HiSeq X Ten paired end sequencing; Hexokinase 2 detects cytokeratin negative circulating tumor cells in lung adenocarcinoma
Peripheral blood and pleural effusion were obtained from lung cancer patients in Shanghai Chest Hospital (Shanghai Jiao Tong University School of Medicine) and cerebrospinal fluid was obtained from lung cancer patients in Huashan Hospital (Fudan University) with written informed consent. The clinical sample collection and experiment were carried out in accordance with guidelines and protocols that approved by the Ethics and Scientific Committees of both hospitals. Blood (5 mL) was re-suspended in an equivalent volume of HBSS and mixed with CTC enrichment antibody cocktail (RosetteSepTM CTC Enrichment Cocktail, StemCell) at room temperature for 20 min. Three volumes (15 mL) of HBSS with 2% FBS (Gibco) was then added and mixed well. The mixture was carefully added along the wall of Sepmate tube after adding 15 mL density gradient liquid (LymphoprepTM, Stemcell) into the tube through the middle hole. After centrifuging at 1200 g for 20 min, the top most supernatant (~10 mL) was discarded, and the remaining liquid (~10 mL) above the barrier of the Sepmate tube was rapidly poured out into a new centrifuge tube. After centrifuging at 600 g for 8 min, the supernatant was removed and 1 mL of red blood cell lysing buffer (BD Biosciences) was then added to lyse red blood cells for 5 min. After centrifuging at 250 g for 5 min, the nucleated cell pellet was re-suspended in HBSS. The cell suspension was then applied onto the 3% BSA -treated microwell chip and waited for 10 min until cells sitting down in the a total of 28,000 microwells. The chip was sealed with a porous membrane to avoid cell loss during subsequent on-chip staining. After cell fixation and permeabilization (0.5% Triton X-100, 15 min), blocking solution consisting of 3% BSA and 10% normal goat serum was applied onto the chip for 1 h of blocking, followed by incubation with APC-conjugated anti-CD45 antibody, PE-conjugated anti-CK antibody and anti-HK2 antibody in PBS overnight at 4℃. After extensive washing with PBS, cells on the chip were treated with FITC-conjugated goat-anti-rabbit secondary antibody in PBS for 1h and DAPI for 10 min. After extensive washing with PBS, an ImageXpress Micro XLS Widefield High Content Screening System (Molecular Devices) scanned the chip and imaged all cells in bright field and four fluorescent colors. A computational algorithm analyzed the images and identified HK2+ and CK+ cells based on the cut-offs generated from HK2 and CK fluorescence intensity of CD45+ leukocytes in the samples. For CTC identification in pleural effusion, 5 mL of sample was first filtered by a membrane with a pore size around 100 μm, followed by centrifuging at 500 g for 5 min to separate cell pellets. 1 mL of red blood cell lysing buffer (BD) was then added to lyse red blood cells for 5 min. After centrifuging at 500 g for 5 min, the nucleated cell pellet was re-suspended in and washed with HBSS. After cell counting, at most 500,000 cells were applied onto a 112,000-well microwell chip for cell fixation, permeabilization and immunostaining. Cerebrospinal fluid samples were added onto microwell chip for cell fixation, permeabilization and immunostaining, directly. Whole genome amplification of single candidate tumor cells were conducted according to MALBAC® Single Cell WGA Kit (Yikon Genomics) protocol. In briefly, retrieved single cell into 5μl cell lysis buffer to lysis cell and added 30μl pre-amplification lysis buffer for DNA pre-amplification. Finally, added 30μl amplification buffer to amplification. Amplified genomic DNA was then purified by Agencourt AMPure XP Beads (Beckman Coulter), and its concentration was determined by Nanodrop. Whole genome amplification DNA were digested with dsDNA fragmentase (New England Biolabs) and 300-500bp fragments were then retrieved with Agencourt AMPure XP Beads (Beckman Coulter). DNA fragments repaired, library adaptor ligation and amplification were performed with NEBNext® Ultra™ DNA Library Prep Kit for Illumina kit (New England Biolabs) and index primers (New England Biolabs) in accordance with the manufacturer's protocol. Library purification was performed with TIANgel Midi Purification Kit (TIANGEN Biotech). The concentration of purified fragmented DNA or libraries was quantified with Qubit HsDNA kits (Invitrogen).