Overexpression of  malignancy-associated laminins and  laminin receptors by angiotropic  human melanoma cells in a chick  chorioallantoic membrane model.
BACKGROUND
As distinct from intravascular/lymphatic dissemination, extravascular migratory metastasis (EVMM) has been described as a potential additional mechanism of melanoma spread in which tumor cells migrate along the external surfaces of vessels. Angiotropic melanoma cells are linked to the endothelium by a matrix containing laminin. In addition, it has been shown that C16 laminin-derived peptide increases extravascular migration of human green fluorescent protein (GFP) melanoma cells along vessels in a chicken chorioallantoic membrane model (CAM). In this study, we have tested the hypothesis that expression levels of some genes related to lamimin and metastasis are differentially expressed in vascularized angiotropic melanoma areas vs. avascular melanoma areas from the same tumor.


METHODS
C8161 human melanoma cells in a shell-less chick CAM assay were used to study EVMM associated with the presence of vascularized angiotropic melanoma areas. For both high-quality histomorphology and RNA preservation in paraffin-embedded tissue, we used a methanol-based fixative coupled with microwave-assisted rapid tissue processing as previously described. Using laser capture microdissection, angiotropic melanoma areas as well as avascular areas were microdissected. Using quantitative real time polymerase chain reaction (QRT-PCR), six genes have been studied: LAMC2 (laminin gamma2 chain), LAMA4 (laminin alpha4 chain), ITGB1 (integrin beta1), ITGB3 (integrin beta3), RSPA (ribosomal protein), and MMP2 (matrix metallopeptidase 2). QRT-PCR data were normalized to human GAPDH housekeeping gene and values were compared against Human Total RNA. Final results were expressed as percentage of expression.


RESULTS
All tumors demonstrated a similar pattern, i.e. EVMM of angiotropic melanoma cells. The microdissected histopathological sections presented both angiotropic areas and avascular areas. All genes were overexpressed in angiotropic melanoma areas vs. avascular melanoma areas, especially LAMC2, LAMA4 and ITGB3 (respectively, 165.18, 208.86, and 483.69%).


CONCLUSIONS
This study shows that several genes related to laminin are overexpressed in angiotropic melanoma areas vs. avascular melanoma areas. Since extravascular migration of melanoma cells along vessels has been demonstrated in the CAM model, taken together these results suggests that some laminins and laminin receptors may play a role in extravascular migratory metastasis. This model may represent a promising strategy to analyze differential gene expression in EVMM.
Lugassy
Claire
C
Department of Pathology, University of Miami Miller School of Medicine, Miami, FL, USA.
Torres-Muñoz
Jorge E
JE
Kleinman
Hynda K
HK
Ghanem
Ghanem
G
Vernon
Stephen
S
Barnhill
Raymond L
RL
D000818:Animals; D045744:Cell Line, Tumor; D002642:Chick Embryo; D049033:Chorioallantoic Membrane; D015870:Gene Expression; D006801:Humans; D007150:Immunohistochemistry; D019012:Integrin beta1 / Q000096:biosynthesis / Q000235:genetics; D039661:Integrin beta3 / Q000096:biosynthesis / Q000235:genetics; D007797:Laminin / Q000096:biosynthesis* / Q000235:genetics; D007834:Lasers; D020778:Matrix Metalloproteinase 2 / Q000096:biosynthesis / Q000235:genetics; D008545:Melanoma / Q000235:genetics / Q000378:metabolism*; D042282:Microdissection; D009361:Neoplasm Invasiveness / Q000235:genetics*; D017474:Receptors, Laminin / Q000096:biosynthesis* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012269:Ribosomal Proteins / Q000096:biosynthesis / Q000235:genetics
C061248:ITGB3 protein, human; D019012:Integrin beta1; D039661:Integrin beta3; C400567:LAMC2 protein, human; D007797:Laminin; D017474:Receptors, Laminin; D012269:Ribosomal Proteins; D020778:Matrix Metalloproteinase 2