Changliang Fu

Two-dimensional kinetics of β2-integrin and ICAM-1 bindings between neutrophils and melanoma cells in a shear flow

Cell adhesion, mediated by specific receptor-ligand interactions, plays an important role in biological processes such as tumor metastasis and inflammatory cascade. For example, interactions between beta 2-integrin (lymphocyte function-associated antigen-1 and/or Mac-1) on polymorphonuclear neutrophils (PMNs) and ICAM-1 on melanoma cells initiate the bindings of melanoma cells to PMNs within the tumor microenvironment in blood flow, which in turn activate PMN-melanoma cell aggregation in a near-wall region of the vascular endothelium, therefore enhancing subsequent extravasation of melanoma cells in the microcirculations. Kinetics of integrin-ligand bindings in a shear flow is the determinant of such a process, which has not been well understood. In the present study, interactions of PMNs with WM9 melanoma cells were investigated to quantify the kinetics of beta 2-integrin and ICAM-1 bindings using a cone-plate viscometer that generates a linear shear flow combined with a two-color flow cytometry technique. Aggregation fractions exhibited a transition phase where it first increased before 60 s and then decreased with shear durations. Melanoma-PMN aggregation was also found to be inversely correlated with the shear rate. A previously developed probabilistic model was modified to predict the time dependence of aggregation fractions at different shear rates and medium viscosities. Kinetic parameters of beta 2-integrin and ICAM-1 bindings were obtained by individual or global fittings, which were comparable to respectively published values. These findings provide new quantitative understanding of the biophysical basis of leukocyte-tumor cell interactions mediated by specific receptor-ligand interactions under shear flow conditions.

Determining β2-Integrin and Intercellular Adhesion Molecule 1 Binding Kinetics in Tumor Cell Adhesion to Leukocytes and Endothelial Cells by a Gas-driven Micropipette Assay

Interactions between polymorphonuclear neutrophils (PMNs) and tumor cells have been reported to facilitate the adhesion and subsequent extravasation of tumor cells through the endothelium under blood flow, both of which are mediated by binding β2-integrin to intercellular adhesion molecule 1 (ICAM-1). Here the adhesions between human WM9 metastatic melanoma cells, PMNs, and human pulmonary microvascular endothelial cells (HPMECs) were quantified by a gas-driven micropipette aspiration technique (GDMAT). Our data indicated that the cellular binding affinity of PMN-WM9 pair was 3.9-fold higher than that of the PMN-HPMEC pair. However, the effective binding affinities per molecular pair were comparable between the two cell pairs no matter whether WM9 cells or HPMECs were quiescent or cytokine-activated, indicating that the stronger adhesion between PMN-WM9 pair is mainly attributed to the high expression of ICAM-1 on WM9 cells. These results proposed an alternative mechanism, where WM9 melanoma cells adhere first with PMNs near vessel-wall regions and then bind to endothelial cells via PMNs under blood flow. In contrast, the adhesions between human MDA-MB-231 metastatic breast carcinoma cells and PMNs showed a comparable cellular binding affinity to PMN-HPMEC pair because the ICAM-1 expressions on MDA-MB-231 cells and HPMECs are similar. Furthermore, differences were observed in the intrinsic forward and reverse rates of the β2-integrin-ICAM-1 bond between PMN-TC and PMN-EC pairs. This GDMAT assay enables us to quantify the binding kinetics of cell adhesion molecules physiologically expressed on nucleated cells. The findings also further the understanding of leukocyte-facilitated tumor cell adhesion from the viewpoint of molecular binding kinetics.

Melanoma upregulates ICAM-1 expression on endothelial cells through engagement of tumor CD44 with endothelial E-selectin and activation of a PKCα–p38–SP-1 pathway

Cancer metastasis involves multistep adhesive interactions between tumor cells (TCs) and endothelial cells (ECs), but the molecular mechanisms of intercellular communication in the tumor microenvironment remain elusive. Using static and flow coculture systems in conjunction with flow cytometry, we discovered that certain receptors on the ECs are up‐regulated on melanoma cell adhesion. Direct contact but not separate coculture between human umbilical endothelial cells (HUVECs) and a human melanoma cell line (Lu1205) increased intercellular adhesion molecule 1 (ICAM‐1) and E‐selectin expression on HUVECs by 3‐ and 1.5‐fold, respectively, compared with HUVECs alone. The nonmetastatic cell line WM35 failed to promote ICAM‐1 expression changes in HUVECs on contact. Enzyme‐linked immunosorbent assay (ELISA) revealed that EC‐TC contact has a synergistic effect on the expression of the cytokines interleukin (IL)‐8, IL‐6, and growth‐related oncogene α (Gro‐α). By using E‐selectin cross‐linking and beads coated with CD44 immunopurified from Lu1205 cells, we showed that CD44/selectin ligation was responsible for the ICAM‐1 up‐regulation on HUVECs. Protein kinase Cα (PKC‐α) activation was found to be the downstream target of the CD44/selectin‐initiated signaling, as ICAM‐1 elevation was inhibited by siRNA targeting PKCα or a dominant negative form of PKCα (PKCα DN). Western blot analysis and electrophoretic mobility shift assays (EMSAs) showed that TC‐EC contact mediated p38 phosphorylation and binding of the transcription factor SP‐1 to its regulation site. In conclusion, CD44/selectin binding signals ICAM‐1 up‐regulation on the EC surface through a PKCα–p38‐SP‐1 pathway, which further enhances melanoma cell adhesion to ECs during metastasis.—Zhang, P., Goodrich, C., Fu, C., Dong, C., Melanoma upregulates ICAM‐1 expression on ECs through engagement of tumor CD44 with endothelial E‐selectin and activation of a PKCα–p38‐SP‐1 pathway. FASEB J. 28, 4591–4609 (2014). www.fasebj.org

CD44 variant, but not standard CD44 isoforms, mediate disassembly of endothelial VE-cadherin junction on metastatic melanoma cells

Loss of endothelial adherens junctions is involved in tumor metastasis. Here, we demonstrate that, in the metastatic Lu1205 melanoma cells, expression of the CD44 variant CD44v8‐v10 induced junction disassembly and vascular endothelial (VE)‐cadherin phosphorylation at Y658 and Y731. Short interfering RNA (siRNA)‐mediated CD44 knockdown or sialic acid cleavage reversed these effects. Moreover, microspheres coated with recombinant CD44v8‐v10 promoted endothelial junction disruption. Overexpression of CD44v8‐v10 but not of standard CD44 (CD44s) promoted gap formation in the non‐metastatic WM35 melanoma cells, whereas CD44 knockdown or neuraminidase treatment dramatically diminished melanoma transendothelial migration. Endothelial cells transfected with the phosphomimetic VE‐cadherin mutant Y658E supported transmigration of CD44‐silenced Lu1205 cells. Our findings imply that CD44 variant isoform (CD44v) but not CD44s regulates endothelial junction loss, promoting melanoma extravasation.

Fibrin serves as a divalent ligand that regulates neutrophil-mediated melanoma cells adhesion to endothelium under shear conditions.

Elevated soluble fibrin (sFn) levels are characteristic of melanoma hematogeneous dissemination, where tumor cells interact intimately with host cells. Melanoma adhesion to the blood vessel wall is promoted by immune cell arrests and tumor-derived thrombin, a serine protease that converts soluble fibrinogen (sFg) into sFn. However, the molecular requirement for sFn-mediated melanoma-polymorphonuclear neutrophils (PMNs) and melanoma-endothelial interactions under physiological flow conditions remain elusive. To understand this process, we studied the relative binding capacities of sFg and sFn receptors e.g., α(v)β(3) integrin and intercellular adhesion molecule-1 (ICAM-1) expressed on melanoma cells, ICAM-1 on endothelial cells (EC), and CD11b/CD18 (Mac-1) on PMNs. Using a parallel-plate flow chamber, highly metastatic melanoma cells (1205Lu and A375M) and human PMNs were perfused over an EC monolayer expressing ICAM-1 in the presence of sFg or sFn. It was found that both the frequency and lifetime of direct melanoma adhesion or PMN-facilitated melanoma adhesion to the EC in a shear flow were increased by the presence of sFn in a concentration-dependent manner. In addition, sFn fragment D and plasmin-treated sFn failed to increase melanoma adhesion, implying that sFn-bridged cell adhesion requires dimer-mediated receptor-receptor cross-linking. Finally, analysis of the respective kinetics of sFn binding to Mac-1, ICAM-1, and α(v)β(3) by single bond cell tethering assays suggested that ICAM-1 and α(v)β(3) are responsible for initial capture and firm adhesion of melanoma cells. These results provide evidence that sFn enhances melanoma adhesion directly to ICAM-1 on the EC, while prolonged shear-resistant melanoma adhesion requires interactions with PMNs.

C6-ceramide nanoliposome suppresses tumor metastasis by eliciting PI3K and PKCζ tumor-suppressive activities and regulating integrin affinity modulation.

Nanoliposomal formulation of C6-ceramide, a proapoptotic sphingolipid metabolite, presents an effective way to treat malignant tumor. Here, we provide evidence that acute treatment (30 min) of melanoma and breast cancer cells with nanoliposomal C6-ceramide (NaL-C6) may suppress cell migration without inducing cell death. By employing a novel flow migration assay, we demonstrated that NaL-C6 decreased tumor extravasation under shear conditions. Compared with ghost nanoliposome, NaL-C6 triggered phosphorylation of PI3K and PKCζ and dephosphorylation of PKCα. Concomitantly, activated PKCζ translocated into cell membrane. siRNA knockdown or pharmacological inhibition of PKCζ or PI3K rescued NaL-C6-mediated suppression of tumor migration. By inducing dephosphorylation of paxillin, PKCζ was responsible for NaL-C6-mediated stress fiber depolymerization and focal adhesion disassembly in the metastatic tumor cells. PKCζ and PI3K regulated cell shear-resistant adhesion in a way that required integrin αvβ3 affinity modulation. In conclusion, we identified a novel role of acute nanoliposomal ceramide treatment in reducing integrin affinity and inhibiting melanoma metastasis by conferring PI3K and PKCζ tumor-suppressive activities.

RacGAP1-driven focal adhesion formation promotes melanoma transendothelial migration through mediating adherens junction disassembly.

Melanoma cell migration across vascular endothelial cells is an essential step of tumor metastasis. Here, we provide evidence that RacGAP1, a cytokinesis-related Rho GTPase-activating protein, contributed to this process. Depletion of RacGAP1 with RacGAP1-targeting siRNA or overexpression of RacGAP1 mutant (T249A) attenuated melanoma cell transendothelial migration and concomitant changes of adherens junctions. In addition, RacGAP1 promoted the activations of RhoA, FAK, paxillin and triggered focal adhesion formation and cytoskeletal rearrangement. By overexpressing FAK-related non-kinase (FRNK) in endothelium, we showed that RacGAP1 mediated endothelial barrier function loss and melanoma transmigration in a focal adhesion-dependent manner. These results suggest that endothelial RacGAP1 may play critical roles in pathogenic processes of cancer by regulating endothelial permeability.

Lysosomal degradation of CD44 mediates ceramide nanoliposome-induced anoikis and diminished extravasation in metastatic carcinoma cells.

Background: Even though ceramide selectively induces apoptosis in multiple cancer models, the mechanisms by which ceramide limits cancer metastasis are unknown. Results: The ceramide nanoliposome (CNL) inhibits cancer cell metastasis by inducing anoikis and inhibiting extravasation via lysosomal degradation of CD44. Conclusion: Ceramide reduces the metastatic potential of multiple cancer cell models. Significance: CNL may have therapeutic utility in preventing and treating metastatic cancers. The ceramide nanoliposome (CNL) has shown promise in being able to treat a variety of primary tumors. However, its potential for treating metastatic cancer remains unknown. In this study, we demonstrate that CNL increases anoikis while preventing cancer cell extravasation under both static and physiological fluid flow conditions. Mechanistically, CNL limits metastases by decreasing CD44 protein levels in human breast and pancreatic cancer cells via lysosomal degradation of CD44, independent of palmitoylation or proteasome targeting. siRNA down-regulation of CD44 mimics CNL-induced anoikis and diminished extravasation of cancer cells. Taken together, our data indicate that ceramide limits CD44-dependent cancer cell migration, suggesting that CNL could be used to prevent and treat solid tumor metastasis.

Localized Modeling of Biochemical and Flow Interactions during Cancer Cell Adhesion

This work focuses on one component of a larger research effort to develop a simulation tool to model populations of flowing cells. Specifically, in this study a local model of the biochemical interactions between circulating melanoma tumor cells (TC) and substrate adherent polymorphonuclear neutrophils (PMN) is developed. This model provides realistic three-dimensional distributions of bond formation and attendant attraction and repulsion forces that are consistent with the time dependent Computational Fluid Dynamics (CFD) framework of the full system model which accounts local pressure, shear and repulsion forces. The resulting full dynamics model enables exploration of TC adhesion to adherent PMNs, which is a known participating mechanism in melanoma cell metastasis. The model defines the adhesion molecules present on the TC and PMN cell surfaces, and calculates their interactions as the melanoma cell flows past the PMN. Biochemical rates of reactions between individual molecules are determined based on their local properties. The melanoma cell in the model expresses ICAM-1 molecules on its surface, and the PMN expresses the β-2 integrins LFA-1 and Mac-1. In this work the PMN is fixed to the substrate and is assumed fully rigid and of a prescribed shear-rate dependent shape obtained from micro-PIV experiments. The melanoma cell is transported with full six-degrees-of-freedom dynamics. Adhesion models, which represent the ability of molecules to bond and adhere the cells to each other, and repulsion models, which represent the various physical mechanisms of cellular repulsion, are incorporated with the CFD solver. All models are general enough to allow for future extensions, including arbitrary adhesion molecule types, and the ability to redefine the values of parameters to represent various cell types. The model presented in this study will be part of a clinical tool for development of personalized medical treatment programs.

Mutant B-Raf(V600E) Promotes Melanoma Paracellular Transmigration by Inducing Thrombin-mediated Endothelial Junction Breakdown.

Background: A prothrombotic state is one of the hallmarks of advanced cancer. Results: B-Raf(V600E)-dependent thrombin release from metastatic melanoma signals focal adherens junction disassembly by triggering VE-cadherin phosphorylation and ubiquitination. Conclusion: Thrombin-mediated junction breakdown allows melanoma extravasation via a paracellular route. Significance: Targeting B-Raf(V600E)-mediated thrombin generation and endothelial barrier dysfunction may represent a novel therapeutic strategy to limit tumor metastasis. Tumor invasiveness depends on the ability of tumor cells to breach endothelial barriers. In this study, we investigated the mechanism by which the adhesion of melanoma cells to endothelium regulates adherens junction integrity and modulates tumor transendothelial migration (TEM) by initiating thrombin generation. We found that the B-Raf(V600E) mutation in metastatic melanoma cells up-regulated tissue factor (TF) expression on cell membranes and promoted thrombin production. Co-culture of endothelial monolayers with metastatic melanoma cells mediated the opening of inter-endothelial spaces near melanoma cell contact sites in the presence of platelet-free plasma (PFP). By using small interfering RNA (siRNA), we demonstrated that B-Raf(V600E) and TF silencing attenuated the focal disassembly of adherens junction induced by tumor contact. Vascular endothelial-cadherin (VE-cadherin) disassembly was dependent on phosphorylation of p120-catenin on Ser-879 and VE-cadherin on Tyr-658, Tyr-685, and Tyr-731, which can be prevented by treatment with the thrombin inhibitor, hirudin, or by silencing the thrombin receptor, protease-activated receptor-1, in endothelial cells. We also provided strong evidence that tumor-derived thrombin enhanced melanoma TEM by inducing ubiquitination-coupled VE-cadherin internalization, focal adhesion formation, and actin assembly in endothelium. Confocal microscopic analysis of tumor TEM revealed that junctions transiently opened and resealed as tumor cells accomplished TEM. In addition, in the presence of PFP, tumor cells preferentially transmigrated via paracellular routes. PFP supported melanoma transmigration under shear conditions via a B-Raf(V600E)-thrombin-dependent mechanism. We concluded that the activation of thrombin generation by cancer cells in plasma is an important process regulating melanoma extravasation by disrupting endothelial junction integrity.