Diagnostic leukapheresis reveals distinct phenotypes of NSCLC circulating tumor cells.

BACKGROUND: Circulating tumor cells (CTCs) hold immense promise for unraveling tumor heterogeneity and understanding treatment resistance. However, conventional methods, especially in cancers like non-small cell lung cancer (NSCLC), often yield low CTC numbers, hindering comprehensive analyses. This study addresses this limitation by employing diagnostic leukapheresis (DLA) to cancer patients, enabling the screening of larger blood volumes. To leverage DLA's full potential, this study introduces a novel approach for CTC enrichment from DLAs. METHODS: DLA was applied to six advanced stage NSCLC patients. For an unbiased CTC enrichment, a two-step approach based on negative depletion of hematopoietic cells was used. Single-cell (sc) whole-transcriptome sequencing was performed, and CTCs were identified based on gene signatures and inferred copy number variations. RESULTS: Remarkably, this innovative approach led to the identification of unprecedented 3,363 CTC transcriptomes. The extensive heterogeneity among CTCs was unveiled, highlighting distinct phenotypes related to the epithelial-mesenchymal transition (EMT) axis, stemness, immune responsiveness, and metabolism. Comparison with sc transcriptomes from primary NSCLC cells revealed that CTCs encapsulate the heterogeneity of their primary counterparts while maintaining unique CTC-specific phenotypes. CONCLUSIONS: In conclusion, this study pioneers a transformative method for enriching CTCs from DLA, resulting in a substantial increase in CTC numbers. This allowed the creation of the first-ever single-cell whole transcriptome in-depth characterization of the heterogeneity of over 3,300 NSCLC-CTCs. The findings not only confirm the diagnostic value of CTCs in monitoring tumor heterogeneity but also propose a CTC-specific signature that can be exploited for targeted CTC-directed therapies in the future. This comprehensive approach signifies a major leap forward, positioning CTCs as a key player in advancing our understanding of cancer dynamics and paving the way for tailored therapeutic interventions.

Ultra-sensitive CTC-based liquid biopsy for pancreatic cancer enabled by large blood volume analysis.

The limited sensitivity of circulating tumor cell (CTC) detection in pancreatic adenocarcinoma (PDAC) stems from their extremely low concentration in the whole circulating blood, necessitating enhanced detection methodologies. This study sought to amplify assay-sensitivity by employing diagnostic leukapheresis (DLA) to screen large blood volumes. Sixty patients were subjected to DLA, with a median processed blood volume of ~ 2.8 L and approximately 5% of the resulting DLA-product analyzed using CellSearch (CS). Notably, DLA significantly increased CS-CTC detection to 44% in M0-patients and 74% in M1-patients, yielding a 60-fold increase in CS-CTC enumeration. DLA also provided sufficient CS-CTCs for genomic profiling, thereby delivering additional genomic information compared to tissue biopsy samples. DLA CS-CTCs exhibited a pronounced negative prognostic impact on overall survival (OS), evidenced by a reduction in OS from 28.6 to 8.5 months (univariate: p = 0.002; multivariable: p = 0.043). Additionally, a marked enhancement in sensitivity was achieved (by around 3-4-times) compared to peripheral blood (PB) samples, with positive predictive values for OS being preserved at around 90%. Prognostic relevance of CS-CTCs in PDAC was further validated in PB-samples from 228 PDAC patients, consolidating the established association between CTC-presence and reduced OS (8.5 vs. 19.0 months, p < 0.001). In conclusion, DLA-derived CS-CTCs may serve as a viable tool for identifying high-risk PDAC-patients and aiding the optimization of multimodal treatment strategies. Moreover, DLA enables comprehensive diagnostic profiling by providing ample CTC material, reinforcing its utility as a reliable liquid-biopsy approach. This high-volume liquid-biopsy strategy presents a potential pathway for enhancing clinical management in this malignancy.

Comparative analysis of EpCAM high-expressing and low-expressing circulating tumour cells with regard to their clonal relationship and clinical value.

BACKGROUND: Circulating tumour cells (CTCs) are mainly enriched based on the epithelial cell adhesion molecule (EpCAM). Although it was shown that an EpCAM low-expressing CTC fraction is not captured by such approaches, knowledge about its prognostic and predictive relevance and its relation to EpCAM-positive CTCs is lacking. METHODS: We developed an immunomagnetic assay to enrich CTCs from metastatic breast cancer patients EpCAM independently using antibodies against Trop-2 and CD-49f and characterised their EpCAM expression. DNA of single EpCAM high expressing and low expressing CTCs was analyzed regarding chromosomal aberrations and predictive mutations. Additionally, we compared CTC-enrichment on the CellSearch system using this antibody mix and the EpCAM based enrichment. RESULTS: Both antibodies acted synergistically in capturing CTCs. Patients with EpCAM high-expressing CTCs had a worse overall and progression-free survival. EpCAM high- and low-expressing CTCs presented similar chromosomal aberrations and mutations indicating a close evolutionary relationship. A sequential enrichment of CTCs from the EpCAM-depleted fraction yielded a population of CTCs not captured EpCAM dependently but harbouring predictive information. CONCLUSIONS: Our data indicate that EpCAM low-expressing CTCs could be used as a valuable tumour surrogate material-although they may be prognostically less relevant than EpCAM high-expressing CTCs-and have particular benefit if no CTCs are detected using EpCAM-dependent technologies.

Mesenchymal-epithelial transition in lymph node metastases of oral squamous cell carcinoma is accompanied by ZEB1 expression.

BACKGROUND: Oral squamous cell carcinoma (OSCC), an HPV-negative head and neck cancer, frequently metastasizes to the regional lymph nodes but only occasionally beyond. Initial phases of metastasis are associated with an epithelial-mesenchymal transition (EMT), while the consolidation phase is associated with mesenchymal-epithelial transition (MET). This dynamic is referred to as epithelial-mesenchymal plasticity (EMP). While it is known that EMP is essential for cancer cell invasion and metastatic spread, less is known about the heterogeneity of EMP states and even less about the heterogeneity between primary and metastatic lesions. METHODS: To assess both the heterogeneity of EMP states in OSCC cells and their effects on stromal cells, we performed single-cell RNA sequencing (scRNAseq) of 5 primary tumors, 9 matching metastatic and 5 tumor-free lymph nodes and re-analyzed publicly available scRNAseq data of 9 additional primary tumors. For examining the cell type composition, we performed bulk transcriptome sequencing. Protein expression of selected genes were confirmed by immunohistochemistry. RESULTS: From the 23 OSCC lesions, the single cell transcriptomes of a total of 7263 carcinoma cells were available for in-depth analyses. We initially focused on one lesion to avoid confounding inter-patient heterogeneity and identified OSCC cells expressing genes characteristic of different epithelial and partial EMT stages. RNA velocity and the increase in inferred copy number variations indicated a progressive trajectory towards epithelial differentiation in this metastatic lesion, i.e., cells likely underwent MET. Extension to all samples revealed a less stringent but essentially similar pattern. Interestingly, MET cells show increased activity of the EMT-activator ZEB1. Immunohistochemistry confirmed that ZEB1 was co-expressed with the epithelial marker cornifin B in individual tumor cells. The lack of E-cadherin mRNA expression suggests this is a partial MET. Within the tumor microenvironment we found immunomodulating fibroblasts that were maintained in primary and metastatic OSCC. CONCLUSIONS: This study reveals that EMP enables different partial EMT and epithelial phenotypes of OSCC cells, which are endowed with capabilities essential for the different stages of the metastatic process, including maintenance of cellular integrity. During MET, ZEB1 appears to be functionally active, indicating a more complex role of ZEB1 than mere induction of EMT.

Proficiency Testing to Assess Technical Performance for CTC-Processing and Detection Methods in CANCER-ID.

BACKGROUND: Multiple technologies are available for detection of circulating tumor cells (CTCs), but standards to evaluate their technical performance are still lacking. This limits the applicability of CTC analysis in clinic routine. Therefore, in the context of the CANCER-ID consortium, we established a platform to assess technical validity of CTC detection methods in a European multi-center setting using non-small cell lung cancer (NSCLC) as a model. METHODS: We characterized multiple NSCLC cell lines to define cellular models distinct in their phenotype and molecular characteristics. Standardized tumor-cell-bearing blood samples were prepared at a central laboratory and sent to multiple European laboratories for processing according to standard operating procedures. The data were submitted via an online tool and centrally evaluated. Five CTC-enrichment technologies were tested. RESULTS: We could identify 2 cytokeratin expressing cell lines with distinct levels of EpCAM expression: NCI-H441 (EpCAMhigh, CKpos) and NCI-H1563 (EpCAMlow, CKpos). Both spiked tumor cell lines were detected by all technologies except for the CellSearch system that failed to enrich EpCAMlow NCI-H1563 cells. Mean recovery rates ranged between 49% and 75% for NCI-H411 and 32% and 76% for NCI-H1563 and significant differences were observed between the tested methods. CONCLUSIONS: This multi-national proficiency testing of CTC-enrichment technologies has importance in the establishment of guidelines for clinically applicable (pre)analytical workflows and the definition of minimal performance qualification requirements prior to clinical validation of technologies. It will remain in operation beyond the funding period of CANCER-ID in the context of the European Liquid Biopsy Society (ELBS).

Spatiotemporally controlled induction of gene expression in vivo allows tracking the fate of tumor cells that traffic through the lymphatics.

Metastasis is a multistep process, during which circulating tumor cells traffic through diverse anatomical locations. Stable inducible marking of tumor cells in a manner that is tightly spatially and temporally controlled would allow tracking the contribution of cells passing through specific locations to metastatic dissemination. For example, tumor cells enter the lymphatic system and can form metastases in regional lymph nodes, but the relative contribution of tumor cells that traffic through the lymphatic system to the formation of distant metastases remains controversial. Here, we developed a novel genetic switch based on mild transient warming (TW) that allows cells to be marked in a defined spatiotemporal manner in vivo. Prior to warming, cells express only EGFP. Upon TW, the EGFP gene is excised and expression of mCherry is permanently turned on. We employed this system in an experimental pancreatic cancer model and used localized TW to induce the genetic switch in tumor cells trafficking through tumor-draining lymph nodes. Thereby we found that tumor cells disseminating via the lymphatics make a major contribution to the seeding of lung metastases. The inducible genetic marking system we have developed is a powerful tool for the tracking of metastasizing cells in vivo.

EGFR/Ras-induced CCL20 production modulates the tumour microenvironment.

BACKGROUND: The activation of the EGFR/Ras-signalling pathway in tumour cells induces a distinct chemokine repertoire, which in turn modulates the tumour microenvironment. METHODS: The effects of EGFR/Ras on the expression and translation of CCL20 were analysed in a large set of epithelial cancer cell lines and tumour tissues by RT-qPCR and ELISA in vitro. CCL20 production was verified by immunohistochemistry in different tumour tissues and correlated with clinical data. The effects of CCL20 on endothelial cell migration and tumour-associated vascularisation were comprehensively analysed with chemotaxis assays in vitro and in CCR6-deficient mice in vivo. RESULTS: Tumours facilitate progression by the EGFR/Ras-induced production of CCL20. Expression of the chemokine CCL20 in tumours correlates with advanced tumour stage, increased lymph node metastasis and decreased survival in patients. Microvascular endothelial cells abundantly express the specific CCL20 receptor CCR6. CCR6 signalling in endothelial cells induces angiogenesis. CCR6-deficient mice show significantly decreased tumour growth and tumour-associated vascularisation. The observed phenotype is dependent on CCR6 deficiency in stromal cells but not within the immune system. CONCLUSION: We propose that the chemokine axis CCL20-CCR6 represents a novel and promising target to interfere with the tumour microenvironment, and opens an innovative multimodal strategy for cancer therapy.

Genetic Analysis of Circulating Tumour Cells.

The classification of human cancers has traditionally relied on the tissue of origin, the histologic appearance and anatomical extent of disease, otherwise referred to as grade and stage. However, this system fails to explain the highly variable clinical behaviour seen for any one cancer. Molecular characterization through techniques such as next-generation sequencing (NGS) has led to an appreciation of the extreme genetic heterogeneity that underlies most human cancers. Because of the difficulties associated with fresh tissue biopsy, interest has increased in using circulating tumour material, such as circulating tumour cells (CTCs), as a non-invasive way to access tumour tissue. CTC enumeration has been demonstrated to have prognostic value in metastatic breast, colon and prostate cancers. Recent studies have also shown that CTCs are suitable material for molecular characterization, using techniques such as reverse transcription-polymerase chain reaction (RT-PCR), fluorescence in situ hybridization (FISH), array comparative genomic hybridization (aCGH) and NGS. Furthermore, genetic analysis of CTCs may be more suitable to study tumour heterogeneity and clonal evolution than fresh tissue biopsy. Whether blood-based biopsy techniques will be accepted as a replacement to fresh tissue biopsies remains to be seen, but there is reason for optimism. While significant barriers to this acceptance exist, blood-based biopsy techniques appear to be reliable and representative alternatives to fresh tissue biopsy.

BRAFV600E mutation: A promising target in colorectal neuroendocrine carcinoma.

To determine the role of BRAFV600E mutation and MAPK signaling as well as the effects of BRAF and MEK directed therapy in gastroenteropancreatic neuroendocrine neoplasia (GEP-NEN), with a focus on highly aggressive gastroenteropancreatic neuroendocrine carcinoma (GEP-NEC). Using Sanger sequencing of BRAF exon 15 we determined the frequency of BRAFV600E mutations in 71 primary GEP-NENs. MEK phosphorylation was examined by immunohistochemistry in corresponding tissue samples. To evaluate the biological relevance of BRAFV600E mutation and MAPK signaling in GEP-NECs, effects of a pharmacological BRAF and MEK inhibition were analyzed in NEC cell lines both in vitro and in vivo. BRAFV600E mutation was detected in 9.9% of all GEP-NENs. Interestingly, only NECs of the colon harbored BRAFV600E mutations, leading to a mutation frequency of 46.7% in this subgroup of patients. In addition, a BRAFV600E mutation was significantly associated with high levels of MEK phosphorylation (pMEK) and advanced tumor stages. Pharmacological inhibition of BRAF and MEK abrogated NEC cell growth, inducing G1 cell cycle arrest and apoptosis only in BRAFV600E mutated cells. BRAF inhibitor dabrafenib and MEK inhibitor trametinib prevented growth of BRAFV600E positive NEC xenografts. High frequencies of BRAFV600E mutation and elevated expression levels of pMEK were detected in biologically aggressive and highly proliferative colorectal NECs. We provide evidence that targeting BRAF oncogene may represent a therapeutic strategy for patients with BRAF mutant colorectal NECs.

Label-Free Enrichment and Molecular Characterization of Viable Circulating Tumor Cells from Diagnostic Leukapheresis Products.

INTRODUCTION: Circulating tumor cells (CTCs) may be used to improve cancer diagnosis, prognosis, and treatment. However, because knowledge regarding CTC biology is limited and the numbers of CTCs and CTC-positive cancer patients are low, progress in this field is slow. We addressed this limitation by combining diagnostic leukapheresis (DLA) and microfluidic enrichment to obtain large numbers of viable CTCs from metastasized breast cancer patients. METHODS: DLA was applied to 9 patients, and 7.5 mL of peripheral blood was drawn. CTCs were enriched with the Parsortix™ system. The quality of CTCs from fresh and cryopreserved DLA products was tested, and CTCs were cultured in vitro. Single uncultured and cultured CTCs were isolated by micromanipulation to determine different parameters, such as genomic aberrations and mutation profiles of selected tumor-associated genes. Expression levels of estrogen receptor and HER2/neu were monitored during in vitro culture. RESULTS: Viable CTCs from peripheral blood and fresh or frozen DLA products could be enriched. DLA increased the likelihood of successful CTC culture. Cryopreserved DLA products could be stored with minimal CTC loss and no overt reduction in the tumor cell quality and viability during an observation period of up to 3 years. The analyzed parameters did not change during in vitro culture. DLA samples with high CTC numbers and lower ratios of apoptotic CTCs were more likely to grow in culture. CONCLUSIONS: The increased CTC numbers from fresh or cryopreserved DLA products facilitate multiple functional and molecular analyses and, thus, could improve our knowledge of their biology.

Diagnostic pathology of early systemic cancer: ERBB2 gene amplification in single disseminated cancer cells determines patient survival in operable esophageal cancer.

Early metastatic dissemination and evolution of disseminated cancer cells (DCCs) outside the primary tumor is one reason for the failure of adjuvant therapies because it generates molecular genotypes and phenotypes different from primary tumors, which still underlie therapy decisions. Since ERBB2 amplification in esophageal DCCs but not in primary tumor cells predict outcome, we aimed to establish an assay with diagnostic reliability for single DCCs or circulating tumor cells. For this, we evaluated copy number alterations of more than 600 single DCCs from multiple cancer types to define reference regions suitable for quantification of target regions, such as ERBB2. We then compared ERBB2 quantitative PCR (qPCR) measurements with fluorescent in situ hybridization (FISH) data of various breast cancer cell lines and identified the aberration-calling threshold. The method was applied to two independent cohorts of esophageal cancer patients from Hamburg (n = 59) and Düsseldorf (n = 53). We found a high correlation between the single cell qPCR assay and the standard FISH assay (R = 0.98) and significant associations between amplification and survival for both patient cohorts (Hamburg (HH), p = 0.033; Düsseldorf (D), p = 0.052; pooled HH + D, p = 0.002) when applied to DCCs of esophageal cancer patients. Detection of a single ERBB2-amplified DCC was the most important risk factor for death from esophageal cancer (relative risk = 4.22; 95% CI = 1.91-9.32; p < 0.001). In our study, we detected ERBB2-amplified cells in 7% of patients. These patients could benefit from anti-ERBB2 targeting therapies.

Toward a real liquid biopsy in metastatic breast and prostate cancer: Diagnostic LeukApheresis increases CTC yields in a European prospective multicenter study (CTCTrap).

Frequently, the number of circulating tumor cells (CTC) isolated in 7.5 mL of blood is too small to reliably determine tumor heterogeneity and to be representative as a "liquid biopsy". In the EU FP7 program CTCTrap, we aimed to validate and optimize the recently introduced Diagnostic LeukApheresis (DLA) to screen liters of blood. Here we present the results obtained from 34 metastatic cancer patients subjected to DLA in the participating institutions. About 7.5 mL blood processed with CellSearch® was used as "gold standard" reference. DLAs were obtained from 22 metastatic prostate and 12 metastatic breast cancer patients at four different institutions without any noticeable side effects. DLA samples were prepared and processed with different analysis techniques. Processing DLA using CellSearch resulted in a 0-32 fold increase in CTC yield compared to processing 7.5 mL blood. Filtration of DLA through 5 µm pores microsieves was accompanied by large CTC losses. Leukocyte depletion of 18 mL followed by CellSearch yielded an increase of the number of CTC but a relative decrease in yield (37%) versus CellSearch DLA. In four out of seven patients with 0 CTC detected in 7.5 mL of blood, CTC were detected in DLA (range 1-4 CTC). The CTC obtained through DLA enables molecular characterization of the tumor. CTC enrichment technologies however still need to be improved to isolate all the CTC present in the DLA.

Diagnostic leukapheresis for CTC analysis in breast cancer patients: CTC frequency, clinical experiences and recommendations for standardized reporting.

Diagnostic leukapheresis (DLA) is based on continuous centrifugation that collects mononuclear cells from peripheral blood with a density of 1.055-1.08 g/ml. As epithelial cells have a similar density, DLA cocollects circulating tumor cell (CTCs) along with the targeted mononuclear cells. Here, we report on our single center experience applying DLA in 40 nonmetastatic and metastatic breast cancer patients and its impact on CTC detection. We found that the use of just 5% of the DLA product (corresponding to a median peripheral blood volume of around 60 ml) in the CellSearch® assay already leads to a significant increase in CTC detection frequency and yield. The implementation of the method was unproblematic, and we did not observe any adverse events in our patient cohort. Extrapolating the CTC counts in the DLA samples to the whole DLA product indicated that enormous CTC numbers could be harvested by this approach (around 205x more CTCs than in the 7.5 ml blood sample in M1 patients). In conclusion, DLA is a clinically safe method to collect CTCs from liters of blood enabling a real liquid biopsy. Yet, further technical developments are required to process whole DLA products and exploit the full potential of this approach. As it is foreseeable that DLA will be used by several groups, and hopefully ultimately brought to the patients in a routine setting, we discuss recommendations on the minimum of required information for reporting on DLAs to allow comparison across different approaches. © 2018 International Society for Advancement of Cytometry.

Minimal Residual Disease in Head and Neck Cancer and Esophageal Cancer.

Malignant epithelial tumors of the upper digestive tract are a major cause of cancer-related death worldwide. The most common of these cancers are head and neck squamous cell carcinomas (HNSCC) and esophageal cancers (EC), which are both characterized by early dissemination and poor prognosis. Although patients with early detected cancers can be subjected to multimodal therapies with curative intention, they are endangered by lethal relapses that frequently occur. These relapses originate from minimal residual cancer (MRD) cells that can only be traced by highly sensitive molecular methods as rare disseminated tumor cells (DTC). The aim of this chapter is to comprehensively inform the reader about the detection, the mode of spread, the clinical relevance, and the biology of DTCs in HNSCC and EC. A better understanding of DTCs will be key to suppress progression of the upper digestive tract cancers more effectively.

Immunohistochemical detection of lymph node-DTCs in patients with node-negative HNSCC.

This study was performed to systematically assess the prevalence, topography and prognostic impact of disseminated tumor cells (DTCs) in lymph nodes (LN) of patients with primary, regional and distant metastasis-free head and neck squamous cell carcinoma (HNSCC) who underwent resection with elective neck dissection. From the routinely processed resection specimen, we could prospectively analyze a total of 1.137 exactly mapped LNs of 50 pN0-HNSCC patients, classified as tumor free by routine histopathology. Three immunohistochemistry (IHC) assays using antibodies directed against CK5/14, a broad spectrum of CKs (1-8, 10, 14-16 and 19), and CD44v6, respectively, were applied on 4.190 LN sections to detect DTCs. The IHC results were correlated with clinicopathologic parameters and clinical follow-up data. We detected seven micrometastases (MM) in five patients and 31 DTCs in 12 patients. Overall, 15 (30%) patients were positive for DTCs or MMs. Strikingly, the anatomical distribution of LN affected with DTCs was not random, but was dependent on the lateralization of the primary tumor and clustered significantly most proximal to the primary tumor. None of the investigated patients developed loco-regional lymphatic or distant metastasis during the mean follow-up period of 71 months. Our results reveal clinically occult tumor cell dissemination as an early and frequent event in HNSCC. Considering that higher rates of recurrences in therapeutic LN dissection concepts have been reported than in elective neck dissection strategies, our DTC-data support to perform elective neck dissections, since they appear to be effective in preventing loco-regional lymphatic recurrence from LN DTCs or MMs.

Disseminated tumour cells with highly aberrant genomes are linked to poor prognosis in operable oesophageal adenocarcinoma.

BACKGROUND: Chromosomal instability (CIN) has repeatedly been identified as a prognostic marker. Here we evaluated the percentage of aberrant genome per cell (PAG) as a measure of CIN in single disseminated tumour cells (DTC) isolated from patients with operable oesophageal adenocarcinoma (EAC), to assess the impact of CINhigh DTCs on prognosis. METHODS: We isolated CK18positive DTCs from bone marrow (BM) or lymph node (LN) preparations of operable EAC patients. After whole-genome amplification, single DTCs were analysed for chromosomal gains and losses using metaphase-based comparative genomic hybridisation (mCGH). We calculated the PAG for each DTC and determined the critical threshold value that identifies high-risk patients by STEPP (Subpopulation Treatment Effect Pattern Plot) analysis in two independent EAC patient cohorts (cohort #1, n=44; cohort #2; n=29). RESULTS: The most common chromosomal alterations observed among the DTCs were typical for EAC, but the DTCs showed a varying PAG between individual patients. Generally, LNDTCs displayed a significantly higher PAG than BMDTCs. STEPP analysis revealed an increasing PAG of DTCs to be correlated with an increased risk for short survival in two independent EAC cohorts as well as in the corresponding pooled analysis. In all three data sets (cohort #1, cohort #2 and pooled cohort), PAGhigh DTCs conferred an independent risk for a significantly decreased survival. CONCLUSIONS: The analysis of PAG/CIN in solitary marker-positive DTCs identifies operable EAC patients with poor prognosis, indicating a more aggressive minimal residual disease.

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.

Peritoneal sarcomatosis: site of origin for the establishment of an in vitro and in vivo cell line model to study therapeutic resistance in dedifferentiated liposarcoma.

Approximately 50-70 % of patients with retroperitoneal or intraabdominal sarcoma develop a relapse after surgical therapy, including peritoneal sarcomatosis, an extremely rare site of metastatic disease which is associated with an extremely poor prognosis. Accordingly, the establishment of a permanent cell line derived from peritoneal sarcomatosis might provide a helpful tool to understand the biological behavior and to develop new therapeutic strategies. Thus, we established and characterized a liposarcoma cell line (Lipo-DUE1) from a peritoneal sarcomatosis that was permanently cultured without showing any morphological changes. Lipo-DUE1 cells exhibited a spindle-shaped morphology and positive staining for S100. Tumorigenicity was demonstrated in vitro by invasion and migration assays and in vivo by using a subcutaneous xenograft mouse model. In addition, aCGH analysis revealed concordant copy number variations on chromosome 12q in the primary tumor, peritoneal sarcomatosis, and Lipo-DUE1 cells that are commonly observed in liposarcoma. Chemotherapeutic sensitivity assays revealed a pronounced drug-resistant phenotype of Lipo-DUE1 cells to conventionally used chemotherapeutic agents. In conclusion, we describe for the first time the establishment and characterization of a liposarcoma cell line derived from a peritoneal sarcomatosis. Hence, in the future, the newly established cell line Lipo-DUE1 might serve as a useful in vitro and in vivo model to investigate the biological behavior of liposarcoma and to assess novel targeted therapies.