{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,4]],"date-time":"2026-10-04T02:47:53Z","timestamp":1791082073759,"version":"4.1.0"},"reference-count":48,"publisher":"Wiley","issue":"7","license":[{"start":{"date-parts":[[2016,4,20]],"date-time":"2016-04-20T00:00:00Z","timestamp":1461110400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"funder":[{"DOI":"10.13039\/501100001659","name":"Deutsche Forschungsgemeinschaft","doi-asserted-by":"publisher","award":["DFG, NE805\/4-1"],"award-info":[{"award-number":["DFG, NE805\/4-1"]}],"id":[{"id":"10.13039\/501100001659","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["febs.onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Molecular Oncology"],"published-print":{"date-parts":[[2016,8]]},"abstract":"<jats:p>The presence of circulating tumor cells (CTCs) in the blood of ovarian cancer patients was shown to correlate with decreased overall survival, whereby CTCs with epithelial\u2013mesenchymal\u2010transition (EMT) or stem\u2010like traits are supposed to be involved in metastatic progression and recurrence. Thus, investigating the transcriptional profiles of CTCs might help to identify therapy resistant tumor cells and to overcome treatment failure. For this purpose, we established a multi\u2010marker panel for the molecular characterization of single CTCs, detecting epithelial (EpCAM, Muc\u20101, CK5\/7), EMT (N\u2010cadherin, Vimentin, Snai1\/2, CD117, CD146, CD49f) and stem cell (CD44, ALDH1A1, Nanog, SOX2, Notch1\/4, Oct4, Lin28) associated transcripts.<\/jats:p><jats:p>First primer specificity and PCR\u2010performance of the multiplex\u2010RT\u2010PCRs were successfully validated on genomic DNA and cDNA isolated from OvCar3 cells. The assay sensitivity of the epithelial panel was evaluated by adding defined numbers of tumor cells into the blood of healthy donors and performing a subsequent immunomagnetic tumor cell enrichment (AdnaTest OvarianCancerSelect), resulting in a 100% concordance for the epithelial markers EpCAM and Muc\u20101 to the AdnaTest OvarianCancerDetect. Additionally, by processing blood from ovarian cancer patients, high assay sensitivity could be verified. In blood of healthy donors no signals for epithelial markers were detected, for EMT and stem cell markers, however, signals were obtained mainly originating from leukocytes which calls for single cell analysis.<\/jats:p><jats:p>To that aim by using the ovarian cancer cell line OvCar3, we successfully established a workflow enabling the characterization of single CTCs. It consists of a density gradient\u2010dependent enrichment for nucleated cells, a depletion of CD45\u2010positive cells of hematopoietic origin followed by immunofluorescent labeling of CTCs by EpCAM and Muc\u20101. Single CTCs are then isolated by micromanipulation and processed for panel gene expression profiling. Finally, fifteen single CTCs from three ovarian cancer patients were analyzed and found to be positive for stem cell (CD44, ALDH1A1, Nanog, Oct4) and EMT markers (N\u2010cadherin, Vimentin, Snai2, CD117, CD146). Albeit, inter\u2010cellular and intra\/inter\u2010patient heterogeneity and co\u2010expression of epithelial, mesenchymal and stem cell transcripts on the same CTC was observed.<\/jats:p><jats:p>We have established a robust workflow to perform sensitive single cell panel gene expression analysis without the need of pre\u2010amplification steps. Our data point towards a heterogeneous expression of stem cell and EMT associated transcripts in ovarian cancer CTCs.<\/jats:p>","DOI":"10.1016\/j.molonc.2016.04.002","type":"journal-article","created":{"date-parts":[[2016,5,1]],"date-time":"2016-05-01T02:42:56Z","timestamp":1462070576000},"page":"1030-1042","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":85,"title":["Gene expression profiling of single circulating tumor cells in\u00a0ovarian cancer \u2013 Establishment of a multi\u2010marker gene panel"],"prefix":"10.1002","volume":"10","author":[{"given":"Christina","family":"Blassl","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jan Dominik","family":"Kuhlmann","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alessandra","family":"Webers","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pauline","family":"Wimberger","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tanja","family":"Fehm","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hans","family":"Neubauer","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2016,4,20]]},"reference":[{"key":"e_1_2_11_2_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.ygyno.2014.06.013"},{"key":"e_1_2_11_3_1","doi-asserted-by":"publisher","DOI":"10.1097\/IGC.0b013e318216cb91"},{"key":"e_1_2_11_4_1","doi-asserted-by":"publisher","DOI":"10.1186\/bcr2333"},{"key":"e_1_2_11_5_1","doi-asserted-by":"publisher","DOI":"10.1158\/0008-5472.CAN-04-3931"},{"key":"e_1_2_11_6_1","doi-asserted-by":"publisher","DOI":"10.1186\/1471-2407-12-114"},{"key":"e_1_2_11_7_1","doi-asserted-by":"publisher","DOI":"10.18632\/oncotarget.2053"},{"key":"e_1_2_11_8_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.ccr.2012.11.009"},{"key":"e_1_2_11_9_1","doi-asserted-by":"publisher","DOI":"10.2353\/ajpath.2006.060312"},{"key":"e_1_2_11_10_1","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkm051"},{"key":"e_1_2_11_11_1","doi-asserted-by":"publisher","DOI":"10.1002\/cncr.24149"},{"key":"e_1_2_11_12_1","doi-asserted-by":"publisher","DOI":"10.1155\/2010\/105269"},{"key":"e_1_2_11_13_1","doi-asserted-by":"publisher","DOI":"10.1097\/IGC.0b013e3182907109"},{"key":"e_1_2_11_14_1","doi-asserted-by":"publisher","DOI":"10.1007\/s10549-010-1163-x"},{"key":"e_1_2_11_15_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.ijgo.2008.12.015"},{"key":"e_1_2_11_16_1","doi-asserted-by":"publisher","DOI":"10.1038\/onc.2011.384"},{"key":"e_1_2_11_17_1","doi-asserted-by":"publisher","DOI":"10.1158\/1535-7163.MCT-12-0460"},{"key":"e_1_2_11_18_1","doi-asserted-by":"publisher","DOI":"10.1002\/cncr.11349"},{"key":"e_1_2_11_19_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.biocel.2007.07.011"},{"key":"e_1_2_11_20_1","doi-asserted-by":"publisher","DOI":"10.1007\/s13577-010-0002-z"},{"key":"e_1_2_11_21_1","doi-asserted-by":"publisher","DOI":"10.1186\/bcr2896"},{"key":"e_1_2_11_22_1","doi-asserted-by":"publisher","DOI":"10.1186\/bcr3099"},{"key":"e_1_2_11_23_1","doi-asserted-by":"publisher","DOI":"10.3332\/ecancer.2013.352"},{"key":"e_1_2_11_24_1","doi-asserted-by":"publisher","DOI":"10.2174\/1871520615666150716104152"},{"key":"e_1_2_11_25_1","doi-asserted-by":"publisher","DOI":"10.1373\/clinchem.2014.224808"},{"key":"e_1_2_11_26_1","doi-asserted-by":"publisher","DOI":"10.1038\/bjc.2013.265"},{"key":"e_1_2_11_27_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2008.03.027"},{"key":"e_1_2_11_28_1","doi-asserted-by":"publisher","DOI":"10.1053\/j.seminoncol.2008.12.003"},{"key":"e_1_2_11_29_1","doi-asserted-by":"publisher","DOI":"10.7150\/jca.5111"},{"key":"e_1_2_11_30_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0002888"},{"key":"e_1_2_11_31_1","doi-asserted-by":"publisher","DOI":"10.15252\/emmm.201404033"},{"key":"e_1_2_11_32_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.ygyno.2011.05.028"},{"key":"e_1_2_11_33_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0033788"},{"key":"e_1_2_11_34_1","doi-asserted-by":"publisher","DOI":"10.1158\/1078-0432.CCR-14-1213"},{"key":"e_1_2_11_35_1","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-60327-933-8"},{"key":"e_1_2_11_36_1","doi-asserted-by":"publisher","DOI":"10.1385\/1-59259-192-2:365"},{"key":"e_1_2_11_37_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0029-7844(98)00334-2"},{"key":"e_1_2_11_38_1","doi-asserted-by":"publisher","DOI":"10.1002\/ijc.26311"},{"key":"e_1_2_11_39_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0144535"},{"key":"e_1_2_11_40_1","unstructured":"2011 R. 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