´╗┐Background The spatial heterogeneity of epithelial to mesenchymal transition (EMT)-related circulating tumor cells (CTCs) within the circulatory system and its potential clinical relevance remain unclear in pancreatic cancer (PC) patients

´╗┐Background The spatial heterogeneity of epithelial to mesenchymal transition (EMT)-related circulating tumor cells (CTCs) within the circulatory system and its potential clinical relevance remain unclear in pancreatic cancer (PC) patients. (RFS) were analyzed. Catharanthine sulfate {Results Both the number median CTC total count, and EMT status of CTCs [median mesenchymal CTC (M-CTC) percentage, 0.33 (0.13C0.52) in PoV 0.2 (0C0.4) in PV, P=0.0211] showed significant spatial heterogeneity during dissemination from the PoV to the PV. Univariate analysis adjusting for patient age and sex revealed that CTC total count and M-CTC percentage in PoV samples could be risk factors for RFS in PC patients (P=0.003 and P=0.001, respectively), and ROC curve analysis found that both of these factors had good performance in distinguishing patients with early distant recurrence (within 6 months), with the optimal cut-off values of 14 cells/mL (AUROC =0.893, sensitivity =0.857, specificity =0.813, P=0.001) and 0.545 (AUROC =0.795, sensitivity =0.714, specificity =0.906, Catharanthine sulfate P=0.016), respectively. Conclusions Multivascular assessment of EMT-related CTCs suggested profound dynamic alterations in total count and phenotypes during dissemination, and the spatial heterogeneity of CTCs in circulation could help establish novel prognosis markers in PC patients. compared CTC counts between portal vein (PoV) and peripheral vein (PV) blood samples collected during pancreaticoduodenectomy in 41 patients, and found that CTCs were detected in 58.5% of portal and 39% of PV samples from patients, and a high CTC count in portal blood was a significant predictor for early postoperative liver metastases (17). However, the majority of such studies focused on CTC counts only, without examining the EMT status of CTCs. Many different methods of CTC isolation have been developed based on the physical properties, such as size exclusion (18) of tumor cells, the positive selection of tumor cell surface markers (19), or the Rabbit Polyclonal to GCNT7 negative removal of white blood cells (20). However, there has not been any unified method so far due to the complexity of CTCs (21). To analyze the distinct EMT status of CTCs, density gradient centrifugation for blood sample pretreatment and CD45+ white blood cell depletion for CTC negative enrichment were applied in our study. Therefore, in this study, we aimed to identify CTCs in PoV and PV samples from 39 PC patients and explore possible dynamic changes in CTCs in circulation. Furthermore, we analyzed the relationship between CTC characteristics in PoV samples and the clinicopathologic data of PC patients, particularly those parameters indicating early recurrence in patients who had undergone resection. Methods Patients and characteristics This study was carried out with the approval of the Institutional Review Board at the Peking University First Hospital (ID: 2018 keyan-15), and in compliance with the guidelines of the Declaration of Helsinki. A total of 39 consecutive PC patients who were treated with curative surgical resection combined with adjuvant therapy at the Peking University First Hospital between March 2018 and April 2019 were recruited, Catharanthine sulfate and informed consent was obtained from all the patients. PV blood (at least 6 mL) was collected prior to the surgery, and PoV blood (also at least 6 mL) was then obtained by direct intraoperative venipuncture before manipulating the tumor during pancreaticoduodenectomy or distal pancreatectomy. Patients were followed with standard postoperative visits every 3 months to monitor tumor recurrence. Follow-up was terminated on October 30, 2019, and recurrence-free survival (RFS) was defined as the time interval between the date of operation and the date of recurrence or the endpoint of follow-up. Isolation and identification of CTCs CTCs were isolated by Catharanthine sulfate the CD45 negative enrichment method. First, peripheral or portal blood mononuclear cells were collected by density gradient centrifugation using Histopaque-1077 (Sigma-Aldrich, St. Louis, MO, USA). Next, mononuclear cells were mixed with microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) conjugated Catharanthine sulfate to monoclonal anti-human CD45 antibodies and incubated for 15 min at 4 C. Subsequently, the cells and microbeads were loaded onto a LS column for magnetic separation (Miltenyi Biotec). The suspensions of unlabeled CD45? cells were collected and spread on a glass slide for immunofluorescence staining. In brief, cells were fixed and then incubated with anti-CK-FITC (Miltenyi Biotec, catalog: 130-080-101) (1:100 diluted in 2% BSA), anti-Vimentin-Alexa Fluor 647 (Cell Signaling Technology, Danvers, MA, USA) (1:200 diluted in 2% BSA) or anti-CD45-PE (Miltenyi Biotec) (1:300 diluted in.