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• W2014261716 abstract "Cell migration in blood flow is mediated by engagement of specialized adhesion molecules that function under hemodynamic shear conditions, and many of the effectors of these adhesive interactions, such as the selectins and their ligands, are well defined. However, in contrast, our knowledge of the adhesion molecules operant under lymphatic flow conditions is incomplete. Among human malignancies, head and neck squamous cell cancer displays a marked predilection for locoregional lymph node metastasis. Based on this distinct tropism, we hypothesized that these cells express adhesion molecules that promote their binding to lymphoid tissue under lymphatic fluid shear stress. Accordingly, we investigated adhesive interactions between these and other cancer cells and the principal resident cells of lymphoid organs, lymphocytes. Parallel plate flow chamber studies under defined shear conditions, together with biochemical analyses, showed that human head and neck squamous cell cancer cells express heretofore unrecognized L-selectin ligand(s) that mediate binding to lymphocyte L-selectin at conspicuously low shear stress levels of 0.07–0.08 dynes/cm2, consistent with lymphatic flow. The binding of head and neck squamous cancer cells to L-selectin displays canonical biochemical features, such as requirements for sialylation, sulfation, and N-glycosylation, but displays a novel operational shear threshold differing from all other L-selectin ligands, including those expressed on colon cancer and leukemic cells (e.g. HCELL). These data define a novel class of L-selectin ligands and expand the scope of function for L-selectin within circulatory systems to now include a novel activity within shear stresses characteristic of lymphatic flow. Cell migration in blood flow is mediated by engagement of specialized adhesion molecules that function under hemodynamic shear conditions, and many of the effectors of these adhesive interactions, such as the selectins and their ligands, are well defined. However, in contrast, our knowledge of the adhesion molecules operant under lymphatic flow conditions is incomplete. Among human malignancies, head and neck squamous cell cancer displays a marked predilection for locoregional lymph node metastasis. Based on this distinct tropism, we hypothesized that these cells express adhesion molecules that promote their binding to lymphoid tissue under lymphatic fluid shear stress. Accordingly, we investigated adhesive interactions between these and other cancer cells and the principal resident cells of lymphoid organs, lymphocytes. Parallel plate flow chamber studies under defined shear conditions, together with biochemical analyses, showed that human head and neck squamous cell cancer cells express heretofore unrecognized L-selectin ligand(s) that mediate binding to lymphocyte L-selectin at conspicuously low shear stress levels of 0.07–0.08 dynes/cm2, consistent with lymphatic flow. The binding of head and neck squamous cancer cells to L-selectin displays canonical biochemical features, such as requirements for sialylation, sulfation, and N-glycosylation, but displays a novel operational shear threshold differing from all other L-selectin ligands, including those expressed on colon cancer and leukemic cells (e.g. HCELL). These data define a novel class of L-selectin ligands and expand the scope of function for L-selectin within circulatory systems to now include a novel activity within shear stresses characteristic of lymphatic flow. All cell-cell and cell-matrix interactions in nature require engagement of adhesion molecules capable of resisting prevailing forces of shear, either present within the organism or in the microenvironmental milieu. Cell lodgment and tissue residence thus require the presence of adhesion molecules capable of mediating binding within the shear stresses inherent to a particular site. Within organisms possessing circulatory systems, the shear force to be resisted in order to establish biologically relevant interactions is smallest within interstitial compartments and greatest within the hemovascular compartment. At present, our knowledge regarding the molecular basis of leukocyte hematogenous trafficking to lymph node, skin, and acute inflammatory sites is relatively robust (1Kannagi R. Curr. Opin. Struct. Biol. 2002; 12: 599-608Crossref PubMed Scopus (145) Google Scholar, 2Sackstein R. Curr. Opin. Hematol. 2005; 12: 444-450Crossref PubMed Scopus (105) Google Scholar). Most commonly, leukocytes exit the vasculature at post-capillary venules, where shear stress ranges from 1 to 4 dynes/cm2 (1Kannagi R. Curr. Opin. Struct. Biol. 2002; 12: 599-608Crossref PubMed Scopus (145) Google Scholar, 2Sackstein R. Curr. Opin. Hematol. 2005; 12: 444-450Crossref PubMed Scopus (105) Google Scholar). The well established “multistep” paradigm of leukocyte trafficking holds that leukocytes in flow must first make contact along the endothelial surface with adhesive interactions of sufficient strength to overcome the shear forces of blood flow. The principal effectors of the initial leukocyte adhesion to endothelium is the selectin family of adhesion molecules. The selectins (E-, P-, and L-selectins, CD62E, CD62P, and CD62L, respectively) are a family of calcium-dependent glycoproteins that are the most efficient mediators of shear-resistant interactions described to date. Each of the selectins displays optimal binding to its respective ligands under physiologic shear conditions, particularly once a shear threshold has been surpassed (3Lawrence M.B. Kansas G.S. Kunkel E.J. Ley K. J. Cell Biol. 1997; 136: 717-727Crossref PubMed Scopus (295) Google Scholar). E- and P-selectin are expressed on the vascular endothelium, and P-selectin is also expressed on platelets (4Springer T.A. Annu. Rev. Physiol. 1995; 57: 827-872Crossref PubMed Scopus (1376) Google Scholar). L-selectin, however, is strictly expressed on leukocytes and is highly expressed on peripheral blood lymphocytes, particularly among naive and central memory lymphocytes (4Springer T.A. Annu. Rev. Physiol. 1995; 57: 827-872Crossref PubMed Scopus (1376) Google Scholar, 5von Andrian U.H. Mempel T.R. Nat. Rev. Immunol. 2003; 3: 867-878Crossref PubMed Scopus (976) Google Scholar). Initial selectin-mediated interaction enables subsequent engagement of chemokine receptors and integrins that promote cellular firm adhesion, endothelial transmigration, and ultimately target site residence. It is known that similar receptor/ligand cascades, also initiated by selectin-mediated interactions, promote homing of hematopoietic stem cells to bone marrow as well as tumor cell hematogenous targeting of distant metastatic sites (2Sackstein R. Curr. Opin. Hematol. 2005; 12: 444-450Crossref PubMed Scopus (105) Google Scholar, 6Brodt P. Fallavollita L. Bresalier R.S. Meterissian S. Norton C.R. Wolitzky B.A. Int. J. Cancer. 1997; 71: 612-619Crossref PubMed Scopus (189) Google Scholar, 7Burdick M.M. McCaffery J.M. Kim Y.S. Bochner B.S. Konstantopoulos K. Am. J. Physiol. 2003; 284: C977-C987Crossref PubMed Scopus (97) Google Scholar, 8Gulubova M.V. Histochem. J. 2002; 34: 67-77Crossref PubMed Scopus (46) Google Scholar). In contrast, however, the process of cellular trafficking within lower fluid shear stress levels such as in lymphatic compartments is poorly understood. Tumor spread to lymph nodes is the culmination of a multistep process that includes tumor cell invasion into the lymphovascular compartment, tumor cell lodgment within the targeted tissue, and tumor cell growth within this new microenvironment. Recent reports have shed light on the initial steps driving the process of lymphatic metastasis (9Skobe M. Hawighorst T. Jackson D.G. Prevo R. Janes L. Velasco P. Riccardi L. Alitalo K. Claffey K. Detmar M. Nat. Med. 2001; 7: 192-198Crossref PubMed Scopus (1472) Google Scholar, 10Mandriota S.J. Jussila L. Jeltsch M. Compagni A. Baetens D. Prevo R. Banerji S. Huarte J. Montesano R. Jackson D.G. Orci L. Alitalo K. Christofori G. Pepper M.S. EMBO J. 2001; 20: 672-682Crossref PubMed Scopus (827) Google Scholar, 11He Y. Kozaki K. Karpanen T. Koshikawa K. Yla-Herttuala S. Takahashi T. Alitalo K. J. Natl. Cancer Inst. 2002; 94: 819-825Crossref PubMed Scopus (446) Google Scholar, 12Hoshida T. Isaka N. Hagendoorn J. di Tomaso E. Chen Y.L. Pytowski B. Fukumura D. Padera T.P. Jain R.K. Cancer Res. 2006; 66: 8065-8075Crossref PubMed Scopus (273) Google Scholar) and of physiologic cellular recruitment into lymphatic vessels (13Johnson L.A. Clasper S. Holt A.P. Lalor P.F. Baban D. Jackson D.G. J. Exp. Med. 2006; 203: 2763-2777Crossref PubMed Scopus (262) Google Scholar). However, the molecular basis of tumor cell lodgment within lymph nodes is uncharacterized. In head and neck squamous cell cancer (HNSCC), 4The abbreviations used are: HNSCC, head and neck squamous cell cancer; PBMC, peripheral blood mononuclear cells; PMA, phorbol 12-myristate 13-acetate; FBS, fetal bovine serum; PBS, phosphate-buffered saline; FBS, fetal bovine serum. 4The abbreviations used are: HNSCC, head and neck squamous cell cancer; PBMC, peripheral blood mononuclear cells; PMA, phorbol 12-myristate 13-acetate; FBS, fetal bovine serum; PBS, phosphate-buffered saline; FBS, fetal bovine serum. as opposed to most other solid tumors, spread of disease is overwhelmingly confined to regional lymph nodes, with distant metastatic disease developing only as a late feature of the most advanced cases (14Forastiere A. Koch W. Trotti A. Sidransky D. N. Engl. J. Med. 2001; 345: 1890-1900Crossref PubMed Scopus (1129) Google Scholar). This distinctive clinical characteristic prompted us to analyze whether HNSCC cells possess unique molecular effectors that mediate adhesive interactions active within lymphovascular shear stress, such as those that may be important for tumor cell lodgment within the lymph node microenvironment. These cellular interactions occur in the setting of lymph flow and in the absence of an endothelial barrier. Thus, cells entering the lymph node via afferent lymphatic flow percolate within the nodal parenchyma, a compartment composed primarily of lymphocytes. Measurements of shear stresses within the lymph node microenvironment have not been reported. However, measurements of flow within human lymphatic capillaries in vivo have recorded lymph median linear velocity to be 0.097 mm/s (15Fischer M. Franzeck U.K. Herrig I. Costanzo U. Wen S. Schiesser M. Hoffmann U. Bollinger A. Am. J. Physiol. 1996; 270: H358-H363PubMed Google Scholar). Based on this value, and the similar dimensions of measured lymphatic capillaries and lymph node sinuses, mean wall physiological shear stress within the lymph node sinuses is estimated to be 0.08 dynes/cm2, 10-fold lower than hematogenous shear stress levels. Within this backdrop of motion, we hypothesized that cells entering the lymph node interstitial microenvironment that express adhesion receptors specialized to function within this shear stress would have an advantage in establishing residence. Thus, we examined whether HNSCC cells are specialized to bind lymphocytes under low fluid shear. Here we show that HNSCC cells interact with L-selectin expressed on lymphocytes under conditions of shear stress, but not under static conditions. This interaction is maximal within low shear stress levels characteristic of lymph flow and is mediated by a novel class of L-selectin ligands expressed on primary HNSCC cells. This adhesive phenotype is characteristic of HNSCC but is not displayed by other solid tumor cells. Our findings unveil a previously unrecognized role for L-selectin in mediating lymphocyte-adhesive interactions under low shear stress (<1 dyne/cm2) and provide new perspectives on shear stress-based biology as may occur within the lymphatic system. Cells and Reagents—Head and neck cancer cell lines JHU-SCC-011, JHU-SCC-013, and JHU-SCC-019 were a gift from Dr. James Rocco (Boston). These cell lines were developed from tumors in patients diagnosed with squamous cell carcinoma of the upper aerodigestive tract (16Rocco J.W. Leong C.O. Kuperwasser N. DeYoung M.P. Ellisen L.W. Cancer Cell. 2006; 9: 45-56Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar, 17Scher R.L. Koch W.M. Richtsmeier W.J. Arch. Otolaryngol. Head Neck Surg. 1993; 119: 432-438Crossref PubMed Scopus (29) Google Scholar). KG1a, LS174T, MCF-7, and MDA-MB-231 cells were purchased from ATCC (Manassas, VA). Cells were maintained in RPMI 1640 medium containing glutamine supplemented with 10% fetal bovine serum (FBS) under standard cell culture conditions. Human lymphocytes were purified from blood by isolation of the mononuclear cell fraction from peripheral blood (PBMCs). PBMCs were prepared by density gradient separation (Histopaque 1077, Sigma) of peripheral blood obtained from healthy donors with full consent as per an Institutional Human Subjects Internal Review Board-approved protocol. Isolated PBMC were >90% T cells (CD3+ cells) and expressed L-selectin as determined by immunofluorescent staining and flow cytometry using a Beckman-Coulter model Cytomics FC 500 MPL (Fullerton, CA). Enzymes and chemical reagents used are as follows: α2–3-neuraminidase from Streptococcus pneumoniae (Calbiochem), α2–3,6,8-neuraminidase from Vibrio cholerae (Roche Diagnostics), O-sialoglycoprotein endopeptidase (Cedarlane, Ontario, Canada), heparinase II (Sigma), sodium chlorate (Sigma), tunicamycin (Sigma), phorbol 12-myristate 13-acetate (Sigma), and bromelain (Sigma). Recombinant human L-selectin/human Ig Fc chimera was purchased from R & D Systems. Antibodies used in the study were as follows: rat anti-human CLA, HECA-452, IgM (Pharmingen); MECA-79, rat IgM (kind gift from Dr. Phillip R. Streeter, Oregon Health Sciences University); murine anti-human PSGL-1, KPL-1, IgG1 (Pharmingen); mouse IgG1,κ isotype control (Pharmingen); rat IgM isotype control (Pharmingen); murine anti-human CD44, 2C5, IgG2a (R & D Systems, Minneapolis, MN); anti-L-selectin antibody LAM1-116, mIgG2a (Research Diagnostics Inc., Concord, MA); murine anti-human cytokeratin antibody, MAB3412, IgG1 (Chemicon, Temecula, CA); Texas red-conjugated goat anti-mouse IgG antibody (Pierce); fluorescein isothiocyanate-conjugated goat anti-mouse Ig and fluorescein isothiocyanate-conjugated goat anti-rat IgM (Pharmingen);. rat anti-human CD44, Hermes-1, IgG2a was a gift of Dr. Brenda Sandmaier (Fred Hutchinson Cancer Research Center; Seattle, WA). Purified and phycoerythrin-conjugated murine anti-human CD34, QBEND10, IgG1, and phycoerythrin-conjugated mouse IgG1,κ isotype control were from Coulter-Immunotech (Miami, FL). Alkaline phosphatase-conjugated anti-rat IgM and anti-mouse Ig were from Southern Biotechnology Associates, Birmingham, AL. Shear-dependent Binding Assays—Cell-cell interactions were studied by growing cells in standard tissue culture-treated 6-well plates. A circular parallel plate flow chamber apparatus (GlycoTech, Gaithersburg, MD) with internal flow chamber dimensions of 2 × 0.5 × 0.025 cm was mounted over the unfixed live cell monolayer (HNSCC cells) or fixed cell monolayer (KG1a and LS174T cells) within a well and sealed using low wall suction as described previously (18Burdick M.M. Chu J.T. Godar S. Sackstein R. J. Biol. Chem. 2006; 281: 13899-13905Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 19Dimitroff C.J. Lee J.Y. Fuhlbrigge R.C. Sackstein R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13841-13846Crossref PubMed Scopus (109) Google Scholar). The system was then equilibrated with Hanks' balanced salt solution (Invitrogen) supplemented with 10 mm HEPES, pH 7.4, and 2 mm CaCl2 (binding buffer). Purified lymphocytes were washed and resuspended in binding buffer at a concentration of 4.0 × 106 cells/ml. Unfixed lymphocytes were drawn into the chamber under defined flow conditions by a precision syringe pump (Harvard Apparatus, Cambridge, MA), and interactions were observed in real time under stable shear force conditions using an inverted phase contrast microscope. For experiments titrating shear stresses, the lymphocytes were first introduced into the chamber at 0.5 ml/min and then flow was momentarily suspended allowing for cells to settle, followed by an incremental increase in flow rate. Runs were recorded onto videotape using a standard CCD camera attached to a VHS recorder. Interactions were scored by review of the videotape. Primary attachments were quantified by determining the number of lymphocytes that interact with the cancer cell monolayer for more than two video frames (0.07 s) during the first 3 min of flow. Secondary interactions from cells that rolled into the field of observation from upstream regions were excluded. Rolling was defined as lateral translation of a bound cell greater than five cell diameters below the hydrodynamic velocity. Firm adhesion was defined as no lateral translation in the setting of hydrodynamic velocity and interaction resistant to shear force. Accumulation was defined as the sum of rolling and firm adhesion interactions and was scored after 4 min of flow. L-selectin specificity of interactions were established by pretreating lymphocytes with anti-L-selectin antibody LAM1-116 or anti-L-selectin polyclonal serum (50 μg/ml, on ice, 30 min) or PMA (10 ng/ml, 37 °C, 1 h). Alternatively, unfixed cancer cells in suspension were also introduced in the flow chamber over L-selectin chimera adsorbed to the tissue culture plate. Cells were grown under standard culture conditions and harvested by incubation with EDTA (5 mm EDTA, 37 °C, 15 min) and washed with binding buffer. Cells were resuspended to a final concentration of 1.0 × 106 cells/ml. Chimera plates were prepared by overnight incubation with L-selectin chimera (0.5 μg/ml in PBS) at 4 °C and blocked with heat-inactivated FBS for 24 h. Attachment was assayed by introducing tumor cells into the parallel flow chamber at 0.5 ml/min (1.4 dynes/cm2) followed by decreasing flow to the desired level. In experiments shown in Fig. 2A, flow was incrementally decreased every 30 s. In other experiments, flow was immediately decreased to the desired level once the cell bolus was visualized in the field. Accumulation was scored at the end of 30 s of flow at a determined shear stress. The specificity of L-selectin interaction was assessed by pretreating the cancer cells with bromelain (0.1%, 37 °C, 1 h), EDTA (10 mm EDTA, 30 min), neuraminidase (0.1 unit/ml at 37 °C, 1 h), O-sialoglycoprotein endopeptidase (24 or 240 μg/ml at 37 °C, 2 h), heparinase (0.05 or 5 units/ml, 37 °C, 4 h), sodium chlorate (10 mm, 37 °C, 20 h), or tunicamycin (15 μg/ml, 37 °C, 20 h.). L-selectin specificity of interactions was established by pretreating the prepared L-selectin chimera plates with anti-L-selectin antibody LAM1-116 or anti-L-selectin polyclonal serum (50 μg/ml) prior to the infusion of the cancer cell suspensions. Mouse isotype antibody was used as negative controls for blocking. Detachment was assayed by first establishing binding at 0.07 dynes/cm2 and then incrementally raising flow rate. Adhesive interactions were scored 30 s after establishing a discrete shear stress level. Rolling velocity was calculated as the distance traveled by the centroid of an attached cell divided by the period of observation (5 s). All experiments were performed on different days with the use of freshly prepared reagents, i.e. newly isolated lymphocytes, newly isolated cancer cell lines, or newly created L-selectin chimera spots. Wall shear stress (T) values were calculated according to the formula T (dynes/cm2) = 3 μQ/2ba2, where μ is the coefficient of viscosity of the solution in the chamber (poise); Q is the volumetric flow rate (cm3/s); b is the channel width (0.5 cm); and a is the half-channel height (0.0127 cm). A value of 0.009 poise (for water) was used for the viscosity of flow buffer at 25 °C. Primary Tumor-based Assays—Fresh primary tumor specimens were dissected from the center of the tumor mass (i.e. not bordering on normal mucosa) and were minced and incubated in RPMI containing 60 μg/ml collagenase (Sigma C9697) at 37 °C for 3–6 h. Undigested tumor pieces were allowed to briefly settle to the bottom of the tube, and cells in suspension were removed with the supernatant. Cells were then pelleted at 200 × g for 10 min and resuspended in 3–6 ml of Hanks' balanced salt solution (Invitrogen) supplemented with 10 mm HEPES, pH 7.4, and 2 mm CaCl2 (binding buffer). Cells were examined for low shear-dependent L-selectin binding as described above using the adherent L-selectin chimera assay. Adhered human immunoglobulin (Miles Laboratories, West Haven, CT) (0.5 μg/ml in PBS) or FBS was used as negative control for binding. Cells were introduced into the parallel plate chamber at 0.07 dynes/cm2 for 5 min, followed by a 1-ml wash with binding buffer introduced at 0.3 dynes/cm2, and fixation with 1 ml of 4% phosphate-buffered paraformaldehyde introduced at 0.3 dynes/cm2. Fixed cells were stored at 4 °C until stained. Plates containing previously fixed cells were treated with PBS, 0.5% Triton X-100 for 5 min, followed by a PBS rinse. Plates were blocked for 15 min in 1× PBS, 5% normal horse serum, 1% bovine serum albumin and then incubated with anti-cytokeratin antibody for 30 min. Following PBS rinses, the plates were incubated with a 1:200 dilution of Texas red-conjugated goat anti-mouse antibody for 30 min at 25 °C. Following a PBS rinse, the plates were incubated with 0.4 μg/ml 4′,6-diamidino-2-phenylindole (Pierce) in PBS for 5 min and rinsed, and coverslips were mounted with Vectashield (Vector Laboratories, Burlingame, CA). Cells were examined, and digital pictures were taken using a Nikon TE2000-U microscope with Photometrics Cool SNAP EZ digital camera (Roper Scientific, Tucson, AZ) and the NIS Elements BR software (Nikon, Melville, NY). Selectin Ligand Expression—The expression of CD34, PSGL-1, HECA-452, and MECA-79 was assessed by standard flow cytometry. Expression of HCELL was assessed by immunoprecipitating CD44 with the Hermes-1 antibody followed by Western blot evaluation of the immunoprecipitate with the HECA-452 antibody as described previously (19Dimitroff C.J. Lee J.Y. Fuhlbrigge R.C. Sackstein R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13841-13846Crossref PubMed Scopus (109) Google Scholar). Briefly, membrane protein lysates of the KG1a and HNSCC cell lines were prepared in 2% Nonidet P-40, Buffer A (1% SDS, 150 mm NaCl, 0.5 mm Tris, pH 10.4, 1 mm EDTA, 20 μg/ml phenylmethylsulfonyl fluoride) and incubated with immunoprecipitating antibodies or with appropriate isotype controls overnight at 4 °C. The antibody/lysate mixture was then immunoprecipitated by the addition of protein G-agarose beads (Invitrogen) followed by extensive washes with immunoprecipitating buffer. The pellet was then treated with 6× reducing gel loading buffer, boiled, and the supernatant recovered. The samples were then resolved by standard SDS-PAGE using 7.5% Tris-HCl pre-cast gels (Bio-Rad). The gels were subsequently transferred onto polyvinylidene difluoride blot membrane (Millipore, Billerica, MA) and blocked using standard Western blot technique. The blots were treated with primary antibody (1 μg/ml) and alkaline phosphatase-linked secondary antibody (1:1000). Bands were visualized using the Western blue alkaline phosphatase substrate system (Promega, Madison, WI). Statistics—Statistical analysis was performed using single factor analysis of variance, as well as Tukey post hoc pairwise test. Statistical significance was accepted for p values less than 0.05. All p values were two-tailed. A minimum of three independent observations were made for each measurement. Lymphocytes Bind to HNSCC Cells under Conditions of Low Fluid Shear—To analyze whether HNSCC cells interact with lymphocytes under shear stress, parallel plate flow chamber binding assays were used. This experimental system has been extensively used for the study of shear stress-dependent adhesion molecule biology (7Burdick M.M. McCaffery J.M. Kim Y.S. Bochner B.S. Konstantopoulos K. Am. J. Physiol. 2003; 284: C977-C987Crossref PubMed Scopus (97) Google Scholar, 20Fuhlbrigge R.C. Alon R. Puri K.D. Lowe J.B. Springer T.A. J. Cell Biol. 1996; 135: 837-848Crossref PubMed Scopus (89) Google Scholar, 21Hanley W.D. Burdick M.M. Konstantopoulos K. Sackstein R. Cancer Res. 2005; 65: 5812-5817Crossref PubMed Scopus (101) Google Scholar, 22Dimitroff C.J. Lee J.Y. Schor K.S. Sandmaier B.M. Sackstein R. J. Biol. Chem. 2001; 276: 47623-47631Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 23Sackstein R. Dimitroff C.J. Blood. 2000; 96: 2765-2774Crossref PubMed Google Scholar, 24Fuhlbrigge R.C. King S.L. Dimitroff C.J. Kupper T.S. Sackstein R. J. Immunol. 2002; 168: 5645-5651Crossref PubMed Scopus (66) Google Scholar). Human PBMCs from normal donors were used as a source of lymphocytes (>90% lymphocytes). Head and neck cancer cells were grown on a solid support, and the lymphocytes were delivered under flow at discrete shear stresses by use of a standard syringe pump to define the latter. This experimental design avoids membrane fixation and thus measures the interaction(s) of the physiologically presented cell surface adhesion molecules, in native conformations, within the lipid bilayer. Adhesive interaction events were captured on videotape by using a standard CCD camera and video recording assembly. Events were scored on video as rolling events or firm adhesion events (see “Experimental Procedures”). Lymphocytes were introduced into the flow chamber at 0.6 dynes/cm2. The flow velocity was incrementally increased every 30 s up to 20 dynes/cm2. Under these conditions, HNSCC cell lines JHU-SCC-011, JHU-SCC-013, and JHU-SCC-019 failed to support binding of the flowing lymphocytes. For comparison, we assessed lymphocyte binding to KG1a (human leukemia) cells and LS174T (colon cancer) cells as reported previously, given their known activity for L-selectin-mediated binding (18Burdick M.M. Chu J.T. Godar S. Sackstein R. J. Biol. Chem. 2006; 281: 13899-13905Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 22Dimitroff C.J. Lee J.Y. Schor K.S. Sandmaier B.M. Sackstein R. J. Biol. Chem. 2001; 276: 47623-47631Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Both cell lines supported lymphocyte rolling at shear stresses between 0.6 and 4.0 dynes/cm2 (data not shown). Having estimated lymphatic shear to be 0.08 dynes/cm2, we next assayed adherent interactions at lower shear stresses (<0.6 dyne/cm2). The lymphocyte bolus was introduced into the chamber, and flow was halted and allowed to equilibrate (i.e. cells in flow stopped moving). Flow was then re-started at 0.01 dynes/cm2 and incrementally increased every 30 s up to a shear stress of 20 dynes/cm2. These experiments revealed adhesive interactions consisting of tethering and transient rolling preceding firm adhesion at shear stresses below 0.6 dynes/cm2 between the flowing human lymphocytes and the HNSCC cells, but not KG1a cells or LS174T cells (Fig. 1A). The HNSCC-lymphocyte adhesive interactions were observed between shear stresses of 0.035–0.9 dynes/cm2, with maximal activity at a wall shear stress of 0.07–0.08 dynes/cm2. A shear threshold was consistently observed at 0.035 dynes/cm2, and, importantly, no binding was observed under static conditions. Lymphocyte-HNSCC Cell Interaction Is Mediated by L-selectin—In initial studies, we sought to determine whether binding was affected by treatment of lymphocytes with PMA, a lymphocyte-activating agent known to cause L-selectin shedding and concomitant activation of integrin binding activity (25Chen A. Engel P. Tedder T.F. J. Exp. Med. 1995; 182: 519-530Crossref PubMed Scopus (162) Google Scholar, 26Jung T.M. Dailey M.O. J. Immunol. 1990; 144: 3130-3136PubMed Google Scholar, 27Kishimoto T.K. Jutila M.A. Berg E.L. Butcher E.C. Science. 1989; 245: 1238-1241Crossref PubMed Scopus (903) Google Scholar). Treatment of lymphocytes with PMA for 30 min at 37 °C resulted in a marked decrease in L-selectin expression (<5% of treated cells were L-selectin+ by flow cytometry). Using the parallel plate chamber, adhesion of PMA-activated lymphocytes with the tumor cell monolayer at 0.07 dynes/cm2 was reduced by 80% (Fig. 1B), indicating a dominant effect of L-selectin receptor-ligand interaction(s) on lymphocyte-HNSCC adhesion. We then incubated lymphocytes with blocking anti-L-selectin antibodies LAM1-116 or polyclonal anti-L-selectin serum to directly test whether L-selectin mediated shear stress-resistant interactions between lymphocytes and HNSCC cells at 0.07 dynes/cm2. Antibody blockade of L-selectin using LAM1-116 or polyclonal anti-L-selectin serum reduced binding by >50% (Fig. 1B). Collectively, these data show that L-selectin is an important effector of lymphocyte binding to HNSCC cells. To further elucidate the discrete contribution of L-selectin in capturing HNSCC cells under flow conditions, unfixed HNSCC cells were perfused at defined shear stresses over human L-selectin/Fc chimera adhered to a solid support. To assess the upper limit of shear that can support initial tethering interactions, we initiated flow at 1.4 dynes/cm2 and then subsequently lowered shear stress in defined increments every 30 s. As shown in Fig. 2A, significant adherence of HNSCC cells to immobilized L-selectin was observed at an upper limit of 0.14 dynes/cm2, with maximal binding seen as shear was lowered to 0.07–0.03 dynes/cm2. No additional cells bound as shear was decreased from 0.03 to 0.02 dyne/cm2 (Fig. 2A), indicating that initial L-selectin-mediated capture of HNSCC is optimal at shear ∼0.03 dyne/cm2. Interestingly, although there was a clear threshold shear requirement to initiate tethering, the previously bound HNSCC did not detach from the L-selectin/Fc chimera at 0.02 dynes/cm2, t" @default.
• W2014261716 created "2016-06-24" @default.
• W2014261716 creator A5024015661 @default.
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• W2014261716 date "2008-06-01" @default.
• W2014261716 modified "2023-10-11" @default.
• W2014261716 title "L-selectin-mediated Lymphocyte-Cancer Cell Interactions under Low Fluid Shear Conditions" @default.
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