P-selectin mediates rolling of neutrophils and other leukocytes on activated endothelial cells and platelets through binding to P-selectin glycoprotein ligand-1 (PSGL-1). Certain PSGL-1 negative tumor cell lines can bind P-selectin under static conditions through the GPI-linked surface mucin, CD24, but the physiological significance of this interaction and whether it can occur under flow conditions is not known. Here, we show that CD24+ PSGL-1- KS breast carcinoma cells attach to and roll on recombinant P-selectin under a continuous wall shear stress, although at a lower density and higher velocity than CD24+ PSGL-1+ cells, such as HL-60. Adding excess soluble CD24 or removing CD24 from the cell surface with phosphatidylinositol-phospholipase C (PI-PLC) significantly reduced KS cell rolling on P-selectin. The ability of KS cells to roll on P-selectin was positively correlated with the CD24 expression level. Comparison with three other CD24+ cell lines established that expression of sialyl-Lewis(x) antigen was also necessary for CD24-mediated rolling on P-selectin. CD24 purified from KS cells supported rolling of P-selectin transfectants, but not L-selectin transfectants. Finally, KS cells rolled on vascular endothelium in vivo in a P-selectin-dependent manner. Together our data show that CD24 serves as a ligand for P-selectin under physiological flow conditions. Interaction of tumor cells with P-selectin via CD24 may be an important adhesion pathway in cancer metastasis.
Purpose: We investigated the antitumor effect of murine/human chimeric CD138-specific monoclonal antibody nBT062 conjugated with highly cytotoxic maytansinoid derivatives against multiple myeloma (MM) cells in vitro and in vivo. Experimental Design: We examined the growth inhibitory effect of BT062-SPDB-DM4, BT062-SMCC-DM1, and BT062-SPP-DM1 against MM cell lines and primary tumor cells from MM patients. We also examined in vivo activity of these agents in murine MM cell xenograft model of human and severe combined immunodeficient (SCID) mice bearing implant bone chips injected with human MM cells (SCID-hu model). Results: Anti-CD138 immunoconjugates significantly inhibited growth of MM cell lines and primary tumor cells from MM patients without cytotoxicity against peripheral blood mononuclear cells from healthy volunteers. In MM cells, they induced G 2 -M cell cycle arrest, followed by apoptosis associated with cleavage of caspase-3, caspase-8, caspase-9, and poly(ADP-ribose) polymerase. Nonconjugated nBT062 completely blocked cytotoxicity induced by nBT062-maytansinoid conjugate, confirming that specific binding is required for inducing cytotoxicity. Moreover, nBT062-maytansinoid conjugates blocked adhesion of MM cells to bone marrow stromal cells. The coculture of MM cells with bone marrow stromal cells protects against dexamethasone-induced death but had no effect on the cytotoxicity of immunoconjugates. Importantly, nBT062-SPDB-DM4 and nBT062-SPP-DM1 significantly inhibited MM tumor growth in vivo and prolonged host survival in both the xenograft mouse models of human MM and SCID-hu mouse model. Conclusion: These results provide the preclinical framework supporting evaluation of nBT062-maytansinoid derivatives in clinical trials to improve patient outcome in MM.The cell surface proteoglycan CD138 (syndecan-1) is an integral membrane protein acting as a receptor for the extracellular matrix. Within the normal human hematopoetic compartment, CD138 is expressed on differentiated plasma cells and is a primary diagnostic marker of multiple myeloma (MM; ref. 1). The large extracellular domain of CD138 binds via its heparin sulfate chains to soluble extracellular molecules, including the growth factors epidermal growth factor, fibroblast growth factor, and hepatocyte growth factor, and to insoluble extracellular molecules, such as collagen and fibronectin (2, 3). CD138 also mediates cell-cell adhesion through interactions with heparinbinding molecules. Studies of plasma cell differentiation show that CD138 is a differentiation antigen (4) and a coreceptor for MM growth factors (5).Several monoclonal antibodies (mAb; i.e.,
P-selectin (CD62P) is a Ca2+-dependent endogenous lectin that can be expressed by vascular endothelium and platelets. The major ligand for P-selectin on leukocytes is P-selectin glycoprotein ligand-1 (PSGL-1). P-selectin can also bind to carcinoma cells, but the nature of the ligand(s) on these cells is unknown. Here we investigated the P-selectin binding to a breast and a small cell lung carcinoma cell line that are negative for PSGL-1. We report that CD24, a mucin-type glycosylphosphatidylinositol-linked cell surface molecule on human neutrophils, pre B lymphocytes, and many tumors can promote binding to P-selectin. Latex beads coated with purified CD24 from the two carcinoma cell lines but also neutrophils could bind specifically to P-selectin-IgG. The binding was dependent on divalent cations and was abolished by treatment with O-sialoglycoprotein endopeptidase but not endoglycosidase F or sialidase. The beads were stained with a monoclonal antibody (MoAb) to CD57 (HNK-1 carbohydrate epitope) but did not react with MoAbs against the sialylLex/a epitope. The carcinoma cells and CD24-beads derived from these cells could bind to activated platelets or P-selectin transfected Chinese hamster ovary cells (P-CHO) in a P-selectin–dependent manner and this binding was blocked by soluble CD24. Transfection of human adenocarcinoma cells with CD24 enhanced the P-selectin–dependent binding to activated platelets. Treatment of the carcinoma cells or the CD24 transfectant with phosphatidylinositol-specific phospholipase C reduced CD24 expression and P-selectin–IgG binding concomitantly. These results establish a role of CD24 as a novel ligand for P-selectin on tumor cells. The CD24/P-selectin binding pathway could be important in the dissimination of tumor cells by facilitating the interaction with platelets or endothelial cells.
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