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Circulatin Rare Cells Application Note P l a s t i n - 3 Tr a n s f o r m e d C i r c u l a t i n Tu m o r C e l l s i n H e p a t i c P o r t a l Ve i n o f P a n c r e a t i c C a n c e r P a t i e n t f o r P r e d i c t i n g L i v e r M e t a s t a s i s Introduction Materials & Methods Pancreatic cancer is an asymptomatic disease which is usually diagnosed during advanced stage with high incidence of liver metastasis [1]. Evidence of liver metastasis is a principal determinant of disease management and clinical outcome in pancreatic cancer patients [2]. Whipple operation is a beneficial surgical procedure for localized pancreatic cancer, whereas palliative procedures are recommended for metastatic disease. Plastin-3, a metastatic-specific gene located on chromosome Xq23, is an epithelial mesenchymal transition (EMT) marker associated with colorectal and breast cancers [3]. Presence of Plastin-3 transformed circulating tumor cells (CTCs) in hepatic portal vein could serve as a clinical predictor of liver metastasis. We applied CytoQuest™ CR positive enrichment system, EpCAM, PanCK and Plastin-3 monoclonal antibodies to capture and identify epithelial CTCs in hepatic portal vein of a pancreatic cancer patient. Hepatic portal vein blood of pancreatic cancer patient was collected in Heparin Tube (02-689-6, BD). 7.5 ml blood was prepared for collecting the peripheral mononuclear cell (PBMC) by density gradient centrifugation using Leucosep® (163290P, Greiner Bio One) and Histopaque®-1077 (10771, Sigma-Aldrich). PBMC fraction was harvested and resuspended in Wash Medium. Resuspended PBMC was loaded into the CytoQuest™ CR System and CTCs were captured by EpCAM (KA4586, Abnova) immobilized CytoChipNano (U0095, Abnova). Immunofluorescence staining for detecting CTCs was performed using PanCK, CD45, Plastin-3 (KA4586, Abnova) and DAPI as the instruction of protocol. Imaging was performed using Nikon Eclipse Ti-E fluorescent inverted microscope. Results CTC Counts: In 7.5 ml blood of pancreatic cancer patient, 2 cells count as CTC (PanCK+, Plastin3+, CD45-, DAPI+). Merged PanCK Plastin3 CD45 Nucleus Figure 1: Representative images of CTC (white arrow) and WBCs (yellow arrow) from pancreatic cancer patient, CTC was detected by using immunofluorescence staining for PanCK (FITC, green), Plastin-3 (Alexa647, red) CD45 (PE, orange) and Nucleus (DAPI, blue). Circulatin Rare Cells Application Note Discussions Effective clinical management of pancreatic cancer patients depends on a complete medical workup to determine exact nature of metastasis. A clinical predictor of liver metastasis would be highly desirable before Whipple procedure for “localized” disease to assess the probability of metastasis before surgery. Access to the hepatic portal vein for sampling and detection of EMT-transformed circulating tumor cells would be a valuable adjunct to the clinical management. A combination of EpCAM, PanCK and Plastin-3 monoclonal antibodies successfully identify circulating pancreatic cancer cells using an antibody-based, positive enrichment. This case study is the first report of Plastin-3 EMT expression in pancreatic CTCs identified with EpCAM and PanCK epithelial cell markers. A larger cohort study will provide clinically evidence for establishing Plastin-3 transformed epithelial CTCs as a predictor of liver metastasis in “localized” pancreatic cancer patients. References 1. Berger HG, Rau B, Gansauge F, Poch B, Link KH. Treatment of pancreatic cancer: challenge of the facts. World journal of surgery, 2003, 27(10):1075-85. 2. Li D, Xie K, Wolff R. Abbruzzese JL Pancreatic cancer, Lancet, 2004, 363(9414):1049-57. 3. Yokobori T, et al. Plastin3 is a novel marker for circulating tumor cells undergoing the epithelial-mesenchymal transition and is associated with colorectal cancer prognosis. Cancer research, 2013, 73(7):2059-69. 4. Lyberopoulou A, et al. Mutational analysis of circulating tumor cells from colorectal cancer patients and correlation with primary tumor tissue. PloS one, 2015, 10(4):e0123902. Date of issue January 2016 Manufactured by D i s t r i b u t e d b y Abnova Corporation 9F, No. 108, Jhouzih St., Neihu Taipei 114, Taiwan Tel: + 886 2 8751 1888 Fax: + 886 2 6602 1218 BioVendor – Laboratorni medicina a.s. Karasek 1767/1, 621 00 Brno, Czech Republic Phone: +420 549 124 185 Fax: +420 549 211 460 E-mail: [email protected] w w w. a b n o va . c o m w w w. b i o v e n d o r. c o m BioVendor GesmbH Nußdorfer Straße 20/10, 1090 Vienna, Austria Phone: +43 1 890 9025 Fax: +43 1 890 9025-15 E-mail: [email protected] Oxford Biosystems Ltd 115J Olympic Avenue, Milton Park, Oxfordshire OX14 4SA, United Kingdom Phone: 01235 431390 E-mail: [email protected] BioVendor GmbH Otto-Hahn-Straße 16, 34123 Kassel, Germany Phone: +49 6221 4339 100 Fax: +49 6221 4339 111 E-mail: [email protected] w w w. o x f o r d b i o s y s t e m s . c o m