Functional E-selectin Ligands Research Articles

Expression of functional E-selectin ligands on the plasma membrane of carcinoma cells correlates with poor prognosis in clear cell renal cell carcinoma

ObjectivesGiven the relatively high frequency of metastatic recurrence of clear cell renal cell carcinoma (ccRCC), reliable prognostic markers of ccRCC, particularly those associated with metastasis, are needed. Here, in search of those factors, we assessed the contribution of sialyl Lewis x (sLex) and sialyl Lewis a (sLea), as well as functional E-selectin ligand carbohydrates expressed on carcinoma cells, to metastasis and consequent poor prognosis in ccRCC. Materials and methodsPatients who underwent surgical resection (curative nephrectomy) for RCC, and whose post-operative pathological diagnosis was ccRCC (n = 117) were enrolled in this study. Expression of sLex/sLea carbohydrate antigens in ccRCC was evaluated by immunohistochemistry with an anti-sLex/sLea monoclonal antibody HECA-452. To evaluate membrane expression of sLex/sLea carbohydrate antigens quantitatively, we employed a histological scoring system used to evaluate membrane expression of human epidermal growth factor receptor 2 (HER2) in breast cancer. We also conducted an E-selectin•IgM chimera in situ binding assay to assess expression of functional E-selectin ligand carbohydrates in ccRCC. We then carried out statistical analysis to determine whether membrane expression of HECA-452-reactive sLex/sLea glycans as well as of E-selectin•IgM-binding functional E-selectin ligand carbohydrates correlates with progression-free, overall, or cancer-specific survival. ResultsBased on HECA-452 immunochemistry, 106 of 117 ccRCC specimens expressed detectable levels of sLex/sLea glycans, primarily on the plasma membrane, and of those, 31 that showed robust membrane expression were judged as HECA-452-positive. Membrane expression of HECA-452-positive sLex/sLea glycans correlated with shortened progression-free and overall survival. Moreover, in in situ analysis, these HECA-452-positive ccRCC tissues were decorated with E-selectin•IgM chimeric proteins, calcium-dependently. Comparable analysis in normal kidney showed both HECA-452 positivity and chimera binding almost exclusively in epithelial cells that constitute proximal tubules. Membrane expression of functional E-selectin ligand carbohydrates, as detected by the E-selectin•IgM chimera, correlated more significantly with poor prognosis of patients, namely, shortened progression-free, overall and cancer-specific survival, than did HECA-452 positivity. ConclusionsExpression of E-selectin•IgM-binding functional E-selectin ligand carbohydrates can serve as a reliable and potentially superior prognostic biomarker of patients with ccRCC.

Functional binding of E-selectin to its ligands is enhanced by structural features beyond its lectin domain

Selectins are key to mediating interactions involved in cellular adhesion and migration, underlying processes such as immune responses, metastasis, and transplantation. Selectins are composed of a lectin domain, an epidermal growth factor (EGF)-like domain, multiple short consensus repeats (SCRs), a transmembrane domain, and a cytoplasmic tail. It is well-established that the lectin and EGF domains are required to mediate interactions with ligands; however, the contributions of the other domains in mediating these interactions remain obscure. Using various E-selectin constructs produced in a newly developed silkworm-based expression system and several assays performed under both static and physiological flow conditions, including flow cytometry, glycan array analysis, surface plasmon resonance, and cell-rolling assays, we show here that a reduction in the number of SCR domains is correlated with a decline in functional E-selectin binding to hematopoietic cell E- and/or L-selectin ligand (HCELL) and P-selectin glycoprotein ligand-1 (PSGL-1). Moreover, the binding was significantly improved through E-selectin dimerization and by a substitution (A28H) that mimics an extended conformation of the lectin and EGF domains. Analyses of the association and dissociation rates indicated that the SCR domains, conformational extension, and dimerization collectively contribute to the association rate of E-selectin–ligand binding, whereas just the lectin and EGF domains contribute to the dissociation rate. These findings provide the first evidence of the critical role of the association rate in functional E-selectin–ligand interactions, and they highlight that the SCR domains have an important role that goes beyond the structural extension of the lectin and EGF domains.

Inhibition of Sialylation Impairs Adhesion on Madcam-1 and E-Selectin and Sensitize Multiple Myeloma Cells to Bortezomib in a Xenograft Mouse Model

IntroductionMultiple myeloma (MM) is a cancer of clonal plasma cells that hijack the bone marrow (BM) niche to create a drug resistant, incurable malignancy. Aberrant sialylation has been linked to immune cell evasion, drug resistance, and metastasis in cancer; indeed sialyltransferases, including ST3GAL1, ST3GAL4 and ST3GAL6, are aberrantly expressed in many cancers (Glavey et al., 2014). We have previously shown that targeting ST3GAL6 in MM cells inhibits their ability to extravasate and colonize the BM in mouse models (Glavey et al., 2014). Moreover, we also showed that a subpopulation of MM cells expresses functional E-Selectin ligands which, upon expansion, gives rise to a more aggressive disease and resistance to bortezomib in mice (Natoni et al., 2017). Based off these findings, we herein investigated whether inhibiting sialylation in E-selectin-enriched MM cells with 3Fax-Neu5Ac, a small molecule sialyltransferase inhibitor, could alter the ability of these cells to home in the BM and restore bortezomib sensitivity in vivo. We hypothesized that inhibiting homing of MM cells to the BM will improve survival and that co-treatment with bortezomib and 3Fax-Neu5Ac will have a synergistic effect.MethodsE-selectin ligands enriched MM1S cells (either positive or negative for GFP/Luciferase) were derived from parental cells by cell sorting using the HECA-452 antibody, which recognize sialofucosylated E-selectin ligands. We then determined the 3Fax-Neu5Ac dose and exposure times needed to decrease sialylation on these MM cells without causing toxicity. HECA-452-enriched MM1S cells were pretreated with 3Fax-Neu5Ac or vehicle for 7 days before being injected into SCID-beige mice and then treated with vehicle or bortezomib (0.3 mg/kg twice a week). Mice were analyzed via bioluminescence imaging (BLI) to monitor tumor progression and weighed twice a week. Mice were euthanized when they began to show paralysis under our IACUC protocol. 3Fax-Neu5Ac pretreated HECA-452 MM1S cells were also tested in vitro for their ability to adhere and roll on VCAM-1, MAdCAM-1 and E-Selectin under shear stress and to respond to bortezomib in co-culture with HS5 cells.ResultsTreatment of HECA-452 MM1S cells with 3Fax-Neu5Ac, at 300 μM for 7 days significantly reduced sialylation on these cells. Importantly, reducing sialylation with 3Fax-Neu5AC reduced tumor burden and increased survival, although this did not reach significance for survival (Figure 1A). Both vehicle- and 3Fax-Neu5Ac-treated cells significantly responded to bortezomib in the first 5 weeks of the in vivo study (Figure 1B). However, the HECA-452 MM1S cells did not show increased survival when treated with bortezomib suggesting an acquired mechanism of resistance in vivo. Importantly, pretreatment of the HECA-452 MM1S with 3Fax-Neu5Ac could improve survival of these mice preventing bortezomib resistance. In vitro, the HS5 stromal cells protected the HECA-452 MM1S cells from bortezomib and pretreatment with 3Fax-Neu5Ac partially reverted this protection. Moreover, the HECA-452 MM1S cells pretreated with 3Fax-Neu5Ac displayed reduced adhesion on MAdCAM-1 and E-selectin.ConclusionsSialylation plays an instrumental role in bone homing, BM colonization, and drug resistance of MM cells. Pretreatment of HECA-452 MM1S cells with 3Fax-Neu5Ac decreased their sialylation, restored sensitivity to bortezomib in vivo and prolonged survival in mice. This is likely because 3Fax-Neu5Ac pretreatment has multiple effects on MM cells including reducing cell adhesion mediated-drug resistance and adhesion to key molecules involved in BM homing such as MAdCAM-1 and E-selectin. The reduced adhesion on E-selectin is most likely due to the disruption of E-selectin ligands on the surface of MM cells as they require Sialyl Lewis X to function. Notably, we also found that de-sialylation impairs adhesion on MAdCAM-1 (3Fax-Neu5Ac vs DMSO P=0.038) which, together with E-selectin, is another critical BM homing receptor. This data suggests for the first time that sialylation may controls the affinity of integrin α4β7 and its counter-receptor MAdCAM-1. In turn, this would reduce BM homing and increase MM cells in the circulation were they are more prone to the cytotoxic effects of bortezomib. This study supports the importance of targeting sialylation in MM and provides a strong rationale for further clinical translation of this novel approach. [Display omitted] DisclosuresO'Dwyer:Glycomimetics: Research Funding; Celgene: Research Funding; BMS: Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Onkimmune: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Abstract 98: Cell membrane cholesterol modulates lung cancer cell adhesion and rolling on E-selectin

Abstract Despite numerous advances in cancer research, lung cancer has remained the major cause of cancer-related mortality worldwide, mainly because of metastasis and the lack of curative systemic therapy. It has been well established that lipid rafts, the membrane microdomains containing cholesterol and glycosphingolipids, play an important role in cell adhesion and spreading. Notably, there are elevated levels of cholesterol-rich lipid rafts observed in cancer cells relative to normal ones. Thus, alterations in lipid rafts, such as through cholesterol depletion, have been considered as a strategy for cancer metastasis prevention. In this study three different treatments - methyl-beta-cyclodextrin (mβCD), sphingomyelinase (smase), and simvastatin (simva) - were used to investigate the effects of cholesterol depletion on the mechanics of adhesion and rolling velocities of multiple non-small cell lung cancer (NSCLC) cell lines (H1299, H23, H460, and A569) and a small cell lung cancer (SCLC) cell line (SHP-77) on E-selectin, a vascular endothelial molecule hypothesized to initiate recruitment of circulating tumor cells at metastatic sites. Under physiological conditions in a parallel plate chamber, it was observed that the number of adherent NSCLC cells significantly decreased through mβCD and simva treatments, while smase treatment did not show significant effect on those cells. In addition, only for NSCLC cells, significant increases in rolling velocities were detected after treatments with mβCD and simva, but not via smase. In contrast, cholesterol depletion did not affect the number of attached cells and rolling velocity of the SCLC cells. Our results show that cholesterol might regulate E-selectin-mediated adhesion of NSCLC cells to endothelium, most likely by disturbing functional E-selectin ligands expressed on the surface of the cancer cells. We have previously reported that CD44 expressed on colon cancer and breast cancer cells are functional E-selectin ligands that mediate cell adhesion and rolling on endothelium. Protein structure analysis by flow cytometry and western blot showed that all NSCLC cell lines strongly expressed CD44. After the cholesterol depletion through the various treatments, the molecular surface expression of CD44 was down regulated for all these cells. In contrast, the SCLC line did not show any expression of CD44. Although further studies must be performed to verify CD44 function as an E-selectin ligand, the data in total imply protein loss or absence due to the defection of the cell membrane integrity could impact cell adhesion and rolling, and consequently metastasis. Therefore, cholesterol depletion could have the potential to help prevent cancer metastasis through down regulation of relevant adhesion molecules. Citation Format: Amina Mohammadalipour, Christian Showalter, Harrison T. Muturi, Vishva Sharma, Amir M. Farnoud, Vishwajeet Puri, Sonia M. Najjar, Monica M. Burdick. Cell membrane cholesterol modulates lung cancer cell adhesion and rolling on E-selectin [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 98.

Abstract 2029: Notch signaling inhibitor increases E-selectin/ligand interactions and alters cell migration of mesenchymal-like breast cancer cells

Abstract Activation of Notch signaling has been shown to promote cell migration properties and hematogenous metastasis by inducing a dramatic phenotypic change in breast cancer cells via the epithelial-to-mesenchymal transition (EMT). In response to these findings, Notch signaling inhibitors are being explored as therapeutics to prevent EMT in cancers. However, research examining the effects of Notch inhibitors on the activity of sialofucosylated ligands used by circulating tumor cells to adhere to vascular endothelial E-selectin during metastasis is lacking. To advance knowledge in this area, our study investigated the use of an indirect Notch signaling inhibitor (DAPT) on modifying E-selectin ligand activity of breast cancer cell lines by shear flow adhesion assays performed at physiological bone marrow shear stress. DAPT exposure to mesenchymal-like MDA-MB-231 and MDA-MB-468 (induced to the mesenchymal-like state by exogenous epidermal growth factor) breast cancer cells increased E-selectin ligand activity. Furthermore, mesenchymal-like MDA-MB-468 cells after DAPT exposure demonstrated E-selectin ligand activity similar to that of epithelial-like MDA-MB-468 cells. This supports the hypothesis by our group that epithelial-like and mesenchymal-like phenotypes determine activity of functional E-selectin ligands in breast cancer cells, as we have previously shown that epithelial cells display high E-selectin ligand activity while mesenchymal cells display little or no ligand activity. Cell migration ability was assessed using wound closure assays. Mesenchymal-like MDA-MB-468 cells exposed to DAPT exhibited decreased migration compared to mesenchymal-like MDA-MB-468 cells exposed to the DMSO diluent control, whereas mesenchymal-like MDA-MB-231 cells demonstrated no difference in migration compared to DMSO exposed cells. Altogether, our results suggest Notch inhibitors may both increase fluid-shear resistant adhesion and alter migration ability of mesenchymal-like breast cancer cells, and that factors present in the blood might impact the metastatic potential of cancer cells via regulating cellular phenotypes. Citation Format: Christian A. Showalter, Monica M. Burdick. Notch signaling inhibitor increases E-selectin/ligand interactions and alters cell migration of mesenchymal-like breast cancer cells [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 2029.

Vascular E-Selectin Mediates Chemo-Resistance in Acute Myeloid Leukemia Initiating Cells Via Canonical Receptors PSGL-1 (CD162) and Hcell (CD44) and AKT Signaling

The vascular adhesion molecule E-selectin is a key component of the bone marrow (BM) vascular niche, awakening otherwise dormant hematopoietic stem cells (HSC) (Winkler et al., Nat Med 2012). Vascular niches also mediate malignant cell survival. We find E-selectin becomes upregulated on BM vasculature during leukemia and that adhesion of BM AML blasts to E-selectin promotes survival signaling. This is unique to E-selectin - not observed with adhesion to P-selectin, PECAM1, or integrin ligands ICAM1, VCAM1. In vivo we find absence (in SELE-/- mice) or therapeutic blockade of E-selectin by administration of E-selectin antagonist GMI-1271 to mice, induces 9-fold greater chemosensitivity in 11q23-rearranged AML Initiating Cells (LIC) to cytarabine therapy in vivo and doubles survival over that achieved by chemotherapy alone. Together these data support the Phase I/II clinical trial of GMI-1271 in combination with intensive chemotherapy to improve efficacy of therapy for AML (NCT02306291), and raise the further questions: 1) what are the AML LIC ligands interacting with E-selectin at the vascular niche and 2) what are the pathways initiated by E-selectin adhesion that mediate chemoresistance.Using murine models of AML generated by retroviral transduction of the 11q23 fusion oncogene MLL-AF9 into HSC, we find leukemic blasts rapidly upregulate E-selectin binding potential upon oncogenic transformation. To investigate if this is due to aberrant glycosylation facilitating the generation of de novo AML cell surface receptors, we generated AML from HSC knocked-out for the two canonical E-selectin receptors PSGL1 and CD44. Surprisingly we found although HSC from CD44-/-PSGL1-/- mice still bound to E-selectin (indicating HSC express additional E-selectin ligands), AML cells generated from these same HSCs no longer did. Thus the functional E-selectin ligands utilized by murine HSC and AML blasts differ.When transplanted into recipient mice CD44-/-PSGL1-/- AML engrafted with similar disease progression to matching wildtype AML but demonstrated ~100-fold greater sensitivity to high-dose cytarabine chemotherapy. When GMI-1271 was co-administered together with chemotherapy no further boost in chemosensitivity was observed in CD44-/-PSGL1-/- AMLs. This complements in vitro data showing E-selectin adhesion-mediated chemoresistance is lost when CD44 and/orPSGL1are absent in AML cells. Together suggesting that CD44 and/or PSGL1 are the key receptors involved in E-selectin-mediated pro-survival signaling.To understand the pro-survival signaling pathways involved, E-selectin antagonist GMI1271 or saline control were administered 5 days to mice with AML and BM KIT+ leukemic blasts sorted. RNA sequencing revealed only a small number (~200) of mRNA transcripts differentially regulated following therapeutic E-selectin blockade. Of note these included down-regulation in several components of the PI3K/AKT/NF-kB signaling pathway frequently involved in cell survival. Indeed AKT Ser473 and NF-kB p65 Ser536 were found to be rapidly phosphorylated within 15 minutes of E-selectin adhesion in AML blasts in vitro. This phosphorylation was specific to E-selectin adhesion and not observed with other vascular adhesion molecules tested suggesting that direct E-selectin-mediated AKT activation in AML cells at the vascular niche may be the potential mechanism driving vascular mediated chemoresistance.To confirm whether therapeutic blockade of E-selectin alone dampens intracellular AKT signaling in AML blasts, cohorts of mice with wildtype or matching CD44-/-PSGL1-/- AML were administered GMI-1271 for 5 days then BM AML blasts collected for signaling studies. Both the administration of E-selectin antagonist or absence of CD44 and/or PSGL-1 dampened AKTSer473 phosphorylation in BM AML blasts in vivo, correlating with their heightened sensitivity to chemotherapy .In summary, these findings suggest E-selectin ligands on specific CD44 and/or PSGL1 glycoforms are the predominant determinants through which the induction/amplification of chemoresistant pathways in AML blasts occur following vascular adhesion and further that these pathways are coupled to an early phosphorylation event of AKT - and finally explain potential mechanism how GMI-1271 administration is able to attenuate vascular-mediated signaling and restore susceptibility of AML to chemotherapy. DisclosuresMagnani:GlycoMimetics, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Selective Expression of CCR10 and CXCR3 by Circulating Human Herpes Simplex Virus-Specific CD8 T Cells

Herpes simplex virus (HSV) infection is restricted to epithelial cells and neurons and is controlled by CD8 T cells. These cells both traffic to epithelial sites of recurrent lytic infection and to ganglia and persist at the dermal-epidermal junction for up to 12 weeks after lesion resolution. We previously showed that cutaneous lymphocyte-associated antigen (CLA), a functional E-selectin ligand (ESL), is selectively expressed on circulating HSV-2-specific CD8 T cells. CLA/ESL mediates adhesion of T cells to inflamed vascular endothelium. Later stages in T-cell homing involve chemokines (Ch) and lymphocyte chemokine receptors (ChR) for vascular wall arrest and diapedesis. Several candidate ChR have been implicated in skin homing. We measured cell surface ChR on HSV-specific human peripheral blood CD8 T cells and extended our studies to HSV-1. We observed preferential cell surface expression of CCR10 and CXCR3 by HSV-specific CD8 T cells compared to CD8 T cells specific for control viruses, Epstein-Barr virus (EBV) and cytomegalovirus (CMV), and compared to bulk memory CD8 T cells. CXCR3 ligand mRNA levels were selectively increased in skin biopsy specimens from persons with recurrent HSV-2, while the mRNA levels of the CCR10 ligand CCL27 were equivalent in lesion and control skin. Our data are consistent with a model in which CCL27 drives baseline recruitment of HSV-specific CD8 T cells expressing CCR10, while interferon-responsive CXCR3 ligands recruit additional cells in response to virus-driven inflammation.IMPORTANCE HSV-2 causes very localized recurrent infections in the skin and genital mucosa. Virus-specific CD8 T cells home to the site of recurrent infection and participate in viral clearance. The exit of T cells from the blood involves the use of chemokine receptors on the T-cell surface and chemokines that are present in infected tissue. In this study, circulating HSV-2-specific CD8 T cells were identified using specific fluorescent tetramer reagents, and their expression of several candidate skin-homing-associated chemokine receptors was measured using flow cytometry. We found that two chemokine receptors, CXCR3 and CCR10, are upregulated on HSV-specific CD8 T cells in blood. The chemokines corresponding to these receptors are also expressed in infected tissues. Vaccine strategies to prime CD8 T cells to home to HSV lesions should elicit these chemokine receptors if possible to increase the homing of vaccine-primed cells to sites of infection.

Abstract 866: The epithelial-to-mesenchymal transition regulates E-selectin ligand activities of breast cancer cells

Abstract The epithelial-to-mesenchymal transition (EMT) leads to specific stem-like and invasive properties in breast cancer cells. However, it remains unclear whether the EMT modifies sialofucosylated ligands that circulating tumor cells use to attach and adhere to vascular endothelial E-selectin during metastasis. To address this issue, immunohistochemistry and E-selectin microsphere dynamic adhesion assays were initially used to assay 110 cases of breast cancer for EMT biomarkers and E-selectin ligand activity. The expression of E-cadherin, but not N-cadherin or vimentin, directly correlated with E-selectin ligand activity. Thus, it was hypothesized that the EMT, and in turn the mesenchymal-to-epithelial transition (MET), regulate expression of functional E-selectin ligands in breast cancer cells. The ectopic expression or shRNA knockdown of transcription factors (i.e., Snail and Twist) in target cell lines were used to induce EMT or MET, respectively. Shear flow adhesion assays and flow cytometry demonstrated that cells with epithelial phenotypes had greater E-selectin ligand activities and greater expression of sialofucosylated carbohydrates compared to cells with mesenchymal phenotypes. Moreover, glycoprotein E-selectin ligand activities of breast cancer cells were more affected by the EMT compared to glycolipid E-selectin ligand activities. Assessing expression of α1,3- and α1,4- fucosyltransferases (FUT) at the RNA level using qRT-PCR revealed that FUT3 and FUT6 gene expression were decreased via the EMT, corresponding to the lowered E-selectin ligand activities observed in mesenchymal phenotype cells. Altogether, the results of this investigation demonstrate that the EMT, as well as the MET, mechanistically regulate the expression of functional E-selectin ligands expressed by breast cancer cells. Citation Format: Grady E. Carlson, Alexander O. Ostermann, Venktesh S. Shirure, Fabian Benencia, Monica M. Burdick. The epithelial-to-mesenchymal transition regulates E-selectin ligand activities of breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 866. doi:10.1158/1538-7445.AM2017-866

An Analysis of Trafficking Receptors Shows that CD44 and P-Selectin Glycoprotein Ligand-1 Collectively Control the Migration of Activated Human T-Cells.

Selectins guide the traffic of activated T-cells through the blood stream by mediating their tethering and rolling onto inflamed endothelium, in this way acting as beacons to help navigate them to sites of inflammation. Here, we present a comprehensive analysis of E-selectin ligands expressed on activated human T-cells. We identified several novel glycoproteins that function as E-selectin ligands. Specifically, we compared the role of P-selectin glycoprotein ligand-1 (PSGL-1) and CD43, known E-selectin ligands, to CD44, a ligand that has not previously been characterized as an E-selectin ligand on activated human T-cells. We showed that CD44 acts as a functional E-selectin ligand when expressed on both CD4+ and CD8+ T-cells. Moreover, the CD44 protein carries a binding epitope identifying it as hematopoietic cell E- and/or L-selectin ligand (HCELL). Furthermore, by knocking down these ligands individually or together in primary activated human T-cells, we demonstrated that CD44/HCELL, and not CD43, cooperates with PSGL-1 as a major E-selectin ligand. Additionally, we demonstrated the relevance of our findings to chronic autoimmune disease, by showing that CD44/HCELL and PSGL-1, but not CD43, from T-cells isolated from psoriasis patients, bind E-selectin.

E-selectin ligands recognised by HECA452 induce drug resistance in myeloma, which is overcome by the E-selectin antagonist, GMI-1271

Multiple myeloma (MM) is characterized by the clonal expansion and metastatic spread of malignant plasma cells to multiple sites in the bone marrow (BM). Recently, we implicated the sialyltransferase ST3Gal-6, an enzyme critical to the generation of E-selectin ligands, in MM BM homing and resistance to therapy. Since E-selectin is constitutively expressed in the BM microvasculature, we wished to establish the contribution of E-selectin ligands to MM biology. We report that functional E-selectin ligands are restricted to a minor subpopulation of MM cell lines which, upon expansion, demonstrate specific and robust interaction with recombinant E-selectin in vitro. Moreover, an increase in the mRNA levels of genes involved in the generation of E-selectin ligands was associated with inferior progression-free survival in the CoMMpass study. In vivo, E-selectin ligand-enriched cells induced a more aggressive disease and were completely insensitive to Bortezomib. Importantly, this resistance could be reverted by co-administration of GMI-1271, a specific glycomimetic antagonist of E-selectin. Finally, we report that E-selectin ligand-bearing cells are present in primary MM samples from BM and peripheral blood with a higher proportion seen in relapsed patients. This study provides a rationale for targeting E-selectin receptor/ligand interactions to overcome MM metastasis and chemoresistance.

The Dual E-Selectin/CXCR4 Inhibitor, GMI-1359, Enhances Efficacy of Anti-Leukemia Chemotherapy in FLT3-ITD Mutated Acute Myeloid Leukemia

Aberrant activation of the FMS-like tyrosine kinase-3 (FLT3) is driven by internal tandem duplication (ITD) mutations in the FLT3 gene, which are commonly observed in patients with acute myeloid leukemia (AML). Hence, FLT3 represents an attractive therapeutic target in AML (Weisberg et al., 2002). Indeed, several small molecule FLT3 inhibitors including sorafenib have showed encouraging efficacy in reducing leukemia blasts in the peripheral blood in FLT3 mutated AML patients. However, these agents have little effect on leukemic stem cells in the bone marrow (BM) microenvironment (Borthakur et al., 2011; Fathi and Chabner, 2011; Zhang et al., 2008).The BM microenvironment is enriched with cytokines and adhesion molecules, such as CXCR4 and E-selectin, which are believed to provide AML cells protection against chemotherapeutic agents (Horacek et al., 2013; Peled and Tavor, 2013). In fact, treatment with sorafenib markedly upregulated CXCR4 levels in FLT3 -mutated cells. In addition, leukemia cells can activate endothelial cells (EC) that induce adhesion of a sub-set of the leukemia cells through E-selectin. The adherent AML cells are sequestered in a nonproliferative state that further protects them from chemotherapy (Pezeshkian et al., 2013). Therefore, blocking CXCR4 and E-selectin in parallel could theoretically eliminate the protection provided by the interaction of leukemic cells with their BM microenvironment and enhance effectiveness of chemotherapy in FLT3-mutant AML patients. In the present study, we evaluated the effectiveness of a dual CXCR4 and E-selectin antagonist, GMI-1359 (GlycoMimetics, Inc., Rockville, MD), in targeting FLT3-ITD-mutant AML in vitro and in vivo. High levels of CXCR4 expression were observed in several human and murine AML cell lines, which was further increased in hypoxic (i.e., 1% oxygen) conditions that mimic the BM microenvironment. These FLT3 -ITD leukemic cell lines also expressed hypoxia-responsive, functional E-selectin ligands identified by reactivity with an antibody (HECA452) that binds the same carbohydrate epitope required for binding to E-selectin. One such E-selectin ligand CD44 increased in FLT3 -ITD cells cultured in hypoxia compared to those cultured in normoxia (i.e. 21% oxygen). In addition, hypoxia also enhanced CXCR4 expression on mesenchymal stem cells (MSC) and EC such as HUVEC. In hypoxic co-cultures of the FLT3 -ITD-mutant leukemia cells MV4-11 or MOLM14 with MSCs and ECs (i.e., HUVEC or TeloHAEC), the presence of the dual E-selectin/CXCR4 inhibitor GMI-1359 effectively reduced leukemic cell adhesion by ~ 50% to the MSC/EC feeder layer compared to the PBS-treated control (p<0.05), even in the presence of TNFa, which induces E-selectin expression in EC. However, an E-selectin specific inhibitor only reduced adhesion of MV4-11 and MOLM14 by ~ 20%. GMI-1359 markedly abrogated the protection provided by the BM microenvironment (i.e., hypoxia and/or MSC and EC) of Baf3-FLT3 -ITD leukemic cells treated with the FLT3 inhibitor sorafenib. Apoptosis was induced in 36.6%, 35.6% and 48.9% of leukemic cells cultured with sorafenib alone, sorafenib and an E-selectin inhibitor or sorafenib and GMI-1359, respectively.The significance of these in vitro findings were studied in vivo. Female SCID beige mice were injected iv with MV4-11 and followed for survival. Beginning 14 days post tumor injection, cohorts of mice (n=10/group) were treated with saline, GMI-1359 (40 mg/kg), standard chemotherapy cytarabine plus daunorubicin, or a combination of GMI-1359 and chemotherapy. Combined treatment of mice with GMI-1359 (40 mg/kg) and chemotherapy demonstrated a profound survival benefit compared to controls or chemotherapy alone at day 135 after leukemia cell injection (i.e., 67% vs. 11% or 30%, p=0.0011 and 0.0406, respectively). Single agent treatment with GMI-1359 was statistically indistinguishable from saline alone or chemotherapy alone. In a separate cohort of MV4.11-engrafted mice, the single administration of GMI-1359 increased circulating WBC and leukemic MV4-11cells, which persisted for at least 8 hrs. This effect was consistent with GMI-1359 disrupting the protective effects of the tumor microenvironment and mobilizing MV4-11 cells from the BM niche.. These findings provide the pre-clinical basis for the evaluation of GMI-1359 in patients with FLT3 -mutant AML. [Display omitted] DisclosuresZhang:Karyopharm: Research Funding. Fogler:GlycoMimetics, Inc.: Employment. Magnani:GlycoMimetics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

E-Selectin Ligand Expression Increases with Progression of Myeloma and Induces Drug Resistance in a Murine Transplant Model, Which Is Overcome By the Glycomimetic E-Selectin Antagonist, GMI-1271

There is increasing evidence that E-selectin and its ligands play an important role in the progression of multiple myeloma (MM) and drug resistance. We reported that the sialyltransferase ST3GAL6 influences homing and survival in MM, and postulated that it may function in the synthesis of E-selectin ligands (Glavey et al Blood, 2014). We also found that a small subpopulation of cells (~ 5%) from MM cell lines express functional E-selectin ligands, which could be expanded under hypoxic conditions typical of the bone marrow (BM) microenvironment. These cells were identified by reactivity with an antibody (HECA452), which binds the same carbohydrate epitope required for binding to E-selectin. Rolling of MM1S cells on E-selectin was blocked by a small molecule glycomimetic antagonist to E-selectin (GMI-1271). Moreover, GMI-1271 significantly enhanced the anti-myeloma activity of bortezomib (BTZ) in an in vivo murine transplant model (Natoni et al Blood, 2014). We now extend these observations to obtain a more complete understanding of the role E-selectin plays in MM biology and chemotherapy resistance for its potential clinical relevance.The parental, heterogeneous MM cell lines MM1S and RPMI8226 (MM1Spar, RPMI8226par, respectively) were sequentially sorted to obtain cell lines highly enriched (>85% and 80%) for the expression of cell surface carbohydrates bound by HECA452, and designated MM1SHECA452 and RPMI8226HECA452. The cell lines could be passaged in vitro and were stable for enriched E-selectin ligand expression. In contrast to parental cells, both MM1SHECA452 and RPMI8226HECA452 showed strong binding to E-selectin in static adhesion assays. Both MM1SHECA452 and RPMI8226HECA452 exhibited strong rolling on E-selectin under shear stress. MM1Spar or RPMI8226par failed to roll well on E-selectin. The addition of GMI-1271 during culture conditions led to a marked reduction in adhesion of MM1SHECA452 and profoundly inhibited rolling on E-selectin of both HECA452 enriched MM cell lines.The significance of these in vitro findings was studied in vivo. MM1Spar or MM1SHECA452 cells were injected i.v. into SCID beige mice. Beginning 5 days post tumor injection, the survival impact of treatment with saline control, GMI-1271, BTZ or a combination of both was determined. Mice transplanted with MM1SHECA452 had more aggressive disease with significantly shorter survival compared to those transplanted with MM1Spar. In contrast to MM1Spar cells, mice engrafted with MM1SHECA452 demonstrated a marked resistance to BTZ treatment. Whereas GMI-1271 treatment alone had no impact on survival, the combination of GMI-1271 and BTZ led to a highly significant improvement in survival of MM1Spar engrafted mice (P=0.0363), and more importantly broke the resistance and restored the anti-myeloma activity of BTZ in MM1SHECA452 engrafted mice (P=0.0123) (figure 1). The number of peripheral blood (PB) human CD138+ cells was increased in MM1SHECA452-engrafted mice within 60 min following a single injection of GMI-1271, and persisted for at least 24 hours (2.37% v. 0.03%, p <0.001). This effect was consistent with GMI-1271 disrupting the tumor microenvironment and mobilizing MM1SHECA-452 cells from the BM niche.Given these findings we wished to see if samples from patients with MM express E-selectin ligands and whether higher levels are seen with disease progression. BM and/or PB were obtained following informed consent from patients with MM and plasma cells (CD38+/CD138+) were analyzed for E-selectin ligand expression by flow cytometry using the HECA452 antibody. To date all primary MM samples (n=25) contained HECA452-reactive cell populations (median 22%). A consistently higher proportion of circulating MM cells express HECA452 when compared with paired BM samples (n=14), with a median difference of 33% (Wilcoxon signed rank test, p=0.02). HECA452 expression of MM in PB was significantly higher (on average 40% higher) in samples taken at relapse vs. diagnosis, (unpaired t test, p = 0.0008)These data provide compelling evidence that E-selectin ligand bearing cells play an important role in disease progression and drug resistance in MM, and a strong rationale for clinical strategies incorporating GMI-1271 to improve patient outcome. [Display omitted] DisclosuresSmith:GlycoMimetics, Inc.: Employment. Locatelli-Hoops:GlycoMimetics, Inc.: Employment. Oliva:GlycoMimetics, Inc.: Employment. Fogler:GlycoMimetics, Inc.: Employment. Magnani:GlycoMimetics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. O'Dwyer:Celgene: Honoraria, Research Funding.

Abstract 342: Breast cancer adhesion cascade is influenced by Galectin-1 and E-selectin receptor-ligand interactions

Abstract Invasion of other tissues during bloodborne metastasis requires the adhesion of cancer cells to the endothelium by specific fluid shear-dependent receptor-ligand interactions. It is hypothesized that ligands are shared between the endothelial adhesion molecule known as E-selectin, a transmembrane glycoprotein, and Galectin-1 (Gal-1), a secretory dimer protein, both of which are upregulated during inflammation and cancer. Our previous data strongly indicate that this shear-dependent interaction involves the same or closely related cancer cell-expressed ligands, such as the glycoprotein Mac-2BP. Initial investigation using immuno-staining methods to locate E-selectin and Gal-1 ligands and determine relative proximity to one another revealed that E-selectin and Gal-1 ligands on ZR-75-1 breast cancer cells and breast cancer tissue microarrays had a high percentage of colocalization, as high as 75% in some cases. These results allude to the possibility that these reactive molecules are in close proximity or are in fact the same molecule. Subsequently, E-selectin microspheres were perfused over Gal-1-treated ZR-75-1 cell monolayers and breast cancer tissue microarrays to determine the functional relationship, if any, between Gal-1 and E-selectin ligands during adhesion from flow. Flow assay data show that Gal-1 treatment enhances E-selectin microsphere interactions to a breast cancer monolayer in a shear-dependent manner, with a shear stress of 0.5 dynes/cm2 having an almost 300% increase in binding compared to the untreated monolayer. This amount of binding at a physiological shear stress gives credence to the hypothesis that Gal-1 influences E-selectin receptor-ligand interactions via the same or similar ligands during breast cancer cell adhesion. Dynamic biochemical tissue analysis (DBTA), a novel flow-based tissue interrogation technique developed in our lab, was then implemented to test human invasive ductal breast carcinoma tissue sections. Similar to ZR-75-1 cell line flow assay data, a 142% increase in shear-dependent binding interactions between the E-selectin microspheres and the Gal-1 treated breast cancer tissue sections at 0.5 dynes/cm2 shear stress was observed. Colocalization of reactive signals and increased shear-dependent flow interactions indicate that metastatic binding in breast cancer is aided through E-selectin to Gal-1 interaction via a shared ligand intermediate. Ascertaining the mechanism behind this interaction would lead to a better understanding of the circulating breast cancer cells’ adhesion cascade during metastasis. Citation Format: Nathan M. Reynolds, Claire R. Hall, Sean E. Thomas, Monica M. Burdick. Breast cancer adhesion cascade is influenced by Galectin-1 and E-selectin receptor-ligand interactions. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 342. doi:10.1158/1538-7445.AM2015-342

Multiple Myeloma Cells Express Functional E-Selectin Ligands Which Can be Inhibited Both in-Vitro and in-Vivo Leading to Prolongation of Survival in a Murine Transplant Model

IntroductionSelectins and their ligands play an important role in the adhesion and trafficking of multiple myeloma (MM) cells. While PSGL-1 is strongly expressed by MM cells, E-selectin binding is seen in a small minority only and its contribution to MM cell trafficking is unclear. As E-selectin is constitutively expressed by BM endothelial cells it may play a critical role in the BM homing and retention of MM cells. We wished to determine (1) if MM cells express functional E-selectin ligands (2) whether hypoxia enhances E-selectin ligand positivity (3) the potential of the E-selectin inhibitor GMI-1271 to prolong survival in a murine model of MM minimal residual disease (4) the ability to identify patients most likely to benefit from E-selectin inhibition.MethodsTo detect E-selectin ligands MM cell lines were stained with an antibody to HECA452, which indicates reactivity with sialofucosylated glycoforms of PSGL-1 and CD44, which can bind E-selectin. To test the effect of hypoxia, MM cells were maintained in either 1% O2 or normoxia at 37¡C for 5 days and then stained with HECA452. MM cells were sorted into E-selectin ligand positive and negative populations based on HECA452 staining. We evaluated rolling of MM cells on E-selectin +/- 10 µM GMI-1271 for 1 h at 37¡C, following which 50 µl of cell suspension were loaded onto a channel of a microfluidic chip (Cellix) pre-coated with 30 µg/ml of recombinant E-Selectin (Peprotech). Cells were perfused into the channel at 0.5 Dyne/cm2 using Mirus Evo Nanopump (Cellix). After 2 min of perfusion, images were acquired at 5 different positions of the channel using an AX10 Vert.A1 microscope (Zeiss) equipped with aQImaging QIClickª digital CCD camera. Thirty frames were acquired at 5 different positions with 0.5 sec between each frame. Each sample was assayed in triplicate. To evaluate in-vivo activity of GMI-1271, SCID-beige mice were injected with luciferase-expressing MM1S cells. Seventy two hours after inoculation, mice were divided into cohorts treated with vehicle, GMI-1271 (40mg/kg bid), bortezomib (0.75 mg/kg IP weekly for 4 weeks), or a combination of GMI-1271 and bortezomib. Tumor burden was determined by bioluminescence imaging and animals were followed for survival. We evaluated gene expression levels of 2 genes involved in selectin ligand expression, the sialyltransferase ST3GAL6 and FUCA1 (fucosidase), which we have previously shown to be important in MM prognosis, in the context of ISS and FISH in the MRCIX dataset. Specifically, we analyzed the interaction of high ST3GAL6 and low FUCA1 with ISS/FISH risk, where patients are considered high risk if ISS=3 along with 17pdel or t(4;14).ResultsTwelve percent (12%) of MM1S and 15.8% of RPMI8226 cells were HECA452 positive. We consistently observed rolling of sorted HECA452 positive RPMI8226 and MM1S cells on E-selectin with absence of rolling in HECA452 negative fraction. Treatment with GMI-1271 completely inhibited rolling on E-selectin (100% inhibition). Prolonged exposure of RPMI8226 cells to hypoxia resulted in a significant increase in HECA452 positive cells (38.8% versus 25.2%, P=0.0215), consistent with our previous observation that hypoxia upregulates ST3GAL6. The survival of mice treated with either GMI-1271 alone or bortezomib alone was significantly greater than vehicle treated control mice. GMI-1271 in combination with bortezomib treatment however, resulted in the longest survival and significantly longer than treatment with bortezomib alone (p=0.0025). The high ST3GAL6/low FUCA1 signature was overrepresented in high risk compared to low risk [39% (6% of whole population) .v. 13.9% p=0.01] patients and conferred poor prognosis with a median survival of 10 months versus 33 months in the remaining 61% of high-risk patients. On multivariate analysis this pattern was independent of ISS/FISH for OS (p=0.013).ConclusionsA small fraction of MM cells express functional E-selectin ligands, which may be higher in the hypoxic BM niche. The binding of these cells to E-selectin is blocked by GMI-1271 and GMI-1271 significantly enhances the activity of bortezomib in vivo. These data indicate the importance of E-selectin ligand expressing MM cells in homing and engraftment and provide a rationale for targeting E-selectin in MM. Furthermore, high ST3GAL6 + low FUCA1 identifies a subgroup of patients with high risk ISS/FISH with particularly poor outcome who may benefit from E-selectin inhibition. [Display omitted] DisclosuresMagnani:GlycoMimetics Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees. Ghobrial:GlycoMimetics: Consultancy, Research Funding. O’Dwyer:GlycoMimetics: Consultancy, Research Funding.

CD44 variant isoforms expressed by breast cancer cells are functional E-selectin ligands under flow conditions.

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.

Mac-2 Binding Protein Is a Novel E-Selectin Ligand Expressed by Breast Cancer Cells

Hematogenous metastasis involves the adhesion of circulating tumor cells to vascular endothelium of the secondary site. We hypothesized that breast cancer cell adhesion is mediated by interaction of endothelial E-selectin with its glycoprotein counter-receptor(s) expressed on breast cancer cells. At a hematogenous wall shear rate, ZR-75-1 breast cancer cells specifically adhered to E-selectin expressing human umbilical vein endothelial cells when tested in parallel plate flow chamber adhesion assays. Consistent with their E-selectin ligand activity, ZR-75-1 cells expressed flow cytometrically detectable epitopes of HECA-452 mAb, which recognizes high efficiency E-selectin ligands typified by sialofucosylated moieties. Multiple E-selectin reactive proteins expressed by ZR-75-1 cells were revealed by immunoprecipitation with E-selectin chimera (E-Ig chimera) followed by Western blotting. Mass spectrometry analysis of the 72 kDa protein, which exhibited the most prominent E-selectin ligand activity, corresponded to Mac-2 binding protein (Mac-2BP), a heretofore unidentified E-selectin ligand. Immunoprecipitated Mac-2BP expressed sialofucosylated epitopes and possessed E-selectin ligand activity when tested by Western blot analysis using HECA-452 mAb and E-Ig chimera, respectively, demonstrating that Mac-2BP is a novel high efficiency E-selectin ligand. Furthermore, silencing the expression of Mac-2BP from ZR-75-1 cells by shRNA markedly reduced their adhesion to E-selectin expressing cells under physiological flow conditions, confirming the functional E-selectin ligand activity of Mac-2BP on intact cells. In addition to ZR-75-1 cells, several other E-selectin ligand positive breast cancer cell lines expressed Mac-2BP as detected by Western blot and flow cytometry, suggesting that Mac-2BP may be an E-selectin ligand in a variety of breast cancer types. Further, invasive breast carcinoma tissue showed co-localized expression of Mac-2BP and HECA-452 antigens by fluorescence microscopy, underscoring the possible role of Mac-2BP as an E-selectin ligand. In summary, breast cancer cells express Mac-2BP as a novel E-selectin ligand, potentially revealing a new prognostic and therapeutic target for breast cancer.

Abstract 5181: Dynamic cellular adhesivity of breast cancer cells conferred by CD44 varies with mesenchymal or epithelial phenotype

Abstract 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

Abstract 4566: Dynamic biochemical tissue analysis: A novel in situ assay that demonstrates potential as a diagnostic/prognostic for cancer

Abstract 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

Expression of E-selectin ligands on circulating tumor cells: cross-regulation with cancer stem cell regulatory pathways?

Although significant progress has been made in the fight against cancer, successful treatment strategies have yet to be developed to combat those tumors that have metastasized to distant organs. Poor characterization of the molecular mechanisms of cancer spread is a major impediment to designing predictive diagnostics and effective clinical interventions against late stage disease. In hematogenous metastasis, it is widely suspected that circulating tumor cells (CTCs) express specific adhesion molecules that actively initiate contact with the vascular endothelium lining the vessel walls of the target organ. This “tethering” is mediated by ligands expressed by CTCs that bind to E-selectin expressed by endothelial cells. However, it is currently unknown whether expression of functional E-selectin ligands on CTCs is related to cancer stem cell regulatory or maintenance pathways, particularly epithelial-to-mesenchymal transition and the reverse, mesenchymal-to-epithelial transition. In this hypothesis and theory article, we explore the potential roles of these mechanisms on the dynamic regulation of selectin ligands mediating CTC trafficking during metastasis.

Gangliosides expressed on breast cancer cells are E-selectin ligands

Cancer cell adhesion to vascular endothelium is a critical process in hematogenous metastasis. We hypothesized that breast cancer cells express ligands that bind under blood flow conditions to E-selectin expressed by endothelial cells. At a hemodynamic wall shear rate, BT-20 and MDA-MB-468 breast cancer cells adhered to cytokine-activated human umbilical cord vein endothelial cells (HUVECs) but not to anti-E-selectin monoclonal antibody treated HUVECs, demonstrating that adhesion was specifically mediated by E-selectin. Characterization of glycans expressed on breast cancer cells by a panel of antibodies revealed that BT-20 cells expressed sialyl Lewis X (sLe x) and sialyl Lewis A (sLe a) but MDA-MB-468 cells did not, suggesting that the former possess classical glycans involved in E-selectin mediated adhesion while the latter have novel binding epitopes. Protease treatment of the breast cancer cells failed to significantly alter the carbohydrate expression profiles, binding to soluble E-selectin–Ig chimera, or the ability of the cells to tether and roll on E-selectin expressed by HUVECs, indicating that glycosphingolipids are functional E-selectin ligands on these cells. Furthermore, extracted breast cancer cell gangliosides supported binding of E-selectin–Ig chimera and adhesion of E-selectin transfected cells under physiological flow conditions. In summary, our results demonstrate that breast cancer cells express sialylated glycosphingolipids (gangliosides) as E-selectin ligands that may be targeted for prevention of metastasis.