Selectin-mediated cell adhesion assay of tumor cells

Authors：

Category

Matrices & cellular trafficking

Protocol Name

Authors

Miyazaki, Keiko
Division of Molecular Pathology, Aichi Cancer Center

Kimura, Naoko
Division of Molecular Pathology, Aichi Cancer Center

Kannagi, Reiji *
Division of Molecular Pathology* / Research Complex for the Medical Frontiers**, Aichi Cancer Center* / Aichi Medical University**

*To whom correspondence should be addressed.

KeyWords

sialyl Lewis A hematogenous metastasis sialyl Lewis X cancer Selectin

Reagents

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Preparation of cells expressing selectins

Appropriate cultured endothelial cells (for instance, human umbilical vein endothelial cells, HUVECs) are grown in the monolayer at the bottom of 24-well plates in the presence of 1.0 ng IL1-β for 4 h. HUVECs at 2–6 passages after isolation will show the best response to IL-1β. Endothelial cells cultured without IL1-β serve as negative control cells.
Cultured cells transfected with E-selectin cDNA (for instance, E-sel/CHO cells) can be used instead of endothelial cells. In such case, mock transfectant cells can serve as control cells.

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Preparation and labeling of cancer cells

Cancer cells are harvested, resuspended in RPMI without FCS at 5×10⁶ cells/mL, and labeled with BCECF-AM (2'7'-bis-(carboxyethyl)-5-(and-6)- carboxyfluorescein acetoxymethyl ester) at a final concentration of 5 nM for 30 min at 37°C in the dark.
After labeling, the cells are washed twice with RPMI with 10% FCS, and are resuspended in the same medium.

Instruments

●	A rotary plate shaker: we use a small-size rotary shaker NR-3 (TAITEC Co., Ltd., Koshigaya, Japan).
●	A 24-well plate reader: we use an Arvo 1420 multi-label counter (Wallac, Gaithersburg, MD).

Methods

Selectin-mediated cell adhesion assay of tumor cells

1)	Remove the medium from the 24 well plate, and add the labeled cancer cells at 0.5–1.0×10⁶ cells/0.5 mL/well.	Comment 0

2)	Place the 24-well plate on a rotating platform (Taitec NR-3) for incubation under shear (90–180 rpm) for 20 min at room temperature in the dark.	Comment 0

3)	Remove the medium containing non-adherent cells, and wash the wells gently twice with PBS containing Ca²⁺ and Mg²⁺.	Comment 0

4)	Add 0.4 mL of 1% NP-40 in RPMI with 10% FCS to each well to lyse the adherent cells.	Comment 0

5)	Measure the fluorescence intensity of each well using a plate reader using a filter set for BCECF-AM (the same filter set as used for FITC).	Comment 0

Calculate the number of adherent cells in each well using a reference curve made from fluorescence intensity of known number of labeled cancer cells.

Comment 0

Notes

This cell adhesion protocol is called a non-static monolayer cell adhesion assay, because of continuous mechanical rotation of the plate applied throughout the incubation period. This is based on that carbohydrate-mediated cell adhesion is relatively resistant to mechanic shear stress, while cell adhesion mediated by proteins such as integrins generally cannot withstand mechanical stress.

During the washing procedures (step 3), the plate can be checked under a microscope, to confirm that the washings were done gently enough, making sure that adherent cells do not detach from endothelial cells at the bottom of the wells. After the washing procedure, the plate can be checked again under a microscope, to make sure that any non-adherent cells do not remain floating in the medium.

Inhibitory antibodies such as anti-E-selectin (for treatment of endothelial cells), or antibodies directed to carbohydrate ligands (for treatment of cancer cells) can be added to the wells (Fig. 1) to ensure which molecules are involved in the adhesion, depending on the purpose of the experiments.

Figure & Legends

Fig. 1. Non-static monolayer cell adhesion assay using human cancer cells and typical inhibition patterns of the adhesion to recombinant IL-1β-activated HUVECs by treatment with anti-sialyl Lewis X, and anti-sialyl Lewis A antibodies.

Cancer cells were treated with the respective antibodies (20 μg/mL) for 30 min prior to the adhesion experiment. Results for QG-56 (lung cancer), and Colo201 (colon cancer) cells are shown. Note that adhesion of QG-56 is inhibited by anti-sialyl Lewis X antibody, while that of Colo201 cells is inhibited by anti-sialyl Lewis A antibody.

This figure was originally published in Cancer Res. Takada A, Kannagi R. et al. “Contribution of carbohydrate antigens sialyl Lewis A and sialyl Lewis X to adhesion of human cancer cells to vascular endothelium” 1993, 53(2):354–61.

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Date of registration：2014-11-12 09:48:40