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The role of interferon-gamma (IFN-γ) in Chronic Myelogenous/Myeloid Leukemia (CML) and in the treatment of CML remains unclear; specifically, the effect of IFN-γ on apoptosis. There is reported interplay between IFN-γ and glycogen synthase kinase–3 (GSK-3), a kinase which has been implicated in both cell death and, conversely, cell survival. Thus, we utilized the CML-derived HAP1 cell line and a mutant HAP1 GSK-3β knocked-down cell line (GSK-3β 31bp) to investigate whether GSK-3 modulates IFN-γ’s action on CML cells. Significantly less GSK-3β 31bp cells, relative to HAP1 cells, were present after 48 h treatment with IFN-γ. IFN-γ treatment significantly decreased GSK-3β 31bp substrate adhesiveness (relative to HAP1 cells); an observation often correlated with cell death. Fluorescence microscopy revealed that IFN-γ induces a modest level of apoptosis in the HAP1 cells and that IFN-γ induced apoptosis is significantly enhanced in GSK-3β 31bp cells. Utilizing a complementary GSK-3β knocked-down cell line (8bp) we found, via flow cytometric analysis, that IFN-γ induced apoptosis is significantly enhanced in GSK-3β 8bp cells relative to HAP1 cells. Combined, our findings suggest that IFN-γ induces apoptosis of CML cells and that loss of GSK-3β significantly augments IFN-γ-induced apoptosis.

Sepsis is a serious condition that can lead to long-term organ damage and death. At the molecular level, the hallmark of sepsis is the elevated expression of a multitude of potent cytokines, i.e. a cytokine storm. For sepsis involving gram-negative bacteria, macrophages recognize lipopolysaccharide (LPS) shed from the bacteria, activating Toll-like-receptor 4 (TLR4), and triggering a cytokine storm. Glycogen synthase kinase-3 (GSK-3) is a highly active kinase that has been implicated in LPS-induced cytokine production. Thus, compounds that inhibit GSK-3 could be potential therapeutics for sepsis. Our group has recently described a novel and highly selective inhibitor of GSK-3 termed COB-187. In the present study, using THP-1 macrophages, we evaluated the ability of COB-187 to attenuate LPS-induced cytokine production. We found that COB-187 significantly reduced, at the protein and mRNA levels, cytokines induced by LPS (e.g. IL-6, TNF-α, IL-1β, CXCL10, and IFN-β). Further, the data suggest that the inhibition could be due, at least in part, to COB-187 reducing NF-κB (p65/p50) DNA binding activity as well as reducing IRF-3 phosphorylation at Serine 396. Thus, COB-187 appears to be a potent inhibitor of the cytokine storm induced by LPS.

Nonalcoholic fatty liver disease (NAFLD) is the hepatic manifestation of both metabolic and inflammatory diseases and has become the leading chronic liver disease worldwide. High-fat (HF) diets promote an increased uptake and storage of free fatty acids (FFAs) and triglycerides (TGs) in hepatocytes, which initiates steatosis and induces lipotoxicity, inflammation and insulin resistance. Activation and signaling of Toll-like receptor 4 (TLR4) by FFAs induces inflammation evident in NAFLD and insulin resistance. Currently, there are no effective treatments to specifically target inflammation associated with this disease. We have established the efficacy of phenylmethimazole (C10) to prevent lipopolysaccharide and palmitate-induced TLR4 signaling. Because TLR4 is a key mediator in pro-inflammatory responses, it is a potential therapeutic target for NAFLD. Here, we show that treatment with C10 inhibits HF diet-induced inflammation in both liver and mesenteric adipose tissue measured by a decrease in mRNA levels of pro-inflammatory cytokines. Additionally, C10 treatment improves glucose tolerance and hepatic steatosis despite the development of obesity due to HF diet feeding. Administration of C10 after 16 weeks of HF diet feeding reversed glucose intolerance, hepatic inflammation, and improved hepatic steatosis. Thus, our findings establish C10 as a potential therapeutic for the treatment of NAFLD.

Inhibition of interleukin–6 (IL-6) holds significant promise as a therapeutic approach for triple negative breast cancer (TNBC). We previously reported that phenylmethimazole (C10) reduces IL-6 expression in several cancer cell lines. We have identified a more potent derivative of C10 termed COB-141. In the present work, we tested the hypothesis that C10 and COB-141 inhibit TNBC cell expressed IL-6 and investigated the potential for classical IL-6 pathway induced signaling within TNBC cells. A panel of TNBC cell lines (MDA-MB-231, Hs578T, MDA-MB-468) was used. Enzyme linked immunosorbent assays (ELISA) revealed that C10 and COB-141 inhibit MDA-MB-231 cell IL-6 secretion, with COB-141 being ∼6.5 times more potent than C10. Therefore, the remainder of the study focused on COB-141 which inhibited IL-6 secretion, and was found, via quantitative real time polymerase chain reaction (QRT-PCR), to inhibit IL-6 mRNA in the TNBC panel. COB-141 had little, if any, effect on metabolic activity indicating that the IL-6 inhibition is not via a toxic effect. Flow cytometric analysis and QRT-PCR revealed that the TNBC cell lines do not express the IL-6 receptor (IL-6Rα). Trans-AM assays suggested that COB-141 exerts its inhibitory effect, at least in part, by reducing NF-κB (p65/p50) DNA binding. In summary, COB-141 is a potent inhibitor of TNBC cell expressed IL-6 and the inhibition does not appear to be due to non-specific toxicity. The TNBC cell lines do not have an intact classical IL-6 signaling pathway. COB-141's inhibitory effect may be due, at least in part, to reducing NF-κB (p65/p50) DNA binding.

Cancers of the digestive tract cause nearly one quarter of the cancer deaths worldwide, and nearly half of these are due to cancers of the esophagus and colon. Early detection of cancer significantly increases the rate of survival, and thus it is critical that cancer within these organs is detected early. In this regard, endoscopy is routinely used to screen for transforming/cancerous (i.e. dysplastic to fully cancerous) tissue. Numerous studies have revealed that the biochemistry of the luminal surface of such tissue within the colon and esophagus becomes altered throughout disease progression. Molecular endoscopic imaging (MEI), an emerging technology, seeks to exploit these changes for the early detection of cancer. The general approach for MEI is as follows: the luminal surface of an organ is exposed to molecular ligands, or particulate probes bearing a ligand, cognate to biochemistry unique to pre-cancerous/cancerous tissue. After a wash, the tissue is imaged to determine the presence of the probes. Detection of the probes post-washing suggests pathologic tissue. In the current review we provide a succinct, but extensive, review of ligands and target moieties that could be, or are currently being investigated, as possible cognate chemistries for MEI. This is followed by a review of the biophysics that determines, in large part, the success of a particular MEI design. The work draws an analogy between MEI and the well-advanced field of cell adhesion and provides a road map for engineering MEI to achieve assays that yield highly selective recognition of transforming/cancerous tissue in situ.

Glycogen synthase kinase-3 (GSK-3) is a multitasking protein kinase that regulates numerous critical cellular functions. Not surprisingly, elevated GSK-3 activity has been implicated in a host of diseases including pathological inflammation, diabetes, cancer, arthritis, asthma, bipolar disorder, and Alzheimer’s. Therefore, reagents that inhibit GSK-3 activity provide a means to investigate the role of GSK-3 in cellular physiology and pathophysiology and could become valuable therapeutics. Finding a potent inhibitor of GSK-3 that can selectively target this kinase, among over 500 protein kinases in the human genome, is a significant challenge. Thus there remains a critical need for the identification of selective inhibitors of GSK-3. In this work, we introduce a novel small organic compound, namely COB-187, which exhibits potent and highly selective inhibition of GSK-3. Specifically, this study 1) utilized a molecular screen of 414 kinase assays, representing 404 unique kinases, to reveal that COB-187 is a highly potent and selective inhibitor of GSK-3; 2) utilized a cellular assay to reveal that COB-187 decreases the phosphorylation of canonical GSK-3 substrates indicating that COB-187 inhibits cellular GSK-3 activity; and 3) reveals that a close isomer of COB-187 is also a selective and potent inhibitor of GSK-3. Taken together, these results demonstrate that we have discovered a region of chemical design space that contains novel GSK-3 inhibitors. These inhibitors will help to elucidate the intricate function of GSK-3 and can serve as a starting point for the development of potential therapeutics for diseases that involve aberrant GSK-3 activity.

Cell adhesion mediated by selectins (expressed by activated endothelium, activated platelets, and leukocytes) binding to their resepective selectin ligands (expressed by cancer cells) may be involved in metastasis. Therefore, methods of characterizing selectin ligands expressed on human tissue may serve as valuable assays. Presented herein is an innovative method for detecting functional selectin ligands expressed on human tissue that uses a dynamic approach, which allows for control over the force applied to the bonds between the probe and target molecules. This new method of tissue interrogation, known as dynamic biochemical tissue analysis (DBTA), involves the perfusion of molecular probe-coated microspheres over tissues. DBTA using selectin-coated probes is able to detect functional selectin ligands expressed on tissue from multiple cancer types at both primary and metastatic sites.

Glycogen synthase kinase-3 (GSK-3) has been implicated in numerous pathologies making GSK-3 an attractive therapeutic target. Our group has identified a compound termed COB-187 that is a potent and selective inhibitor of GSK-3. In this study, we probed the mechanism by which COB-187 inhibits GSK-3β. Progress curves, generated via real-time monitoring of kinase activity, indicated that COB-187 inhibition of GSK-3β is time-dependent and subsequent jump dilution assays revealed that COB-187 binding to GSK-3β is reversible. Further, a plot of the kinetic constant (kobs) versus COB-187 concentration suggested that, within the range of concentrations studied, COB-187 binds to GSK-3β via an induced-fit mechanism. There is a critical cysteine residue at the entry to the active site of GSK-3β (Cys-199). We generated a mutant version of GSK-3β wherein Cys-199 was substituted with an alanine. This mutation caused a dramatic decrease in the activity of COB-187; specifically, an IC50 in the nM range for wild type versus >100 µM for the mutant. A screen of COB-187 against 34 kinases that contain a conserved cysteine in their active site revealed that COB-187 is highly selective for GSK-3 indicating that COB-187′s inhibition of GSK-3β via Cys-199 is specific. Combined, these findings suggest that COB-187 inhibits GSK-3β via a specific, reversible, time and Cys-199-dependent mechanism.

Esophageal cancer has a 5 year survival rate of ∼20%. This dismal prognosis is due, in part, to the fact that esophageal cancer often presents at a late stage. Thus, there is a critical need for assays that enable the early detection of cancerous tissue within the esophagus. The luminal surface of the esophagus expresses signature molecule(s) at sites of transformation providing an avenue for the development of in situ assays that detect neoplastic growth within the esophagus. An attractive approach, receiving increased attention, is the endoscopic administration of particles conjugated with ligands to signature molecules present on transforming tissue. Detection of the particles within the esophagus, post-washing, would indicate the presence of the signature molecule and thus transforming tissue. In this work, we utilized cancerous and normal esophageal cells to provide in vitro proof of principle for this approach utilizing ligand-conjugated microspheres and demonstrate the need, and provide the framework for, engineering this technology. Specifically, the study (i) reveals selective increased expression of signature molecules on cancerous esophageal cells relative to normal cells; (ii) demonstrates selective binding of ligand-conjugated microspheres to cancerous esophageal cells relative to normal cells; (iii) demonstrates that the selective recognition of cancerous, relative to normal esophageal cells, is highly dependent on the biophysical design of the assay; and (iv) advocates utilizing the knowledge from the field of cell adhesion as a guide for the effective development of ligand-conjugated particle-based schemes that seek to detect esophageal oncogenesis in situ.

Previous studies revealed that phenylmethimazole (C10) inhibits IRF3 signaling, preventing dsRNA-induction of type 1 interferon gene expression, production, and downstream signaling. In the present study, we investigated the molecular basis for C10 inhibition of dsRNA-stimulated IRF3 signaling. IRF-3 Trans-AM assays were used to measure C10 effects on dsRNA induction of IRF3 DNA binding. Green fluorescent protein-labeled IRF3 was used to measure C10 effects on dsRNA-induced IRF3 nuclear translocation. Native PAGE, SDS PAGE, and western blotting were used to identify effects of C10 on IRF3 homodimer formation and phosphorylation, respectively. There was a significant impairment of dsRNA-induced IRF3 DNA binding activity in human embryonic kidney and pancreatic cancer cells with C10 treatment. C10 also blocked dsRNA-induced IRF3 nuclear translocation and homodimer formation without blocking serine 396 phosphorylation of IRF3. Together, these results indicate that C10 interferes with IRF3 signaling by blocking dsRNA-induced IRF3 homodimer formation, a prerequisite for nuclear translocation and DNA binding activities.

Objective: Wnt5a is a secreted glycoprotein highly present in atherosclerotic lesions. Uptake of oxidized-low density lipoprotein (ox-LDL) by monocytes/macrophages plays a critical role in atherosclerosis. The objective of this study was to determine if Wnt5a mRNA expression correlates with the severity of atherosclerotic lesions, and if, ox-LDL can induce Wnt5a mRNA in macrophages. Methods: Wnt5a mRNA in tissue sections from carotid arteries of patients undergoing endarterectomy was quantified via RT-PCR and correlated with plaque severity. Human monocyte-derived macrophages and differentiated THP-1 cells, a human monocytic cell line, were treated with ox-LDL or native-LDL. Subsequently, Wnt5a transcripts were quantified by RT-PCR. Results: Regions of the arteries with more severe plaques had detectable and significant levels of Wnt5a mRNA, while regions of the arteries containing less vulnerable plaques had low or non-detectable Wnt5a. Ox-LDL, but not native-LDL, induced Wnt5a mRNA in both human monocyte-derived macrophages and differentiated THP-1 cells. Conclusion: Our results demonstrate that the expression of Wnt5a correlates with the severity of atherosclerotic lesions, and that ox-LDL induces Wnt5a mRNA expression in human macrophages. These findings are consistent with the hypothesis that Wnt5a plays a critical role in atherosclerosis progression and that a source of Wnt5a is ox-LDL stimulated macrophages.

The transition to an era of functional glycomics has created a need for the development of novel techniques to assess complex glycoconjugate cellular adhesion molecules (e.g., selectin ligands) that may be involved in capturing and mediating the adhesion circulating tumor cells to distant sites. Cell line, knockout, and selectin antagonist evidence suggests that selectin/selectin-ligand interactions are involved in metastasis. However, identification and characterization of functional selectin ligand expression on cancer tissue (i.e., CTC origin site) remains challenging due to the inherent heterogeneity of tissues magnified by the "catch-slip" and stochastic nature of selectin/selectin-ligand bonds. We have developed a flow-based assay known as dynamic biochemical tissue analysis (DBTA) that involves the perfusion of selectin-coated microprobes over tissues at physiologic wall shear rates. Using DBTA, the expression of functional selectin ligands has been detected (i.e., specific binding and rolling of selectin-coated microprobes) on tissue from multiple solid tumors at primary and metastatic sites. Although DBTA provides evidence that multiple types of cancer can express complicated glycoconjugate structures (selectin ligands) that are able to bind to selectins in a shear-dependent manner, the challenge of the assay is in the analysis of the large amounts of image sequence data generated. This analysis is further complicated by the dynamic behavior of selectin-coated microprobe adhesion to selectin ligands expressed on a heterogeneous cancer tissue surface. To extract the biophysical underpinnings towards the development of this prospective in vitro diagnostic, a mathematical model was devised to refine the design space of this flow assay. The model is semi-empirical and consists of a coupled set of stochastic, 4D partial differential equations that account for the transport of probes to the tissue surface as well as the dynamics of probe adsorption to a heterogeneous tissue surface. Empirical data were obtained by processing image sequences with machine-learning, template-matching algorithms to conduct image segmentation, followed by probe tracking over time using a program written in Python. The empirical data displayed a stochastic adsorption profile and showed agreement with the model equations that were solved numerically using a finite difference scheme written in C++. Altogether, this approach is able to successfully bridge the gap between the seemingly random rolling interactions observed in the flow assay and the underlying physical mechanisms. Citation Format: Eric Martin, Douglas Goetz, Monica Burdick. Modeling selectin-coated microprobe adhesion to selectin ligands heterogeneously expressed on cancer tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4277.

P-selectin glycoprotein ligand-1 (PSGL-1) has been proposed as an important tethering ligand for E-selectin and is expressed at a modest level on human leukocytes. Sialyl Lewis x (sLex)-like glycans bind to E-selectin and are expressed at a relatively high level on circulating leukocytes. It is unclear whether PSGL-1 has unique biochemical attributes that contribute to its role as an E-selectin ligand. To probe this issue, we conjugated microspheres with either sLexor PSGL-1 purified from myeloid cells (neutrophils and HL-60) and compared their adhesion to endothelial expressed E-selectin under defined shear conditions. We found that both sLexand PSGL-1 microspheres adhere to 4 h of IL-1β-activated human umbilical vein endothelial cells predominantly through E-selectin. Analysis of the adhesion revealed that the rate of initial tethering of the PSGL-1 microspheres to E-selectin was significantly greater than the rate of initial tethering of the sLexmicrospheres despite the fact that the sLexmicrospheres tested had higher ligand densities than the PSGL-1 microspheres. We also found that pretreatment of the PSGL-1 or sLexmicrospheres with HECA-452 had no significant effect on initial tethering to E-selectin. These results support the hypotheses that 1) PSGL-1 is a high-efficiency tethering ligand for E-selectin, 2) ligand biochemistry can significantly influence initial tethering to E-selectin, and 3) PSGL-1 tethering to E-selectin can occur via non-HECA-452 reactive epitopes.

Toll-like receptors (TLRs) initiate an innate immune response. TLR3 on dendritic cells recognize double-stranded (ds) RNA and then signal increases in cytokines and recognition molecules important for immune cell interactions. In this report, we demonstrate TLR3 mRNA and protein are expressed on Fisher rat thyroid cell line-5 (FRTL-5) thyroid cells and are functional because incubating cells with polyinosine-polycytidylic acid causes 1) transcriptional activation of both the nuclear factor kappaB (NF-kappaB)/Elk1 and interferon (IFN) regulatory factor-3/IFN-beta signal paths, 2) posttranscriptional activation of NF-kappaB and ERK1/2, and 3) increased IFN-beta mRNA. TLR3 can be overexpressed, along with dsRNA-dependent protein kinase, major histocompatibility complex-I or II, and IFN regulatory factor-1, by transfecting dsRNA into the cells, infection with Influenza A virus, or incubation with IFN-beta, but not by incubation with dsRNA or IFNgamma, or by dsDNA transfection. A methimazole (MMI) derivative, phenylmethimazole, to a significantly greater degree than MMI, prevents overexpression by inhibiting increased transcriptional activation of IRF-3 and of IFN-stimulated response elements, phosphorylation of signal transducers and activation of transcription (STAT-1), but not NF-kappaB activation. TLR3 can be functionally overexpressed in cultured human thyrocytes by dsRNA transfection or IFN-beta treatment. Immunohistochemical studies show that TLR3 protein is overexpressed in human thyrocytes surrounded by immune cells in 100% of patients with Hashimoto's thyroiditis examined, but not in normal or Graves' thyrocytes. We conclude that functional TLR3 are present on thyrocytes; TLR3 downstream signals can be overexpressed by pathogen-related stimuli; overexpression can be reversed by phenylmethimazole to a significantly greater extent than MMI by inhibiting only the IFN regulatory factor-3/IFN-beta/signal transducers and activation of transcription arm of the TLR3 signal system; and TLR3 overexpression can induce an innate immune response in thyrocytes, which may be important in the pathogenesis of Hashimoto's thyroiditis and in the immune cell infiltrates.

Proinflammatory cytokine (e.g., TNF-α)-induced expression of endothelial cell adhesion molecules (ECAMs) on the lumenal surface of the vascular endothelium and a consequent increase in leukocyte adhesion are key aspects of pathological inflammation. A promising therapeutic approach to diminish aberrant leukocyte adhesion is, therefore, to inhibit cytokine-induced ECAM expression at the transcription level. Several studies suggest that methimazole, a compound used clinically to treat autoimmune diseases, such as Graves’ disease, may also diminish pathological inflammation by suppressing ECAM expression. In this study we probed the hypothesis that a derivative of methimazole, phenyl methimazole (compound 10), can reduce cytokine-induced ECAM expression and consequent leukocyte adhesion. We found that compound 10 1) dramatically inhibits TNF-α-induced VCAM-1 mRNA and protein expression in human aortic endothelial cells (HAEC), has a relatively modest inhibitory effect on TNF-α induced E-selectin expression and has no effect on ICAM-1 expression; 2) significantly reduces TNF-α-induced monocytic (U937) cell adhesion to HAEC under in vitro flow conditions similar to that present in vivo; 3) inhibits TNF-α-induced IFN regulatory factor-1 binding to VCAM-1 promoter; and 4) reduces TNF-α-induced IRF-1 expression in HAEC. Combined, the results indicate that phenyl methimazole can reduce TNF-α-induced VCAM-1 expression in an IFN regulatory factor-1-dependent manner and that this contributes significantly to reduced monocytic cell adhesion to TNF-α-activated HAEC.

The expression of vascular cell adhesion molecule-1 (VCAM-1) on the vascular endothelium can be increased by pro-inflammatory cytokines [e.g. tumor necrosis factor – α (TNF-α)]. VCAM-1 contributes to leukocyte adhesion to, and emigration from, the vasculature which is a key aspect of pathological inflammation. As such, a promising therapeutic approach for pathological inflammation is to inhibit the expression of VCAM-1. Methimazole [3-methyl-1, 3 imidazole-2 thione (MMI)] is routinely used for the treatment of Graves’ disease and patients treated with MMI have decreased levels of circulating VCAM-1. In this study we used cultured human umbilical vein endothelial cells (HUVEC) to investigate the effect of MMI structural modifications on TNF-α induced VCAM-1 expression. We found that addition of a phenyl ring at the 4-nitrogen of MMI yields a compound that is significantly more potent than MMI at inhibiting 24 h TNF-α-induced VCAM-1 protein expression. Addition of a para methoxy to the appended phenyl group increases the inhibition while substitution of a thiazole ring for an imidazole ring in the phenyl derivatives yields no clear difference in inhibition. Addition of the phenyl ring to MMI appears to increase toxicity as does substitution of a thiazole ring for an imidazole ring in the phenyl MMI derivatives. Each of the compounds reduced TNF-α-induced VCAM-1 mRNA expression and had a functional inhibitory effect, i.e. each inhibited monocytic cell adhesion to 24 h TNF-α-activated HUVEC under fluid flow conditions. Combined, these studies provide important insights into the design of MMI-related anti-inflammatory compounds.

P-selectin glycoprotein ligand-1 (PSGL-1) is present on leukocytes and is the major ligand for endothelial expressed P-selectin. A variety of studies strongly suggests that the N-terminal region of PSGL-1 contains the binding site for P-selectin. We hypothesized that this relatively small N-terminal peptide of PSGL-1 is sufficient to support adhesion to P-selectin in vivo. To test this hypothesis, we coated 2 μm–diameter microspheres with a recombinant PSGL-1 construct, termed 19.ek.Fc. The 19.ek.Fc construct consists of the first 19 N-terminal amino acids of mature PSGL-1 linked to an enterokinase cleavage site that, in turn, is linked to human immunoglobulin G Fc. The 19.ek.Fc-coated microspheres were injected into the jugular vein of mice. Intravital microscopy of postcapillary venules within the cremaster muscle of mice revealed that a significantly greater number of 19.ek.Fc microspheres rolled compared with control microspheres. The number of rolling 19.ek.Fc microspheres was significantly diminished by pretreatment of the mice with a monoclonal antibody to P-selectin or by pretreatment of the 19.ek.Fc microspheres with a monoclonal antibody to PSGL-1. Combined, the results indicate that the N-terminal peptide of PSGL-1 can mediate adhesion to trauma-activated microvascular endothelium via P-selectin in vivo.

A growing body of evidence suggests that L-selectin ligands presented on circulating tumor cells facilitate metastasis by binding L-selectin presented on leukocytes. Commonly used methods for detecting L-selectin ligands on tissues, e.g., immunostaining, are performed under static, no-flow conditions. However, such analysis does not assay for functional L-selectin ligands, specifically those ligands that promote adhesion under shear flow conditions. Recently our lab developed a method, termed dynamic biochemical tissue analysis (DBTA), to detect functional selectin ligands in situ by probing tissues with L-selectin-coated microspheres under hemodynamic flow conditions. In this investigation, DBTA was used to probe human colon tissues for L-selectin ligand activity. The detection of L-selectin ligands using DBTA was highly specific. Furthermore, DBTA reproducibly detected functional L-selectin ligands on diseased, e.g., cancerous or inflamed, tissues but not on noncancerous tissues. In addition, DBTA revealed a heterogeneous distribution of functional L-selectin ligands on colon cancer tissues. Most notably, detection of L-selectin ligands by immunostaining using HECA-452 antibody only partially correlated with functional L-selectin ligands detected by DBTA. In summation, the results of this study demonstrate that DBTA detects functional selectin ligands to provide a unique characterization of pathological tissue.

Adhesion of circulating tumor cells to vascular endothelium is mediated by specialized molecules that are functional under shear forces exerted by hematogenous flow. Endothelial E-selectin binding to glycoforms of CD44 mediates shear-resistant cell adhesion in numerous physiological and pathological conditions. However, this pathway is poorly understood in breast cancer and is the focus of the present investigation. All breast cancer cell lines used in this study strongly expressed CD44. In particular, BT-20 cells expressed CD44s and multiple CD44v isoforms, whereas MDA-MB-231 cells predominantly expressed CD44s but weakly expressed CD44v isoforms. CD44 expressed by BT-20, but not MDA-MB-231, cells possessed E-selectin ligand activity as detected by Western blotting and antigen capture assays. Importantly, CD44 expressed by intact BT-20 cells were functional E-selectin ligands, regulating cell rolling and adhesion under physiological flow conditions, as found by shRNA-targeted silencing of CD44. Antigen capture assays strongly suggest greater shear-resistant E-selectin ligand activity of BT-20 cell CD44v isoforms than CD44s. Surprisingly, CD44 was not recognized by the HECA-452 MAb, which detects sialofucosylated epitopes traditionally expressed by selectin ligands, suggesting that BT-20 cells express a novel glycoform of CD44v as an E-selectin ligand. The activity of this glycoform was predominantly attributed to N-linked glycans. Furthermore, expression of CD44v as an E-selectin ligand correlated with high levels of fucosyltransferase-3 and -6 and epithelial, rather than mesenchymal, cell phenotype. Together, these data demonstrate that expression of CD44 as a functional E-selectin ligand may be important in breast cancer metastasis.

The diameter of circulating cells that may adhere to the vascular endothelium spans an order of magnitude from approximately 2 microm (e.g., platelets) to approximately 20 microm (e.g., a metastatic cell). Although mathematical models indicate that the adhesion exhibited by a cell will be a function of cell diameter, there have been few experimental investigations into the role of cell diameter in adhesion. Thus, in this study, we coated 5-, 10-, 15-, and 20-microm-diameter microspheres with the recombinant P-selectin glycoprotein ligand-1 construct 19.ek.Fc. We compared the adhesion of the 19.ek.Fc microspheres to P-selectin under in vitro flow conditions. We found that 1) at relatively high shear, the rate of attachment of the 19.ek.Fc microspheres decreased with increasing microsphere diameter whereas, at a lower shear, the rate of attachment was not affected by the microsphere diameter; 2) the shear stress required to set in motion a firmly adherent 19.ek.Fc microsphere decreased with increasing microsphere diameter; and 3) the rolling velocity of the 19.ek.Fc microspheres increased with increasing microsphere diameter. These results suggest that attachment, rolling, and firm adhesion are functions of particle diameter and provide experimental proof for theoretical models that indicate a role for cell diameter in adhesion.

Lu-ECAM-1 is a lung-derived, venular endothelial cell adhesion molecule. It promotes the selective adhesion of lung-metastatic B16-F10 melanoma cells to endothelium under static conditions and mediates colonization of the lungs by the same tumor cells. To test whether Lu-ECAM-1 by itself is sufficient to cause vascular arrest of B16-F10 cells, we measured here under conditions of flow tumor cell adhesion to endothelia that express different amounts of Lu-ECAM-1 on their surfaces. At physiological shear stresses, adhesion of B16-F10 melanoma cells to endothelia correlates positively with the amount of Lu-ECAM-1 expression on the endothelial cell surface and inversely with the level of the applied shear stress. Tumor cell trajectories are biphasic; i.e., B16-F10 melanoma cells initially move along the endothelial surface with a velocity similar to the theoretical velocity, then arrest within a fraction of a second. Arrest is permanent for most B16-F10 melanoma cells at all shear stresses tested. Tumor cells never engaged in a rolling motion prior to arrest. Masking of the Lu-ECAM-1 ligand on the surface of B16-F10 melanoma cells with soluble Lu-ECAM-1 impedes arrest of tumor cells on the surface of the test endothelium. Purified Lu-ECAM-1 also mediates B16-F10 arrest, but arrest is mostly transient at shear stresses of 0.59 dynes/cm2 and higher, implying adhesion by single receptor/ligand bonds. Our data suggest that Lu-ECAM-1 plays a critical role in the recognition and initial arrest of murine melanoma cells in lung venules.

The presence of functional P-selectin ligands is well-documented for human colon cancer cell lines, but not in situ on human colon carcinoma tissue. Presently, immunostaining with antibodies is used to detect critical components of selectin ligands, e.g., sialofucosylated moieties. However, this static biochemical tissue analysis (SBTA) cannot ascertain if a potential selectin ligand is able to mediate (rolling) adhesion. Due to the immense difficulty in detecting functional selectin ligands using traditional methods, we have developed a flow-based assay known as dynamic biochemical tissue analysis (DBTA) for detecting functional selectin ligands expressed on human tissue. DBTA using P-selectin microspheres was performed on colon cancer tissue sections from multiple cases, in conjunction with SBTA using P-selectin, antibodies against purported selectin ligand carbohydrate moieties sLeX and sLeA (HECA-452, CSLEX-1, and KM-231), and antibodies against peptide structures of putative P-selectin ligands (CD24, CD44, and PSGL-1). Examination of serial sections, in the same regions of tissue displaying DBTA probe adhesion, revealed significant detection inconsistencies with SBTA. Subsequently, due to the well-documented force-dependency of selectin ligands, DBTA was conducted with microspheres coated with either HECA-452, CSLEX-1, or KM-231 to determine the effect of applied force on the detection capabilities of these antibodies. Analysis of signet ring cell colon carcinoma tissue revealed microspheres coated with HECA-452, CSLEX-1, or KM-231 antibodies all displayed significantly lower amounts of adhesion than the DBTA P-selectin microspheres. Interestingly, although microspheres coated with these antibodies adhered to signet ring cell carcinoma tissue, these DBTA probes did not interact with all regions of tissue that displayed adhesion with P-selectin microspheres. Specificity of interaction was validated using corresponding isotype control coated microspheres. Taken together, these results show DBTA with P-selectin coated microspheres is able to unequivocally detect functional P-selectin ligands, in contrast to SBTA (immunostaining) and DBTA using microspheres coated with antibodies. In summary, DBTA using P-selectin coated microspheres is able to detect functional P-selectin ligands expressed on colon cancer tissue, data that may provide valuable diagnostic and prognostic information for malignant tumors. Citation Format: Eric W. Martin, Ramiro Malgor, Vicente A. Resto, Douglas J. Goetz, Monica M. Burdick. Detection of functional P-selectin ligands expressed on colon cancer tissue using a novel flow-based assay. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4944.

E-selectin, expressed on inflamed endothelium, and sialyl Lewis x (sLe(x)), present on the surface of leukocytes, play a key role in leukocyte-endothelial interactions during leukocyte recruitment to sites of inflammation. HECA-452 is a monoclonal antibody (mAb) that recognizes sLe(x) and is routinely used by investigators from diverse fields who seek to unravel the mechanisms of leukocyte adhesion. The data regarding the ability of HECA-452 to inhibit carbohydrate-mediated leukocyte adhesion to E-selectin remains conflicted, in part due to the presence of a variety of potential E-selectin reactive moieties on leukocytes. Recognizing this, we utilized a complementary approach to gain insight into HECA-452 adhesion assays. Specifically, we used sLe(x) microspheres to investigate the hypothesis that HECA-452 is a non-function blocking mAb for isolated sLe(x) mediated adhesion to endothelial expressed E-selectin. Flow cytometric analysis revealed that HECA-452 recognizes and binds to the sLe(x) microspheres. Perfusion of the sLe(x) microspheres over human umbilical vein endothelial cells (HUVEC) at 1.5 dyn/cm² revealed that the microspheres attach to 4h interleukin (IL)-1β activated HUVEC specifically via E-selectin. Pretreatment of the sLe(x) microspheres with HECA-452 did not influence sLe(x) microsphere initial tethering and accumulation on IL-1β activated HUVEC. Neuraminidase and fucosidase treatments of sLe(x) microspheres revealed that sialic acid and fucose are required for E-selectin binding, whereas HECA-452 recognition of sLe(x) does not depend on the fucose moiety to the extent required for E-selectin recognition. This latter finding suggests there are potential subtle differences between the sLe(x) antigens for E-selectin and HECA-452. Combined, the data indicate that HECA-452 is a non-inhibitor of sLe(x)-mediated adhesion to endothelial expressed E-selectin.

Prior to extravasation at sites of acute inflammation, neutrophils roll over activated endothelium. Neutrophil rolling is often characterized by the average rolling velocity. An additional dynamic feature of rolling that has been identified but not extensively studied is the fluctuation in the rolling velocity about the average. To analyze this characteristic further, we have measured the instantaneous velocity of bovine neutrophils interacting with lipopolysaccharide-stimulated bovine aortic endothelium at shear stresses of 1, 2, 3, and 4 dynes/cm2. The average velocities are quantitatively similar to those reported for human neutrophils rolling over reconstituted P-selectin at a surface density of 400 sites/microns 2. At all shear stresses tested, the population average variance in the instantaneous velocity is at least 2 orders of magnitude higher than the theoretical variance generated from experimental error, indicating that the neutrophils translate with a nonconstant velocity. Possible sources of the variance are discussed. These include "macroscopic" sources such as topological heterogeneity in the endothelium and microscopic sources, such as inherent stochastic formation and breakage of the receptor-ligand bonds that mediate the rolling. Regardless of the ultimate source of the variance, these results justify the use of mathematical models that incorporate stochastic processes to describe bond formation and breakage between the neutrophil and the endothelium and hence are able to generate variable velocity trajectories.

Wnt5a is a noncanonical member of the Wnt family of signaling molecules that has been linked to various physiological and pathological processes including cell differentiation, cell migration, cell growth, vascular remodeling, cancer and chronic inflammation. To understand the role of Wnt5a in these processes, it is necessary to determine the function and expression level of Wnt5a. In this study we developed a sensitive and specific sandwich enzyme-linked immunosorbent assay (ELISA) for detecting Wnt5a. We found that a rabbit anti-human Wnt5a is a suitable capture antibody for establishing a sandwich ELISA. We used two systems to detect Wnt5a: (1) goat anti-mouse Wnt5a and horseradish peroxidase (HRP) conjugated F(ab’)2 donkey anti-goat IgG as detection and enzyme-linked antibodies respectively, or (2) biotinylated goat anti-mouse Wnt5a and HRP-streptavidin as detection antibody and enzyme-linked avidin respectively. A sandwich ELISA using either of these systems failed to detect recombinant mouse (rm) - Wnt5a diluted in Hank’s balanced salt solution supplemented with Ca2+ and Mg2+ and 1% bovine serum albumin (HBBS+, 1% BSA). Addition of polyethylene glycol (PEG) to the HBBS+, 1% BSA buffer during the binding stage of rm-Wnt5a, afforded the detection of rm-Wnt5a. The use of PEG during both the binding of rm-Wnt5a and detection antibody stages of the assay yielded the maximum signal for rm-Wnt5a. The relationship between the ELISA signal and concentration of Wnt5a was linear with an R2 of 0.9934. In summary, we have developed a specific and sensitive sandwich ELISA that detects rm-Wnt5a.

Atherosclerosis is an inflammatory disease involving the accumulation of macrophages in the intima. Wnt5a is a noncanonical member of the Wnt family of secreted glycoproteins. Recently, human macrophages have been shown to express Wnt5a upon stimulation with bacterial pathogens in vitro and in granulomatous lesions in the lung of Mycobacterium tuberculosis-infected patients. Wnt5a expression has also been liked to Toll-like receptor-4 (TLR-4), an innate immune receptor implicated in atherosclerosis. These observations, along with the fact that Wnt5a is involved in cell migration and proliferation, led us to postulate that Wnt5a plays a role in atherosclerosis. To investigate this hypothesis, we characterized Wnt5a expression in murine and human atherosclerotic lesions. Tissue sections derived from the aortic sinus to the aortic arch of apolipoprotein E-deficient mice and sections derived from the carotid arteries of patients undergoing endarterectomy were subjected to immunohistochemical analysis. All samples were found to be positive for Wnt5a with predominant staining in the areas of macrophage accumulation within the intima. In parallel, we probed for the presence of TLR-4 and found coincident TLR-4 and Wnt5a expression. For both the Wnt5a and TLR-4 staining, consecutive tissue sections treated with an isotype- and species-matched Ig served as a negative control and exhibited little, if any, reactivity. Quantitative RT-PCR revealed that Wnt5a mRNA expression in RAW264.7 murine macrophages can be induced by stimulation with LPS, a known ligand for TLR-4. Combined, these findings demonstrate for the first time Wnt5a expression in human and murine atherosclerotic lesions and suggest that cross talk between TLR-4 and Wnt5a is operative in atherosclerosis.

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.

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.

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.

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

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