CD301 and LSECtin glycan-binding receptors of innate immune cells serve as prognostic markers and potential predictors of immune response in breast cancer subtypes.

Glycosylation is a prominent posttranslational modification, and alterations in glycosylation are a hallmark of cancer. Glycan-binding receptors, primarily expressed on immune cells, play a central role in glycan recognition and immune response. Here, we used the recombinant C-type glycan-binding receptors CD301, Langerin, SRCL, LSECtin, and DC-SIGNR to recognize their ligands on tissue microarrays (TMA) of a large cohort (n = 1859) of invasive breast cancer of different histopathological types to systematically determine the relevance of altered glycosylation in breast cancer. Staining frequencies of cancer cells were quantified in an unbiased manner by a computer-based algorithm. CD301 showed the highest overall staining frequency (40%), followed by LSECtin (16%), Langerin (4%) and DC-SIGNR (0.5%). By Kaplan-Meier analyses, we identified LSECtin and CD301 as prognostic markers in different breast cancer subtypes. Positivity for LSECtin was associated with inferior disease-free survival in all cases, particularly in estrogen receptor positive (ER+) breast cancer of higher histological grade. In triple negative breast cancer, positivity for CD301 correlated with a worse prognosis. Based on public RNA single-cell sequencing data of human breast cancer infiltrating immune cells, we found CLEC10A (CD301) and CLEC4G (LSECtin) exclusively expressed in distinct subpopulations, particularly in dendritic cells and macrophages, indicating that specific changes in glycosylation may play a significant role in breast cancer immune response and progression.

Systematic analysis of the human tumor cell binding to human vs. murine E- and P-selectin under static vs. dynamic conditions.

Endothelial E- and P-selectins promote metastasis formation by interacting with sialyl-Lewis X and A (sLeX/sLeA) on circulating tumor cells. This interaction precedes extravasation and can take place under dynamic and static conditions. Metastasis formation is often studied in xenograft models. However, it is unclear whether species differences exist in the ligand specificity of human (h) vs. murine (m) selectins and whether different ligands are functional under dynamic vs. static conditions. We systematically compared the h vs. m E- and P-selectin (ESel/PSel) binding of a range of human tumor cells under dynamic vs. static conditions. The tumor cells were categorized by their sLeA/X status (sLeA+/sLeX+, sLeA-/sLeX+ and sLeA-/sLeX-). The general biological nature of the tumor-selectin interaction was analyzed by applying several tumor cell treatments (anti-sLeA/X blockade, neuraminidase, pronase and inhibition of O/N-glycosylation). We observed remarkable differences in the static vs. dynamic interaction of tumor cells with h vs. m ESel/PSel depending on their sLeA/X status. The tumor cell treatments mostly affected either static or dynamic as well as either h- or m-selectin interaction. mESel showed a higher diversity of potential ligands than hESel. Inhibition of O-GalNAc-glycosylation also affected glycosphingolipid synthesis. Summarized, different ligands on human tumor cells are functional under static vs. dynamic conditions and for the interaction with human vs. murine ESel/PSel. Non-canonical selectin ligands lacking the sLeA/X glycan epitopes exist on human tumor cells. These findings have important implications for the current development of glycomimetic, antimetastatic drugs and encourage the development of immunodeficient mice with humanized selectins.

Immature O-glycans recognized by the macrophage glycoreceptor CLEC10A (MGL) are induced by 4-hydroxy-tamoxifen, oxidative stress and DNA-damage in breast cancer cells.

BACKGROUND: Ligands of the C-type lectin CLEC10A such as Tn and sialyl-Tn representing early intermediates of O-glycosylation are hallmarks of many human malignancies. A variety of regulatory mechanisms underlying their expression are being discussed. METHODS: CLEC10A ligands were detected in various tissues and cells using the recombinant glycan-binding domain of CLEC10A. In normal breast and endometrium, presence of ligands was correlated to the female cycle. Estrogen- and stress dependent induction of CLEC10A ligands was analyzed in MCF7 and T47D cells exposed to 4-hydroxy-tamoxifen (Tam), zeocin and hydrogen peroxide. The expression and localization of CLEC10A ligands was analyzed by Western blot and immunofluorescence. In breast cancer patients CLEC10A ligand expression and survival was correlated by Kaplan-Meyer analysis. RESULT: We observed binding of CLEC10A in normal endometrial and breast tissues during the late phase of the female hormonal cycle suggesting a suppressive effect of female sex hormones on CLEC10A ligand expression. Accordingly, CLEC10A ligands were induced in MCF7- and T47D breast cancer cells after Tam treatment and accumulated on the cell surface and in the endosomal/lysosomal compartment. Phagocytosis experiments indicate that macrophages preferentially internalize CLEC10A ligands coated beads and Tam treated MCF7 cells. CLEC10A ligands were also expressed after the addition of zeocin and hydrogen-peroxide. Each substance induced the production of ROS indicating reactive oxygen species as a unifying mechanism of CLEC10A ligand induction. Mechanistically, increased expression of GalNAc-transferase 6 (GalNT6) and translocation of GalNT2 and GalNT6 from cis- towards trans-Golgi compartment was observed, while protein levels of COSMC and T-synthase remained unaffected. In breast cancer patients, positivity for CLEC10A staining in tumor tissues was associated with improved outcome and survival. CONCLUSION: CLEC10A ligands are inducible by hormone depletion, 4-hydroxy-tamoxifen and agents inducing DNA damage and oxidative stress. Our results indicate that CLEC10A acts as a receptor for damaged and dead cells and may play an important role in the uptake of cell debris by macrophages and dendritic cells.

CEACAM1 in Liver Injury, Metabolic and Immune Regulation.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is a transmembrane glycoprotein that is expressed on epithelial, endothelial and immune cells. CEACAM1 is a differentiation antigen involved in the maintenance of epithelial polarity that is induced during hepatocyte differentiation and liver regeneration. CEACAM1 regulates insulin sensitivity by promoting hepatic insulin clearance, and controls liver tolerance and mucosal immunity. Obese insulin-resistant humans with non-alcoholic fatty liver disease manifest loss of hepatic CEACAM1. In mice, deletion or functional inactivation of CEACAM1 impairs insulin clearance and compromises metabolic homeostasis which initiates the development of obesity and hepatic steatosis and fibrosis with other features of non-alcoholic steatohepatitis, and adipogenesis in white adipose depot. This is followed by inflammation and endothelial and cardiovascular dysfunctions. In obstructive and inflammatory liver diseases, soluble CEACAM1 is shed into human bile where it can serve as an indicator of liver disease. On immune cells, CEACAM1 acts as an immune checkpoint regulator, and deletion of Ceacam1 gene in mice causes exacerbation of inflammation and hyperactivation of myeloid cells and lymphocytes. Hence, hepatic CEACAM1 resides at the central hub of immune and metabolic homeostasis in both humans and mice. This review focuses on the regulatory role of CEACAM1 in liver and biliary tract architecture in health and disease, and on its metabolic role and function as an immune checkpoint regulator of hepatic inflammation.

Björn J. Öbrink (1943-2015).

Family members, friends and colleagues of Prof. Dr. Dr. h.c. Björn Öbrink were deeply saddened to learn of his sudden death on August 21, 2015. Björn was one of the pioneers in cell adhesion research. Reading an article written by Björn, one quickly recognizes his outstanding qualities as a scientist: knowledgeable, innovative, precise and detailed in order to enable reproduction of his reported results. When meeting Björn, you were captured by his personality: warm-hearted, modest, and, above all, devoted to truth.

COSMC knockdown mediated aberrant O-glycosylation promotes oncogenic properties in pancreatic cancer.

BACKGROUND: Human pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and lethal malignancies in the world and despite great efforts in research types of treatment remain limited. A frequently detected alteration in PDACs is a truncated O-linked N-acetylgalactosamine (GalNAc) glycosylation with expression of the Tn antigen. Changes in O-glycosylation affect posttranslationally modified O-GalNAc proteins resulting in profound cellular alterations. Tn antigen is a tumor associated glycan detected in 75-90 % of PDACs and up to 67 % in its precursor lesions. Since the role of Tn antigen expression in PDAC is insufficiently understood we analyzed the impact of COSMC mediated Tn antigen expression in two human PDAC cell lines on cellular oncogenic properties. METHODS: Forced expression of Tn antigen on O-glycosylated proteins in pancreatic cancer cells was induced by lentiviral-mediated knockdown of the COSMC chaperone, which prevented O-glycan elongation beyond the initial GalNAcα1- residue on O-linked glycoproteins. Altered O-GalNAc glycosylation was analyzed in human pancreatic cancer cell lines Panc-1 and L3.6pl using Western and Far-Western blot as well as immunocytochemical techniques. To assess the biological implications of COSMC function on oncogenic properties, cell viability assays, scratch assays combined with live cell imaging, migration and apoptosis assays were performed. Lectin based glycoprotein enrichment with subsequent mass spectrometric analysis identified new cancer O-GalNAc modified proteins. Expression of Tn antigen bearing Nucleolin in patient derived PDAC tumor specimens was evaluated and correlated with clinicopathological data. RESULTS: Tn antigen expression was induced on various O-GalNAc glycoproteins in COSMC deficient cell lines compared to the control. Proliferation was reduced (p < 0.001) in COSMC knockdown cells, whereas migration was increased (p < 0.001) and apoptosis was decreased (p = 0.03), highlighting the importance of Tn antigen expression on metastatic and anti-apoptotic behavior of PDAC derived cells. Nucleolin was identified as O-GalNAc modified protein in COSMC deficient PDAC cell lines. Interestingly, immunohistochemical staining and co-localization studies of patient derived PDACs revealed poor survival for patients with strong co-localization of Tn antigen and Nucleolin (p = 0.037). CONCLUSION: This study substantiates the influence of altered O-glycan (Tn/STn) expression on oncogenic properties in pancreatic cancer and identifies O-GalNAc modified Nucleolin as novel prognostic marker.

Carcinoembryonic antigen-related cell adhesion molecule 1 inhibits MMP-9-mediated blood-brain-barrier breakdown in a mouse model for ischemic stroke.

RATIONALE: Blood-brain-barrier (BBB) breakdown and cerebral edema result from postischemic inflammation and contribute to mortality and morbidity after ischemic stroke. A functional role for the carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) in the regulation of reperfusion injury has not yet been demonstrated. OBJECTIVE: We sought to identify and characterize the relevance of CEACAM1-expressing inflammatory cells in BBB breakdown and outcome after ischemic stroke in Ceacam1(-/-) and wild-type mice. METHODS AND RESULTS: Focal ischemia was induced by temporary occlusion of the middle cerebral artery with a microfilament. Using MRI and Evans blue permeability assays, we observed increased stroke volumes, BBB breakdown and edema formation, reduction of cerebral perfusion, and brain atrophy in Ceacam1(-/-) mice. This translated into poor performance in neurological scoring and high poststroke-associated mortality. Elevated neutrophil influx, hyperproduction, and release of neutrophil-related matrix metalloproteinase-9 in Ceacam1(-/-) mice were confirmed by immune fluorescence, flow cytometry, zymography, and stimulation of neutrophils. Importantly, neutralization of matrix metalloproteinase-9 activity in Ceacam1(-/-) mice was sufficient to alleviate stroke sizes and improve survival to the level of CEACAM1-competent animals. Immune histochemistry of murine and human poststroke autoptic brains congruently identified abundance of CEACAM1(+)matrix metalloproteinase-9(+) neutrophils in the ischemic hemispheres. CONCLUSIONS: CEACAM1 controls matrix metalloproteinase-9 secretion by neutrophils in postischemic inflammation at the BBB after stroke. We propose CEACAM1 as an important inhibitory regulator of neutrophil-mediated tissue damage and BBB breakdown in focal cerebral ischemia.

Selectin binding is essential for peritoneal carcinomatosis in a xenograft model of human pancreatic adenocarcinoma in pfp--/rag2-- mice.

BACKGROUND AND OBJECTIVE: E- and P-selectins expressed on the luminal surface of mesodermally derived endothelial cells play a crucial role in the formation of haematogenous metastases in a number of malignancies. As peritoneal mesothelial cells are also derived form the mesoderm, it was hypothesised that selectins are also of importance in peritoneal tumour spread. METHODS: Immunohistochemistry was used to identify selectin expression on normal human peritoneum and isolated mesothelial cells. E- and P-selectin interactions with human pancreatic adenocarcinoma cells were investigated in dynamic flow assays and flow cytometry; the latter was also used to determine the main selectin ligands on pancreatic adenocarcinoma cell lines PaCa 5061, BxPC-3 and PaCa 5072, and selectin expression on human mesothelial cells. All cell lines were xenografted into the peritoneum of E- and P-selectin-deficient pfp/rag2 mice and selectin wild-type controls. Peritoneal carcinomatosis was quantified using MRI or a scoring system. RESULTS: E- and P-selectin were constitutively expressed on human mesothelial and endothelial cells in the peritoneum. PaCa 5061 and BxPC-3 cells interacted with E- and P-selectins in dynamic flow assays and flow cytometry, with CA19-9 (Sialyl Lewis a) being the main E-selectin ligand. For xenografted PaCa 5061 and BxPC-3 cells, peritoneal metastasis was significantly reduced in E- and P-selectin double knockout mice compared with wild-type pfp/rag2 animals. In contrast, PaCa 5072 cells were almost devoid of selectin binding sites and no intraperitoneal tumour growth was observed. CONCLUSION: Interactions of tumour cells with peritoneal selectins play an important role in the peritoneal spread of pancreatic adenocarcinoma.

Tumor-associated Neu5Ac-Tn and Neu5Gc-Tn antigens bind to C-type lectin CLEC10A (CD301, MGL).

In human tumors, glycoproteins often exhibit abnormal glycosylation patterns, e.g. certain Lewis structures, TF antigen, Tn antigen and/or their sialylated forms, creating additional binding sites for glycoreceptors. In the present study, we have analyzed the carbohydrate specificity of the C-type lectin CLEC10A using glycan profiling by enzyme-linked immunosorbent assay (ELISA). In addition to the known ligands, we show binding to two tumor-associated antigens, namely Neu5Acα2,6-Tn and Neu5Gcα2,6-Tn, with an affinity of CLEC10A in the micromolar range. Detailed analyses of the glycan-lectin interactions were carried out by surface plasmon resonance (SPR) and saturation transfer difference (STD) NMR. CLEC10A binds Neu5Acα2,6-Tn and Neu5Gcα2,6-Tn with dissociation constants of 297 and 80 µM, respectively, as determined by SPR. Comparison of the STD nuclear magnetic resonance (NMR) binding epitopes of Tn and Neu5Acα2,6-Tn revealed a constant binding mode of the N-acetylgalactosamine moiety. This finding is in good agreement with binding studies of CLEC10A transfectomas, which show a well-defined interaction of transmembrane CLEC10A with 6-sialylated-Tn structures. Since both Neu5Acα2,6-Tn and Neu5Gcα2,6-Tn together with the previously known Tn antigen are expressed in human tumors such as mammary carcinoma, the interaction with CLEC10A expressed by macrophages and dendritic cells could be of major functional significance in tumor progression.

Transcription factors link mouse WAP-T mammary tumors with human breast cancer.

Mouse models are important tools to decipher the molecular mechanisms of mammary carcinogenesis and to mimic the respective human disease. Despite sharing common phenotypic and genetic features, the proper translation of murine models to human breast cancer remains a challenging task. In a previous study we showed that in the SV40 transgenic WAP-T mice an active Met-pathway and epithelial-mesenchymal characteristics distinguish low- and high-grade mammary carcinoma. To assign these murine tumors to corresponding human tumors we here incorporated the analysis of expression of transcription factor (TF) coding genes and show that thereby a more accurate interspecies translation can be achieved. We describe a novel cross-species translation procedure and demonstrate that expression of unsupervised selected TFs, such as ELF5, HOXA5 and TFCP2L1, can clearly distinguish between the human molecular breast cancer subtypes--or as, for example, expression of TFAP2B between yet unclassified subgroups. By integrating different levels of information like histology, gene set enrichment, expression of differentiation markers and TFs we conclude that tumors in WAP-T mice exhibit similarities to both, human basal-like and non-basal-like subtypes. We furthermore suggest that the low- and high-grade WAP-T tumor phenotypes might arise from distinct cells of tumor origin. Our results underscore the importance of TFs as common cross-species denominators in the regulatory networks underlying mammary carcinogenesis.

Low-grade and high-grade mammary carcinomas in WAP-T transgenic mice are independent entities distinguished by Met expression.

Mammary carcinomas developing in SV40 transgenic WAP-T mice arise in two distinct histological phenotypes: as differentiated low-grade and undifferentiated high-grade tumors. We integrated different types of information such as histological grading, analysis of aCGH-based gene copy number and gene expression profiling to provide a comprehensive molecular description of mammary tumors in WAP-T mice. Applying a novel procedure for the correlation of gene copy number with gene expression on a global scale, we observed in tumor samples a global coherence between genotype and transcription. This coherence can be interpreted as a matched transcriptional regulation inherited from the cells of tumor origin and determined by the activity of cancer driver genes. Despite common recurrent genomic aberrations, e.g. gain of chr. 15 in most WAP-T tumors, loss of chr. 19 frequently occurs only in low-grade tumors. These tumors show features of "basal-like" epithelial differentiation, particularly expression of keratin 14. The high-grade tumors are clearly separated from the low-grade tumors by strong expression of the Met gene and by coexpression of epithelial (e.g. keratin 18) and mesenchymal (e.g. vimentin) markers. In high-grade tumors, the expression of the nonmutated Met protein is associated with Met-locus amplification and Met activity. The role of Met as a cancer driver gene is supported by the contribution of active Met signaling to motility and growth of mammary tumor-derived cells. Finally, we discuss the independent origin of low- and high-grade tumors from distinct cells of tumor origin, possibly luminal progenitors, distinguished by Met gene expression and Met signaling.

Acceleration of collateral development by carcinoembryonic antigen-related cell adhesion molecule 1 expression on CD11b/⁺Gr-1⁺ myeloid cells--brief report.

OBJECTIVE: Previously, we demonstrated the relevance for endothelial carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) expression in collateral formation. However, a proarteriogenic role for CEACAM1(+) myeloid cells is unknown. Here, we investigated the contribution of CEACAM1(+) myeloid cells on collateral formation. METHODS AND RESULTS: Collateral growth and vascular remodeling were analyzed in CEACAM1-competent and CEACAM1 null mice after femoral artery ligation in hindlimb ischemia. Reperfusion of the adductor muscles was evaluated by Laser Doppler measurements and microcomputed tomography imaging. In CEACAM1 null mice, poor reperfusion and reduced collateral formation were observed, accompanied by reduction in arterial diameters. Using flow cytometry, we identified an increase of the muscle-resident CD11b(+)/granulocyte receptor-1+ (Gr-1+) population in CEACAM1 null mice only, pointing toward a CEACAM1-dependent functional deviation. Direct and reciprocal bone marrow transplantations between CEACAM1-competent and CEACAM1 null mice, and antibody-mediated depletion of the CD11b(+)/Gr-1(+) population, confirmed the requirement of CEACAM1 expression on the CD11b(+)/Gr-1(+) population for reestablishment of perfusion after arterial occlusion. CONCLUSIONS: CEACAM1 expression on CD11b(+)/Gr-1(+) myeloid cells is a prerequisite for adequate collateral formation.

Combining lectin affinity chromatography and immunodepletion - A novel method for the enrichment of disease-specific glycoproteins in human plasma.

Drastic enrichment of potential disease-specific glycoprotein markers in human plasma can be achieved by the combination of affinity- and immuno-depletion. In the affinity-fractionation step all glycoproteins carrying a certain glycostructure are isolated by lectin affinity chromatography, thus depleting other components. Against the respective glycoprotein fraction isolated from the plasma of healthy individuals antibodies are raised in llamas. The llama heavy chain antibodies (which are particularly stable) directed at the isolated plasma glycoprotein fraction are immobilized and the immunoaffinity column thus obtained is used to deplete the respective glycoprotein fraction of patient plasma samples. Depletion of proteins normally found in human plasma by 99.8-99.9% can be achieved, resulting in a 800-1000-fold enrichment of potential disease-specific proteins in the flow-through of the immunoaffinity column.

Determination of hemolysis thresholds by the use of data loggers in pneumatic tube systems.

BACKGROUND: Pneumatic tube systems (PTSs) for the transport of blood samples are regaining popularity in medical centers after earlier reports that their use could introduce preanalytical distortions such as hemolysis and changes in blood gases. METHODS: We drew duplicate blood samples from 30 volunteers. One sample was hand transported, and the other sample was transported through a PTS together with a mini-data logger that provided continuous measurements of temperature, humidity, pressure, and acceleration. After transport the samples were analyzed at the same time. We looked for possible relationships of the transport method and the parameters measured by the data loggers with differences in hematological parameters, standard clinical chemistry analyses, blood coagulation, erythrocyte sedimentation rate, and blood gas analysis. RESULTS: There were no significant differences in temperature, humidity, and pressure between the methods of transport, but we observed significant differences in 3-axis accelerations. The combined effect of these forces could be described by the right-tailed area under the vector sum acceleration distribution. Our data show that this area correlated with PTS speed and that PTS speed and the area under the curve exhibited a direct relation to the degree of hemolysis. CONCLUSIONS: Assessment of 3-axis acceleration by use of data loggers can be used to identify preanalytical deviations that result from the transportation of blood samples in PTSs. Our approach could be used for the evaluation and regular control of PTSs without the need for repeated blood drawing and laboratory analyses.

CEACAM1+ myeloid cells control angiogenesis in inflammation.

Local inflammation during cutaneous leishmaniasis is accompanied by accumulation of CD11b(+) cells at the site of the infection. A functional role for these monocytic cells in local angiogenesis in leishmaniasis has not been described so far. Here, we show that CD11b(+) cells express high levels of the myeloid differentiation antigen carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1). In experimental cutaneous leishmaniasis in C57BL/6 wild-type (B6.WT) and B6.Ceacam1(-/-) mice, we found that only B6.Ceacam1(-/-) mice develop edemas and exhibit impairment of both hemangiogenesis and lymphangiogenesis. Because CEACAM1 expression correlates with functional angiogenesis, we further analyzed the role of the CD11b(+) population. In B6.Ceacam1(-/-) mice, we found systemic reduction of Ly-6C(high)/CD11b(high) monocyte precursors. To investigate whether CEACAM1(+) myeloid cells are causally related to efficient angiogenesis, we used reverse bone marrow transplants (BMTs) to restore CEACAM1(+) or CEACAM1(-) bone marrow in B6.Ceacam1(-/-) or B6.WT recipients, respectively. We found that angiogenesis was restored by CEACAM1(+) BMT only. In addition, we observed reduced morphogenic potential of inflammatory cells in Matrigel implants in CEACAM1(-) backgrounds or after systemic depletion of CD11b(high) macrophages. Taken together, we show for the first time that CEACAM1(+) myeloid cells are crucial for angiogenesis in inflammation.

Tandem affinity depletion: a combination of affinity fractionation and immunoaffinity depletion allows the detection of low-abundance components in the complex proteomes of body fluids.

Protein biomarker discovery in the low concentration range of human body fluids requires the enrichment of the proteins of interest. Here we report on a tandem affinity strategy: In the first step, we isolated a human plasma glyco-subproteome of healthy individuals by wheat germ agglutinin (WGA) lectin affinity chromatography. In the second step, the proteins of this subproteome were used to raise antibodies in llama (Lama glama). The heavy-chain fraction of the llama antibodies was used to deplete from the WGA lectin binding fraction all proteins normally found in human plasma. In this way, we selectively enriched the glycoprotein, CEA, a known cancer marker which had been spiked into normal plasma. As a proof of concept, we applied this method to the analysis of plasma sample from colon cancer patients. We could demonstrate the selective enrichment of CEA by a factor of 600-800.

Carcinoembryonic antigen-related cell adhesion molecule 1 modulates vascular remodeling in vitro and in vivo.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), a cellular adhesion molecule of the Ig superfamily, is associated with early stages of angiogenesis. In vitro, CEACAM1 regulates proliferation, migration, and differentiation of murine endothelial cells. To prove that CEACAM1 is functionally involved in the regulation of vascular remodeling in vivo, we analyzed 2 different genetic models: in Ceacam1-/- mice, the Ceacam1 gene was deleted systemically, and in CEACAM1(endo+) mice, CEACAM1 was overexpressed under the control of the endothelial cell-specific promoter of the Tie2 receptor tyrosine kinase. In Matrigel plug assays, Ceacam1-/- mice failed to establish new capillaries whereas in CEACAM1(endo+) mice the implants were vascularized extensively. After induction of hind limb ischemia by femoral artery ligation, Ceacam1-/- mice showed significantly reduced growth of arterioles and collateral blood flow compared with their WT littermates. In agreement with a causal role of CEACAM1 in vascular remodeling, CEACAM1(endo+) mice exhibited an increase in revascularization and collateral blood flow after arterial occlusion. Our findings indicate that CEACAM1 expression is important for the establishment of newly formed vessels in vivo. Hence CEACAM1 could be a future target for therapeutic manipulation of angiogenesis in disease.

Approaching clinical proteomics: current state and future fields of application in cellular proteomics.

Recent developments in proteomics technology offer new opportunities for clinical applications in hospital or specialized laboratories including the identification of novel biomarkers, monitoring of disease, detecting adverse effects of drugs, and environmental hazards. Advanced spectrometry technologies and the development of new protein array formats have brought these analyses to a standard, which now has the potential to be used in clinical diagnostics. Besides standardization of methodologies and distribution of proteomic data into public databases, the nature of the human body fluid proteome with its high dynamic range in protein concentrations, its quantitation problems, and its extreme complexity present enormous challenges. Molecular cell biology (cytomics) with its link to proteomics is a new fast moving scientific field, which addresses functional cell analysis and bioinformatic approaches to search for novel cellular proteomic biomarkers or their release products into body fluids that provide better insight into the enormous biocomplexity of disease processes and are suitable for patient stratification, therapeutic monitoring, and prediction of prognosis. Experience from studies of in vitro diagnostics and especially in clinical chemistry showed that the majority of errors occurs in the preanalytical phase and the setup of the diagnostic strategy. This is also true for clinical proteomics where similar preanalytical variables such as inter- and intra-assay variability due to biological variations or proteolytical activities in the sample will most likely also influence the results of proteomics studies. However, before complex proteomic analysis can be introduced at a broader level into the clinic, standardization of the preanalytical phase including patient preparation, sample collection, sample preparation, sample storage, measurement, and data analysis is another issue which has to be improved. In this report, we discuss the recent advances and applications that fulfill the criteria for clinical proteomics with the focus on cellular proteomics (cytoproteomics) as related to preanalytical and analytical standardization and to quality control measures required for effective implementation of these technologies and analytes into routine laboratory testing to generate novel actionable health information. It will then be crucial to design and carry out clinical studies that can eventually identify novel clinical diagnostic strategies based on these techniques and validate their impact on clinical decision making.

Expression of CEACAM-1 in pulmonary adenocarcinomas and their metastases.

BACKGROUND: CEACAM-1 is involved in intercellular adhesion and is expressed in a variety of human tissues. In cases of malignant transformation, a down-regulation or loss of CEACAM-1 has been shown. In contrast, CEACAM-1 is not expressed in normal lung tissue or melanocytes. It has been demonstrated that an expression in these tissues is associated with the development of metastatic disease. The aim of the present investigation was to analyze a possible association between the expression of CEACAM-1 in pulmonary adenocarcinomas and their lymph node and hematogenous metastatic cells. PATIENTS AND METHODS: CEACAM-1 expression was immunhistochemically evaluated in primary tumors, lymph nodes and distant metastases of 96 patients with metastatic pulmonary adenocarcinoma who had undergone surgery between 1999 and 2002. RESULTS: Expression of CEACAM-1 was shown in 78 out of 96 primary tumors (81.3%). A significant positive correlation was found between CEACAM-1 expression on cells of the primary tumor, lymph node metastases (p < 0.005) and hematogenous metastases (p = 0.03). CEACAM-1 expression did not correlate with stage, gender, grading or patients' age. Compared to patients with tumors not expressing CEACAM-1, patients with a CEACAM-1-expressing tumor had a shorter median overall survival (21 vs. 28 months) and progression-free survival (11.7 vs. 16.3 months). CONCLUSION: CEACAM-1 is expressed in most primary pulmonary adenocarcinomas. This investigation demonstrates that its expression is preserved in lymph node and hematogenous metastases, indicating that its expression is of functional significance for both metastatic sites. These results support the prognostic relevance of the expression of CEACAM-1 in pulmonary adenocarcinoma.

Mutual Regulation of TLR/NLR and CEACAM1 in the Intestinal Microvasculature: Implications for IBD Pathogenesis and Therapy.

Background: Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) displays multiple activities, among which pathogen binding and angiogenesis are particularly prominent. These same functions are also exerted by Toll- and NOD-like receptors (TLRs and NLRs), which are critical mediators of innate immune responses. We investigated whether a functional inter-relationship exists between CEACAM1 and TLRs and NLRs and its potential impact on induction of intestinal angiogenesis. Methods: This hypothesis was tested using human intestinal microvascular endothelial cells, a unique cell population exposed to microbial products under physiological and pathological conditions. Results: The results show that activation of TLR2/4, TLR4, NOD1, and NOD2 by specific bacterial ligands selectively and differentially upregulates the levels of cellular and soluble CEACAM1 produced by intestinal microvascular endothelial cells. The results also show that CEACAM1 regulates the migration, transmigration, and tube formation of these endothelial cells and mediates vessel sprouting induced by specific TLR and NLR bacterial ligands. Combined, these results demonstrate a close and reciprocal regulatory interaction between CEACAM1 and bacterial products in mediating multiple functions essential to new vessel formation in the gut mucosa. Conclusions: A coordinated and reciprocal interaction of CEACAM1 and microbiota-derived factors is necessary to optimize angiogenesis in the gut mucosa. This suggests that a coordination of endogenous and exogenous innate immune responses is necessary to promote intestinal angiogenesis under physiological and inflammatory conditions such as inflammatory bowel disease.