title: Comparison of invasion by human microvascular endothelial cell lines in response to vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in a threedimensional (3D) cell culture system.
abstract: BACKGROUND
  Immortalized human endothelial cells are widely used as in vitro models for debilitating conditions such as cancer, cardiovascular and ocular diseases. Human microvascular endothelial cell (HMEC-1) is immortalized via stable transfection with a gene encoding SV40 large antigen whilst telomerase-immortalized human microvascular endothelial (TIME) cells is immortalized by engineering the human telomerase catalytic protein (hTERT) into primary microvascular endothelial cells. Here, we established a three-dimensional (3D) spheroid invasion assay with HMEC-1 and TIME and compared the difference in their ability to invade through the collagen matrix in response to exogenous growth factors, namely vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF).
  
  
  METHODS
  TIME and HMEC-1 spheroids were embedded in a collagen matrix. The spheroids were stimulated with exogenous growth factors, namely VEGF (50 ng/mL) and bFGF (200 ng/mL). Twelve points of invasion length from a spheroid was measured using image analysis software, Image J. Three independent experiments were conducted and data was analysis by GraphPad Instat software, version 3.05.
  
  
  RESULTS
  TIME spheroid invasion was 16.5 fold higher with exogenous VEGF (50 ng/mL) and bFGF (200 ng/mL) treatment as compared to those cultured in complete growth medium only. In contrast, no significant difference was observed between HMEC-1 spheroids stimulated with and without exogenous growth factors, VEGF and bFGF.
  
  
  CONCLUSIONS
  This is the first report on the establishment of a 3D-spheroid invasion assay with TIME cells. The requirement of VEGF and bFGF for TIME spheroids invasion is a novel finding. In addition, this assay offers an advantage over HMEC-1 for testing novel angiogenic agents since it is not affected by endogenously secreted growth factors.
authors.affiliation: Universiti Putra Malaysia, Faculty of Medicine and Health Sciences, Department of Pathology, 43400 UPM Serdang, Selangor, Malaysia. shf@upm.edu.my.
authors.forename: Chin Tat
authors.initials: CT
authors.lastname: Ng
authors.forename: Wai Kien
authors.initials: WK
authors.lastname: Yip
authors.forename: Norhafizah
authors.initials: N
authors.lastname: Mohtarrudin
authors.forename: Heng Fong
authors.initials: HF
authors.lastname: Seow
mesh_terms: D018929:Cell Culture Techniques / Q000379:methods*; D042783:Endothelial Cells / Q000187:drug effects / Q000473:pathology*; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism* / Q000494:pharmacology; D005818:Genetic Engineering; D006801:Humans; D009361:Neoplasm Invasiveness; D009389:Neovascularization, Pathologic / Q000473:pathology*; D019098:Telomerase; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism* / Q000494:pharmacology
chemical_list: C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D016222:Fibroblast Growth Factor 2; C509186:TERT protein, human; D019098:Telomerase