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Leukocyte retention in lung capillaries is observed in normal physiology and following a bacterial infection. It has been hypothesized that cells either become mechanically trapped or adhere to capillary endothelial cells via adhesion molecules. We propose that retention involves both mechanical and adhesive forces and that the biochemical adhesive force is modulated by mechanical forces that alter the area of contact between leukocytes and endothelium.To probe this hypothesis, an adhesion assay has been developed in which individual HL-60 cells were aspirated into micropipettes pre-coated with P-selectin. Following aspiration, cells were exposed to physiological pressure differences.Little adhesion was seen in micropipettes coated with BSA, whereas significant adhesion was observed in micropipettes coated with P-selectin. The frequency of cell arrest on P-selectin in the micropipette was much greater than on P-selectin in a parallel plate flow chamber even though the disruptive force in the micropipette assay exceeds that in the parallel plate flow chamber. These results demonstrate that receptor-ligand interactions can enhance adhesion in a capillary geometry and that differences in capillary geometry vs. venule geometry can significantly influence the adhesive phenotype.Taken together, these observations support the hypothesis that an interplay between mechanical and biochemical adhesive forces can play a major role in retention.

In vitro studies were performed to characterize the relative performance of candidate receptors to target microparticles to inflammatory markers on vascular endothelium. To model the interactions of drug-bearing microparticles or imaging contrast agents with the vasculature, 6 micron polystyrene particles bearing antibodies, peptides, or carbohydrates were perfused over immobilized E- or P-selectin in a flow chamber. Microparticles conjugated with HuEP5C7.g2 (HuEP), a monoclonal antibody (mAb) specific to E- and P-selectin, supported leukocyte-like rolling and transient adhesion at venular shear rates. In contrast, microparticles conjugated with a higher affinity mAb specific for P-selectin (G1) were unable to form bonds at venular flow rates. When both HuEP and G1 were conjugated to the microparticle, HuEP supported binding to P-selectin in flow which allowed G1 to form bonds leading to stable adhesion. While the microparticle attachment and rolling performance was not as stable as that mediated by the natural ligands P-selectin Glycoprotein Ligand-1 or sialyl Lewis(x), HuEP performed significantly better than any previously characterized mAb in terms of mediating microparticle binding under flow conditions. HuEP may be a viable alternative to natural ligands to selectins for targeting particles to inflamed endothelium.

Several mechanisms have been proposed to explain the arrest of leukocytes in capillaries. It has been hypothesized that cells become mechanically trapped in these small vessels or, alternatively, that leukocytes adhere to capillary endothelial cells via endothelial cell adhesion molecules (ECAMs) in a manner similar to that in venules. Recent work using glass microcapillary tubes coated with adhesion molecules supports the proposition that leukocyte arrest in capillaries involves both mechanical and adhesive forces and that the biochemical adhesive force is strongly modulated by mechanical forces that alter the area of contact between leukocytes and the endothelium. These in vitro microcapillary flow chambers allow the effects of vessel geometry, cell deformation, and ECAM chemistry to be studied in a well-controlled manner. The work promises to provide important insights into the process of leukocyte retention in capillaries in the same way that parallel-plate flow chamber assays helped to make possible a detailed understanding of leukocyte adhesion in the venular circulation.

Little is known about the time course and magnitude of the up-regulation of endothelial cell adhesion molecules (ECAMs) in irradiated brain vasculature and the mechanisms by which dexamethasone modulates this up-regulation. We used antibody-conjugated microspheres and a rat closed cranial window model to determine the time course of functional up-regulation of radiation (20 Gy)-induced ICAM1, E-selectin and P-selectin in the pial vasculature of the rat brain and to determine the relationship between suppression of inflammation by dexamethasone and the expression of these ECAMs. The results indicate that ICAM1, E-selectin and P-selectin were up-regulated to a functional level in the microvasculature with distinct time-course patterns. The number of adherent anti-E-selectin and anti-P-selectin microspheres was 5- 12 times greater than that of IgG microspheres 3-6 h postirradiation, and their expression returned to normal at 48 h. The number of adherent anti-ICAM1 microspheres was five and nine times greater than that of IgG at 24 and 48 h, respectively, and returned to baseline by 7 days. Dexamethasone significantly reduced the number of adhering leukocytes and the number of adhering anti-ICAM1, anti-E-selectin and anti-P-selectin microspheres to background levels. Our findings partially identify a key sequence in radiation-induced inflammatory response and provide a potential means to limit radiation-induced inflammatory responses and their potential side effects in the brain.

We exploited leukocyte–endothelial cell adhesion chemistry to generate biodegradable particles that exhibit highly selective accumulation on inflamed endothelium in vitro and in vivo . Leukocyte–endothelial cell adhesive particles exhibit up to 15-fold higher adhesion to inflamed endothelium, relative to noninflamed endothelium, under in vitro flow conditions similar to that present in blood vessels, a 6-fold higher adhesion to cytokine inflamed endothelium relative to non-cytokine-treated endothelium in vivo , and a 10-fold enhancement in adhesion to trauma-induced inflamed endothelium in vivo due to the addition of a targeting ligand. The leukocyte–inspired particles have adhesion efficiencies similar to that of leukocytes and were shown to target each of the major inducible endothelial cell adhesion molecules (E-selectin, P-selectin, vascular cell adhesion molecule 1, and intercellular adhesion molecule 1) that are up-regulated at sites of pathological inflammation. The potential for targeted drug delivery to inflamed endothelium has significant implications for the improved treatment of an array of pathologies, including cardiovascular disease, arthritis, inflammatory bowel disease, and cancer.

Hyaluronan is a naturally occurring polymer that has enjoyed wide successes in biomedical and cosmetic applications as coatings, matrices, and hydrogels. For controlled delivery applications, formulating native hyaluronan into microspheres could be advantageous but has been difficult to process unless organic solvents are used or hyaluronan has been modified by etherification. Therefore, we present a novel method of preparing hyaluronan microspheres using adipic dihydrazide mediated crosslinking chemistry. To evaluate their potential for medical applications, hyaluronan microspheres are incorporated with DNA for gene delivery or conjugated with an antigen for cell-specific targeting. The results show that our method, originally developed for preparing hyaluronan hydrogels, generates robust microspheres with a size distribution of 5–20 μm. The release of the encapsulated plasmid DNA can be sustained for months and is capable of transfection in vitro and in vivo. Hyaluronan microspheres, conjugated with monoclonal antibodies to E- and P-selectin, demonstrate selective binding to cells expressing these receptors. In conclusion, we have developed a novel microsphere preparation using native hyaluronan that delivers DNA at a controlled rate and adaptable for site-specific targeting.

Leukocyte adhesion to the vascular endothelium involves a disruptive force exerted on the leukocyte by the flow of the blood and an adhesive force that forms at the leukocyte-endothelial interface. The relative strengths of these two competing forces govern leukocyte adhesion.

The upregulation of cell adhesion molecules plays an important role in inflammatory response of normal tissue following radiation treatment, and also provides a potential avenue for targeting drugs to select tissues. The detailed time course of the expression of these molecules in vivo has not been well studied. To explore the dynamic consequences of radiation induced intracellular adhesion molecule-1 (ICAM-1) and E-selectin in vivo, a rat cranial window model was established. We coated microspheres with a mAb to ICAM-1 or E-selectin, and tested the adhesion of these microspheres to brain microvasculature following a single dose of 20 Gy radiation. IgG conjugated microspheres were used as negative control. The number of adherent anti-E-selectin microspheres was 6 and 4 times that of IgG microspheres at 3 hr and 6 hr post-irradiation, respectively. The number of adherent anti-ICAM-1 microspheres in irradiated cerebral tissue was 8 and 13 times that of IgG microspheres at 24 hr and 48 hr postirradiation, respectively, and returned to baseline 7 days postirradiation. The data indicate that ICAM-1 is upregulated over a prolonged period whereas E-selectin responds in a very early stage and acts transiently.

Proteasome inhibitors (e.g. lactacystin) have been shown to decrease cytokine-induced up-regulation of endothelial cell adhesion molecules and consequent leukocyte adhesion. The majority of these studies have focused on the effects of proteasome inhibitors on endothelial cells exposed to a single cytokine (TNF alpha) for 4 hrs. However, in the in vivo setting, multiple cytokines (e.g. IL-1 beta and TNF alpha) may be present and their levels may be increased for extended periods off time. In addition, the effects of IL-1 beta (IL-1) and TNF alpha (TNF) on endothelial cells are not identical. Thus, we studied the effect of lactacystin on IL-1 and TNF induced adhesion molecule expression and myeloid cell (HL-60 cells) adhesion to human umbilical vein endothelial cells (HUVEC). We found that (a) lactacystin inhibits 4 hr. IL-1 or TNF induced expression of E-selectin, ICAM-1, VCAM-1 and HL-60 cell adhesion to HUVEC, (b) lactacystin differentially inhibits transient adhesive contacts of HL-60 cells to 4 hr. IL-1 and TNF activated HUVEC, (c) lactacystin inhibits 4 hr. TNF induced expression of VCAM-1, E-selectin and HL-60 cell adhesion to IL-1 sefractory HUVEC and (d) lactacystin inhibits HL-60 cell adhesion to 24 hr. TNF activated HUVEC.

A promising approach for reducing aberrant leukocyte-endothelial adhesion during pathological inflammation is to inhibit endothelial cell adhesion molecule (ECAM) expression at the transcription level. Several compounds have been shown to decrease cytokine-induced upregulation of ECAMs primarily by modulating the activity of transcription factors [e.g., nuclear factor-kappa B (NF-kappa B)]. The majority of the in vitro studies have focused on the effect of transcription inhibitors on endothelial cells exposed to a single cytokine [primarily tumor necrosis factor-alpha (TNF-alpha)] for a relatively short period of time (primarily 4-6 h). However, in the in vivo setting, multiple cytokines [e.g., interleukin-1 beta (IL-1 beta) and TNF-alpha] may be present for extended periods of time. Thus we studied the effects of a transcription inhibitor, the proteasome inhibitor lactacystin, on ECAM expression and myeloid (HL60) cell adhesion to human umbilical vein endothelial cells (HUVEC) activated by concurrent, sequential, and long-term (24 h) treatment with IL-1 beta and TNF-alpha. We show, for the first time, that lactacystin inhibits 1) 4-h concurrent IL-1 beta- and TNF-alpha-induced expression of E-selectin, VCAM-1, ICAM-1, and HL60 cell adhesion to HUVEC; 2) 4-h TNF-alpha-induced expression of E-selectin, VCAM-1, and HL60 cell adhesion to HUVEC that have become desensitized to IL-1 beta activation; 3) 24-h TNF-alpha-induced expression of E-selectin and VCAM-1 but not ICAM-1; and 4) 24-h TNF-alpha-induced HL60 cell adhesion to HUVEC. Combined, our results demonstrate that a proteasome inhibitor can reduce concurrent, sequential, and long-term IL-1 beta- and TNF-alpha-induced ECAM expression and myeloid cell adhesion.

The parallel plate flow chamber is the most widely used device for measuring adhesion to substrates under flow. Typically, cells or particles are perfused through the flow chamber which contains an adhesive substrate at the bottom surface. The concentration of particles at the substrate, Cs, is a function of particle size, position in the flow chamber, chamber geometry and sedimentation velocity. We developed a mathematical model, which relates Cs to these parameters. Under a variety of experimental conditions, we found that Cs predicted by the model is close to that observed experimentally. We used this model to compare the rates of attachment of 10 /spl mu/m and 5 /spl mu/m diameter PSGL-1 microspheres to P-selectin. We found that the uncorrected rates of attachment were significantly different. However, normalization of the data with the developed model revealed that the rates of attachment were similar.

We have investigated the expression of E-selectin and ICAM-1 on human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells (HMEC) at 5 hr, 24 hr, 48 hr and 72 hr post-irradiation. Cells were irradiated (IR) with a single dose of 10 Gy and expression of molecules quantified by flow cytometric analysis. Preliminary results indicate that ICAM-1 is upregulated at 48 hr. Post-IR in both cell types. We did not observe upregulation of E-selectin post-IR. Consistent with this latter finding, in vitro parallel plate flow chamber assays (shear stress of 0.5- 2.0 dynes/cm/sup 2/) revealed a lack of HL-60 rolling on HUVECs and HMECs monolayers 5 hr. post-IR. Thus, our finding suggest that, in response to radiation ICAM-1 is upregulated, but not E-selectin.

Certain endothelial cell adhesion molecules are up regulated in tissue that has been irradiated for therapeutic purposes. This up-regulation of adhesion molecules provides a potential avenue for targeting drugs to select tissues.Microspheres were coated with a mAb to ICAM-1 and the level of adhesion of the anti-ICAM-1 microspheres to irradiated tissue in vitro and in vivo was quantified.Under in vitro flow conditions, the number of adherent microspheres on irradiated HUVEC was 4.8 +/- 0.9 times that of control; the adhesion of anti-ICAM-1 microspheres on irradiated HUVEC could be enhanced by more than 170% in the presence of RBC (20% hematocrit) in the medium. In vivo in a rat cranial window model, the number of adherent anti-ICAM-1 microspheres in locally irradiated cerebral tissue was 8 and 13 times that of IgG microspheres at 24 h and 48 h post-irradiation, respectively and returned to baseline 7 days post-irradiation. In locally irradiated animals, the number of adhering microspheres in unirradiated tissue remained at the basal level.Radiation-induced up-regulation of endothelial cell adhesion molecules may be exploited to target drugs and/or genes to select segments of the endothelium.

Accumulating evidence suggests that selectin/selectin ligand interactions are involved in mediating the adhesion of bloodborne circulating tumor cells at distant sites during the later stages of cancer metastasis. Presently, immunostaining is the standard method for detecting selectin ligands expressed on tissue, but this approach cannot ascertain if the selectin ligands are functional. That is, detection of purported selectin ligands by static (no-force) immunostaining under equilibrium conditions does not necessitate that the reactive antigens are able to mediate cell adhesion with hemodynamic flow (force) under non-equilibrium conditions. The relevance of functional selectin ligands as a biomarker is presently unclear because of the lack of adequate detection methods. Recently, we developed a method to detect functional selectin ligands in situ by perfusing selectin-coated microspheres (probes) over cancer tissues in a microfluidic device. This method of tissue interrogation is termed dynamic biochemical tissue analysis (DBTA). Preliminary analysis of signet ring cell colon carcinoma tissue revealed the highest amount of specific probe adhesion relative to negative control probes occurred with a murine E-selectin probe, rather than human E-, P-, or L-selectin probes. Therefore, to investigate the relevance of functional selectin ligand expression on cancer tissue as a biomarker, 196 cases of various solid tumors at primary and metastatic sites were investigated with DBTA using murine E-selectin. Probe adhesion occurred on multiple cases of breast, colon, liver, lung, ovarian, pancreatic, prostate, and stomach cancers, demonstrating the presence of functional selectin ligands. In addition, probe adhesion occurred on multiple cases of breast, colon, liver, lung, ovarian, pancreatic, rectal, and stomach cancer tissues sampled from metastatic sites. Overall, probe adhesion levels for the tissues investigated ranged from 10 specifically adherent probes/mm2 on lung adenosquamous carcinoma to 1170 specifically adherent probes/mm2 on ovarian endometrioid adenocarcinoma, indicating broad levels of functional selectin ligand expression. In summary, DBTA detects functional selectin ligands expressed on tissue from multiple cancer types at both the primary and metastatic sites, data that may provide valuable diagnostic and prognostic information for malignant tumors. Citation Format: Eric Martin, Ramiro Malgor, Douglas Goetz, Monica Burdick. Dynamic biochemical tissue analysis detects functional selectin ligands expressed on breast, colorectal, liver, lung, ovarian, pancreatic, prostate, and stomach cancer tissues. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4017. doi:10.1158/1538-7445.AM2014-4017

In a variety of disease settings the expression of the endothelial selectins E- and P-selectin appears to be increased. This feature makes these molecules attractive targets around which to design directed drug-delivery schemes. One possible approach for achieving such delivery is to use polymeric biodegradable microspheres bearing a humanized monoclonal antibody (MAb) for E- and P-selectin, MAb HuEP5C7.g2. Perhaps the simplest technique for “coupling” HuEP5C7.g2 to the microspheres is via nonspecific adsorption. Previous studies suggest, however, that the adsorption of proteins onto microspheres fabricated in the presence of a stabilizer such as poly(vinyl alcohol) (PVA) is limited. It is unclear to what extent this limited level of adsorbed HuEP5C7.g2 would be able to support adhesion to E- and P-selectin under flow conditions. To explore this issue, we prepared microspheres from the biodegradable polymer, poly(ϵ-caprolactone) (PCL), using a single emulsion process and PVA as a stabilizer. We then incubated the PCL microspheres with HuEP5C7.g2 and studied the adhesion of the resulting HuEP5C7.g2 microspheres to E- and P-selectin under in vitro flow conditions. We found that the HuEP5C7.g2 PCL microspheres exhibit specific adhesion to Chinese hamster ovary cells stably expressing P-selectin (CHO-P) and 4-h IL-1β–activated human umbilical vein endothelial cells (HUVEC). In contrast, HuEP5C7.g2 PCL microspheres exhibit little adhesion to parental CHO cells or unactivated HUVEC. The attachment efficiency to the selectin substrates was quite low, with appreciable attachment occurring only at low shear (0.3 dyn/cm2). Other supporting data strongly suggest that the limited attachment efficiency is due to a low level of HuEP5C7.g2 adsorbed to the PCL microspheres. Although the attachment was limited, a significant percentage of the HuEP5C7.g2 PCL microspheres were able to remain adherent at relatively high shear (8 dyn/cm2). Combined, our data suggest that HuEP5C7.g2 PCL microspheres exhibit selective limited adhesion to cellular substrate expressing E- and P-selectin. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 73: 500–509, 2001.

Neutrophils can attach to E-selectin under flow. Proposed ligands for E-selectin carry SLex-type glycans. The leukocyte β2 integrins are glycosylated with SLex. Thus, we speculated that β2 integrins could support attachment to E-selectin. To test this hypothesis, we coated 10–diameter microspheres with purified CD11b/CD18 (αMβ2) and investigated the adhesion of the resulting αMβ2 microspheres to E-selectin. Under in vitro flow conditions, the αMβ2 microspheres attached to Chinese hamster ovary cells expressing E-selectin (CHO-E) and 4-h interleukin-1 β-activated human umbilical vein endothelial cells (HUVEC). At a shear stress of 1.8 dynes/cm2, the attachment events were eliminated by pretreatment of the cellular monolayers with a mAb to E-selectin. microspheres did not attach to untransfected CHO cells or unactivated HUVEC at 1.8 dynes/cm2. Taken together, the results strongly suggest that the CD11b/CD18-E-selectin bond has sufficient biophysical properties to mediate attachment of neutro-phil-sized particles to E-selectin under flow. J. Leukoc. Biol. 67: 196–205; 2000.

Leukocyte adhesion to vascular endothelium under flow involves an adhesion cascade consisting of multiple receptor pairs that may function in an overlapping fashion. P-selectin glycoprotein ligand-1 (PSGL-1) and L-selectin have been implicated in neutrophil adhesion to P- and E-selectin under flow conditions. To study, in isolation, the interaction of PSGL-1 with P-and E-selectin under flow, we developed an in vitro model in which various recombinant regions of extracellular PSGL-1 were coupled to 10-μm-diameter microspheres. In a parallel plate chamber with well defined flow conditions, live time video microscopy analyses revealed that microspheres coated with PSGL-1 attached and rolled on 4-h tumor necrosis factor-α–activated endothelial cell monolayers, which express high levels of E-selectin, and CHO monolayers stably expressing E-or P-selectin. Further studies using CHO-E and -P monolayers demonstrate that the first 19 amino acids of PSGL-1 are sufficient for attachment and rolling on both E- and P-selectin and suggest that a sialyl Lewis x–containing glycan at Threonine-16 is critical for this sequence of amino acids to mediate attachment to E- and P-selectin. The data also demonstrate that a sulfated, anionic polypeptide segment within the amino terminus of PSGL-1 is necessary for PSGL-1–mediated attachment to P- but not to E-selectin. In addition, the results suggest that PSGL-1 has more than one binding site for E-selectin: one site located within the first 19 amino acids of PSGL-1 and one or more sites located between amino acids 19 through 148.

A growing body of evidence suggests that E-,P-, and L-selectin, a family of cell adhesion molecules, engage their ligands on colon cancer cells to initiate adhesion with the blood vessel wall at the secondary site during metastasis. This initial adhesion is a “rolling” adhesion in which selectin/selectin ligand bonds break and reform under forces in hemodynamic flow. Current standards for tissue analyses, such as immunostaining, do not assay for functional in situ selectin ligands, i.e., those that promote rolling. Recently, our lab developed a novel tissue analysis, termed dynamic biochemical tissue analysis (DBTA), which detects functional selectin ligands in situ by probing pathological tissues with selectin-coated microspheres. Due to the application of hydrodynamic flow in the assay, the selectin coated microspheres “roll” on tissue sections presenting functional selectin ligands. When colon cancer tissue sections of diverse histological classifications were probed with L-selectin-coated microspheres in DBTA, functional L-selectin ligands were detected (i.e., L-selectin microspheres rolled on the surface of the tissue sections). Rolling velocities were a function of hemodynamic shear stress, illustrating the unique kinetic and tensile properties of force-dependent L-selectin/L-selectin ligand bonds. More specifically, DBTA preserved the catch-slip behavior of selectin/selectin ligand bonds. That is, rolling became stabilized at physiologic shear stresses between 0.5-0.75 dyne/cm2, but outside this range microsphere rolling was increasingly erratic as bond breakage exceeded bond formation necessary to preserve adhesion. Differences in microsphere rolling velocities varied with position on the tissue surface and thereby demonstrated the heterogeneous expression of functional L-selectin ligands on colon cancer tissues. In summary, DBTA provides a unique method to detect functional in situ L-selectin ligands and unveils intercellular heterogeneity such that, with further development, DBTA may promote potential diagnostics and prognostics for cancer characterization. Citation Format: Grady E. Carlson, Venktesh S. Shirure, Vicente A. Resto, Ramiro Malgor, Douglas J. Goetz, Monica M. Burdick. Dynamic biochemical tissue analysis provides a unique method to detect functional in situ L-selectin ligands and unveils intercellular heterogeneity in colon cancer tissues. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 203. doi:10.1158/1538-7445.AM2013-203

Selectin ligands are known to be upregulated in metastatic tumor cell lines, and growing evidence suggests selectin/selectin-ligand interactions mediate the adhesion of circulating tumor cells to distant sites. Thus, novel methods of characterizing selectin ligands expressed on human tissue may serve as valuable assays with significant diagnostic and prognostic potential. Because the kinetic and tensile properties of selectin/selectin-ligand bonds are influenced by local physiological conditions, i.e., wall shear stress due to hydrodynamic force, traditional immunohistochemical approaches cannot adequately detect selectin ligands. That is, though immunohistochemistry is very successful in identifying high affinity interactions, selectin/selectin-ligand interactions exhibit high affinity only when the external force applied on the bond is at an optimal value. This affinity is considerably weaker when applied force on the bond is significantly larger than this optimal value, or else is low or zero, as in traditional immunohistochemistry. Therefore, we have developed a method for detecting selectin ligands expressed on human tissue using a dynamic approach that allows for precise control of the force applied to the bonds between probe and target molecules. More specifically, we conjugated recombinant P-selectin to polystyrene microspheres and perfused this probe over tissue sections of colon cancer - which is known to express P-selectin ligands. Selectin/selectin-ligand adhesive interaction in the form of microsphere rolling on tissue was observed, and specific interaction was confirmed using 10 mM EDTA as a negative control. Increased probe surface coverage (i.e., higher density of P-selectin molecules coated on the surface of the microspheres) resulted in increased interaction with colon cancer tissue. In addition, rolling velocities measured between physiological wall shear stresses 0.25-2.0 dyne/cm2 indicated adhesive interaction of the selectin-conjugated probe with ligands in colon cancer tissues was stabilized at 0.75 dyne/cm2. Analysis of distinct regions within colon cancer tissue sections subjected to this dynamic biochemical tissue analysis revealed different levels of microsphere adhesion in discrete areas, evidence of heterogeneous cellular expression of P-selectin ligands. Taken together, these results demonstrate that functional P-selectin ligands are detectable using dynamic biochemical tissue analysis. Citation Format: Eric W. Martin, Venktesh S. Shirure, Ramiro Malgor, Vicente A. Resto, Douglas J. Goetz, Monica M. Burdick. Dynamic biochemical tissue analysis of P-selectin ligands expressed on colon cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 45. doi:10.1158/1538-7445.AM2013-45

Selections mediate transient adhesion of neutrophils to stimulated endothelial cells at sites of inflammation by binding counter-receptors that present carbohydrates such as sialyl Lewis(x). We have developed a cell-free adhesion assay using sialyl Lewis(x)-coated microspheres and E-selection-IgG chimera-coated substrates to investigate the premise that rolling primarily results from functional properties of selection-carbohydrate bonds, whereas cellular morphology and signaling act as secondary effects. Sialyl Lewis(x)-coated microspheres attach to and roll over E-selectin-IgG chimera-coated substrates between the physiological wall shear stresses of 0.7 and 2 dynes/cm2. Rolling velocities vary with time and depend on E-selectin-IgG chimera site density and wall shear stress. Our results show that sialyl Lewis(x) is a minimal functional recognition element required for rolling on E-selectin under flow.

Identification and characterization of molecular markers on cancerous tissue can lead to novel diagnostics and prognostics for cancer. Markers of interest include sialofucosylated molecules such as sialyl Lewis X and sialyl Lewis A (CA19-9), which are expressed on both lipids and proteins, and are often upregulated on certain cancerous tissues. Mounting evidence suggests that circulating cancer cells use these molecules to bind E-, P- and L- selectin (adhesion molecules present on leukocytes, platelets, and endothelial cells), to facilitate dissemination indicating that the presence of selectin ligands may correlate with metastatic potential. The hallmark of selectin-mediated binding is “rolling,” i.e. adhesion governed by the unique kinetic and tensile properties of selectin-selectin ligand bonds occurring under dynamic (flow) conditions. At present, the analysis of pathological tissue (e.g. immunohistochemistry) is performed under static conditions that only reveal high affinity interactions between the probing molecule and the tissue. Traditional biochemical tissue analysis is woefully inadequate for investigating selectin ligands on cancerous tissue, as it does not allow a detailed exploration of ligand chemistry in situ under biophysical conditions fit for selectin binding. Recently, we established a new assay for probing tissue, termed dynamic biochemical tissue analysis (DBTA), in which the probing molecule is conjugated to microspheres that are contacted with tissue samples under fluid shear. In our current investigation, E-, P- and L-selectin microspheres rolled on colon cancer tissue microarrays in DBTA. The adhesive interactions appeared to be specific, as the microspheres were released from the tissue samples and no further adhesion events were observed when EDTA was perfused through the system. Additionally, the adhesion of the microspheres with a given tissue section was dependent on the selectin. E-selectin microspheres were the most promiscuous, binding at high levels to many of the tissue samples, while the adhesion of the P- and L-selectin microspheres was more select. For a given type of selectin microsphere, the observed adhesion was not homogeneous; it was localized on discrete regions of the tissue sample. Notably, HECA-452 staining (an antibody that recognizes sialyl Lewis X and related moieties) in conjunction with selectin microsphere rolling revealed that HECA-452 reactivity only partially correlated with selectin binding. This observation strongly suggests that DBTA yields tissue characterization that is distinct compared to a high affinity immunohistochemistry assay. In conclusion, DBTA provides a controlled environment in which the shear-dependent selectin-selectin ligand interactions may be observed, providing a unique characterization of cancerous tissue applicable to developing novel diagnostic and prognostic strategies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 441. doi:1538-7445.AM2012-441

Work in the field of cell adhesion has undeniably shown that the full spectrum of molecular bond properties is germane to biological recognition and communication in physiologic and pathologic conditions, including cancer. For example, the unique kinetic and tensile properties of E-selectin bonds allow it to mediate cancer cell adhesion to the endothelium and leukocyte recruitment to sites of inflammation. Typically in a biochemical tissue analysis (e.g., immunohistochemistry), a molecular probe (e.g., antibody) detects antigens by utilizing only one property of the probe-antigen bond, specifically affinity, and other aspects of the bond (e.g., dynamic biophysical properties) remain unexploited. Therefore, we developed a dynamic biochemical tissue analysis (DBTA) that allows exquisite control over the interaction between the probe and the antigen to enhance characterization of molecular recognition in situ. Specifically, we conjugated a recombinant E-selectin construct to polystyrene microspheres and used the resulting microspheres as the probe to investigate E-selectin ligand activity on colon cancer, breast cancer, and normal tissues using DBTA. When E-selectin microspheres were perfused over invasive colon adenocarcinoma tissue sections at a wall shear stress of 0.80 dynes/ cm2, the microspheres exhibited robust adhesion with the tissue. The adhesion of E-selectin microspheres was significantly higher than that of negative control (IgG conjugated) microspheres, demonstrating the specificity of interaction of E-selectin microspheres with the tissue sections. To demonstrate that modulating easily controlled assay parameters results in discernible changes in adhesion, E-selectin microspheres of 10 and 15 µm diameters were perfused over the colon adenocarcinoma tissue sections at different levels of shear stress, achieved by varying the volumetric flow rate. The results showed that with increasing shear stress or particle diameter, the adhesion of E-selectin microspheres to the tissue sections decreased as predicted. To demonstrate that DBTA can be used for high throughput analysis, colon and breast cancer tissue microarrays were used. These microarray slides consisted of tissues from various histopathological classifications. E-selectin microspheres exhibited higher adhesion than the IgG microspheres to the majority of the cancer tissues, showing the preferential expression of functional E-selectin ligands on cancer tissues. Combined the results (i) convincingly demonstrate the specificity of the DBTA assay, (ii) reveal the critical role physical factors play in tissue analysis and (iii) demonstrate that DBTA allows facile control over these key physical factors. Thus, we have established a new approach for characterizing tissue that will lead to novel diagnostic and prognostic assays for a large spectrum of pathologies including cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4566. doi:1538-7445.AM2012-4566

CD44 is a highly pleiotropic molecule whose many structural variants and glycan modifications determine function, particularly with respect to cell adhesion. In this study, we investigated the E-selectin ligand function of CD44 and its relationship with the mesenchymal versus epithelial cell phenotypes of breast cancer cells. Real time RT-PCR analysis for epithelial and mesenchymal cell markers showed that MDA-MB-231 and Hs-578T breast cancer cells are mesenchymal-like cells, but BT-20 and MDA-MB-468 breast cancer cells are epithelial-like cells. Flow cytometry revealed that all cell lines expressed CD44. Further exploration of the expression of specific CD44 isoforms at the mRNA level (by real time RT-PCR) and at the protein level (by flow cytometry) showed that mesenchymal-like breast cancer cells predominantly expressed CD44s but nearly undetectable levels of CD44v isoforms. On the other hand, epithelial-like cells expressed CD44s as well as CD44v isoforms. These results indicate that there are divergent CD44 isoform expression patterns in mesenchymal-like versus epithelial-like breast cancer cell lines. Further distinctions were observed in CD44 function in cells of the two phenotypes. Western blot analysis demonstrated that CD44 expressed by BT-20 cells were positive for E-selectin ligand activity, whereas CD44 of MDA-MB-231 cells failed to exhibit such functionality. Moreover, CD44v isoforms expressed by BT-20 cells, but not CD44s by MDA-MB-231 cells, mediated E-selectin adhesion under physiological flow conditions. Despite the positive ligand activity, none of the CD44v isoforms expressed by BT20 cells possessed HECA-452 reactivity by Western blot analysis. This finding indicates the absence of conventional sialofucosylated carbohydrates and the presence of potentially novel E-selectin reactive glycans. Collectively, these results show that CD44 expressed by epithelial-like cells, but not mesenchymal-like cells, are functional E-selectin ligands. Our investigation therefore implies an association of breast cancer cell adhesivity with the epithelial phenotype. Thus, future studies mechanistically linking the E-selectin ligand activity of CD44 isoforms with epithelial-to-mesenchymal transition may provide targets for novel breast cancer diagnostics, prognostics, and therapeutics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5181. doi:1538-7445.AM2012-5181

Transient, heterotypic adhesion between leukocytes and the endothelium is an essential part of many immune and inflammatory reactions (Stoolman, 1989; Osborn 1990; Springer, 1990; McEver, 1991; Lasky, 1992; Bevilacqua, 1993). It is generally believed adhesive interactions between leukocytes and endothelium are mediated by specific complementary binding between couterreceptors on both leukocytes and endothelium (Springer, 1990). Of particular interest are the adhesive interactions between neutrophils and endothelium near tissue sites of inflammation, where neutrophils are recruited to the inflammatory site to eliminate foreign invaders (Atherton and Born, 1972, 1973). A great deal of research is focused on understanding the molecular and physical requirements of adhesion molecules in this system which provide for local sequestration of neutrophils.

Objective and design Atherosclerosis (ATH) is a chronic inflammatory disease that involves cascades of signaling events mediated by various effector proteins. Here we sought to determine if the expression of Wnt5a, a secreted glycoprotein, is altered in discrete regions of the arterial plaque. Methods Atherosclerotic plaque tissues from 14 human subjects undergoing elective carotid endarterectomy were used in this study. Immunohistochemistry and laser capture microdissection combined with quantitative real-time PCR were used to determine the expression of Wnt5a and Toll-like receptors (TLRs) in different sections of the arterial lesions. Atherosclerotic serum samples (n = 30) and serum from healthy subjects (n = 16) were quantified for Wnt5a using an enzyme-linked immunosorbent assay (ELISA). Results The data analysis revealed that Wnt5a transcripts and protein were elevated in advanced arterial lesions relative to less advanced arterial lesions; that Wnt5a expression correlated with the presence of TLR4 and TLR2 transcripts; and that the average amount of Wnt5a protein present in atherosclerotic patient serum was significantly higher compared to healthy controls. Conclusions This study is the first to provide evidence that the expression of Wnt5a increases as the disease progresses to a more advanced stage, and that this expression is coincident with that of TLR2 and TLR4. In addition, we found that the average Wnt5a levels in the serum of atherosclerotic patients are elevated relative to healthy controls, which is consistent with the hypothesis that Wnt5a plays a role in ATH.