Molecular Medicine Tri-Con February 15 - 20, 2015, San Francisco, CA
Multiplex Mesofluidic Device for Simultaneous Detection of Heterogeneous Viable Subpopulations of Circulating Tumor Cells (CTCs) in Cancer Patients Phuong Lan Tran Stanislas Ropert[1]; François Goldwasser[1]; Patrick Tauc[2]; Frank Griscelli[3]; Jérome Darbon[4]; Marc Sigelle[5] FISHingCELL, 25 Rue du Moulin de la Vierge, Paris, F-75014 France [1] Cochin-Broca-Hotel Dieu Teaching Hospital, Department of Medical Oncology, Paris Descartes University, Paris, 75014 France; [2] LBPA, Institut d’Alembert, ENS Cachan, Cachan, 94235 France; [3] INSERM U935, Villejuif, 94800 France; [4] CMLA, ENS Cachan, Cachan, 94235 France; [5] Telecom ParisTech, Paris, 75014 France
Introduction: Circulating Tumor Cells (CTCs) shed from primary tumors and metastases have become a helpful tool as liquid biopsy marker in cancer diagnostics. Evaluation of CTCs in cancer patients has a potential clinical value for correlation in disease staging or response to therapy. CTCs may comprise heterogeneous subpopulations, epithelial and non-epithelial cells: endothelial (CEC), stem cells (CSC), and those that have undergone the epithelial to mesenchymal transition (EMT). We previously described the development of a new mesofluidic multiplex immunosensor device for selective capture and downstream analysis of CTCs from blood samples of metastatic breast cancer patients (Breton et al, 2011). We now report a considerable improvement of the multiplex device performance in simultaneously capturing selective and specific epithelial and non-epithelial CTCs from a single patient blood sample. This multiplex detection was achieved for several types of metastatic carcinoma and on samples collected before and after chemotherapeutic treatment. Blood samples from healthy individuals were analyzed in parallel and used as controls. Methods: Blood samples from 21 patients with metastatic carcinomas under chemotherapy were used. Two blood samples were drawn immediately before and two at the end of chemotherapy infusion for two repeated cycles. Four to 6 hours after collection, all fresh blood samples were submitted to a Ficoll-Hypaque density gradient centrifugation. Then the interphase leukocyte cells can be used immediately as fresh or frozen in calf fetal serum plus 10% DMSO at -80 °C for subsequent cell capture experiments. To perform simultaneously capture of two subpopulations: epithelial CTC and CEC, each standard microscopic glass slide comprised four silanized surfaces of dimensions 6x16 mm2 of which two surfaces were coated with the anti-EpCAM antibody for epithelial cell detection and both others with the anti-CD146 antibody for CEC detection. Downstream analyses of each subpopulation on the same slide were
performed: first, by immunostaining to identify cells of interest, and then processed for fluorescence in situ hybridization (FISH). Viable CTCs were also suitable for labeling with mRNA and miRNA nanoprobes. Results: The cohort of cancer patients included colonic (5), pancreatic (3), prostatic (3), lung (1), liver (1), gastric (1), ovarian (2), kidney (1), bladder (2), cholangiohepatoma (1), and sweat gland (1) carcinomas. Cell captures were performed for each blood sample onto functionalized slides in duplicate. A concomitant and selective detection in parallel of both CTCs and CECs subpopulations in each blood sample was obtained. CTCs were scored as pan-cytokeratin positive and DAPI for DNA content, and CECs as CD144 positive and DAPI for DNA content. All identified CTCs and CECs stained negative for the pan-leukocyte anti-CD45 antibody. Cell numbers were manually scored across fluorescence scanning microscopy images of the full device. Cell numbers were highly consistent across duplicate chambers. Genomic analyses on surfaces containing epithelial CTCs were accomplished, for example, with EGF-R FISH probes in colonic samples and with PTEN and TMPRSS2-ERG FISH probes in prostatic samples. FISH in the epithelial CTCs identified by immunostaining as cells of interest was thus optimized with commercial FISH probes. Conclusion: This study was successful in addressing a technical challenge. It will help making sense of CTCs subpopulations as liquid biomarkers in cancer therapy. For information: PL Tran
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