CD15s/CD62E Interaction Mediates the Adhesion of Non-Small Cell Lung Cancer Cells on Brain Endothelial Cells: Implications for Cerebral Metastasis

Abstract

Expression of the cell adhesion molecule (CAM), Sialyl Lewis X (CD15s) correlates with cancer metastasis, while expression of E-selectin (CD62E) is stimulated by TNF-α. CD15s/CD62E interaction plays a key role in the homing process of circulating leukocytes. We investigated the heterophilic interaction of CD15s and CD62E in brain metastasis-related cancer cell adhesion. CD15s and CD62E were characterised in human brain endothelium (hCMEC/D3), primary non-small cell lung cancer (NSCLC) (COR-L105 and A549) and metastatic NSCLC (SEBTA-001 and NCI-H1299) using immunocytochemistry, Western blotting, flow cytometry and immunohistochemistry in human brain tissue sections. TNF-α (25 pg/mL) stimulated extracellular expression of CD62E while adhesion assays, under both static and physiological flow live-cell conditions, explored the effect of CD15s-mAb immunoblocking on adhesion of cancer cell-brain endothelium. CD15s was faintly expressed on hCMEC/D3, while high levels were observed on primary NSCLC cells with expression highest on metastatic NSCLC cells (p < 0.001). CD62E was highly expressed on hCMEC/D3 cells activated with TNF-α, with lower levels on primary and metastatic NSCLC cells. CD15s and CD62E were expressed on lung metastatic brain biopsies. CD15s/CD62E interaction was localised at adhesion sites of cancer cell-brain endothelium. CD15s immunoblocking significantly decreased cancer cell adhesion to brain endothelium under static and shear stress conditions (p < 0.001), highlighting the role of CD15s-CD62E interaction in brain metastasis.

Keywords: CD15s; CD62E; Sialyl Lewis X; adhesion; brain metastasis.

Figure 1

Expression of CD15s in brain endothelial and lung cancer cell lines: (A) Representative ICC images showing CD15s in hCMEC/D3, NCI-H1299, SEBTA-001, A549 and COR-L105. Hoechst blue was used as a nuclear counterstain. Scale bar = 20 µm; (B) Western blot analysis of CD15s of proteins from the cell lines showed highest CD15s expression in NCI-H1299 and SEBTA-001 followed by hCMEC/D3, COR-L105 and A549. Cyclophilin A was used as a protein loading control; (C) representative flow cytometric histograms (Green: control, purple: hCMEC/D3, black: NCI-H1299, red: SEBTA-001, blue COR-L105 and yellow: A549); (D) quantitative flow cytometric analysis of CD15s expression on hCMEC/D3, NCI-H1299, SEBTA-001, COR-L105 and A549. CD15s was overexpressed in NCI-H1299 and SEBTA-001 with less expression in hCMEC/D3, COR-L105 and A549. n = 3, *** p < 0.001 and ** p < 0.01. Data is expressed as ±SE.

Figure 2

The role of CD62E in adhesion of NSCLC cells to brain endothelium: (A) Qualitative adhesion of NSCLC cells onto brain endothelium monolayer. Green fluorescently tagged NSCLC cells were applied onto the hCMEC/D3 monolayer and incubated for 90 min with and without activation via TNF-α. Non-adherent cells were washed and co-cultures were fixed and examined by confocal microscopy; (B) quantitative adhesion of NSCLC cells. hCMEC/D3 cells were seeded into 96-well plate followed by seeding of green fluorescently tagged NSCLC cells on the hCMEC/D3 monolayer and incubated for 90 min incubation. Non-adherent cells were washed out and adherent cells were lysed followed by quantification via a microplate reader at 480–520 nm. Results showed a strong decrease in adhesion caused by absence of TNF-α (White bar) compared to TNF-α stimuli. n = 3, *** p < 0.001 and ** p < 0.01. Scale bar = 20 µm. Data is expressed as ±SE.

Figure 3

(A) CD15s immunoblocking reduced the adhesion of lung cancer cells under static conditions. Confocal images (top panel) showing adhesion of green fluorescently labelled NSCLCs on a brain endothelial cell monolayer (blue) and semi-quantitative analysis of confocal images (lower panel) showed a significant decrease in adhesion ability of NSCLC cells to adhere to hCMEC/D3 cells. n = 3, *** p < 0.001 and ** p < 0.01. Scale bar = 20 µm. Data is expressed as ±SE. We used the same concentration of TNF-α as optimised in a previous study [5] to activate the expression of CD62E on brain endothelial cells. Brain endothelial cells (hCMEC/D3) were exposed to 25 pg/mL of TNF-α for 18 h and levels of CD62E were detected using flow cytometry (Data not shown); (B) blocking with CD15s mAb significantly decreased the adhesion of NSCLC to brain endothelium. Quantitative adhesion assay of human primary and metastatic lung cancer (NSCLC) cells blocked with CD15s mAb (blue bar), non-specific isotype IgM (white bar) to assess the efficiency and specificity of CD15s mAb-blocking efficiency and NSCLC cells on a monolayer of hCMEC/D3 (grey bar) as a negative control. n = 3, *** p < 0.001. Data is expressed as ±SE.

Figure 4

Immunoblocking with CD15s mAb significantly decreased the adhesion ability of NSCLC under dynamic conditions (2.5 dyn/cm²). A dynamic cell adhesion assay was carried out on highly metastatic brain cells (SEBTA-001) using an AxioVert 200 M microscope. (Top panel) Cancer cells were incubated with isotype control (IgM) or CD15 mAb followed by perfusion of 1 × 10⁶ cancer cells over a monolayer of hCMEC/D3 cells at 2.5 dyn/cm² for 90 min. Phase contrast and fluorescent images were acquired at real time every 10 min with a ×5 objective using Volocity software. Scale bar = 20 µm. (Lower panel) Results are shown in relative fluorescent units of adherent cancer cells with and without CD15 mAb for 10, 20, 30, 40, 60 and 90 min time points. * p < 0.05. Data is expressed as ±SE.

Figure 5

Localisation of CD15s on the surface of adherent SEBTA-001 cells at the site of adhesion. Confocal image of green fluorescently-labelled adherent brain to lung metastatic cancer cells (SEBTA-001) on a monolayer of activated hCMEC/D3 cells (blue). (A) Expression of CD15s (red) (white arrows) was seen on the edges of adherent cells SEBTA-001 (green) at the site of adhesion on brain endothelial cells; (B) expression of CD15s (red) (white arrows) was observed on surface of adherent cancer cells SEBTA-001 (green) and CD62E (purple) (white arrows) was seen on the monolayer of activated brain endothelial cells (hCMEC/D3) at adhesion site of cancer cell–brain endothelium. Scale bar = 20 µm.

Figure 6

Representative immunohistochemistry images from one patient with lung–brain metastasis. (Left panel) CD15s was detected in tumour core and infiltrated into non-neoplastic host brain tissue. No expression was seen in a normal brain section. (Right panel) CD62E was expressed on the inner lining of brain endothelial cells with no expression seen in normal brain sections. Images were obtained using an Ariol microscope (Leica, Wetzlar, Germany) at 20× and 40× magnification. Scale bar = 20 µm. n = 1.