Shear stress modulates the interaction of platelet-secreted matrix proteins with tumor cells through the integrin α_vβ₃

Abstract

The precise interaction among metastatic cells, circulating platelets, the vessel wall, and physiological flow conditions remains to be determined. In this study, we investigated the interaction of shear on metastatic cell lines adherent to lipopolysaccharide (LPS)-treated endothelium. Tumor cells were perfused over LPStreated human umbilical vein endothelial cells (HUVECs) at incremental venous shear rates from 50 to 800 s Ϫ1 . At a venous shear rate of 400 s Ϫ1 , 3% of adherent tumor cells formed pseudopodia under shear, a process we termed shear-induced activation. Because platelets promote tumor dissemination, we then investigated the effect of pretreating tumor cells with platelet releasate collected from activated platelet concentrate. We found that in the presence of platelet releasate, the number of tumor cells adhering to HUVECs increased and tumor "activation" occurred at a significantly lower shear rate of 50 s Ϫ1 . This was inhibited with acetylsalicylic acid. Depletion of fibronectin or vitronectin from the platelet releasate resulted in significantly less adhesion at higher venous shear rates of 600 and 800 s Ϫ1 . The integrin ␣v␤3 has been shown to mediate cell adhesion primarily through vitronectin and fibronectin proteins. Inhibition of ␣v␤3, followed by the addition of platelet releasate to the tumor cells, resulted in significantly less adhesion at higher venous shear rates of 600 and 800 s Ϫ1 . Collectively, our data suggest that ␣v␤3 promotes the metastatic phenotype of tumor cells through interactions with the secreted platelet proteins vitronectin and fibronectin under venous shear conditions. platelet releasate; vitronectin; fibronectin ESOPHAGEAL CANCER IS ONE of the least studied and deadliest cancers worldwide. At the time of diagnosis, Ͼ50% of patients have radiographically visible metastases (12). Metastases arise through a complex series of sequential steps involving a variety of tumor and host properties. A key step is emigration from the circulation. Metastasis occurs primarily in the venous circulation (20). This relatively lowshear venous environment facilitates the arrest of tumor cells to the vessel wall, eventually leading to penetration of the vascular wall (21). Previous experiments suggested that cancer cell binding to endothelium has a key role in metastases; however, these experiments were performed under static conditions (32). The interaction of metastatic cells with the endothelium under fluid flow conditions is not well understood. Recent evidence suggests that tumor adherence occurring in the presence of shear stress is analogous to the manner in which platelets (6) and leukocytes tether, adhere, and roll (4).In the present investigation, we studied a shear-specific effect on metastatic cells and further characterized the interaction of tumor cells, platelets, and inflamed endothelium. Successful cell arrest in the circulation is dependent on the balance between adhesive and antiadhesive forces and on the rate at which adhesive interactions are broken (33). The init...

Mobility and invasiveness of metastatic esophageal cancer are potentiated by shear stress in a ROCK- and Ras-dependent manner

“…To examine the effects of shear on actin and tubulin in metastatic cells, adherent OC-1 cells were exposed to static (no shear) or sheared conditions (continuous shear rate of 200 s Ϫ1 , corresponding to a low venous shear rate). Fn was used as a ligand-binding substrate, because OC-1 cells bind to Fn through the integrin ␣ V␤3 (16). Glass slides were coated with Fn (100 g/ml) for 2 h at room temperature, blocked with 1% BSA in PBS for 1 h, and then washed three times with PBS before use.…”

“…The behavior of OC-1 cells under physiologically relevant shear conditions was assayed as previously described (16). In brief, OC-1 cells in the presence and absence of Y-27632 were resuspended in DMEM (1 ϫ 10 6 cells/ml) and allowed to settle for 5 min on LPS-stimulated HUVECs in the parallel-plate flow chamber before being exposed to shear rates of 50 -800 s Ϫ1 .…”

“…Y-27632 caused no toxicity at a concentration of 10 M as assessed by Trypan blue staining (data not shown). To simulate physiological shear conditions, we investigated the morphology of OC-1 cells adherent to LPS-stimulated HUVECs under shear conditions in the parallel-plate flow chamber as previously described (16). The tumor cells were allowed to settle on the inflamed endothelium for 5 min, and then perfusion was initiated.…”

An Intracellular Signal Pathway That Regulates Cancer Cell Adhesion in Response to Extracellular Forces

“…Cells swirling through the lymphatic or venous circulation or around a body cavity or surgical site contact endothelial or other cells or extracellular matrix proteins, adhering if the correct membrane receptors are deployed at the right place to bind to adhesion sites and if the energy of cell movement is not so great that it overcomes binding affinity. However, work from our laboratory (1-3) and others (4,5) suggests that cancer cells may regulate their own adhesion to matrix proteins, endothelial cells, or surgical wounds by intracellular signals that regulate the binding affinity of matrix receptors including h 1 -integrin heterodimers. At least one pathway by which this occurs seems to involve focal adhesion kinase (FAK) and Src activation (2) and paxillin (6,7) and has been shown to be active in breast (8), head and neck (6), and colon (1) cancer cell lines, as well as in primary human colon cancer cells isolated directly from surgical specimens (2).…”

“…At least one pathway by which this occurs seems to involve focal adhesion kinase (FAK) and Src activation (2) and paxillin (6,7) and has been shown to be active in breast (8), head and neck (6), and colon (1) cancer cell lines, as well as in primary human colon cancer cells isolated directly from surgical specimens (2). These signals mediating increased adhesion are stimulated by increased extracellular pressure (2) as well as by laminar or nonlaminar shear stress (4,5,9).…”