Anti-human EpCAM antibody produced in rabbit was purchased from Sigma-Aldrich; Anti-human CD45-PE produced in mouse was purchased from eBioscience; PE-labeled CK antibody purchased from Johnson & Johnson Veridex; anti-human hMAM antibody produced in rabbit was purchased from Proteintech; Hoechst 33342 Staining Solution staining solutions was purchased from Beyotime; magnetic nanoparticles was purchased from Nanjing XF NANO; DSPE-PEG2K, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and DSPE-PEG2K-Maleimide (DSPE-PEG-Mal) were purchased from Xi’an Ruixi; cholesterol, methylene chloride, and other commonly used reagents purchased from Sinopharm. QDream Antibody Labeling Kit was purchased from Najingbio.
Human mammary gland cell MCF-10A cells, Cervical cancer Hela cells, breast cancer cell MDR-MB-231, SK-BR-3, MCF-7 and 293T were purchased from ATCC cell bank and kept in the cell lab of Changzheng Hospital; DMEM culture medium, fetal bovine serum and trypsin purchased from Gibco.
Clinical samples collection
This clinical study was approved by Zibo Central Hospital. All the patient and healthy volunteer consents were written informed consents, and that this was conducted in accordance with the Declaration of Helsinki. Peripheral blood was collected from patient and healthy volunteer, each person was collected two tubes of venous blood, 8 ml per tube at Zibo Central Hospital, including 20 normal blood samples from healthy volunteers, 100 blood samples from breast cancer patient. All breast cancer patients were confirmed by breast ultrasonography and MRI, 60 of the 100 breast cancer patients have complete clinical case information and follow-up records. All blood samples were performed CTC separation and identification within 48 h after the blood collected in anticoagulant vessel collection.
ELMP was prepared refer to the reverse evaporation method. First of all, DSPE-PEG2K-MAL 0.5 mg and DSPE-PEG2K 2.0 mg was dissolved in 2.5 mL ddH2O, DOPC 3.0 mg, raw magnetic beads 1.0 mg and Chol 2.0 mg was dissolved in 1.5 mL chloroform, the water phase and the organic phase was mixed, followed by ultrasonic treatment to obtain emulsion. Then liposome solution was obtained under reduced pressure by means of rotary evaporator. Secondly, ELMP was prepared by the method in reference[28, 29], the reaction steps are as follows, 5.0 mg liposome solution was added into 4 mL HEPES buffer (50 mM, pH 6.5), then 40 µg EpCAM Antibody solution was added and stirred for 12 h at room temperature. The reaction mixture was dialyzed in a 3,500 Da dialysis bag for 12 h at 4 ℃ to obtain ELMP.
Characterization of ELMP
The surface potential and particle size of ELMP was measured by particle size potential analyzer (Zetasizer Nano ZS 90). The morphology of ELMP was observed by transmission electron microscopy (TEM), ELMP was dropped onto the copper mesh and observed after drying. Determination of saturation magnetization of ELMP, after the machines were warmed up, instructions and software prompts were followed to calibrate the machine first, and then the right amount of dry sample of magnetic ball powder was taken to be installed and to start measurements. Intrinsic coercivity HCJ value can be drawn by a magnetic regression line that can come from the test. The saturation magnetization value of the sample can be obtained by its mass values.
hMAM-QDs was reparented by hMAM antibody labeled with quantum dot
The rabbit anti-human hMAM antibody was labeled with a quantum dot labeling kit (QDream Antibody Labeling Kit), the brief steps are as follows, 10 × reaction solution and DEionized water were used to dilute the antibody concentration to 2 mg/ml, and the buffer system was 1 × reaction solution; according to the molar ratio of activating reagent: antibody = 20:1, the activating reagent was added to the above diluted antibody solution, mixed completely, and incubated for 30 minutes at room temperature; according to the molar ratio of antibody: quantum dots = 2:1, the quantum dot solution was added to the above reaction mixture, fully mixed, and incubated at room temperature for 30 minutes, labeled step was end.
hMAM expression and analysis
The expression of hMAM in tumor tissues was detected by immunostaining. Immunohistochemical diagnosis results of different types of tumor tissues provided by the pathology department of Zibo Central Hospital.
Immunofluorescence analysis of tumor markers
The expression of hMAM in different cell lines was detected by immunostaining, take the cell culture slides into a 24-well plate, plate MCF-10A or tumor cells at a density of 20,000/well, wash twice with PBS after overnight attachment, fix with 4% paraformaldehyde for 20 min, and wash with PBS twice. Add Hoechst 33342 and CK19-FITC or Hmam-QDs to stain for 15 min, wash twice with PBS, invert the slide on the glass slide covered with the mounting solution, and observe under the laser confocal microscope (Olympus FV10i).
Flow cytometry was used to detect the immunostaining of breast cancer cells by CK19 and hMAM at the same time. MDA-MB-231 cells were plated at a density of 80,000/well in a 12-well plate, and CK19-FITC and hMAM- were added after overnight adhesion. QDs were used for 1 h, washed twice with PBS, digested with trypsin, centrifuged at 1000 rpm for 5 min, collected cells, resuspended with 300 µL of PBS containing 1% FBS, and analyzed by flow cytometry.
Process of isolation and identification breast cancer CTC
The steps for CTC separation and identification of breast cancer were as follows: (1) collect 7.5 ml blood of breast cancer patients in an anticoagulant tube and centrifuge it at 1000 r/min for 10 min;(2) take the pelagic liquid and place it in EP tube, add the equivalent amount of PBS and mix it thoroughly;(3) add the immune lipid magnetic sphere 30 µL, incubate it at room temperature for 30 min, and mix it every 5 min;(4) The EP tube was inserted into the magnetic separation rack for 15 min to absorb and discard the superfluid; (5) wash it once in PBS;(6) Hoechst 33342 staining solution 30 µL, anti-CK19-FITC staining solution 10 µL, and anti-CD45-PE staining solution 10 µL or hMAM-QDs staining solution 10 µL was added to blend and stain for 15 min;(7) wash with double steam water once;(8) 30 µL ddH2O was added to the EP tube and resuspended, the droplets were evenly coated on the anti-slide slide. Then, the sample is observed and counted under a fluorescence microscope until the droplets dry in the dark.
CTC isolation efficiency analysis of ELMP
Breast cancer cells MDA-MB-231 was used as CTC model to study the CTC separation efficiency in use of ELMP. Different amount (10, 20, 50, 100, 200) MDA -MB-231 cells were added into 7.5 mL anticoagulant venous blood separately, the subsequent CTC separation and identification steps was carried out according to the above steps.
Clinical study on isolation and identification of circulating tumor cells
ELMP was used to separate CTC from 120 peripheral blood samples (20 normal and 100 patient samples), Hoechst 33342, anti-CK19-FITC, and PE-labeled CD45 antibody were used for the separated CTC identification in single tumor marker method. Hoechst 33342, anti-CK19-FITC, and hMAM-QDs were used for the separated CTC identification in double tumor marker method. The CTC positive determination criteria are greater than 5 in 7.5 mL blood.
Data were analyzed using descriptive statistics, for multiple comparisons, a one-way ANOVA test was performed, and Data are expressed as mean ± standard deviation (S.D.) derived averaged across three to at least three independent measurements. Comparisons among different groups were performed by Chi-square tes. Differences were considered as significant and the null hypothesis of no difference among treatment groups was rejected for P values < 0.05.