Method for enriching and identifying tumor cells with different phenotypes based on double-antibody double-particle-size magnetic beads

Abstract

The invention discloses a method for enriching and identifying tumor cells with different phenotypes based on double-antibody double-particle-size magnetic beads. Epithelial cell specific cell adhesion molecules and interstitial cell specific waveform proteins are combined with magnetic beads with different particle sizes, so that epithelial, interstitial and mixed specific markers of the same cell in a living cell state can be enriched and identified at one time. The magnetic beads specifically capture target cells with different phenotypes, the captured target cells are separated in a cell sorter under the action of a magnetic field, the phenotypes of the target cells are identified according to different particle sizes of the magnetic beads adhered to the target cells, and on the basis, subsequent analysis of more markers can be further performed. According to the invention, epithelial, mesenchymal and mixed target cells can be simultaneously captured from various body fluids in a living cell state, typing and counting can be rapidly carried out through the particle sizes of the magnetic beads adhered to the surfaces of the cells, the living target cells are analyzed more accurately, and the clinical and scientific research application values are improved.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a method for enriching and identifying tumor cells of different phenotypes based on double-antibody double-size magnetic beads. Background Art

[0002] Emerging liquid biopsy technologies such as circulating tumor cell (CTCs) detection technology have become effective means for early cancer screening, auxiliary diagnosis, prognosis monitoring and efficacy evaluation due to its non-invasiveness and repeatable detection. As this technology has entered the national and Chinese cancer diagnosis and treatment guidelines, its clinical application has gradually become more widespread.

[0003] Circulating tumor cells (CTCs) are a general term for various types of tumor cells present in peripheral blood. They can be released from solid tumors or metastatic lesions into the peripheral blood circulation spontaneously or due to diagnostic and treatment procedures. They are an important cause of postoperative recurrence and distant metastasis in patients with malignant tumors, and an important factor leading to death in tumor patients. They are the most direct indicator of cancer in liquid biopsy. CTC detection enriches and tests CTCs in trace amounts in peripheral blood, monitors the trend of changes in the number of CTCs, so as to monitor tumor dynamics in real time, evaluate treatment effects, and achieve real-time individual treatment. Most CTCs undergo apoptosis or are phagocytosed after entering the peripheral blood, and a few escape and develop into metastatic lesions, increasing the risk of death in patients with malignant tumors. Therefore, detecting rare CTCs from patients' peripheral blood samples has become a new research hotspot.

[0004] A large number of studies have shown that CTCs undergo epithelial to mesenchymal transition (EMT) during their entry into the peripheral blood circulation, resulting in different phenotypes, including epithelial, mesenchymal, and mixed epithelial-mesenchymal types. Mesenchymal CTCs have stronger metastatic potential, drug resistance, and invasive ability. Therefore, in clinical applications, while CTCs are isolated, identified, and counted, it is also necessary to analyze the phenotype of CTCs to fully evaluate the risk of tumor metastasis, disease progression, and prognosis.

[0005] The epithelial-mesenchymal transition process is often accompanied by overexpression of mesenchymal markers (such as CSV and Twist1), while the expression of epithelial markers (such as CK and EpCAM) is reduced. Epithelial cell adhesion molecule (EpCAM) is the most commonly used marker for epithelial CTC enrichment, and vimentin (CSV) can be used as a marker for enrichment of mesenchymal CTCs.

[0006] At present, CTCs enrichment technologies mainly include positive or negative enrichment through cell surface markers, membrane filtration based on cell size, density gradient centrifugation, etc. The most common methods in the market are mainly immune positive capture method and negative enrichment method. The positive enrichment method based on immunomagnetic beads is currently the only CTCs detection technology approved by the Food and Drug Administration (FDA) for marketing. EpCAM-expressing target cells are captured by immunomagnetic beads coated with EpCAM antibodies. The most commonly used marker in traditional positive enrichment methods is epithelial cell adhesion molecule (EpCAM), which can capture epithelial and epithelial-stromal mixed CTCs in the blood. In theory, it should not be able to capture EpCAM-negative interstitial CTCs, which may lead to missed detection of interstitial CTCs.

[0007] The multi-antibody magnetic bead enrichment method provided by the present invention overcomes the shortcomings of the traditional positive enrichment method. On the one hand, magnetic beads with different antibodies coupled to the surface are used, namely EpCAM and CSV antibody magnetic beads. On the other hand, magnetic beads of different particle sizes are selected for distinction, so that the cells are captured under the action of a directional magnetic field after binding to the magnetic beads. It ensures that all phenotypic CTCs in peripheral blood samples, including epithelial, interstitial, and mixed types, can be enriched and separated, and then accurately identified by immunofluorescence staining, so as to achieve the purpose of accurately analyzing the cytological characteristics of CTCs. Summary of the invention

[0008] In order to solve the defects of existing EpCAM antibody magnetic beads that they cannot enrich mesenchymal tumor cells and have a low detection rate of mixed tumor cells, and at the same time solve the limitation that they cannot directly identify epithelial, mesenchymal and mixed target cells in the living cell state, the present invention provides a method for simultaneously enriching and identifying epithelial / mesenchymal / mixed tumor cells based on double-antibody double-particle size magnetic beads.

[0009] In order to achieve the above object, the present invention adopts the following technical solutions:

[0010] The method for enriching and identifying tumor cells of different phenotypes based on dual-antibody dual-size magnetic beads comprises the following steps:

[0011] (1) preparing magnetic beads that bind epithelial cell-specific cell adhesion molecules and magnetic beads that bind mesenchymal cell-specific vimentin, wherein the two magnetic beads have different particle sizes;

[0012] (2) mixing magnetic beads binding to epithelial cell adhesion molecules and magnetic beads binding to vimentin with a sample containing tumor cells, wherein the magnetic beads specifically capture tumor cells of different phenotypes, and separating the captured target cells using a magnetic field in a cell sorter;

[0013] (3) Under a microscope, the phenotypic type of the captured target cells is identified based on the size of the magnetic beads attached to them under bright field light or fluorescence. Magnetic beads bound to epithelial cell adhesion molecules specifically capture epithelial tumor cells, magnetic beads bound to vimentin specifically capture mesenchymal tumor cells, and mixed tumor cells bind to magnetic beads of both sizes at the same time.

[0014] Preferably, the particle size of the large magnetic beads is preferably 3 μm to 5 μm, and the particle size of the small magnetic beads is preferably 300 nm to 1 μm.

[0015] Preferably, the captured target cells are further analyzed for markers in combination with immunofluorescence staining.

[0016] The tumor cells are derived from blood, urine, saliva, pleural effusion, abdominal effusion, pulmonary effusion or stool dilution.

[0017] Through the above technical scheme, the present invention mixes two kinds of antibody magnetic beads, EpCAM and CSV, with the tumor to be tested, wherein EpCAM magnetic beads recognize and specifically adhere to epithelial tumor cells, CSV magnetic beads recognize and specifically adhere to mesenchymal tumor cells, and mixed tumor cells adhere to the above two kinds of magnetic beads at the same time. Through the action of the directional magnetic field, the cells adhering to the magnetic beads are separated and purified from hundreds of millions of cell samples, and can be stabilized in the container and not easily lost. The target cells are stained with markers using immunofluorescence staining reagents, and tumor cells can be observed and identified under a fluorescent microscope, and tumor cells can be typed and counted by the type of magnetic beads adhered to the cell surface. Compared with using EpCAM magnetic beads alone, the method of the invention can simultaneously capture epithelial, mesenchymal and mixed tumor cells in the sample to be tested, separate the tumor cells in the sample to the greatest extent, prevent missed detection, and has strong practicality in clinical and scientific research.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] Most existing clinical tumor cell detection technologies are positive magnetic bead enrichment detection technologies, the main drawback of which is the loss of interstitial tumor cells during the enrichment stage and the low detection rate of mixed tumor cells. The dual-antibody magnetic bead positive enrichment typing method described in the present invention not only improves the detection sensitivity of interstitial and mixed tumor cells, but also can simply analyze and identify the phenotype of target cells, especially can preserve the activity of target cells, achieving multiple goals at one stroke. Compared with the negative enrichment method, the present invention is simpler to operate, the target cell purity obtained is higher, and the interference caused by leukocytes to target cell analysis is eliminated; compared with the membrane filtration method, it can avoid missing smaller sizes; compared with the density gradient centrifugation method, there is a higher cell recovery rate, and cell clusters (CTM) will not be lost; compared with the microfluidic chip method coated with antibodies, the consumables cost is lower, it is not limited by the sample volume, and a sample of 5 to 30 ml volume can be analyzed at a time, and the separated cells are suspended in a six-well cell culture plate filled with a buffer solution as a single cell, which makes it easier to conduct single cell related research such as single cell sequencing, tumor cell in vitro culture, etc.; compared with the EpCAM antibody magnetic bead positive enrichment method, not only epithelial and mixed tumor cells can be captured, but also mesenchymal tumor cells can be captured, which avoids the target cell leakage to the greatest extent. The target cell cell activity obtained by the present invention is high, the analysis is kept in the liquid phase throughout the whole process, and the cell morphology remains complete and three-dimensional, which is more conducive to clearly observing, tracking and further molecular biological analysis of the cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is a fluorescence staining image of epithelial / stromal / mixed A549 lung cancer cells enriched and captured by the double antibody double magnetic beads in Example 2 of the present invention. Figure 1-A is a fluorescence staining image of epithelial A549 lung cancer cells captured by double antibody magnetic beads, Figure 1-B is a fluorescence staining image of mesenchymal A549 lung cancer cells captured by double antibody magnetic beads, and Figure 1-C is a fluorescence staining image of mixed A549 lung cancer cells captured by double antibody magnetic beads.

[0021] Figure 2 is a fluorescence staining image of epithelial / stromal / mixed intestinal cancer cells captured by dual antibody magnetic beads in Example 3 of the present invention. Figure 2-A is a fluorescence staining image of epithelial intestinal cancer cells captured by dual antibody magnetic beads, and Figure 2-B is a fluorescence staining image of mixed intestinal cancer cells captured by dual antibody magnetic beads.

[0022] FIG3 is a fluorescence staining image of epithelial / mesenchymal / mixed lung cancer cells captured by the double antibody magnetic beads in Example 4 of the present invention. Figure 3A This is a fluorescent staining image of epithelial lung cancer cells captured by dual antibody magnetic beads. Figure 3B Fluorescence staining of mixed lung cancer cells captured by dual antibody magnetic beads. Figure 3C This is a fluorescent staining image of mesenchymal lung cancer cells captured by dual antibody magnetic beads.

[0023] Figure 4This is a fluorescence staining image of mesenchymal A549 cells captured by CSV magnetic beads in Example 5 of the present invention. DETAILED DESCRIPTION

[0024] The present invention will be described in detail below in conjunction with specific examples. The materials used in the examples can be obtained from commercial channels.

[0025] Example 1

[0026] This embodiment provides a specific preparation method combining EpCAM magnetic beads (4.5 μm) and CSV magnetic beads (1 μm), comprising the following steps:

[0027] S1. Add 4.5 μm or 1 μm streptavidin magnetic beads to 1 mL PBS buffer (containing 0.5 μl Tween-20) and mix by inversion;

[0028] S2. Transfer the 4.5 μm or 1 μm streptavidin magnetic bead solution to a six-well cell culture plate, place it in a cell sorter for magnetic separation, select the "capture" program, and after three collection, washing, and release operations, the weakly magnetic beads remain in the collection tank, and the strong magnetic beads are enriched in the release tank;

[0029] S3. After separation, discard most of the supernatant from the edge of the release tank, take out the six-well cell culture plate, transfer the remaining magnetic bead solution to a new 1.5 ml EP with a pipette, centrifuge at 1500 rpm for 5 min to collect the magnetic beads, and discard the supernatant;

[0030] S4. Add 10 μg / mL EpCAM antibody to the collected 4.5 μm magnetic beads and 20 μg / mL CSV antibody to the collected 1 μm magnetic beads. Mix them well and place them on a biological mixer. Incubate at 15°C and 6 rpm for 60 min.

[0031] S5. After incubation, centrifuge briefly, separate magnetically and remove the supernatant, then wash twice with PBS buffer (containing 0.5 μl Tween-20), and finally resuspend the magnetic beads (4.5 μm) bound to EpCAM antibody and the magnetic beads (1 μm) bound to CSV antibody in PBS buffer.

[0032] Example 2

[0033] In this example, a known number of tumor cells are placed in a fresh blood sample of a healthy person to prepare a peripheral blood sample to be tested, which is used to verify the tumor cell capture rate of the double antibody magnetic beads prepared in Example 1, and specifically includes the following steps:

[0034] S1. A549 non-small cell lung cancer cells were digested with trypsin to prepare a cell suspension and the cell concentration was counted;

[0035] S2. Dilute the cell suspension to 10 cells / μl, take 50 μl of the cell suspension (containing about 500 A549 cells) and add it to 4 ml of the healthy blood sample, invert and mix to prepare the peripheral blood sample to be tested;

[0036] S3. Place the peripheral blood sample in a low-speed centrifuge and centrifuge at 1500 rpm for 5 min, and carefully discard the upper plasma;

[0037] S4. Add 2 times the volume of cell preservation solution of blood cells, invert 4 to 5 times to mix, centrifuge at 1900 rpm for 10 min, discard the supernatant, and then add cell preservation solution to restore the original volume of the sample to obtain a blood cell sample;

[0038] S5. Take 4 μl of EpCAM (4.5 μm) magnetic beads and 4 μl of CSV (1 μm) magnetic beads and add them to the above blood cell sample. Place the sample tube on a biological mixer, tilt at 15°C, and incubate at 6 rpm for 60 min.

[0039] S6. The incubated blood cell sample is transferred to an anti-cell adhesion six-well plate, the six-well plate is placed in a cell sorter for magnetic separation, and the "capture" program is selected. After three collection, washing, and release operations, the A549 non-small cells adhered to the antibody magnetic beads in the blood cell sample are enriched and separated into the release tank;

[0040] S7. Aspirate the buffer solution from the edge of the release groove of the six-well plate, use a pipette to draw 100 μl of cell fixative solution into the release groove, let it stand at room temperature for 10 minutes, and aspirate the cell fixative solution from the edge of the release groove;

[0041] S8. Preparation of immunofluorescence staining solution: add 68 μl of cell preservation solution to a 1.5 ml EP tube, use a pipette to take 20 μl of cytokeratin antibody, 2 μl of CD45 antibody, 5 μl of AF488 fluorescent antibody, and 5 μl of AF568 fluorescent antibody, respectively, and add them to the EP tube, and shake to mix; the concentration of cytokeratin antibody is 25 μg / mL, the concentration of CD45 antibody is 500 μg / mL, the concentration of AF488 fluorescent antibody is 40 μg / mL, and the concentration of AF568 fluorescent antibody is 40 μg / mL;

[0042] S9. Add the above immunofluorescence staining solution into the release tank of the six-well plate, place the six-well plate on a horizontal shaker, mix and incubate at 50 rpm for 30 min;

[0043] S10. Use a pipette to draw 100 μl of DAPI solution into the release tank of the six-well plate, place the six-well plate on a horizontal shaker, mix and incubate at 50 rpm for 5 min;

[0044] S11. Aspirate the staining solution from the edge of the six-well plate release groove and add 1 ml of cell resuspension solution for easy microscopic observation;

[0045] S12. Observe the stained target cells using an inverted fluorescence microscope.

[0046] In Figure 1, BF represents the detection results of antibody magnetic beads combined with A549 cells under a fluorescence microscope, DAPI represents the detection results of cell nuclei stained with DAPI under a fluorescence microscope (cell nuclei are blue in the figure), CK represents the detection results of cytokeratin stained with CK antibody under a fluorescence microscope (cells are green in the figure), CD45 represents the results of detecting white blood cell markers under a fluorescence microscope, and Merge represents the image obtained by superimposing four images marked with EpCAM, DAPI, CK, and CD45.

[0047] As shown in Figure 1-A, after immunofluorescence staining, EpCAM magnetic beads (4.5 μm) were specifically adhered to the cell surface under bright field, with blue fluorescence (cell nucleus stained by DAPI), green fluorescence (keratin stained by CK), and no red fluorescence (leukocytes identified by CD45), indicating that the detected cells were epithelial tumor cells.

[0048] As shown in Figure 1-B, after immunofluorescence staining, CSV magnetic beads (1 μm) were specifically adhered to the cell surface under bright field, with blue fluorescence (cell nucleus stained by DAPI), green fluorescence (keratin stained by CK), and no red fluorescence (leukocytes identified by CD45), indicating that the detected cells were mesenchymal tumor cells.

[0049] As shown in Figure 1-C, after immunofluorescence staining, EpCAM magnetic beads (4.5 μm) and CSV magnetic beads (1 μm) were specifically adhered to the cell surface under bright field, with blue fluorescence (cell nuclei stained with DAPI), green fluorescence (keratin stained with CK), and no red fluorescence (leukocytes identified by CD45), indicating that the detected cells were epithelial-stromal mixed tumor cells.

[0050] Table 1 shows the capture rate of A549 cells in blood samples by dual antibody magnetic beads. The data in the table include the initial target cell number, the captured target cell number, the target cell capture rate of four repeated tests, and the average capture rate, where capture rate = captured target cell number / initial target cell number. It can be seen from the table that the overall capture rate of A549 cells in blood samples by dual antibody magnetic beads reached an average of 95%, and the highest was 98%.

[0051] Table 1 Capture efficiency of A549 lung cancer cells in blood samples by EpCAM and CSV dual antibody magnetic beads

[0052]

[0053] In order to compare the difference in the capture rate of A549 cells by EpCAM magnetic beads and the above-mentioned dual antibody magnetic beads, a control experiment was set up according to the above method, that is, only EpCAM magnetic beads (4.5μm) were added. Table 2 shows the capture rate of A549 cells in blood samples by EpCAM magnetic beads in the control experiment. It can be seen that the average capture rate of A549 cells in blood samples by single EpCAM magnetic beads is 82%, and the highest can reach 84%, which is lower than the capture rate of dual antibody magnetic beads (average 95%, the highest can reach 98%), indicating that EpCAM magnetic beads cannot capture interstitial cells, and dual antibody magnetic beads can capture interstitial cells. Therefore, dual antibody magnetic beads can effectively improve the overall capture rate of tumor cells in peripheral blood.

[0054] Table 2 Capture efficiency of EpCAM magnetic beads for A549 cells in blood samples

[0055]

[0056] Example 3

[0057] This embodiment takes the detection of CTCs in peripheral blood samples of patients with colorectal cancer as an example, and provides a dual magnetic bead capture method for enriching epithelial / stromal / mixed CTCs, which specifically includes the following steps:

[0058] S1. Place 4 ml of peripheral blood sample from a colorectal cancer patient in a low-speed centrifuge and centrifuge at 1500 rpm for 5 min, and carefully discard the upper plasma;

[0059] S2. Add 2 times the volume of cell preservation solution of blood cells, invert 4-5 times to mix, centrifuge at 1900 rpm for 10 min, discard the supernatant, and then add cell preservation solution to restore the original volume of the sample to 4 ml to obtain a blood cell sample;

[0060] S3. Take 4 μl of EpCAM magnetic beads (4.5 μm) and 4 μl of CSV magnetic beads (1 μm) and add them to the above blood cell samples. Place the sample tube on a biological mixer, tilt at 15°C, and incubate at 6 rpm for 30 min.

[0061] S4. The incubated blood cell sample is transferred to an anti-cell adhesion six-well plate, the six-well plate is placed in a cell sorter for magnetic separation, and the "capture" program is selected. After three collection, washing, and release operations, the target cells adhered to the antibody magnetic beads in the blood cell sample are enriched and separated into the release tank;

[0062] S5. Prepare a staining solution kit for blood cell analysis, and perform liquid phase immunofluorescence staining on the enriched and separated target cells;

[0063] S6. Aspirate the buffer solution from the edge of the release groove of the six-well plate, use a pipette to draw 100 μl of cell fixative solution into the release groove, let it stand at room temperature for 10 minutes, and aspirate the cell fixative solution from the edge of the release groove;

[0064] S7. Preparation of immunofluorescence staining solution: add 68 μl of cell preservation solution to a 1.5 ml EP tube, use a pipette to take 20 μl of cytokeratin antibody, 2 μl of CD45 antibody, 5 μl of AF488 fluorescent antibody, and 5 μl of AF568 fluorescent antibody, respectively, and add them to the EP tube, and shake to mix;

[0065] S8. Add the above immunofluorescence staining solution into the release tank of the six-well plate, place the six-well plate on a horizontal shaker, mix and incubate at 50 rpm for 30 min;

[0066] S9. Use a pipette to draw 100 μl of DAPI solution into the release tank of the six-well plate, place the six-well plate on a horizontal shaker, mix and incubate at 50 rpm for 5 min;

[0067] S10. Aspirate the staining solution from the edge of the six-well plate release groove and add 1 ml of cell resuspension solution for easy microscopic observation;

[0068] S11. Observe the stained target cells using an inverted fluorescence microscope.

[0069] In Figure 2, BF represents the detection results of antibody magnetic beads combined with colorectal cancer cells under a fluorescence microscope, DAPI represents the detection results of cell nuclei stained with DAPI under a fluorescence microscope (cell nuclei are blue in the figure), CK represents the detection results of cytokeratin stained with CK antibody under a fluorescence microscope (cells are green in the figure), CD45 represents the results of detecting white blood cell markers under a fluorescence microscope, and Merge represents the image obtained by superimposing four images marked with EpCAM, DAPI, CK, and CD45.

[0070] As shown in Figure 2-A, after immunofluorescence staining, EpCAM beads were specifically adhered to the cell surface under bright field, with blue fluorescence (cell nucleus stained by DAPI), green fluorescence (keratin stained by CK), and no red fluorescence (leukocytes identified by CD45), indicating that the detected cells were epithelial tumor cells.

[0071] As shown in Figure 2-B, after immunofluorescence staining, specific adhesion of EpCAM beads and CSV beads to the cell surface was visible under bright field, with blue fluorescence (cell nuclei stained with DAPI), green fluorescence (keratin stained with CK), and no red fluorescence (leukocytes identified by CD45), indicating that the detected cells were epithelial-stromal mixed tumor cells.

[0072] Example 4

[0073] This embodiment takes the detection of CTCs in peripheral blood samples of lung cancer patients as an example, and provides a dual magnetic bead capture method for enriching epithelial / stromal / mixed CTCs, which specifically includes the following steps:

[0074] S1. Place 4 ml of peripheral blood sample from lung cancer patients in a low-speed centrifuge and centrifuge at 1500 rpm for 5 min, and carefully discard the upper plasma;

[0075] S2. Add 2 times the volume of cell preservation solution of blood cells, invert 4-5 times to mix, centrifuge at 1900 rpm for 10 min, discard the supernatant, and then add cell preservation solution to restore the original volume of the sample to 4 ml to obtain a blood cell sample;

[0076] S3. Take 4 μl of EpCAM (4.5 μm) magnetic beads and 4 μl of CSV (1 μm) magnetic beads and add them to the above blood cell samples. Place the sample tube on a biological mixer, tilt it at 15°, and incubate it at 6 rpm for 30 min.

[0077] S4. The incubated blood cell sample is transferred to an anti-cell adhesion six-well plate, the six-well plate is placed in a cell sorter for magnetic separation, and the "capture" program is selected. After three collection, washing, and release operations, the A549 cells adhered to the antibody magnetic beads in the blood cell sample are enriched and separated into the release tank;

[0078] S5. Prepare a staining solution kit for blood cell analysis, and perform liquid phase immunostaining on the enriched and separated target cells;

[0079] S6. Aspirate the buffer solution from the edge of the release groove of the six-well plate, use a pipette to draw 100 μl of cell fixative solution into the release groove, let it stand at room temperature for 10 minutes, and aspirate the cell fixative solution from the edge of the release groove;

[0080] S7. Preparation of immunofluorescence staining solution: add 68 μl of cell preservation solution to a 1.5 ml EP tube, use a pipette to take 20 μl of cytokeratin antibody, 2 μl of CD45 antibody, 5 μl of AF488 fluorescent antibody, and 5 μl of AF568 fluorescent antibody, respectively, and add them to the EP tube, and shake to mix;

[0081] S8. Add the above immunofluorescence staining solution into the release tank of the six-well plate, place the six-well plate on a horizontal shaker, mix and incubate at 50 rpm for 30 min;

[0082] S9. Use a pipette to draw 100 μl of DAPI solution into the release tank of the six-well plate, place the six-well plate on a horizontal shaker, mix and incubate at 50 rpm for 5 min;

[0083] S10. Aspirate the staining solution from the edge of the six-well plate release groove and add 1 ml of cell resuspension solution for easy microscopic observation;

[0084] S11. Observe the stained target cells using an inverted fluorescence microscope.

[0085] In Figure 3, BF represents the detection results of antibody magnetic beads combined with lung cancer cells under a fluorescence microscope, DAPI represents the detection results of cell nuclei stained with DAPI under a fluorescence microscope (cell nuclei are blue in the figure), CK represents the detection results of cytokeratin stained with CK antibody under a fluorescence microscope (cells are green in the figure), CD45 represents the results of detecting white blood cell markers under a fluorescence microscope, and Merge represents a picture obtained by superimposing four pictures marked with EpCAM, DAPI, CK, and CD45.

[0086] As shown in Figure 3-A, after immunofluorescence staining, EpCAM (4.5 μm) magnetic beads were observed to be specifically adhered to the cell surface under bright field, with blue fluorescence (cell nucleus stained by DAPI), green fluorescence (keratin stained by CK), and no red fluorescence (leukocytes identified by CD45), indicating that the detected cells were epithelial tumor cells.

[0087] As shown in Figure 3-B, after immunofluorescence staining, specific adhesion of EpCAM (4.5 μm) magnetic beads and CSV (1 μm) magnetic beads to the cell surface was visible under bright field, with blue fluorescence (cell nucleus stained by DAPI), green fluorescence (keratin stained by CK), and no red fluorescence (leukocytes identified by CD45), indicating that the detected cells were epithelial-stromal mixed tumor cells.

[0088] As shown in Figure 3-C, after immunofluorescence staining, CSV 1μm magnetic beads were specifically adhered to the cell surface under bright field, with blue fluorescence (cell nucleus stained by DAPI), green fluorescence (keratin stained by CK), and no red fluorescence (leukocytes identified by CD45), indicating that the detected cells were mesenchymal tumor cells.

[0089] Example 5

[0090] This embodiment provides a magnetic bead staining method, which performs immunofluorescence staining on vimentin antibodies on CSV magnetic beads, so that the magnetic beads appear yellow under the fluorescent channel, which is convenient for further identification of small magnetic beads, and specifically includes the following steps:

[0091] S1. Prepare magnetic bead staining solution: add 97 μl of cell preservation solution to a 1.5 ml EP tube, use a pipette to take 2 μl of vimentin antibody (200 μg / ml) and 1 μl of AF647 fluorescent antibody (2 mg / ml) and add them to the EP tube, and shake to mix;

[0092] S2. Take 4 μl of CSV magnetic beads (particle size 1 μm) and add them to the above EP tube, shake and mix, and stain for 1 hour in the dark;

[0093] S3. Place the EP tube on the magnetic rack, let it stand for 1 min, and discard the staining solution;

[0094] S4. Remove the EP tube, add 1 ml of cell resuspension solution, invert 4 to 5 times to wash the magnetic beads, then place it on the magnetic rack to remove the supernatant, and resuspend the magnetic beads with 100 μl of cell resuspension solution;

[0095] S5. A549 non-small cell lung cancer cells were digested with trypsin to prepare a cell suspension, which was diluted to 10 cells / μl for later use;

[0096] S6. Take 50 μl of cell suspension (containing about 500 A549 cells) and add it to 1 ml of cell resuspension solution, invert and mix to prepare the cell sample to be tested;

[0097] S7. Add the fluorescent CSV magnetic beads prepared in step S4 to the above cell sample, place the sample tube on a biological mixer, tilt at 15°C, and incubate at 6 rpm for 60 min;

[0098] S8. After the incubation is completed, take an appropriate amount of cell solution and add it to the six-well plate, and discard the liquid in the magnetic field;

[0099] S9. Preparation of immunofluorescence staining solution: add 68 μl of cell preservation solution to a 1.5 ml EP tube, use a pipette to take 20 μl of cytokeratin antibody, 2 μl of CD45 antibody, 5 μl of AF488 fluorescent antibody, and 5 μl of AF568 fluorescent antibody, respectively, and add them to the EP tube, and shake to mix;

[0100] S10. Add the above immunofluorescence staining solution to the six-well plate in step S8, place the six-well plate on a horizontal shaker, mix and incubate at 50 rpm for 30 min;

[0101] S11. Use a pipette to draw 100 μl of DAPI solution into the release tank of the six-well plate, place the six-well plate on a horizontal shaker, mix and incubate at 50 rpm for 5 min;

[0102] S12. Aspirate the staining solution from the edge of the six-well plate release groove and add 1 ml of cell resuspension solution for easy microscopic observation;

[0103] S13. Observe the stained A549 cells under an inverted fluorescence microscope.

[0104] Figure 4In the figure, BF represents the detection result of antibody magnetic beads combined with A549 cells under a fluorescence microscope, DAPI represents the detection result of cell nuclei stained with DAPI under a fluorescence microscope (cell nuclei are blue in the figure), CK represents the detection result of cytokeratin stained with CK antibody under a fluorescence microscope (cells are green in the figure), CD45 represents the result of detecting white blood cell markers under a fluorescence microscope, CSV magnetic bead fluorescence represents the detection result of CSV magnetic beads under a fluorescence microscope, and Merge represents the image obtained by superimposing four images marked with EpCAM, DAPI, CK, and CD45.

[0105] like Figure 4 As shown, after immunofluorescence staining, CSV magnetic beads (1 μm) can be seen to be specifically adhered to the cell surface under bright field, with blue fluorescence (cell nucleus stained by DAPI), green fluorescence (keratin stained by CK), no red fluorescence (leukocytes recognized by CD45), and CSV magnetic beads showing yellow fluorescence, indicating that the detected cells are mesenchymal tumor cells.

Claims

1. A method for enriching and identifying tumor cells of different phenotypes based on dual-antibody dual-size magnetic beads, characterized in that: The following steps are involved: (1) preparing magnetic beads that bind epithelial cell-specific cell adhesion molecules and magnetic beads that bind mesenchymal cell-specific vimentin, wherein the two magnetic beads have different particle sizes; (2) The magnetic beads binding to epithelial cell adhesion molecules and the magnetic beads binding to vimentin are uniformly mixed with a sample containing tumor cells, wherein the magnetic beads specifically capture tumor cells of different phenotypes, and the captured target cells are separated by using a magnetic field in a cell sorter; (3) Under a microscope, identify the phenotypic type of the captured target cells based on the size of the magnetic beads attached to them under bright field light or fluorescence.

2. The method according to claim 1, characterized in that Magnetic beads bound to epithelial cell adhesion molecules specifically capture epithelial tumor cells, magnetic beads bound to vimentin specifically capture mesenchymal tumor cells, and mixed tumor cells bind to magnetic beads of both particle sizes at the same time.

3. The use according to claim 1, characterized in that: The particle size of large magnetic beads is preferably 3μm to 5μm, and the particle size of small magnetic beads is preferably 300nm to 1μm.

4. The method according to claim 1, characterized in that The captured target cells were further analyzed for markers using immunofluorescence staining.

5. The method according to claim 1, characterized in that The tumor cells are derived from blood, urine, saliva, pleural effusion, abdominal effusion, pulmonary effusion or stool dilution.

Images