Hydrodynamic shear rate regulates melanoma-leukocyte aggregation, melanoma adhesion to the endothelium, and subsequent extravasation - PubMed (original) (raw)

Hydrodynamic shear rate regulates melanoma-leukocyte aggregation, melanoma adhesion to the endothelium, and subsequent extravasation

Shile Liang et al. Ann Biomed Eng. 2008 Apr.

Abstract

Adhesion to and subsequent extravasation through the endothelial lining of blood vessels is critical for tumor cells to establish metastases. Recent studies have indicated that polymorphonuclear neutrophils (PMNs) may enhance melanoma adhesion to the endothelium (EC) and subsequent extravasation under dynamic flow conditions. However, little is known about hydrodynamics involved in the tumor microenvironment within the microcirculation. In this study, effects of hydrodynamic flow on regulating melanoma cell adhesion to the EC have been investigated. Results indicate that under flow conditions, interactions between melanoma cells and the EC are distinctly different from PMN-EC interactions. Without expressions of surface integrins or sialylated molecules, most melanoma cells that express a high-level of intercellular adhesion molecule (ICAM-1) are not able to effectively adhere to the inflamed EC by themselves. Binding of melanoma cells and PMNs through ICAM-1 on melanoma cells and beta(2) integrins on PMNs has been shown to enhance melanoma cell arrest on the EC. Although PMN tethering on the EC is regulated by both the shear rate and shear stress, melanoma cell adhesion to the EC and subsequent extravasation via tethering PMN on the EC is predominantly regulated by shear rate, which partly is due to the shear-rate-dependent PMN-melanoma aggregation in shear flow. These findings provide a rationale and mechanistic basis for understanding of leukocyte-tumor cell interactions under flow conditions during tumor cell extravasation and metastasis.

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Figures

FIGURE 1

FIGURE 1

Effects of shear rate and shear stress on melanoma extravasation through EI monolayer. Shear rate and shear stress were isolated by varying viscosity with dextran-supplemented medium (0-4%). (a) Cross-section view of the flow-migration chamber shows the schematic of PMN-facilitated melanoma extravasation through EI monolayer in a shear flow. (b) Migration varies under constant shear stress but increasing shear rate. (c) Migration is unchanged over an order of magnitude of shear stress when shear rate is constant. All values are mean ± SEM for N ≥ 3.

FIGURE 2

FIGURE 2

Effects of shear rate and shear stress on melanoma adhesion to the EI monolayer. (a) PMN tethering frequency under flow conditions. (b) Melanoma cell adhesion to the EI monolayer under flow conditions. WM9 adhesion efficiency and data correction procedure are defined in “Materials and Methods”. All values are mean ± SEM for N ≥ 3.

FIGURE 3

FIGURE 3

The kinetics of PMN-WM9 aggregation under different shear conditions. (a) Detection of PMN-tumor cell aggregates by two-color flow cytometry. LDS-571-labeled PMNs (1 × 106/mL), TRITC-stained WM9 melanoma cells (1 × 106/mL), or both were sheared at 62.5 s-1 for 120 s in a cone-plate viscometer in the presence of 1 _μ_M fMLP. Upon termination of shear, aliquots were immediately fixed with 2% formaldehyde and subsequently analyzed in a GUAVA flow cytometer. Left panel shows PMN only using flow cytometry; middle panel shows WM9 only; and right panel shows WM9-PMN heterotypic aggregates. The population of WM9 cells was resolved into singlet and aggregates composed of a single WM9 cell bound to one, two, or more than two PMNs. The concentrations of these aggregates were represented by [WM9], [TP1], [TP2], and [TP3+], respectively. The gating was based on the specific fluorescence channel where each population fell in. (b) The percentage of melanoma cell in the heterotypic aggregations at different shear rates with a medium viscosity 1.0 cP. (c) The percentage of melanoma cells in the heterotypic aggregations at a fixed shear rate 62.5 s-1 while shear stress varied from 2 to 6.4 dyn/cm2. (d) The percentage of melanoma cells in the heterotypic aggregations under a fixed shear stress 2 dyn/cm2 while shear rate varied from 62.5 to 100 s-1. Values are mean ± S.E.M. for N ≥ 3. *p < 0.05.

FIGURE 4

FIGURE 4

Effects of _β_2 integrin-ICAM-1 binding on PMN-WM9 aggregation. Blocking of _β_2 integrin on PMNs significantly reduced PMN-WM9 aggregation compared with control. *p < 0.05 compared with the other two blocking cases. Values are mean ± SEM for N ≥ 3.

FIGURE 5

FIGURE 5

Effects of PMN tethering on melanoma cell adhesion to the EI monolayer. PMNs were treated with mAb to functionally block LFA-1 and Mac-1 (30 min, 4 °C); EI monolayer was treated with mAb against ICAM-1 (30 min, 4 °C); WM9 cells were treated with mAb against ICAM-1 (30 min, 4 °C). After each treatment, the excess of mAb was washed out by centrifuging cells down and re-suspending them in fresh medium. Cells were then injected into the parallel plate flow chamber for the adhesion assay. Blocking ICAM-1 on WM9 significantly reduced melanoma adhesion efficiency compared with both the control and anti-ICAM-1 on EI cases. *p < 0.05 compared with control samples. #p < 0.05 with respect to concurrent ICAM-1 blocking on the EI cells under the same shear condition. Values are mean ± SEM for N ≥ 3.

FIGURE 6

FIGURE 6

Population ratio effects on: (a) PMN-WM9 aggregation; (b) WM9 adhesion to the EI monolayer; and (c) C8161 extravasation under various shear conditions. Increase in ratio of PMN to WM9 significantly promotes PMN-WM9 aggregation, WM9 adhesion to the EI monolayer; and increase in ratio of PMN to C8161 increases subsequent extravasation through the EI monolayer. Values are mean ± SEM for N ≥ 3.

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