Kit for detecting circulating tumor cells with high metastasis potential and application thereof

Abstract

The invention discloses a kit for detecting circulating tumor cells with high metastasis potential and application of the kit. The kit for detecting circulating tumor cells with high metastasis potential comprises: (a) a detection reagent for circulating tumor cells; (b) a detection reagent of a high transfer potential marker; (c) a detection reagent of immune cells; and (d) a nucleus staining agent.

Description

Technical Field

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

[0002] Circulating tumor cells (CTC) refer to tumor cells that fall off from the primary or metastatic lesions 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 clinical liquid biopsy tool for auxiliary diagnosis, dynamic monitoring, and prognosis evaluation of diseases. CTC has great heterogeneity, which brings certain difficulties to detection. Previous studies have classified CTCs based on their metabolism, epithelial-mesenchymal or PD-L1 expression, and other characteristics. This fixed combination of indicators cannot be applied to all types of tumors, especially the inability to capture CTCs that lack the above-mentioned surface markers, which can easily lead to false negatives.

[0003] During the process of blood flow dissemination, CTCs face challenges such as mechanical damage, anoikis, and immune attack, and only a few CTCs survive. Studies have shown that most CTCs are not single cells that spread alone in the blood, but are accompanied by a large number of blood cells. Our team has previously found in multiple cancer types that platelet adhesion to CTCs is a common phenomenon and is potentially related to CTC resistance to immune attack and distant metastasis. However, CTCs may carry platelet markers due to membrane fusion with platelets or phagocytosis of platelets. Detection of platelet-related CTCs will increase the false positive results of the experiment, and there is an urgent need for CTC markers that can better reflect the patient's prognosis.

[0004] Immunomagnetic particles are highly manipulable magnetic microspheres whose surface can be modified to express antibodies against multiple indicators. When targeting CTCs, they can achieve enrichment and detection of CTCs through antigen-antibody binding and magnetic separation. However, there is no method in the art to accurately detect CTCs with high metastatic potential using immunomagnetic particles.

[0005] The number of CTCs in peripheral blood is extremely rare, accounting for only 1 / 10^6-1 / 10^7 of peripheral blood leukocytes, so the practical application of CTCs has always been restricted by its effective enrichment and detection methods. In addition, there are many types of existing CTCs and there are technical limitations that cannot be effectively and comprehensively enriched. There is no method in the field that can accurately detect CTCs with high metastatic potential, such as platelet adhesion / related CTCs.

[0006] There is still an urgent need in the art for a method that can accurately and sensitively detect circulating tumor cells with high metastatic potential. Summary of the invention

[0007] In order 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) Detection reagents for circulating tumor cells;

[0009] (b) a detection reagent for a high metastatic potential marker;

[0010] (c) immune cell detection reagents; and

[0011] (d) Nuclear staining reagent.

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

[0013] (a) Detection reagents for circulating tumor cells;

[0014] (b) a detection reagent for a high metastatic potential marker;

[0015] (c) immune cell detection reagents; and

[0016] (d) Nuclear staining reagent.

[0017] In order 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 detects EpCAM / CK19-positive, HBA2 / NEXN-positive, and CD45-negative CTCs through specific enrichment, and also leaves blank sites that are linked to biotin-streptavidin, thereby achieving high-sensitivity detection of circulating tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0019] Figure 1 This is the result of screening genes that are highly expressed in CTCs compared to primary cancer and upregulated after platelets adhere to tumor cells;

[0020] Figure 2 In vitro and ex vivo qRT-PCR experiments confirmed that platelets upregulated the expression of HBA2 and NEXN after adhesion to CTCs;

[0021] Figure 3 In vivo experiments in mice have demonstrated that platelets adhere to CTCs and increase the systemic metastasis and colonization potential of CTCs by upregulating the expression of HBA2 and NEXN.

[0022] Figure 4This is a result diagram of using the kit according to one embodiment of the present application, which shows the expression of HBA2 and NEXN on CTCs of HCC patients with cancer metastasis, with scale bars of 5 μm and 2 μm, respectively;

[0023] Figure 5 A schematic diagram of immunomagnetic beads according to one embodiment of the present application. DETAILED DESCRIPTION

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

[0025] The kit for detecting circulating tumor cells with high metastatic potential also comprises: (b) a detection reagent for a high metastatic potential marker. In the present application, the detection reagent for a high metastatic potential marker may include any reagent that can be used to detect a high metastatic potential specific marker for tumor cells. In one embodiment, the detection reagent for the high metastatic potential marker includes a detection reagent for hemoglobin subunit α2 (HBA2) and / or a detection reagent for nexilin F-actin binding protein (NEXN). In a preferred embodiment, the detection reagent for the high metastatic potential marker includes a detection reagent for hemoglobin subunit α2 (HBA2) and a detection reagent for nexilin F-actin binding protein (NEXN). In one embodiment, the detection reagent for the high metastatic potential marker includes an antibody. In one embodiment, the detection reagent for hemoglobin subunit α2 (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 anthocyanin 3 (Cy3), succinimidyl ester 647 (AF647) or fluorescein isothiocyanate (FITC). In a preferred embodiment, the fluorescent staining reagent of the detection reagent of hemoglobin subunit α2 (HBA2) includes Cy3. In a preferred embodiment, the fluorescent staining reagent of the detection reagent of nexilinF-actin binding protein (NEXN) includes Cy3.

[0026] The kit for detecting circulating tumor cells with high metastatic potential also includes: (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 immune cell-specific markers. In one embodiment, the detection reagent for the immune cells includes a detection reagent for a common leukocyte antigen (CD45). In one embodiment, the detection reagent for the immune cells includes an antibody. In one embodiment, the detection reagent for a common leukocyte 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 anthocyanin 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 cell nucleus staining reagent. In one embodiment, the cell nucleus staining reagent comprises DAPI or Hoechst.

[0028] The kit for detecting circulating tumor cells with high metastatic potential may also include a solid carrier. In one embodiment, the solid carrier includes particles. In a preferred embodiment, the particles include spherical particles. In a preferred embodiment, the particles include magnetic particles. In one embodiment, the magnetic particles include ferrosoferric oxide 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 carrier includes one or more of the components of the above-mentioned embodiments. In one embodiment, one or more of the components of the above-mentioned embodiments are coupled to the surface of the solid carrier. In one embodiment, one or more of the components of the above-mentioned embodiments are covalently bound to the surface of the solid carrier. In one embodiment, one or more of the components of the above-mentioned embodiments are physically adsorbed to the surface of the solid carrier. In one embodiment, one or more of the components of the above-mentioned embodiments are fixed to the surface of the solid carrier by reactive functional groups on the surface of the solid carrier, preferably carboxyl functional groups. In one embodiment, the surface of the solid carrier includes (b) of the above-mentioned embodiment. In one embodiment, the surface of the solid carrier includes (a) and (b) of the above-mentioned embodiment. In one embodiment, the surface of the solid carrier comprises (a), (b) and (c) of the above-mentioned embodiments. In one embodiment, the surface of the solid carrier is coated with streptavidin. In one embodiment, the solid carrier further comprises a blank binding site. In one embodiment, the blank binding site comprises a reactive functional group. In a preferred embodiment, the blank binding site comprises a carboxyl functional group. In one embodiment, the blank binding site comprises a reactive functional group on the surface coated with streptavidin. In a preferred embodiment, the blank binding site comprises a carboxyl functional group on the surface coated with streptavidin. In one embodiment, the blank binding site is used to bind to a detection reagent for detecting other targets. In one embodiment, the blank binding site is used to bind to a detection reagent for amplifying the detection signal. In one embodiment, the kit further comprises reagents and consumables related to particle coupling. In one embodiment, the reagent comprises an activation reagent, a reaction reagent, a chemical reagent, a blocking agent and a protective solution. In one embodiment, the chemical reagent comprises EDC and NHS. In one embodiment, the consumables comprise an EP tube and a magnetic separation device. In one embodiment, the EP tube comprises a 1.5ml EP tube. 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 also include a labeled detection reagent. In a preferred embodiment, the labeled detection reagent includes a fluorescent dye-labeled detection reagent or a biotin-labeled detection reagent. In one embodiment, the detection reagent includes an antibody. In one embodiment, the fluorescent dye includes an 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 the interpretation of luminescent signals by immunofluorescence of magnetic particles that can be assembled independently in combination with fluorescent dyes. In one embodiment, the luminescent signal interpretation includes the interpretation of luminescent signals under the microscope of the Operetta CLS High Content Analysis System platform combined with Harmony software. In one embodiment, cells with DAPI+EpCAM / CK19+ and adhered to magnetic particles are judged as CTCs with high metastatic potential. In one embodiment, cells with DAPI+EpCAM / CK19+NEXN / HBA2+ are judged as CTCs with high metastatic potential. In one embodiment, DAPI+EpCAM / CK19+NEXN / HBA2+CD45- cells are judged as 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 lysing solution.

[0031] The present application also relates to the use of a kit as described in 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 comprises a body fluid. In a preferred embodiment, the sample comprises blood. In one embodiment, the tumor cells comprise liver cancer cells. In one embodiment, the tumor cells comprise platelet adhesion / associated CTCs.

[0032] Example

[0033] The present 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 the inventions of the present application. It should be noted that the spirit of the present application and the scope of protection of the claims are not limited by the following specific examples.

[0034] List of reagents and equipment used

[0035]

[0036]

[0037] Unless otherwise specified, the patients in this application were from Zhongshan Hospital Affiliated to Fudan University and met the ethical and informed consent requirements.

[0038] Example 1

[0039] In this example, RNA-seq sequencing technology and single-cell transcriptome sequencing were used to simultaneously detect platelets after co-culture with tumor cell lines. It was found that NEXN and HBA2 molecules were highly expressed on CTCs that were adhered to platelets and more likely to survive and metastasize in the blood circulation, such as Figure 1 shown.

[0040] After in vitro hepatoma cell lines and in vitro hepatoma organoids were co-cultured with platelets, qRT-PCR technology was used to detect that the expression of NEXN and HBA2 genes was significantly increased compared with that before co-culture. Figure 2 shown. Figure 2 It is a heat map showing the changes in HBA2 and NEXN gene expression before and after culture of organoids, tumor cell lines and platelets. The leftmost label shows the grouping, which are: Hep3B+PLT (Hep3B cell and platelet co-culture group), Hep3B Con (Hep3B cell control group, only culture medium was added, no platelets), PLC / PRF / 5+PLT (PLC / PRF / 5 cell and platelet co-culture group), PLC / PRF / 5Con (PLC / PRF / 5 cell control group, only culture medium was added, no platelets), PDO+PLT (organoid and platelet co-culture group), 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-culture of organoids and tumor cells with platelets.

[0041] The specific experimental steps are as follows:

[0042] Hepatoma cell lines: Hep3B (catalog number: TCHu106), PLC / PRF / 5 (catalog number: SCSP-5095) were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China).

[0043] Liver cancer cell tissue samples: derived from tumor tissue removed during surgery in patients with liver cancer, collected by liver surgeons at Zhongshan Hospital during surgery. All patients signed informed consent forms, and the study was approved by the Ethics Committee (IRB) of Zhongshan Hospital Affiliated to Fudan University (Ethics Approval Number: B2019-059R).

[0044] 1. Organoid (PDO) preparation:

[0045] To generate HCC organoids, HCC tissue samples (0.25-1 cm3 ) were chopped and, depending on the degree of liver fibrosis, enzymatically digested at 37°C for 2-5 hours using 2.5mg / mL collagenase D (Roche) and 0.1mg / mL DNase (Sigma). When no tissue fragments remained, the digestion process was terminated, and the resulting suspension was filtered through a 100μm nylon cell strainer and then centrifuged at 300-400g for 5 minutes. The resulting pellet was washed with cold advanced DMEM / F12 (GIBCO) and then mixed with BME (basement membrane extract, Type 2, Pathclear). Subsequently, 2000-5000 cells were inoculated per well in a 24-well plate. After BME solidified, organoid-specific isolation medium was added. The organoid-specific isolation medium was 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 record, sodium citrate anticoagulated whole blood of hepatocellular carcinoma (HCC) patients (who had not received anticoagulant therapy in the past two weeks) was selected to separate platelets from the whole blood. The specific steps are as follows: 2 ml of whole blood was centrifuged at 150g for 12 minutes at room temperature to obtain platelet-rich plasma (PRP). Centrifuge at 900g for 5 minutes at room temperature and discard the supernatant. The platelet microspheres were resuspended in Tyrode buffer (pH 7.4, calcium-free) and prostaglandin E1 (PGE1, 0.3μg / mL) was added to prevent platelet activation.

[0048] 3. Co-culture Model Establishment

[0049] 1) Take 1 x 10^5 PDO and 1 x 10^8 platelets at a ratio of 1:1000 and co-culture them in a 24-well plate. After 48 hours of culture, 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, remove the supernatant, and the pellet is the PDO co-cultured with platelets.

[0050] 2) 3x 10^5 tumor cell lines (Hep3B and PLC / PRF / 5) and 3x 10^8 platelets were co-cultured in a 24-well plate at a ratio of 1:1000. After 48 hours of co-culture, the platelets were removed by gently washing with PBS using a Pasteur tube. The adherent cells were digested with 0.05% trypsin and neutralized with DMEM medium containing 10% fetal bovine serum. The cells were centrifuged at 1000 rpm for 5 minutes to obtain a cell pellet, which was washed twice with PBS and the supernatant was removed. The pellet was the tumor cells co-cultured with platelets.

[0051] Further in vivo experiments showed that platelet adhesion to CTCs increased the systemic metastasis and colonization potential of CTCs by upregulating the expression of HBA2 and NEXN. Figure 3 shown. Figure 3 The live imaging of small animals shows the systemic tumor metastasis of the model mice injected by 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 show that compared with the control group, the systemic metastasis of mice with platelet removal was significantly reduced, indicating that without the protective effect of platelets, tumor cells are easily removed during blood dissemination; the systemic metastasis of mice with HBA2 or NEXN knockdown tumor cells injected by tail vein was better than that of the control group, and there was no significant difference in the number of metastatic foci between the groups, which was equivalent 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] Liver cancer cell lines: H22-luc Con, H22-luc sh-HBA2, H22-luc sh-NEXN (mouse liver cancer cell line H22-luc (H22 cells with luciferase). Con is the control group, and sh is the gene knocked down. The control group and sh-HBA2, sh-NEXN are cell lines stably constructed using lentivirus based on H22-luc, and were provided by the Liver Cancer Institute of Zhongshan Hospital Affiliated to Fudan University).

[0054] Mouse information: strain: Balb / c (BALB / cCrSlcNifdc mice, purchased from Biocytogen Jiangsu Gene Biotechnology Co., Ltd., SPF level, mice were housed in a barrier at the Department of Laboratory Animal Science of Fudan University, raised under SPF conditions, and treated humanely throughout the study. Animal experiments were performed in strict 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; sex: male mice.

[0055] Reagents: Platelet depletion reagent iPLT, antibody for platelet depletion in mice (#R300, EmfretAnalytics).

[0056] Instrument: PerkinElmer IVIS Spectrum.

[0057] 1. Drug intervention:

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

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

[0060] 2x 10^6 H22-luc Con (control group) and H22-luc sh-HBA2 and H22-luc sh-NEXN cell lines with knockdown of HBA2 or NEXN were dissolved in 200μl PBS and injected into Balb / c mice through the tail vein. After 3 weeks, the mice were intraperitoneally injected with the luciferase reaction substrate XenoLight D-luciferin potassium salt (#122799, PerkinElmer) (150mgD-luciferin / kg mouse body weight). After anesthesia (isofluorane (R510-22-10, RWD), the mice were anesthetized with 4% gas at a flow rate of 300ml / min. Steps: After the anesthetic fills the induction box, the animal is placed in the induction box and waits for the animal's body to fall to the side and does not try to return to its lying position, which means that the animal is fully anesthetized), and imaging is performed using IVIS. Data analysis was performed using Living Image (v4.1) (Caliper Life Sciences, Inc). Figure 3 The results showed that compared with the control group, the number of metastatic lesions in the mice after platelet removal was significantly reduced. Knocking down the expression of HBA2 or NEXN in cells can achieve a similar effect of inhibiting tumor metastasis as platelet removal.

[0061] Example 2

[0062] In this example, a self-built immunomagnetic microsphere kit is used to enrich CTCs with high metastatic potential. The CTCs in this example are from patients with multi-organ distant metastasis after liver cancer surgery (high metastasis patients) and patients with primary hepatocellular carcinoma who come to the hospital for the first time and have no metastasis. The kit includes DAPI reagent and immunomagnetic microspheres, which are coated with 5 antibodies at the same time, 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 showed that, using this kit, the presence of DAPI+ CTC cells with magnetic particles attached was observed in the peripheral blood of patients with high metastasis (whole blood samples of patients anticoagulated with EDTA-K2; patient type: patients who came to the hospital again after surgical resection of hepatocellular carcinoma, and imaging and other evidence showed that the patients had multiple organ metastases and were determined to be patients with high metastasis) on the Operetta CLS High Content Analysis System platform. The CTCs expressed (EpCAM-AF647) red light, (CK19-AF647) red light, and (HBA2 / NEXN-Cy3) yellow fluorescence around them, and did not express (CD45-FITC) green fluorescence (CD45 negative (magnetic beads did not emit green fluorescence) to exclude cells expressing CD45); while the peripheral blood of patients without metastasis (whole blood samples of patients anticoagulated with EDTA-K2; patient type: patients who came to the hospital for the first time, the pathological results showed hepatocellular carcinoma and there was no evidence to support the occurrence of metastasis, and the patients had not received any cancer-related treatment, that is, patients without metastasis) CTCs were only DAPI+, without magnetic particles attached (such as Figure 4 In addition, the patient's peripheral blood (whole blood samples of patients anticoagulated with EDTA-K2; patient type: hepatocellular carcinoma patients) leukocytes (DAPI+CD45+) had no magnetic particles adhered, which served as a negative control.

[0063] The specific experimental steps are as follows:

[0064] 1. CTC Negative Enrichment

[0065] Manually mix the whole blood sample tube up and down slowly, transfer 5ml of whole blood to a 15ml centrifuge tube, mix with an equal volume of PBS buffer, and gently blow the Pasteur tube to mix. Slowly add the diluted blood along the wall of the 15ml centrifuge tube to 3.5ml of Ficoll-Paque gradient density separation solution. Centrifuge at 1200g for 10 minutes, keep the white film layer in a new 15ml centrifuge tube, add an equal volume of PBS, centrifuge at 300g for 10 minutes, and remove the supernatant. Add 1ml of red blood cell lysis buffer, incubate at 4℃ for 5 minutes, centrifuge at 3000rpm for 5 minutes, discard the supernatant, and obtain CTC and immune cells.

[0066] 2. Preparation of Antibody-coupled Magnetic Particles

[0067] Take 3 μl Dynabeads TM MyOne TM Carboxylic Acid magnetic particles were placed in 500μl activator, gently pipetted, and separated on a magnetic stand for 20 seconds. The supernatant was removed and resuspended in 100μl activator. 1mg EDC and 1mg NHS were dissolved in 200μl activator, mixed, and added to the magnetic particle suspension. Reacted on a rotating mixer at 25℃ 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, remove the supernatant, add 100 μl protein solution containing 1 mg / ml NEXN antibody, HBA2 antibody and CD45 antibody (prepared using a reaction activator), 5 μl EpCAM-AF647 reagent and 5 μl CK19-AF647 reagent. Protect from light, react on a rotating mixer at 25°C for 2 hours to couple the antibodies.

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

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

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

[0072] 4.Observation under Harmony software

[0073] The cell suspension after immunofluorescence staining and magnetic bead incubation in step 3 was transferred to a blocked 96-well plate, allowed to stand for 5 minutes, and then observed on the Operetta CLS High Content Analysis System platform and interpreted by Harmony software.

[0074] like 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., magnetic particles were observed to be adsorbed on CTC cells under white light BF); while there was no adsorption of magnetic particles on CTCs in the peripheral blood of primary HCC patients (first visit, imaging and other tests determined no tumor metastasis), that is, no expression of NEXN or HBA2. Using patients' peripheral blood leukocytes for negative control, it was found that magnetic particles would not be non-specifically adsorbed on leukocytes.

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

[0076] 1. The present invention is the first to use hemoglobin subunit α2 (HBA2) and nexilin F-actin binding protein (NEXN) as circulating tumor cells with high metastatic potential typing markers, thereby improving the experimental accuracy and detection rate.

[0077] 2. The present invention leaves blank sites connected with biotin-streptavidin, which is convenient for users to add targets of interest to be detected, and uses bioluminescence to amplify the captured circulating tumor cell signals, thereby achieving high-sensitivity detection of circulating tumor cells.

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

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

[0080] 5. In hepatocellular carcinoma specimens, NEXN / HBA2 + CTC subtypes are more related to tumor metastasis and postoperative recurrence of patients. They can be used as auxiliary diagnostic markers for tumor metastasis and are an important supplement to CTC counting and epithelial-mesenchymal classification. They have good application prospects in tumor diagnosis and metastasis detection.

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

[0082] 7. The kit of the present invention eliminates the interference of mixed immune cells (such as monocytes) 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 using EpCAM / CK19 in combination with NEXN / HBA2.

Claims

1. A kit for detecting circulating tumor cells with high metastatic potential, the kit comprising: (a) Detection reagents for circulating tumor cells; (b) a detection reagent for a high metastatic potential marker; (c) immune cell detection reagents; and (d) Nuclear staining reagent.

2. The kit according to claim 1, wherein the detection reagent for circulating tumor cells comprises a detection reagent for epithelial cell adhesion molecule (EpCAM) and / or a detection reagent for cytokeratin 19 (CK19); and / or The detection reagents of the high metastatic potential markers include detection reagents of hemoglobin subunit α2 (HBA2) and / or detection reagents of nexilin F-actin binding protein (NEXN).

3. The kit according to claim 1, wherein the immune cell detection reagent comprises a detection reagent for leukocyte common antigen (CD45); and / or The cell nucleus staining reagent includes DAPI or Hoechst.

4. The kit of claim 1, wherein the kit further comprises a solid carrier, preferably particles, more preferably magnetic particles, wherein the surface of the solid carrier comprises (a) and (b), and optionally, the surface of the solid carrier further comprises (c).

5. A kit as claimed in claim 4, wherein the solid support further comprises a blank binding site, preferably, the blank binding site comprises a reactive functional group, preferably a carboxyl functional group.

6. The kit of claim 1, wherein one or more of (a), (b) and (c) comprises an antibody; and / or One or more of (a), (b) and (c) include a fluorescent staining reagent, preferably, the fluorescent staining reagent includes anthocyanin 3 (Cy3), succinimidyl ester 647 (AF647) or fluorescein isothiocyanate (FITC).

7. The kit as claimed in claim 1, wherein the kit further comprises a labeled detection reagent, preferably, a fluorescent dye-labeled detection reagent or a biotin-labeled detection reagent, preferably, the labeled detection reagent comprises an antibody, preferably, the fluorescent dye comprises an acridinium ester.

8. 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 lysing solution.

9. Use of the kit according to any one of claims 1 to 8 in the preparation of a reagent for detecting circulating tumor cells with high metastatic potential in a sample.

10. The use according to claim 9, wherein the sample comprises a body fluid, preferably blood, and / or the tumor cells comprise liver cancer cells, preferably, the tumor cells comprise platelet-adherent / associated CTCs.

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