Kit for detecting circulating tumor cells with high metastatic potential and its use

Abstract

The present application discloses a kit for detecting circulating tumor cells with high metastatic potential and its use. The kit for detecting circulating tumor cells with high metastatic potential comprises: (a) a detection reagent for circulating tumor cells; (b) a detection reagent for high metastatic potential markers; (c) a detection reagent for immune cells; and (d) a nuclear staining reagent.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering, specifically relates to the detection of circulating tumor cells, and more specifically relates to a kit for detecting circulating tumor cells with high metastatic potential and its uses. Background Art

[0002] Circulating tumor cells (CTC) refer to tumor cells that detach from the primary or metastatic focus and enter the blood circulation during tumor formation or progression, and have the potential to develop into metastatic lesions. Therefore, CTC can be used as a new tool for clinical liquid biopsy such as disease auxiliary diagnosis, dynamic monitoring, and prognosis evaluation. There is a large heterogeneity in CTC, which brings certain difficulties to detection. Previous studies classified CTC according to characteristics such as metabolism, epithelial-mesenchymal or PD-L1 expression. This fixed combination of indicators cannot be applied to all types of tumors, especially cannot capture CTC lacking the above surface markers, and is prone to false negatives.

[0003] During the blood dissemination process of CTC, it faces challenges such as mechanical damage, anoikis, and immune attack, and only a few CTC survive. Research shows that most CTC are not single cells spreading alone in the blood, but are accompanied by a large number of blood cells. Our team previously found in multiple cancer types that platelet adhesion to CTC is a common phenomenon and is potentially related to CTC resistance to immune attack and distant metastasis. However, CTC may fuse with platelets or phagocytize platelets to carry platelet markers. Detecting platelet-related CTC will increase the false positives of experimental results, and there is an urgent need for CTC markers that can better reflect the prognosis of patients.

[0004] Immunomagnetic particles are a type of highly operable magnetic microspheres, whose surface can be modified to express antibodies against multiple indicators. When targeting CTC, enrichment and detection of CTC are achieved through antigen-antibody binding and magnetic separation. However, there has not yet appeared a method in this field to accurately detect CTC with high metastatic potential using immunomagnetic particles.

[0005] The number of CTC in peripheral blood is extremely scarce, only accounting for 1 / 10^6 - 1 / 10^7 of peripheral blood white blood cells. Therefore, the practical application of CTC has been restricted by its effective enrichment and detection methods. In addition, there are technical limitations in the existing CTC types where they cannot be effectively and comprehensively enriched. There has not yet appeared a method in this field to accurately detect CTC with high metastatic potential, such as platelet adhesion / related CTC.

[0006] There is still an urgent need in this field for a method to accurately and sensitively detect circulating tumor cells with high metastatic potential. Summary of the Invention

[0007] To solve the above technical problems, in one aspect, the present application provides a kit for detecting circulating tumor cells with high metastatic potential, the kit comprising:

[0008] (a) A detection reagent for circulating tumor cells;

[0009] (b) A detection reagent for high-metastatic potential markers;

[0010] (c) A detection reagent for immune cells; and

[0011] (d) A nuclear staining reagent.

[0012] In another aspect, the present application provides the use of a kit for detecting circulating tumor cells with high metastatic potential in the preparation of a reagent for detecting circulating tumor cells with high metastatic potential in a sample, the kit for detecting circulating tumor cells with high metastatic potential comprising:

[0013] (a) A detection reagent for circulating tumor cells;

[0014] (b) A detection reagent for high-metastatic potential markers;

[0015] (c) A detection reagent for immune cells; and

[0016] (d) A nuclear staining reagent.

[0017] To overcome the technical limitation that there are many types of existing CTCs and they cannot be effectively and comprehensively enriched, the kit of the present application specifically enriches and detects CTCs that are EpCAM / CK19 positive, HBA2 / NEXN positive, and CD45 negative, and can also leave blank sites linked to biotin-streptavidin, thereby realizing highly sensitive detection of circulating tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The present application will be described in more detail below with reference to the accompanying drawings, in which:

[0019] Figure 1 It is a result diagram for screening genes that are highly expressed in CTCs compared to primary cancer and upregulated after platelet adhesion to tumor cells;

[0020] Figure 2 It is an in vitro and ex vivo qRT-PCR experiment to confirm that platelet adhesion to CTCs upregulates the expression of HBA2 and NEXN;

[0021] Figure 3 It is an in vivo experiment in mice to prove that platelet adhesion to CTCs increases the systemic metastatic colonization potential of CTCs by upregulating the expression of HBA2 and NEXN;

[0022] Figure 4Result diagram of using the kit according to an embodiment of the present application, which shows that HBA2 and NEXN are expressed on CTCs of HCC patients with cancer metastasis, and the scale bars are 5μm and 2μm respectively;

[0023] Figure 5 Schematic diagram of the immunomagnetic beads according to an embodiment of the present application. Detailed implementation mode

[0024] The present application relates to a kit for detecting circulating tumor cells with high metastatic potential, and the kit includes: (a) reagents for detecting circulating tumor cells. In the present application, the reagents for detecting circulating tumor cells may include any reagents that can be used to detect specific markers of circulating tumor cells. In one embodiment, the reagents for detecting circulating tumor cells include reagents for detecting epithelial cell adhesion molecule (EpCAM) and / or reagents for detecting cytokeratin 19 (CK19). In a preferred embodiment, the reagents for detecting circulating tumor cells include reagents for detecting epithelial cell adhesion molecule (EpCAM) and reagents for detecting cytokeratin 19 (CK19). In one embodiment, the reagents for detecting circulating tumor cells include antibodies. In one embodiment, the reagents for detecting epithelial cell adhesion molecule (EpCAM) include antibodies. In one embodiment, the reagents for detecting cytokeratin 19 (CK19) include antibodies. In one embodiment, the antibody includes a monoclonal antibody, for example, a murine monoclonal antibody. In one embodiment, the detection reagent includes a fluorescent staining reagent. In one embodiment, the fluorescent staining reagent includes cyanine 3 (Cy3), succinimidyl ester 647 (AF647) or fluorescein isothiocyanate (FITC). In a preferred embodiment, the fluorescent staining reagent of the reagent for detecting epithelial cell adhesion molecule (EpCAM) includes AF647. In a preferred embodiment, the fluorescent staining reagent of the reagent for detecting cytokeratin 19 (CK19) includes Cy3.

[0025] The kit for detecting circulating tumor cells with high metastatic potential further comprises: (b) a detection reagent for high-metastatic-potential markers. In the present application, the detection reagent for high-metastatic-potential markers may include any reagent that can be used to detect specific markers of the high metastatic potential of tumor cells. In one embodiment, the detection reagent for high-metastatic-potential markers includes a detection reagent for hemoglobin subunit alpha 2 (HBA2) and / or a detection reagent for nexilin F-actin binding protein (NEXN). In a preferred embodiment, the detection reagent for high-metastatic-potential markers includes a detection reagent for hemoglobin subunit alpha 2 (HBA2) and a detection reagent for nexilin F-actin binding protein (NEXN). In one embodiment, the detection reagent for high-metastatic-potential markers includes an antibody. In one embodiment, the detection reagent for hemoglobin subunit alpha 2 (HBA2) includes an antibody. In one embodiment, the detection reagent for nexilin F-actin binding protein (NEXN) includes an antibody. In one embodiment, the antibody includes a monoclonal antibody, for example, a rabbit monoclonal antibody. In one embodiment, the detection reagent includes a fluorescent staining reagent. In one embodiment, the fluorescent staining reagent includes cyanine 3 (Cy3), succinimidyl ester 647 (AF647), or fluorescein isothiocyanate (FITC). In a preferred embodiment, the fluorescent staining reagent for the detection reagent of hemoglobin subunit alpha 2 (HBA2) includes Cy3. In a preferred embodiment, the fluorescent staining reagent for the detection reagent of nexilin F-actin binding protein (NEXN) includes Cy3.

[0026] The kit for detecting circulating tumor cells with high metastatic potential further comprises: (c) a detection reagent for immune cells. In the present application, the detection reagent for immune cells may include any reagent that can be used to detect specific markers of immune cells. In one embodiment, the detection reagent for immune cells includes a detection reagent for leukocyte common antigen (CD45). In one embodiment, the detection reagent for immune cells includes an antibody. In one embodiment, the detection reagent for leukocyte common antigen (CD45) includes an antibody. In one embodiment, the antibody includes a monoclonal antibody. In one embodiment, the detection reagent includes a fluorescent staining reagent. In one embodiment, the fluorescent staining reagent includes cyanine 3 (Cy3), succinimidyl ester 647 (AF647), or fluorescein isothiocyanate (FITC). In a preferred embodiment, the fluorescent staining reagent includes FITC.

[0027] The kit for detecting circulating tumor cells with high metastatic potential further comprises: (d) a nuclear staining reagent. In one embodiment, the nuclear staining reagent includes DAPI or Hoechst.

[0028] The kit for detecting circulating tumor cells with high metastatic potential may further comprise a solid support. In one embodiment, the solid support comprises particles. In a preferred embodiment, the particles comprise spherical particles. In a preferred embodiment, the particles comprise magnetic particles. In one embodiment, the magnetic particles comprise magnetite particles. In one embodiment, the diameter of the particles is 0.1-10 μm. In one embodiment, the diameter of the particles is 0.5-2 μm. In a preferred embodiment, the diameter of the particles is about 1 μm. In one embodiment, the surface of the solid support comprises one or more of the components in the above embodiments. In one embodiment, one or more of the components in the above embodiments are coupled to the surface of the solid support. In one embodiment, one or more of the components in the above embodiments are covalently bound to the surface of the solid support. In one embodiment, one or more of the components in the above embodiments are physically adsorbed to the surface of the solid support. In one embodiment, one or more of the components in the above embodiments are immobilized on the surface of the solid support through reactive functional groups on the surface of the solid support, preferably carboxyl functional groups. In one embodiment, the surface of the solid support comprises (b) in the above embodiment. In one embodiment, the surface of the solid support comprises (a) and (b) in the above embodiment. In one embodiment, the surface of the solid support comprises (a), (b) and (c) in the above embodiment. In one embodiment, the surface of the solid support is coated with streptavidin. In one embodiment, the solid support further comprises blank binding sites. In one embodiment, the blank binding sites comprise reactive functional groups. In a preferred embodiment, the blank binding sites comprise carboxyl functional groups. In one embodiment, the blank binding sites comprise reactive functional groups on the streptavidin-coated surface. In a preferred embodiment, the blank binding sites comprise carboxyl functional groups on the streptavidin-coated surface. In one embodiment, the blank binding sites are used to bind detection reagents for detecting other targets. In one embodiment, the blank binding sites are used to bind detection reagents for amplifying detection signals. In one embodiment, the kit further comprises particle conjugation-related reagents and consumables. In one embodiment, the reagents comprise activation reagents, reaction reagents, chemical reagents, blocking agents and protective solutions. In one embodiment, the chemical reagents comprise EDC and NHS. In one embodiment, the consumables comprise EP tubes and magnetic separation devices. In one embodiment, the EP tubes comprise 1.5 ml EP tubes. In one embodiment, the magnetic separation device comprises a magnetic bead separation rack.

[0029] The kit for detecting circulating tumor cells with high metastatic potential may further comprise a labeled detection reagent. In a preferred embodiment, the labeled detection reagent includes a detection reagent labeled with a fluorescent dye or a detection reagent labeled with biotin. In one embodiment, the detection reagent includes an antibody. In one embodiment, the fluorescent dye includes acridinium ester. In one embodiment, the biotin-labeled detection reagent includes a detection reagent for detecting other targets. In one embodiment, the fluorescent dye-labeled detection reagent includes a detection reagent for amplifying the detection signal. In one embodiment, the detection includes qualitative detection. In one embodiment, the qualitative detection includes interpreting the luminescence signal by the immunofluorescence of self-assembled magnetic particles combined with a fluorescent dye. In one embodiment, the interpretation of the luminescence signal includes interpreting the luminescence signal under a microscope using the Operetta CLS High Content Analysis System platform in combination with the Harmony software. In one embodiment, cells that are DAPI+EpCAM / CK19+ and adhered with magnetic particles are determined to be CTCs with high metastatic potential. In one embodiment, cells that are DAPI+EpCAM / CK19+NEXN / HBA2+ are determined to be CTCs with high metastatic potential. In one embodiment, cells that are DAPI+EpCAM / CK19+NEXN / HBA2+CD45- are determined to be CTCs with high metastatic potential. In one embodiment, CTCs with high metastatic potential include platelet adhesion / associated CTCs.

[0030] The kit for detecting circulating tumor cells with high metastatic potential may further comprise a cell fixative, a blocking agent, an anti-fluorescence quenching agent, and / or a red blood cell lysate.

[0031] This application also relates to the use of the kit according to any of the above embodiments in the preparation of a reagent for detecting circulating tumor cells with high metastatic potential in a sample. In one embodiment, the sample includes a body fluid. In a preferred embodiment, the sample includes blood. In one embodiment, the tumor cells include liver cancer cells. In one embodiment, the tumor cells include platelet adhesion / associated CTCs.

[0032] Examples

[0033] This application will be described in detail by the following exemplary specific examples. The following examples are only used to help those skilled in the art better understand various inventions of this application. It should be pointed out that the spirit of this application and the protection scope of the claims are not limited by the following specific examples.

[0034] List of reagents and equipment used

[0035]

[0036]

[0037] Unless otherwise specified, all patients in this application are from Zhongshan Hospital, Fudan University and meet the requirements of ethics and informed consent.

[0038] Example 1

[0039] In this example, through co-culturing platelets with tumor cell lines and simultaneous detection by RNA-seq sequencing technology and single-cell transcriptome sequencing, it was found that NEXN and HBA2 molecules were highly expressed on CTCs to which platelets adhered and were more likely to survive and metastasize in the blood circulation, as Figure 1 shown.

[0040] After co-culturing in vitro hepatocellular carcinoma cell lines and ex vivo hepatocellular carcinoma organoids with platelets, the qRT-PCR technology was used to detect that the expression of NEXN and HBA2 genes was significantly increased compared with that before co-culturing, as Figure 2 shown. Figure 2 is a heat map showing the changes in the expression of HBA2 and NEXN genes before and after the culture of organoids, tumor cell lines and platelets. The leftmost label shows the grouping, which are: Hep3B+PLT (co-culture group of Hep3B cells and platelets), Hep3B Con (control group of Hep3B cells, only medium was added, no platelets), PLC / PRF / 5+PLT (co-culture group of PLC / PRF / 5 cells and platelets), PLC / PRF / 5Con (control group of PLC / PRF / 5 cells, only medium was added, no platelets), PDO+PLT (co-culture group of organoids and platelets), PDO Con (pure organoid group). The parallel experiment was repeated 3 times, and each row represents an independent experiment. The results showed that the expression of HBA2 and NEXN genes was significantly increased after co-culturing organoids and tumor cells with platelets.

[0041] The specific experimental steps are as follows:

[0042] Hepatocellular carcinoma cell lines: Hep3B (Catalog No.: TCHu106), PLC / PRF / 5 (Catalog No.: SCSP-5095), purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China).

[0043] Hepatocellular carcinoma tissue samples: Derived from tumor tissues resected during hepatectomy of hepatocellular carcinoma patients, collected by hepatobiliary surgeons during the operation in Zhongshan Hospital. All patients signed informed consent forms, and this study was approved by the Ethics Committee (IRB) of Zhongshan Hospital, Fudan University (Ethical Approval Number: B2019-059R).

[0044] 1. Preparation of organoids (PDO):

[0045] To generate hepatocellular carcinoma organoids, hepatocellular carcinoma tissue samples (0.25 - 1 cm3 ) Chop up, according to the degree of liver fibrosis, and digest with 2.5 mg / mL collagenase D (Roche) and 0.1 mg / mL DNase (Sigma) at 37 °C for 2 - 5 hours. When there are no remaining tissue fragments, terminate the digestion process. Filter the resulting suspension through a 100 μm nylon cell filter, and then centrifuge at 300 - 400 g for 5 minutes. Wash the obtained pellet with cold advanced DMEM / F12 (GIBCO), and then mix it with BME (basement membrane extract, Type 2, Pathclear). Subsequently, seed 2000 - 5000 cells per well in a 24-well plate. After the BME solidifies, add the organoid-specific isolation medium. The organoid-specific isolation medium is advanced DMEM / F12 supplemented with 1% penicillin / streptomycin, 1% glutamine, 10 mM HEPES, 1:50 B27 supplement (without vitamin A), 1:100 N2 supplement, 1.25 mM N-acetyl-L-cysteine, 10 mM nicotinamide, 10 nM recombinant human [Leu15]-gastrin I, 50 ng / ml recombinant human EGF, 100 ng / ml recombinant human FGF10, 25 ng / ml recombinant human HGF, 10 μM forskolin, 5 μM A8301, 10 μM Y27632, and 3 nM dexamethasone.

[0046] 2. Platelet extraction

[0047] According to the patient's hospitalization records, select sodium citrate anticoagulated whole blood from hepatocellular carcinoma (HCC) patients (who have not received anticoagulant treatment in the past two weeks) and isolate platelets from the whole blood. The specific steps are as follows: Centrifuge 2 ml of whole blood at 150 g for 12 minutes at room temperature to obtain platelet-rich plasma (PRP). Centrifuge at 900 g for 5 minutes at room temperature and discard the supernatant. Resuspend the platelet microspheres in Tyrode buffer (pH 7.4, calcium-free), and add prostaglandin E1 (PGE1, 0.3 μg / mL) to prevent platelet activation.

[0048] 3. Establishment of co-culture model

[0049] 1) Co-culture 1 x 10^5 PDOs and 1 x 10^8 platelets at a ratio of 1:1000 in a 24-well plate. After culturing for 48 hours, transfer all the liquid in the well to a 15 ml centrifuge tube, centrifuge at 1000 rpm for 5 minutes to obtain the cell pellet, and wash it twice with PBS. Discard the supernatant, and the pellet is the PDOs that have been co-cultured with platelets.

[0050] 2) Take 3x10^5 tumor cell line cells (Hep3B and PLC / PRF / 5) and 3x10^8 platelets and co-culture them in a 24-well plate at a ratio of 1:1000. After 48 hours of co-culture, gently wash with PBS using a Pasteur pipette to remove platelets. Digest the adherent cells with 0.05% trypsin, neutralize with DMEM medium containing 10% fetal bovine serum, centrifuge at 1000 rpm for 5 minutes to obtain cell pellets, wash twice with PBS, discard the supernatant, and the pellets are the tumor cells co-cultured with platelets.

[0051] Further in vivo experiments showed that after platelet adhesion, CTCs increased the potential of systemic metastasis and colonization of CTCs by upregulating the expression of HBA2 and NEXN, as Figure 3 shown. Figure 3 The small animal in vivo imaging diagram shows the systemic tumor metastasis of model mice injected via the tail vein in different treatment groups (left), and the statistical results of the average fluorescence radiation values of different treatment groups (right). The experimental results showed that compared with the control group, the systemic metastatic foci of mice with platelets removed were significantly reduced, indicating that without the protection of platelets, tumor cells are easily cleared during blood dissemination; the systemic metastasis of mice injected with tumor cells with HBA2 or NEXN knockdown via the tail vein was better than that of the control group, and there was no significant difference in the number of metastatic foci between groups, which was comparable to the platelet removal group, indicating that the protective effect of platelets on tumor cells may be exerted by affecting the expression of HBA2 or NEXN. Reducing the expression of HBA2 or NEXN on tumor cells can inhibit tumor metastasis in the presence of platelets.

[0052] The specific steps of the in vivo experiment are as follows:

[0053] Hepatocarcinoma cell lines: H22-luc Con, H22-luc sh-HBA2, H22-luc sh-NEXN (mouse hepatocarcinoma cell line H22-luc (H22 cells with luciferase). Con, that is, the control group, sh means gene knockdown. The control group, sh-HBA2, and sh-NEXN are all cell lines stably transfected with lentivirus based on H22-luc, provided by the Liver Cancer Research Institute of Zhongshan Hospital, Fudan University).

[0054] Mouse information: Strain: Balb / c (BALB / cCrSlcNifdc mice, purchased from Beijing Biocytogen Co., Ltd., SPF level. The mice were housed in the barrier of the Department of Laboratory Animal Science of Fudan University, raised under SPF conditions, and treated humanely throughout the study. Animal experiments were carried out strictly in accordance with institutional guidelines. All animal handling procedures followed the guidelines of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and the Animal Welfare Act).; Age: 6 weeks old; Gender: male mice.

[0055] Reagents: Platelet scavenger iPLT, an antibody for platelet depletion in mice (#R300, Emfret Analytics).

[0056] Instrument: PerkinElmer IVIS Spectrum.

[0057] 1. Drug intervention:

[0058] One day before the experiment (Day 0), 2 μg / g of platelet inhibitor (#R300, Emfret) dissolved in 100 μl of PBS was injected into the control group and the experimental group via the tail vein respectively. After cell tail vein injection on the next day (Day 1), the same dose of platelet inhibitor was injected via the tail vein every 3 days until the second week.

[0059] 2. Small animal in vivo imaging (in vivo imaging system (IVIS)):

[0060] 2 x 10^6 H22-luc Con (control group) and H22-luc sh-HBA2 or H22-luc sh-NEXN cell lines with HBA2 or NEXN knocked down were dissolved in 200 μl of PBS and injected into Balb / c mice via the tail vein. After 3 weeks, the luciferase reaction substrate XenoLight D-luciferin potassium salt (#122799, PerkinElmer) (150 mg D-luciferin / kg mouse body weight) was injected into the mice intraperitoneally. After anesthesia (isoflurane (R510-22-10, RWD), at a flow rate of 300 ml / min and a concentration of 4% gas to anesthetize the mice. Procedure: After the anesthetic fills the induction chamber, place the animal in the induction chamber and wait for the animal's body to fall to the side position and not attempt to resume its lying position, indicating that the animal is fully anesthetized), imaging was performed using IVIS. Data analysis was performed using Living Image (v4.1) (Caliper Life Sciences, Inc). As Figure 3 , it was found that compared with the control group, the number of systemic metastatic foci in mice was significantly reduced after platelet clearance. Knocking down the expression of HBA2 or NEXN in cells could achieve a similar effect of inhibiting tumor metastasis as platelet clearance.

[0061] Example Ⅱ

[0062] In this embodiment, a self-built immunomagnetic microsphere kit is used to enrich CTCs with high metastatic potential. The CTCs in this embodiment are from patients with multiple organ distant metastases after liver cancer surgery (high-metastasis patients) and primary hepatocellular carcinoma patients who come to the hospital for the first time and have no metastases. The kit includes DAPI reagent and immunomagnetic microspheres, and the immunomagnetic microspheres are coated with 5 antibodies, specifically including: EpCAM-AF647 antibody, CK19-AF647 antibody, CD45 antibody, HBA2 antibody, and NEXN antibody, which can be used for the identification and enrichment of highly metastatic CTCs. The implementation results show that using this kit, on the Operetta CLS High Content Analysis System platform, CTC cells that are DAPI+ and adhered with magnetic particles are observed in the peripheral blood of high-metastasis patients (peripheral blood whole blood samples of patients anticoagulated with EDTA-K2; patient type: patients who come to the hospital again after surgical resection of hepatocellular carcinoma, and imaging and other evidence indicate that the patients have multiple organ metastases, determined to be high-metastasis patients). Red fluorescence (EpCAM-AF647), red fluorescence (CK19-AF647), and yellow fluorescence (HBA2 / NEXN-Cy3) are expressed around the CTCs, and green fluorescence (CD45-FITC) is not expressed (CD45 negative (magnetic beads do not emit green fluorescence) excludes cells expressing CD45); while in the peripheral blood of non-metastatic patients (peripheral blood whole blood samples of patients anticoagulated with EDTA-K2; patient type: patients who come to the hospital for the first time, the pathological results show hepatocellular carcinoma and there is no evidence to support the patients having metastases, and the patients have not received any cancer-related treatments, that is, non-metastatic patients), the CTCs are only DAPI+, and there are no magnetic particles attached (as Figure 4 shown). In addition, in the peripheral blood of patients (peripheral blood whole blood samples of patients anticoagulated with EDTA-K2; patient type: hepatocellular carcinoma patients), white blood cells (DAPI+CD45+) have no magnetic particle adhesion, which is used as a negative control.

[0063] The specific experimental steps are as follows:

[0064] 1. Negative enrichment of CTCs

[0065] Manually mix the whole blood sample tube slowly up and down, transfer 5 ml of whole blood to a 15 ml centrifuge tube, mix it with an equal volume of PBS buffer, and gently pipette and mix with a Pasteur pipette. Slowly add the diluted blood along the wall of the 15 ml centrifuge tube onto 3.5 ml of Ficoll-Paque gradient density separation liquid. Centrifuge at 1200 g for 10 minutes, collect the buffy coat layer in a new 15 ml centrifuge tube, add an equal volume of PBS, centrifuge at 300 g for 10 minutes, and remove the supernatant. Add 1 ml of red blood cell lysate, incubate at 4 °C for 5 minutes, centrifuge at 3000 rpm for 5 minutes, and discard the supernatant to obtain CTCs and immune cells.

[0066] 2. Preparation of Magnetic Particles Coupled with Antibodies

[0067] Take 3 μl of Dynabeads TM MyOne TM Carboxylic Acid magnetic particles in 500 μl of activator, gently pipette and mix, then place on a magnetic stand for 20 seconds to separate, remove the supernatant, and resuspend in 100 μl of activator. Take 1 mg of EDC and 1 mg of NHS and dissolve them in 200 μl of activator, mix well, and add to the magnetic particle suspension. React at 25 °C on a rotary mixer for 40 minutes to activate the carboxyl functional groups on the surface of the magnetic particles.

[0068] Separate on a magnetic stand for 20 seconds to remove the supernatant. Add 100 μl of protein solution containing 1 mg / ml of NEXN antibody, HBA2 antibody, and CD45 antibody (prepared using reaction activator), 5 μl of EpCAM-AF647 reagent, and 5 μl of CK19-AF647 reagent. React at 25 °C on a rotary mixer for 2 hours in the dark to couple the antibodies.

[0069] After the reaction, place on a magnetic stand for 20 seconds to separate, remove the supernatant. Add 500 μl of reactant to resuspend the magnetic beads, place on a magnetic stand for 20 seconds to separate, remove the supernatant, and repeat this step once. Add 200 μl of blocking reagent, react at 25 °C in the dark on a rotary mixer for 1 hour to block the unbound sites on the magnetic beads. After the reaction, resuspend in 100 μl of reaction reagent.

[0070] 3. Immunofluorescence Staining and Reaction of Magnetic Particle Attachment

[0071] Resuspend the cell pellet obtained in step 1 in 4% paraformaldehyde, after 15 minutes, centrifuge at 1000 rpm for 5 minutes to remove the supernatant. Resuspend in 200 μl of PBS, carefully pipette and mix well. Add 5 μl of the magnetic particle suspension obtained in step 2, 1 μl of goat anti-mouse IgG-FITC, and 1 μl of goat anti-rabbit IgG-Cy3 fluorescent secondary antibody. Incubate at room temperature in the dark for 20 minutes, then add 3 μl of DAPI reagent and react at room temperature in the dark for 5 minutes.

[0072] 4. Observation under Harmony Software Microscope

[0073] Transfer the cell suspension after immunofluorescence staining and magnetic bead incubation in step 3 to a pre-blocked 96-well plate, let it stand for 5 minutes, then observe on the Operetta CLS High Content Analysis System platform and interpret with Harmony software.

[0074] As Figure 4As shown, CTC cells expressing NEXN and HBA2 were detected in the peripheral blood of patients with distant metastasis after liver cancer surgery (i.e., under white light BF, magnetic particles were observed to be adsorbed on the CTC cells); while there was no adsorption of magnetic particles on the CTCs in the peripheral blood of primary HCC patients (first visit, imaging and other tests determined no tumor metastasis), that is, they did not express NEXN or HBA2. Using the patient's peripheral blood leukocytes as a negative control, it was found that magnetic particles would not non-specifically adsorb on leukocytes.

[0075] In summary, compared with the prior art, the present invention has the following advantages:

[0076] 1. The present invention first uses hemoglobin subunit α2 (HBA2) and nexilin F-actin binding protein (NEXN) simultaneously as the application of circulating tumor cell high-metastasis potential classification markers, improving the experimental accuracy and detection rate.

[0077] 2. The present invention leaves blank sites linked to biotin-streptavidin, facilitating users to add the target to be detected of interest, and using bioluminescence to amplify the signal of the captured circulating tumor cells, thereby achieving high-sensitivity detection of circulating tumor cells.

[0078] 3. The present invention provides a method to maximize the enrichment of CTCs with high-metastasis potential and resistance to immune killing, and for the differences between different cancer types, biological targets that can be self-assembled by users are reserved on the microparticles, such as Figure 5 as shown.

[0079] 4. The kit of the present invention determines the CTC subtype from the perspective of the immune escape of circulating tumor cells with the help of platelets, and can better reflect the prognosis of cancer patients.

[0080] 5. In hepatocellular carcinoma specimens, it is shown that compared with the previous epithelial-mesenchymal classification or metabolic classification, the NEXN / HBA2 + CTC subtype is more relevant to tumor metastasis and postoperative recurrence of patients, can be used as an auxiliary diagnostic marker for tumor metastasis, is an important supplement to CTC counting and epithelial-mesenchymal and other classifications, and has good application prospects in tumor diagnosis and metastasis detection.

[0081] 6. The final result of the present invention determines the CTCs with "high-metastasis potential" as cells that are DAPI+EpCAM / CK19+, NEXN / HBA2+ (i.e., adhered with magnetic particles) and CD45-. This not only avoids the possibility of false positives in the experimental results caused by multiple staining steps and fluorescence crosstalk in multi-color immunofluorescence staining, but also can obtain targeted CTCs through magnetic separation for subsequent multi-omics analysis.

[0082] 7. The kit of the present invention excludes the interference of immune cells (such as monocytes) mixed during gradient density separation of CTCs by detecting the CD45 index, thereby improving the analysis accuracy.

[0083] 8. The kit of the present invention expands the acquisition / detection rate of target cells by the combined use of EpCAM / CK19 and NEXN / HBA2.

Claims

1. A kit for detecting circulating tumor cells with high metastatic potential, wherein the tumor cells are liver cancer cells, and the kit comprises: (a) A reagent for detecting circulating tumor cells, wherein the reagent for detecting circulating tumor cells includes a reagent for detecting epithelial cell adhesion molecule and / or a reagent for detecting cytokeratin 19; (b) A reagent for detecting high metastatic potential markers, wherein the reagent for detecting high metastatic potential markers includes a reagent for detecting hemoglobin subunit alpha 2 and / or a reagent for detecting nexilin F-actin binding protein; (c) A reagent for detecting immune cells; and (d) A nuclear staining reagent.

2. The kit according to claim 1, wherein the reagent for detecting immune cells includes a reagent for detecting leukocyte common antigen; and / or the nuclear staining reagent includes DAPI or Hoechst.

3. The kit according to claim 1, wherein the kit further comprises a solid support, and the surface of the solid support comprises the (a) and the (b).

4. The kit according to claim 3, wherein the solid support comprises particles.

5. The kit according to claim 3, wherein the solid support comprises magnetic particles.

6. The kit according to claim 3, wherein the surface of the solid support further comprises the (c).

7. The kit according to claim 3, wherein the solid support further comprises blank binding sites.

8. The kit according to claim 7, wherein the blank binding sites include reactive functional groups.

9. The kit according to claim 7, wherein the blank binding sites include carboxyl functional groups.

10. The kit according to claim 1, wherein one or more of (a), (b) and (c) include antibodies; and / or wherein one or more of (a), (b) and (c) include fluorescent staining reagents.

11. The kit according to claim 10, wherein the fluorescent staining reagent includes cyanine 3, succinimidyl ester 647 or fluorescein isothiocyanate.

12. The kit according to claim 1, wherein the kit further comprises labeled detection reagents.

13. The kit according to claim 12, wherein the labeled detection reagents include fluorescent dye-labeled detection reagents or biotin-labeled detection reagents.

14. The kit according to claim 12, wherein the labeled detection reagents include antibodies.

15. The kit according to claim 13, wherein the fluorescent dye includes acridinium ester.

16. The kit according to claim 1, wherein the kit further comprises a cell fixative, a blocking agent, an anti-fluorescence quenching agent and / or a red blood cell lysate.

17. Use of the kit according to any one of claims 1-16 in the preparation of a reagent for detecting circulating tumor cells with high metastatic potential in a sample, wherein the tumor cells are liver cancer cells.

18. The use according to claim 17, wherein the sample includes body fluids.

19. The use according to claim 17, wherein the sample includes blood.

20. The use according to claim 17, wherein the tumor cells include platelet-adherent CTCs.

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