Karyotyping of circulating tumor cells for predicting chemotherapeutic sensitivity and efficacy in patients with esophageal cancer

Seventy nine patients with newly diagnosed esophageal cancer were enrolled in The Second Hospital of Shandong University from February 2015 to February 2018. Patients who had not been treated with chemotherapy were recruited in this study. Six ml peripheral blood were taken 2 weeks before chemotherapy. After then these patients received first-line PTX or DDP chemotherapy with 4 cycles, another 6 ml peripheral blood were taken 2 weeks after first 4-cycles chemotherapy.

The therapeutic effect of esophageal cancer patients was assessed after every 2 cycles of chemotherapy by computed tomography based on the RECIST1.1 criteria, including PR (Partial Response), PD (Progressive Disease), SD (Stable Disease).

This study was approved by the Human Research Ethical Committee of the Department of Clinical Laboratory, The Second Hospital of Shandong University, Jinan, Shandong, with protocol No.KYLL-2019 (LW)-001. Written informed consent was obtained from each participants included in this study.

CTCs karyotyping was detected by SET-iFISH according to the manufacture’s updated instruction (Cytelligen, San Diego, CA, USA), as previously described [15].

Briefly, ACD blood vessels were used to collect 6 ml of peripheral blood. All operations were performed at room temperature. Samples were kept and must be processed within 48 h. Samples were centrifuged at 200×g for 15 min. The supernatant was discarded. Then added buffer to make up 6 ml, followed by loading on the non-hematopoietic cell separation matrix in a 50 ml tube, and subsequent centrifugation at 450×g for 5 min. The supernatant and white membrane layer were collected into a 50 ml centrifuge tube containing white blood cells and tumor cells, and then incubated with 300 μl of immuno-magnetic beads conjugated to a cocktail of anti-leukocyte mAbs, for 30 min with gentle shaking. White blood cells were removed using a magnetic rack. The solution was added to a 15 ml centrifuge tube and 14 ml was replenished with buffer. The solution was centrifuged at 500×g for 5 min. Supernatants were aspirated down to 50 μl. Sedimented cells in 50 μl solution were gently resuspended, followed by subjection to immunofluorescence staining, and drying monolayer of cells mixed with the special fixative on the Cytelligen coated and formatted CTC slides for subsequent iFISH analyses.

Briefly, dried monolayer cells on the coated and formatted CTC slides were rinsed and incubated with PBS for 3 min, followed by hybridization with Vysis chromosome 8 centromere probe (CEP8) SpectrumOrange for 4 h. Samples were subsequently incubated with Alexa Fluor (AF) 488(green), 594(red), respectively conjugated to the mAbs recognizing indicated targets, including CD31, CD45, for 20 min in dark. After washing, samples were mounted with mounting media containing DAPI, and subjected to automated CTC image scanning and analyses.

CTCs identified by SET-iFISH are DAPI+ (blue)/FISH+ (aneuploid chromosome 8, range)/CD31- (green) and CD45-(red).

We define the karyotype based on the number of chromosomes 8.The number of chromosome 8 is 3, and the cell is triploidy; The number of chromosome 8 is 4, and the cell is tetraploidy; the number of chromosome 8 is 5, and the cell is pentaploidy; the number of chromosome 8 is more than 5, and the cell is multiploidy.