title: Technical Insights into Highly Sensitive Isolation and Molecular Characterization of Fixed and Live Circulating Tumor Cells for Early Detection of Tumor Invasion.
abstract: Circulating Tumor Cells (CTC) and Circulating Tumor Microemboli (CTM) are Circulating Rare Cells (CRC) which herald tumor invasion and are expected to provide an opportunity to improve the management of cancer patients. An unsolved technical issue in the CTC field is how to obtain highly sensitive and unbiased collection of these fragile and heterogeneous cells, in both live and fixed form, for their molecular study when they are extremely rare, particularly at the beginning of the invasion process. We report on a new protocol to enrich from blood live CTC using ISET® (Isolation by SizE of Tumor/Trophoblastic Cells), an open system originally developed for marker-independent isolation of fixed tumor cells. We have assessed the impact of our new enrichment method on live tumor cells antigen expression, cytoskeleton structure, cell viability and ability to expand in culture. We have also explored the ISET® in vitro performance to collect intact fixed and live cancer cells by using spiking analyses with extremely low number of fluorescent cultured cells. We describe results consistently showing the feasibility of isolating fixed and live tumor cells with a Lower Limit of Detection (LLOD) of one cancer cell per 10 mL of blood and a sensitivity at LLOD ranging from 83 to 100%. This very high sensitivity threshold can be maintained when plasma is collected before tumor cells isolation. Finally, we have performed a comparative next generation sequencing (NGS) analysis of tumor cells before and after isolation from blood and culture. We established the feasibility of NGS analysis of single live and fixed tumor cells enriched from blood by our system. This study provides new protocols for detection and characterization of CTC collected from blood at the very early steps of tumor invasion.
authors.affiliation: Rarecells Diagnostics, Paris, France.
authors.forename: Sophie
authors.initials: S
authors.lastname: Laget
authors.affiliation: Rarecells Diagnostics, Paris, France.|Unité INSERM U1151 (Eq 13), Faculté de Médecine Paris Descartes, Paris, France.
authors.forename: Lucile
authors.initials: L
authors.lastname: Broncy
authors.affiliation: Rarecells Diagnostics, Paris, France.
authors.forename: Katia
authors.initials: K
authors.lastname: Hormigos
authors.affiliation: Thermo Fisher Scientific, South San Francisco, California, United States of America.
authors.forename: Dalia M
authors.initials: DM
authors.lastname: Dhingra
authors.affiliation: Unité INSERM U1151 (Eq 13), Faculté de Médecine Paris Descartes, Paris, France.
authors.forename: Fatima
authors.initials: F
authors.lastname: BenMohamed
authors.affiliation: Unité INSERM U1151 (Eq 13), Faculté de Médecine Paris Descartes, Paris, France.
authors.forename: Thierry
authors.initials: T
authors.lastname: Capiod
authors.affiliation: Fasteris SA, Geneva, Switzerland.
authors.forename: Magne
authors.initials: M
authors.lastname: Osteras
authors.affiliation: Fasteris SA, Geneva, Switzerland.
authors.forename: Laurent
authors.initials: L
authors.lastname: Farinelli
authors.affiliation: Thermo Fisher Scientific, South San Francisco, California, United States of America.
authors.forename: Stephen
authors.initials: S
authors.lastname: Jackson
authors.affiliation: Unité INSERM U1151 (Eq 13), Faculté de Médecine Paris Descartes, Paris, France.
authors.forename: Patrizia
authors.initials: P
authors.lastname: Paterlini-Bréchot
mesh_terms: D000818:Animals; D000951:Antigens, Neoplasm / Q000276:immunology / Q000378:metabolism; D014408:Biomarkers, Tumor; D045744:Cell Line, Tumor; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival; D003599:Cytoskeleton / Q000378:metabolism; D055088:Early Detection of Cancer / Q000379:methods* / Q000592:standards; D005820:Genetic Testing / Q000379:methods / Q000592:standards; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D007150:Immunohistochemistry; D018189:Immunomagnetic Separation / Q000379:methods; D017404:In Situ Hybridization, Fluorescence; D051379:Mice; D009361:Neoplasm Invasiveness; D009369:Neoplasms / Q000175:diagnosis* / Q000378:metabolism*; D009360:Neoplastic Cells, Circulating / Q000378:metabolism* / Q000473:pathology*; D015203:Reproducibility of Results
chemical_list: D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor