title: Quantitative determination of tumor cell intravasation in a real-time polymerase chain reaction-based assay.
abstract: Tumor cells acquire the ability to enter blood vessels surrounding the primary tumor, endowing them with the capacity to disseminate and become established in distant sites, originating a metastasis. Determination of the intravasation ability of tumor cells is thus important, as it can be correlated with their potential malignancy. To analyze the intravasation phenotype of human tumor cells in vivo, we performed chick embryo chorioallantoic membrane (CAM) assays. Cells were inoculated on the CAM of 9-day-old chick embryos and the membrane at the opposite side of the egg was recovered after 48 h incubation. To measure intravasation ability, we calculated the amount of human DNA in each CAM sample by real-time PCR of Alu sequences and SYBR Green 1 fluorescence detection. This analysis showed a detection limit of 1 human cell per 10(5) total cells, and we were able to distinguish between tumor cells of distinct invasive capacity. This assay has several advantages over current methods to measure intravasation ability, including the elimination of post-PCR analysis, sensitivity and easy scale-up of sample numbers.
authors.affiliation: Department of Immunology and Oncology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Madrid, Spain. emira@cnb.uam.es
authors.forename: Emilia
authors.initials: E
authors.lastname: Mira
authors.forename: Rosa Ana
authors.initials: RA
authors.lastname: Lacalle
authors.forename: Concepción
authors.initials: C
authors.lastname: Gómez-Moutón
authors.forename: Esther
authors.initials: E
authors.lastname: Leonardo
authors.forename: Santos
authors.initials: S
authors.lastname: Mañes
mesh_terms: D000230:Adenocarcinoma / Q000473:pathology; D000482:Allantois / Q000473:pathology; D020087:Alu Elements; D000818:Animals; D052160:Benzothiazoles; D001943:Breast Neoplasms / Q000473:pathology; D002642:Chick Embryo; D002823:Chorion / Q000473:pathology; D003199:Computer Systems; D004273:DNA, Neoplasm / Q000032:analysis*; D003959:Diamines; D004996:Ethidium / Q000032:analysis; D005347:Fibroblasts; D005456:Fluorescent Dyes / Q000032:analysis; D006801:Humans; D009361:Neoplasm Invasiveness; D009930:Organic Chemicals; D010641:Phenotype; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D011804:Quinolines; D014407:Tumor Cells, Cultured; D001749:Urinary Bladder Neoplasms / Q000473:pathology
chemical_list: D052160:Benzothiazoles; D004273:DNA, Neoplasm; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I; D004996:Ethidium