression von EpCAM in der normalen Ösophagusmukosa von 26 Patienten und analysierten zudem 10 Fälle einer Barrett-Metaplasie. Die immunhistochemisch erhobene Stärke der EpCAM-Expression wurden bei einem Teil der Proben mittels quantitativer PCR verifiziert.

Einleitung: Die Ösophagusperforation stellt nach wie vor ein lebensbedrohliches Krankheitsbild dar. Die Therapieentscheidung ist abhängig von Ursache, Zeitspanne nach dem auslösenden Ereignis, Grund- bzw. Nebenerkrankungen. Nach wie vor besteht keine Einigung über ein standardisiertes Behandlungskonzept. Um einen Therapiealgorhythmus zu entwickeln, wurden retrospektiv die Ergebnisse von uns behandelter Patienten ausgewertet und mit der aktuellen Literatur verglichen.

Introduction: Compared to other tumor entities the prognosis of patients with colorectal carcinoma (CRC) improved significantly due to advances in surgery and in multimodal therapies. Here we investigated systematically the proliferation, invasion and migration of CRC cell lines as well as their morphology in 3D culture to enable the functional characterisation of novel therapeutic targets. Material and methods: We embedded several CRC cell lines SW480, HT29, DLD-1, Lovo, CaCo, Colo 205 and Colo 206F in matrigel 3D-cell culture systems and investigated the morphology and proliferation characteristics in comparison to 2D- cell culture models. In addition we analysed expression differences between 2D- and 3D- cell cultures on the RNA and protein level. The invasion properties of the cell lines was investigated via Matrigel- coated Transwell-systems (Boyden chamber) while the capability of migration was determined with the Fence assay. The establishment and cultivation of tumour spheroids enables to gain insight into the spatial expression of potential therapeutic targets. Results: The investigated CRC- cell lines differed, in part considerably, among each other with respect to their morphology, proliferation and invasion. Some cell lines showed a solid tumour cell formation while others exhibited budding structures. Changes in the proliferation were obtained among 2D- and 3D-cultures. Furthermore, differences in the expression level of the investigated genes and proteins were apparent among the two culture systems. Conclusion: Compared to conventional 2D cell culture, the 3D-cell culture model offers the opportunity to investigate potential molecular targets under more realistic conditions. The differences in morphology and function among the investigated tumour cell lines might be a representative model for the individual tumor heterogeneity in CRC.

Prior in vivo studies supported the concept that Mallory bodies (MBs) are aggresomes of cytokeratins 8 and 18. However, to test this hypothesis an in vitro model is needed to study the dynamics of MB formation. Such a study is difficult because MBs have never been induced in tissue culture. Therefore, MBs were first induced in vivo in drug-primed mice and then primary cultures of hepatocytes from these mice were studied. Two approaches were utilized: 1. Primary cultures were transfected with plasmids containing the sequence for cytokeratin 18 (CK 18) tagged with green fluorescent protein (GFP). 2. Immunofluorescent staining was used to localize the ubiquitin-proteasome pathway components involved in MB-aggresome complex formation in primary hepatocyte cultures. The cells were double stained with a ubiquitin antibody and one of the following antibodies: CK 8, CK 18, tubulin, mutant ubiquitin (UBB+1), transglutaminase, phosphothreonine, and the 20S and 26S proteasome subunits P25 and Tbp7, respectively. In the first approach, fluorescence was observed in keratin filaments and MBs 48 h after the cells were transfected with the CK 18 GFP plasmid. Nascent cytokeratin 18 was preferentially concentrated in MBs. Less fluorescence was observed in the normal keratin filaments. This indicated that MBs continued to form in vitro. The immunofluorescent staining of the hepatocytes showed that CK 8 and 18, ubiquitin, mutant ubiquitin (UBB+1), P25, Tbp7, phosphothreonine, tubulin, and transglutaminase were all located at the border or the interior of the MB. These results support the concept that MBs are aggresomes of CK 8 and CK18 and are a result of inhibition of the ubiquitin-proteasome pathway of protein degradation possibly caused by UBB+1.

While significant progress has been made to advance our knowledge of microvascular lesion formation, yet the investigation of how stem-like cells may contribute to the pathogenesis of microvascular diseases is still in its infancy. We assessed whether the inhibitor of DNA binding and differentiation 3 (ID3) contributes to the acquisition of a molecular stem cell-like signature in microvascular endothelial cells. The effects of stable ID3 overexpression and SU5416 treatment — a chemical inducer of microvascular lesions, had on the stemness signature were determined by flow cytometry, immunoblot, and immunohistochemistry. Continuous ID3 expression produced a molecular stemness signature consisting of CD133+ VEGFR3+ CD34+ cells. Cells exposed to SU5416 showed positive protein expression of ID3, VEGFR3, CD34 and increased expression of pluripotent transcription factors Oct-4 and Sox-2. ID3 overexpressing cells supported the formation of a 3-D microvascular lesion co-cultured with smooth muscle cells. In addition, in vivo microvascular lesions from SuHx rodent model showed an increased expression of ID3, VEGFR3, and Pyk2 similar to SU5416 treated human endothelial cells. Further investigations into how normal and stem-like cells utilize ID3 may open up new avenues for a better understanding of the molecular mechanisms which are underlying the pathological development of microvascular diseases.

The aim of the present study was to identify differentially expressed genes and their related biological pathways in the secretory phase endometrium from patients with recurrent miscarriage (RM) and fertile subjects. Endometrial samples from RM and fertile patients were analyzed using the Affymetrix GeneChip® ST Array. The bioinformatic analysis using the Partek Genomic Suite revealed 346 genes that were differentially expressed (175 up-regulated and 171 down-regulated) in the endometrium of RM patients compared to the fertile subjects (fold change ≥1.5, p<0.005). Validation step using quantitative real-time polymerase chain reaction (qPCR) confirmed a similar expression pattern of four exemplary genes: one up-regulated gene (fibroblast growth factor 9, FGF9) and three down-regulated genes: integrin β3 (ITGB3), colony stimulating factor 1 (CSF1) and matrix-metalloproteinases 19 (MMP19). The Gene Set Enrichment Analysis (GSEA) and the Pathway Studio Software have found 101 signaling pathways (p<0.05) associated with the affected genes including the FGFR3/signal transducer and activator of transcription (STAT) pathway and the CSF1R/STAT pathway. Cell adhesion, cell differentiation and angiogenesis were among biological processes indicated by this system. In conclusion, microarray technique is a useful tool to study gene expression in the secretory phase-endometrium of RM patients. The differences in endometrial gene expressions between healthy and RM subjects contribute to an increase in our knowledge on molecular mechanisms of RM development and may improve the outcome of pregnancies in high-risk women with RM.

EpCAM represents a promising therapeutic target in antibody-based tumor therapy. In our study, we assessed the potential of EpCAM as a therapeutic molecular target in the treatment of esophageal adenocarcinoma (Barrett’s carcinoma) by analyzing the expression of EpCAM using a standardized immunohistochemistry staining assay on a tissue microarray (TMA) comprising the primary tumor tissue of 98 patients. In addition to the expression in the primary tumors, EpCAM protein expression was also determined in the normal esophageal mucosa of 26 patients and 10 cases of Barrett’s metaplasia.

Early disseminated tumor cells (DTC) are potential precursor cells of later metastases and therefore the ultimate target for adjuvant therapies. To assess if EpCAM is a useful target for elimination of DTC in lymph nodes (LN) and bone marrow (BM), we determined the frequency of EpCAM+ and CK+DTC in these entities. Methods: After establishment of a protocol for double immunofluorescence labeling for simultaneous visualization of EpCAM expression on CK+ cells, we applied this protocol to a series of BM and LN preparations from operable esophageal cancer patients. After isolation of positively stained cells, their genomic DNA was globally amplified using the Mse-adapter PCR method. Then we applied comparative genomic hybridization (CGH) for the genome-wide screening of DNA-gains and-losses.

EpCAM represents a relevant therapeutic target in antibody-based tumor therapy. In our study, we investigated the potential of EpCAM as a therapeutic molecular target in the treatment of esophageal squamous cell carcinoma (ESCC) by assessing the expression of EpCAM using a tissue microarray (TMA) comprising the primary tumor tissue of 118 patients. The EpCAM protein expression in the tumors was analyzed by immunohistochemistry and verified by quantitative real-time PCR in a subgroup of cases. Whereas no expression was detected in normal esophageal squamous epithelium, 64% of the investigate tumors expressed EpCAM on a low 1+(12%), moderate 2+(19%), and strong 3+ (32%) level, respectively. Expression was predominantly heterogeneous, while subgroup exhibited an enhanced EpCAM expression at the tumor invasion front. Strong (3+) expression of EpCAM in the primary tumor tissue correlated significantly with a limited overall survival of the patients (logrank test, p=0.0479). Aside from invasion depth (pT) and nodal involvement (pN), strong (3+) expression of EpCAM in the primary tumor was established as an independent progrnostic marker in the multivariate analysis. Consistently, strong (3+) EpCAM protein expression in the tumor tissue correlated with an increased detection of the transcript in the corresponding subset of cases as determined by qPCR. Thus, the prognostic relevance of its overexpression strongly suggests EpCAM as a promising molecular target for antibody-based adjuvant therapy in ESCC.

Ähnlich wie bei anderen Karzinomen lassen sich Modulationen bei der Integrin-Expression auch beim ESCC häufig nachweisen, wobei insbesondere die Integrin-Untereinheiten α6, β1 und β4 bei der Progression des ESCC eine Rolle zu spielen scheinen. Darüber hinaus war die reduzierte Expression von α6 in der multivariaten Analyse mit einem signifikant verkürzten rezidivfreien Überleben korreliert.

Einleitung: Integrine sind heterodimere Adhäsionsmoleküle, die sich aus einer alpha- und einer beta-Untereinheit zusammensetzen. Sie tragen als Zell-Zell-Adhäsionsmoleküle zur Stabilität von Zellverbänden bei, ermöglichen durch dynamische Zell-Matrix-Interaktion die Zellmigration und beeinflussen als Signaltransduktoren die Zelldifferenzierung und -proliferation. Modulationen der Integrinexpression können bei vielen soliden Tumoren nachgewiesen werden, wobei aberrante Integrin-Expressionsmuster häufig mit einer schlechten Prognose assoziiert sind. Zur Integrin-Expression beim Ösophaguskarzinom ist bislang nur wenig bekannt. Material und Methoden: Die Primärtumoren von 36 kurativ resezierten (R0) Patienten mit einem Ösophagus-Plattenepithelkarzinom (ESCC) wurden mittels Immunfluoreszenz-Färbung auf die Expression der Integrin-Untereinheiten alpha2, alpha3, alpha6, beta1 und beta4 untersucht. Dabei wurde sowohl die Expressionsstärke als auch das Expressionsmuster berücksichtigt und mit der Integrin-Expression der normalen Ösophagusmukosa verglichen. Mithilfe einer hierarchischen Ähnlichkeitsanalyse wurden die Primärtumoren anhand ihrer Expressionsprofile auf Subgruppen mit gemeinsamer Merkmalsausprägung untersucht. Ergebnisse: Die alpha6- und beta4-Expression am unmittelbaren Invasionsrand hob sich in über 90% der Tumoren mit einer verstärkten Expression vom übrigen Tumorgewebe ab. Dabei korrelierte die Stärke der alpha6-Expression auf den Tumorzellen mit direktem Kontakt zum umgebenden Stroma mit dem rezidivfreien Überleben der Patienten (p<0,005). Des Weiteren überlebten Patienten, die im Primärtumorgewebe ein polarisiertes Expressionsmuster aufwiesen, wie es in der Normalmukosa beobachtet wurde, tendenziell länger rezidivfrei als Patienten, deren Tumoren eine ungeordnete Integrin-Expression aufwiesen (p<0,08). Schlussfolgerung: Während sich die Expressionsstärken der untersuchten Integrine in Tumorgewebe und Normalmukosa nicht signifikant voneinander unterschieden, scheint der Nachweis eines aberranten Integrin-Expressionsmusters beim ESCC mit dem raschen Progress der Tumorerkrankung zu korrelieren. Wie bei anderen Karzinomen könnte die alpha6beta4-Expression am Invasionsrand einen potentiellen Indikator zur Beurteilung der Aggressivität von ESCC darstellen.