Non-small cell lung cancer is one of the most common primary tumours to metastasise to the brain in adults. The underlining molecular mechanisms of brain metastasis are still not fully understood. Interactions between brain endothelial cells and cancer cells play key roles in brain metastasis. CD15 and CD15s are cell-cell adhesion molecules which interact with E-selectin which is expressed on endothelial cells and known to be involved in the leukocyte homing process as well as being implicated in metastasis with many non-CNS neoplasms. The aim of this project was to investigate the role of CD15 and CD15s in cancer cell adhesion to brain endothelial cells and transendothelial migration of lung cancer cells during brain metastasis. Expression of CD15, CD15s and CD62E was characterised in human primary and brain metastatic lung cancer cells using immunocytochemistry, flow cytometry, Western blot and immunohistochemistry in human tissue sections. Effects of CD15 and CD15s expression on NSCLC metastatic to brain were assessed using genetic manipulation (overexpression and knockdown) followed by functional assays. Both CD15 and CD15s were overexpressed and knockdowned and cell-cell
adhesion was then examined using qualitative and quantitative adhesion assays, under both static and flow physiological conditions. Transendothelial migration potential was also assessed using a voltometer, Electric Cell-Substrate Impedance sensing system and cell-monitoring system CellZscope™. Findings showed that CD15 and CD15s were prominently expressed on metastatic lung cancer cells (SEBTA-001, SEBTA-005 and NCI-H1299) and weakly expressed on both primary lung cancer cells (COR-L105 and A549) and brain endothelium (hCMEC/D3). The highest expression of CD62E was observed on brain endothelium stimulated with TNF-α (25pg/ml) (p<0.001). CD15, CD15s and CD62E expression was detected in human metastatic tissues. The absence of CD62E and immunoblocking and knockdown of CD15 and CD15s significantly reduced the adhesion of cancer cells under both static and shear stress conditions (p<0.0001).
Overexpression of CD15 and CD15s significantly increased their adhesion on an endothelial monolayer (p<0.001). Metastatic cancer cells were able to transmigrate through a brain endothelial monolayer compared to primary and glioblastoma multiforme (GBM) cells. Knockdown of CD15 and CD15s decreased the transendothelial migration potential of cancer cells while even primary lung cancer cells and GBM cells transmigrated following overexpression of CD15 and CD15s. These results confirmed the correlation between CD15 and CD15s in adhesion as well as transendothelial migration of cancer cells during cerebral metastasis.
|Date of Award||Jan 2016|
|Supervisor||Geoff Pilkington (Supervisor), Helen Fillmore (Supervisor) & Zaynah Maherally (Supervisor)|