Venktesh S. Shirure

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.

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.

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.

Adhesion to substrates induces dendritic cell endothelization and decreases immunological response.

Dendritic cells (DCs) are antigen presenting cells capable of inducing specific immune responses against microbial infections, transplant antigens, or tumors. DCs have been shown to possess a high plasticity showing different phenotypes in response to their microenvironment. For example, tumor-associated DCs can acquire an angiogenic phenotype thus promoting tumor growth. Further, DCs cultured in vitro under different conditions are able to upregulate the expression of endothelial markers and to express angiogenic factors. Indeed, it has been shown that soluble factors such as VEGF of PGE-2, that are present in the microenvironment of several tumors, affect the biology of these cells. We hypothesize that in addition to soluble factors the adhesion to different substrates will also define the phenotype and function of DCs. Herewith we demonstrate that murine myeloid(m) DCs upregulate endothelial markers such as VE-Cadherin, and to a lesser extent TIE-2, and decrease their immune capabilities when cultured on solid surfaces as compared with the same cells cultured on ultra-low binding (ULB) surfaces. On the other hand, the expression of angiogenic molecules at the level of RNA was not different among these cultures. In order to further investigate this phenomenon we used the murine ID8 model of ovarian cancer which can generate solid tumors when cancer cells are injected subcutaneously or a malignant ascites when they are injected intraperitoneally. This model gave us the unique opportunity to investigate DCs in suspension or attached to solid surfaces under the influence of the same tumor cells. We were able to determine that DCs present in solid tumors showed higher levels of expression of endothelial markers and angiogenic molecules but were not able to respond to inflammatory stimuli at the same extent as DCs recovered from ascites. Moreover, mDCs cultured on ULB surfaces in the presence of tumor factors do not expressed endothelial markers. Taking into account all these data we consider that tumor factors might be responsible for inducing angiogenic properties in DCs, but that in some settings the expression of endothelial markers such as VE-Cadherin and TIE-2 might be a function of attachment to solid surfaces and independent of the angiogenic properties of these cells.

UVB-irradiation regulates VLA-4-mediated melanoma cell adhesion to endothelial VCAM-1 under flow conditions.

The major aspect contributing to the mortality of melanoma is its ability to spread, or metastasize. Ultraviolet B light (UVB) is considered an indirect cause of melanoma formation. However, little is known about the potential effects of UVB to melanoma metastasis. Integrins, a large family of cell adhesion molecules (CAMs) expressed on the melanoma cell surface, are important for cell signaling, growth, and migration during metastasis. Most critically, tumor cell tissue invasion is dependent on the initial interaction of tumor cells with vascular endothelium at the target organ, and there is increasing evidence for a prominent role of melanoma very late antigen‐4 (VLA‐4) integrin binding to its endothelial ligand vascular cell adhesion molecule‐1 (VCAM‐1) in this process. This research focuses on the quantitative modulation of VLA‐4 integrin expression and function on melanoma cells after UVB irradiation. The present data show that at 3, 12, and 18 h post‐UVB irradiation, VLA‐4 expression was unchanged relative to untreated cells, but adhesion to VCAM‐1 decreased significantly. Immunofluorescence studies implied that the spatial organization of VLA‐4 on the melanoma cell surface contributed to the changes in avidity for VCAM‐1 upon UVB irradiation. With increased understanding of the molecular mechanisms underlying melanoma–endothelial interactions upon UVB irradiation, clinical advances for melanoma may be developed.

HECA-452 is a non-function blocking antibody for isolated sialyl Lewis x adhesion to endothelial expressed E-selectin under flow conditions.

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.

Dynamic biochemical tissue analysis detects functional L-selectin ligands on colon cancer tissues

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.

Abstract 1609: The epithelial to mesenchymal transition regulates the expression of E-selectin ligands on breast cancer cell lines

Breast cancer cells garner invasive and stem-like characteristics through the epithelial to mesenchymal transition (EMT). Consequently, the EMT has been implicated in cancer metastasis. While many of the effects of the EMT have been elucidated, it is currently unknown how the EMT modifies the expression of E-selectin ligands on breast cancer cells. These ligands are often sialofucosylated glycans that mediate cell adhesion with E-selectin molecules presented by endothelial cells lining the blood vessel walls at the metastatic site, which is commonly bone marrow. Although E-selectin is widely recognized for its role in promoting adhesion during tumor cell extravasation, it also maintains the quiescence of hematopoietic stem cells in the vascular niche of the bone marrow. Thus in this investigation, we studied the effects of the EMT on the E-selectin ligand activities of breast cancer cell lines and their behavior in an E-selectin rich environment. The EMT was induced in breast cancer cell lines by ectopic expression of either the Snail or Twist transcription factors. Through the EMT breast cancer cells upregulated the expression of vimentin and N-cadherin, yet reduced the expression of CD24, E-cadherin, and E-selectin ligands. Breast cancer cell lines were assayed for E-selectin ligand activity using laminar flow assays, flow cytometry, qPCR, and were cultured on E-selectin-coated tissue culture plates. In laminar flow assays breast cancer cells were perfused over E-selectin substrates using physiological flow conditions. Breast cancer cells expressing Snail or Twist adhered to E-selectin from the free fluid stream in significantly fewer numbers and demonstrated fewer E-selectin ligands in flow cytometry than the vector controls. Additionally, qPCR revealed that in comparison to vector controls breast cancer cells expressing Snail or Twist downregulated the expression of α-(1,3) fucosyltransferase and α-(1,4) fucosyltransferase, which catalyze terminal fucosylations necessary for E-selectin ligand function. Interestingly, on E-selectin coated plates breast cancer cells expressing Snail or Twist avoided prolonged exposure to E-selectin by forming mammospheres, yet vector controls did not. Collectively, these data demonstrate that the EMT regulates the expression of E-selectin ligands on breast cancer cells, and causes the cells to become sensitive to culture in an E-selectin rich environment. Citation Format: Grady Earl Carlson, Venktesh Shirure, Alexander O. Ostermann, Emily A. Blaha, Louis F. Delgadillo, David F.J. Tees, Fabian Benencia, Monica Burdick. The epithelial to mesenchymal transition regulates the expression of E-selectin ligands on breast cancer cell lines. [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 1609.

Abstract 45: Dynamic biochemical tissue analysis of P-selectin ligands expressed on colon cancer.

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/cm 2 indicated adhesive interaction of the selectin-conjugated probe with ligands in colon cancer tissues was stabilized at 0.75 dyne/cm 2 . 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

Abstract 203: Dynamic biochemical tissue analysis provides a unique method to detect functional in situ L-selectin ligands and unveils intercellular heterogeneity in colon cancer tissues.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC 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