All cells were kept at low passages and were not further authenticated. metastasis. These findings provide evidence that endothelial E-selectin is a novel factor contributing to endothelial retraction required for efficient lung metastasis. Introduction Hematogenous metastasis is a multistep process in which diverse interactions between tumor cells and their microenvironment allow the malignant cells to cross physical boundaries, disseminate and colonize distant organs. Specifically, Amyloid b-Peptide (10-20) (human) cell-cell interactions between tumor cells and blood constituents, such as platelets, leukocytes and endothelial cells, are initially mediated by selectins at different steps of the metastatic cascade (1C3). Selectins are vascular cell adhesion receptors which are responsible for initial rolling and attachment of leukocytes to the endothelium; enabling leukocyte homeostasis (2, 4). Selectins bind to sialylated and fucosylated lactosamine terminal glycan structures displayed on leukocytes, platelets, and endothelium, or tumor cells. It is accepted that malignant transformation is associated with altered carbohydrate structure presentation on tumor cells, which are potential ligands for selectins, and correlates with poor prognosis due to metastasis (5, 6). E-selectin is the major leukocyte adhesion receptor that is present only on Amyloid b-Peptide (10-20) (human) endothelial cells upon endothelial activation and requires expression. E-selectin has been investigated as the primary receptor mediating tumor cell metastasis through facilitating adhesion of tumor cells on the endothelium shown (1, 7, 8). In addition, E-selectin upregulation was observed during metastatic liver colonization (9, 10). E-selectin was detected in the tumor cell microenvironment Amyloid b-Peptide (10-20) (human) Amyloid b-Peptide (10-20) (human) several hours after their arrest, indicating an inflammatory-like endothelial activation (11C13). E-selectin expression in the pre-metastatic Mouse monoclonal antibody to Pyruvate Dehydrogenase. The pyruvate dehydrogenase (PDH) complex is a nuclear-encoded mitochondrial multienzymecomplex that catalyzes the overall conversion of pyruvate to acetyl-CoA and CO(2), andprovides the primary link between glycolysis and the tricarboxylic acid (TCA) cycle. The PDHcomplex is composed of multiple copies of three enzymatic components: pyruvatedehydrogenase (E1), dihydrolipoamide acetyltransferase (E2) and lipoamide dehydrogenase(E3). The E1 enzyme is a heterotetramer of two alpha and two beta subunits. This gene encodesthe E1 alpha 1 subunit containing the E1 active site, and plays a key role in the function of thePDH complex. Mutations in this gene are associated with pyruvate dehydrogenase E1-alphadeficiency and X-linked Leigh syndrome. Alternatively spliced transcript variants encodingdifferent isoforms have been found for this gene lungs correlated with increased tumor cell homing to these tissues and with enhanced recruitment of myeloid cells (14). Although there is accumulating evidence that selectins contribute to the metastatic microenvironment (12, 15), the mechanism of E-selectin contribution to cancer progression requires further studies. The endothelium in blood vessels controls the extravasation of cells, e.g. leukocytes, and the egress of soluble factors from the plasma. Most Amyloid b-Peptide (10-20) (human) leukocytes extravasate from circulation through the paracellular route by opening endothelial junctions at sites of inflammation (16, 17). The maintenance of the vascular barrier function is dependent on the stability of endothelial adherence junctions mediated by vascular endothelial cadherin; VE-cadherin (18). Phosphorylation of Tyr residues of VE-cadherin regulates vascular permeability and the capacity of leukocytes to transmigrate through the endothelium (19, 20). Breast cancer cells were shown to induce disruption of endothelial adherence junctions by inducing phosphorylation of VE-cadherin (21). Yet, tumor cell extravasation is significantly promoted by myeloid cells recruited to the metastatic sites through chemokines such as CCL2 and CCL5 (11, 15, 22C26), albeit the cellular and molecular mechanism of this process remains unclear. Since E-selectin expression upon tumor cell injection has been frequently observed, the involvement of E-selectin in metastasis could be anticipated. The present study describes the mechanism of E-selectin dependent recruitment and activation of monocytes, which drives the dissociation of VE-cadherin junctions and thereby promotes tumor cell extravasation required for metastasis. Material and Methods Cell culture Mouse colon carcinoma cell line, MC-38 was originally provided by Dr. J. Schlom, NIH Bethesda. MC-38 cells stably expressing GFP (MC-38GFP) were characterized as described (27). B16-BL6 melanoma cells provided by Dr. I. Vlodavsky, Technion Haifa Israel, were grown in DMEM/10% FCS as described (24). Lewis lung carcinoma cells (3LL) were grown in RPMI/10% FCS (12). All cells were kept at low passages and were not further authenticated. Lewis lung carcinoma-LLC1 cells (ATCC) and grown in DMEM/10% FCS. Endothelial cells bEnd.3 (ATCC) were grown in DMEM/10%FCS. Mice Animal experiments were performed according to the guidelines of the Swiss Animal Protection Law, and approved by Veterinary Office of Kanton Zurich. C57BL/6, Ccl2 deficient (double deficient mice bone marrow (10×106) with or without MC-38GFP tumor cells (5×106) in a T75 flask were co-cultured.
