Christina S. Alves

Carcinoembryonic Antigen and CD44 Variant Isoforms Cooperate to Mediate Colon Carcinoma Cell Adhesion to E- and L-selectin in Shear Flow

Selectin-mediated adhesion of tumor cells to platelets, leukocytes, and endothelial cells may regulate their hematogenous dissemination in the microvasculature. We recently identified CD44 variant isoforms (CD44v) as functional P-, but not E- or L-, selectin ligands on colon carcinoma cells. Moreover, an ∼180-kDa sialofucosylated glycoprotein(s) mediated selectin binding in CD44-knockdown cells. Using immunoaffinity chromatography and tandem mass spectrometry, we identify this glycoprotein as the carcinoembryonic antigen (CEA). Blot rolling assays and flow-based adhesion assays using microbeads coated with CEA immunopurified from LS174T colon carcinoma cells and selectins as substrate reveal that CEA possesses E- and L-, but not P-, selectin ligand activity. CEA on CD44-knockdown LS174T cells exhibits higher HECA-452 immunoreactivity than CEA on wild-type cells, suggesting that CEA functions as an alternative acceptor for selectin-binding glycans. The enhanced expression of HECA-452 reactive epitopes on CEA from CD44-knockdown cells correlates with the increased CEA avidity for E- but not L-selectin. Through the generation of stable knockdown cell lines, we demonstrate that CEA serves as an auxiliary L-selectin ligand, which stabilizes L-selectin-dependent cell rolling against fluid shear. Moreover, CEA and CD44v cooperate to mediate colon carcinoma cell adhesion to E- and L-selectin at elevated shear stresses. The novel finding that CEA is an E- and L-selectin ligand may explain the enhanced metastatic potential associated with tumor cell CEA overexpression and the supportive role of selectins in metastasis.

The dual role of CD44 as a functional P-selectin ligand and fibrin receptor in colon carcinoma cell adhesion

Selectins and fibrin(ogen) play key roles in the hematogenous dissemination of tumor cells, and especially of colon carcinomas. However, the fibrin(ogen) receptor(s) on colon carcinoma cells has yet to be defined along with its relative capacity to bind fibrinogen versus fibrin under flow. Moreover, the functional P-selectin ligand has yet to be validated using intact platelets rather than purified selectin substrates. Using human CD44-knockdown and control LS174T cells, we demonstrate the pivotal involvement of CD44 in the P-selectin-mediated binding to platelets in shear flow. Quantitative comparisons of the binding kinetics of LS174T versus P-selectin glycoprotein ligand-1 (PSGL-1)-expressing THP-1 cells to activated platelets reveal that the relative avidity of P-selectin-CD44 binding is more than sevenfold lower than that of P-selectin-PSGL-1 interaction. Using CD44-knockdown LS174T cells and microspheres coated with CD44 immunoprecipitated from control LS174T cells, and purified fibrin(ogen) as substrate, we provide the first direct evidence that CD44 also acts as the major fibrin, but not fibrinogen, receptor on LS174T colon carcinoma cells. Interestingly, binding of plasma fibrin to CD44 on the colon carcinoma cell surface interferes with the P-selectin-CD44 molecular interaction and diminishes platelet-LS174T heteroaggregation in the high shear regime. Cumulatively, our data offer a novel perspective on the apparent metastatic potential associated with CD44 overexpression on colon carcinoma cells and the critical roles of P-selectin and fibrin(ogen) in metastatic spread and provide a rational basis for the design of new therapeutic strategies to impede metastasis.

Biomolecular Characterization of CD44-Fibrin(ogen) Binding DISTINCT MOLECULAR REQUIREMENTS MEDIATE BINDING OF STANDARD AND VARIANT ISOFORMS OF CD44 TO IMMOBILIZED FIBRIN(OGEN)

CD44 and fibrin(ogen) play critical roles in the hematogenous dissemination of tumor cells, including colon carcinomas. We recently reported that CD44 is the primary fibrin, but not fibrinogen, receptor on LS174T colon carcinomas. However, the biochemical nature of this interaction and the roles of CD44 standard (CD44s) versus CD44 variant (CD44v) isoforms in fibrin(ogen) recognition have yet to be delineated. Microspheres, coated with CD44 immunopurified from LS174T or T84 colon carcinoma cells, which express primarily CD44v, effectively bind to immobilized fibrin, but not fibrinogen, in shear flow. In contrast, CD44s from HL-60 cells binds to both immobilized fibrin and fibrinogen under flow. Use of highly specific enzymes and metabolic inhibitors reveals that LS174T CD44 binding to fibrin is dependent on O-glycosylation of CD44, whereas CD44s-fibrin(ogen) interaction has an absolute requirement for N-, but not O-, linked glycans. The presence of chondroitin and dermatan sulfate on CD44 standard and variant isoforms facilitates fibrin recognition. Use of the anti-CD44 function-blocking monoclonal antibody Hermes-1 nearly abolishes binding of LS174T CD44 to fibrin, although it has no effect on CD44s-fibrin(ogen) interaction. The CD44-binding site is localized within the N-terminal portion of the fibrin β chains, including amino acid residues (β15-66). Surface plasmon resonance experiments revealed high affinity binding of immobilized CD44 with solubilized fibrin but not fibrinogen. Collectively, these data suggest that immobilization of fibrinogen exposes a cryptic site that mediates binding to CD44s but not CD44v. Our findings may provide a rational basis for designing novel therapeutic strategies to combat metastasis.

Single-molecule binding of CD44 to fibrin versus P-selectin predicts their distinct shear-dependent interactions in cancer

P-selectin and fibrin(ogen) have pivotal roles in the hematogenous dissemination of tumor cells. CD44 variant isoforms, CD44v, have been identified as the major functional P-selectin ligands and fibrin receptors on metastatic colon carcinoma cells. The molecular recognition of CD44v by fibrin mediates firm adhesion at low shear, whereas CD44v–P-selectin binding supports transient rolling interactions at elevated shear stresses and low site densities of P-selectin. We used single-molecule force spectroscopy to provide a molecular interpretation for these two distinct adhesion events. The CD44v–P-selectin bond has a longer unstressed equilibrium lifetime, a lower reactive compliance and a higher tensile strength relative to the CD44v–fibrin bond. These intrinsic differences confer the ability to the CD44v–P-selectin pair to mediate binding at higher shear stresses. Increasing the duration of receptor–ligand contact (2–200 milliseconds) did not affect the micromechanical properties of the CD44v–P-selectin bond, but it increased the tensile strength and the depth of the free energy barrier of the CD44v–fibrin bond and decreased its reactive compliance. This bond strengthening at longer interaction times might explain why CD44v binding to immobilized fibrin occurs at low shear. Single-molecule characterization of receptor–ligand binding can predict the shear-dependent adhesive interactions between cells and substrates observed both in vitro and in vivo.

Multifunctional Surfaces with Discrete Functionalized Regions for Biological Applications

In this paper we describe a method for creating multifunctional glass surfaces presenting discrete patches of different proteins on an inert PEG-functionalized background. Microcontact printing is used to stamp the substrate with octadecyltrichlorosilane to define the active regions. The substrate is then back-filled with PEG-silane {[[2-methoxypoly(ethyleneoxy)]propyl]trimethoxysilane} to define passive regions. A microfluidics device is subsequently affixed to the substrate to deliver proteins to the active regions, with as many channels as there are proteins to be patterned. Examples of trifunctional surfaces are given which present three terminating functional groups, i.e., protein 1, protein 2, and PEG. These surfaces should be broadly useful in biological studies, as patch size is well established to influence cell viability, growth, and differentiation. Three examples of cellular interactions with the surfaces are demonstrated, including the capture of cells from a single cell suspension, the selective sorting of cells from a mixed suspension, and the adhesion of cells to ligand micropatches at critical shear stresses. Within these examples, we demonstrate that the patterned immobilized proteins are active, as they retain their ability to interact with either antibodies in solution or receptors presented by cells. When appropriate (e.g., for E-selectin), proteins are patterned in their physiological orientations using a sandwich immobilization technique, which is readily accommodated within our method. The protein surface densities are highly reproducible in the patches, as supported by fluorescence intensity measurements. Potential applications include biosensors based on the interaction of cells or of marker proteins with protein patches, fundamental studies of cell adhesion as a function of patch size and shear stress, and studies of cell differentiation as a function of surface cues.

Distinct Kinetic and Molecular Requirements Govern CD44 Binding to Hyaluronan versus Fibrin(ogen)

CD44 is a multifunctional glycoprotein that binds to hyaluronan and fibrin(ogen). Alternative splicing is responsible for the generation of numerous different isoforms, the smallest of which is CD44s. Insertion of variant exons into the extracellular membrane proximal region generates the variant isoforms (CD44v). Here, we used force spectroscopy to delineate the biophysical and molecular requirements of CD44-HA and CD44-fibrin(ogen) interactions at the single-molecule level. CD44v-HA and CD44s-HA single bonds exhibit similar kinetic and micromechanical properties because the HA-binding motif on CD44 is common to all of the isoforms. Although this is the primary binding site, O- and N-linked glycans and sulfation also contribute to the tensile strength of the CD44-HA bond. The CD44s-fibrin pair has a lower unstressed dissociation rate and a higher tensile strength than CD44s-fibrinogen but is weaker than the CD44-HA bond. In contrast to CD44-HA binding, the molecular interaction between CD44 and fibrin(ogen) is predominantly mediated by the chondroitin sulfate and dermatan sulfate on CD44. Blocking sulfation on CD44s modestly decreases the tensile strength of CD44s-fibrin(ogen) binding, which is in stark contrast to CD44v-fibrin interaction. Collectively, the results obtained by force spectroscopy in conjunction with biochemical interventions enable us to delineate the biophysical parameters and molecular constituents of CD44 binding to hyaluronan and fibrin(ogen).

PDGF Suppresses the Sulfation of CD44v and Potentiates CD44v-Mediated Binding of Colon Carcinoma Cells to Fibrin under Flow

Fibrin(ogen) mediates sustained tumor cell adhesion and survival in the pulmonary vasculature, thereby facilitating the metastatic dissemination of tumor cells. CD44 is the major functional fibrin receptor on colon carcinoma cells. Growth factors, such as platelet-derived growth factor (PDGF), induce post-translational protein modifications, which modulate ligand binding activity. In view of the roles of PDGF, fibrin(ogen) and CD44 in cancer metastasis, we aimed to delineate the effect of PDGF on CD44-fibrin recognition. By immunoprecipitating CD44 from PDGF-treated and untreated LS174T colon carcinoma cells, which express primarily CD44v, we demonstrate that PDGF enhances the adhesion of CD44v-coated beads to immobilized fibrin. Enzymatic inhibition studies coupled with flow-based adhesion assays and autoradiography reveal that PDGF augments the binding of CD44v to fibrin by significantly attenuating the extent of CD44 sulfation primarily on chondroitin and dermatan sulfate chains. Surface plasmon resonance assays confirm that PDGF enhances the affinity of CD44v-fibrin binding by markedly reducing its dissociation rate while modestly increasing the association rate. PDGF mildly reduces the affinity of CD44v-hyaluronan binding without affecting selectin-CD44v recognition. The latter is attributed to the fact that CD44v binds to selectins via sialofucosylated O-linked residues independent of heparan, dermatan and chondroitin sulfates. Interestingly, PDGF moderately reduces the sulfation of CD44s and CD44s-fibrin recognition. Collectively, these data offer a novel perspective into the mechanism by which PGDF regulates CD44-dependent binding of metastatic colon carcinoma cells to fibrin(ogen).