A method for extracting tumor cells from urine

Abstract

The application provides a urine tumor cell extraction method and belongs to the technical field of biological detection. In the application, cells are first separated from a urine sample to prepare a cell precipitate into a cell suspension, then EpCAM antibody magnetic beads are added to the cell suspension, so that tumor cells are combined with the EpCAM antibody magnetic beads and are stably in a cell culture plate under the action of a magnetic field, thereby separating the tumor cells from the urine; finally, tumor cells are identified through a fluorescence in situ hybridization method, and immunotherapy drugs are guided through tumor cell immunofluorescence staining. The method is not affected by red blood cells, white blood cells, dead cell fragments, tubules and crystals and other factors in the urine, and can specifically recognize and capture epithelial cells in the urine through the EpCAM antibody magnetic beads, so that the separation is convenient, the purification degree is high, the cell morphology is complete, and false positives or false negatives caused by cell missed detection can be avoided.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a method for extracting tumor cells from urine. Background Technology

[0002] Bladder cancer is the most common malignant tumor of the urinary system, with a 5-year recurrence risk of over 50%. Traditional bladder cancer screening methods include ultrasound / CT scans, cystoscopy, and urine cytology. However, imaging techniques often struggle to detect tumors smaller than 5mm. Early-stage cancer patients often experience no symptoms or only mild symptoms, leading to neglect. In the early stages, even small lesions can shed a significant number of cancer cells into the urine. Using exfoliative cytology to detect cancer cells allows for early detection and diagnosis. Compared to cystoscopy, urine cytology is painless, non-invasive, and allows for repeated sampling. Its detection rate is over 60%, and the collected cells represent a wide range of mucosal exfoliated cells, including cancer cells from the renal pelvis, ureter, and bladder, all of which can be detected in urine cytology smears, making it widely used. However, urine cytology also has some drawbacks, such as a 10-40% false negative rate and the occasional misdiagnosis of atypical benign cells as malignant cells (with a 1-3% probability). Misdiagnosis occurs due to two main reasons: firstly, the limitations of cytological examination, which only examines single or small clusters of cells; and secondly, the greater difficulty in diagnosing exfoliative cytology, requiring re-examination by experienced physicians. The preparation and staining of urine exfoliative cytology smears are also complex procedures, demanding experience from the operator. Exfoliative cytology requires careful observation of cytoplasmic and nuclear morphological abnormalities. Examination of nuclear abnormalities involves examining the size, shape, chromatin distribution, whether the nuclear margin is thickened, and the regularity of the nuclear boundary. Furthermore, the significance of squamous cells differs between male and female patients. Urine exfoliative cytology lacks sensitivity in diagnosing low-grade tumors, and the results are easily affected by other symptoms such as inflammation, as well as subjective factors such as the experience of the examining physician. While urine exfoliative cytology can roughly determine the possible origin of tumor cells by analyzing the morphology of atypical cells, accurate diagnosis requires a comprehensive assessment in conjunction with imaging or biomarker results.

[0003] More than 95% of malignant tumors of the urinary tract originate from epithelial tissue. However, epithelial cells sloughed off from the urinary system are prone to degeneration or autolysis in urine, which poses certain difficulties for cytological diagnosis. Therefore, it is necessary to study methods that can rapidly separate tumor cells from urine. Summary of the Invention

[0004] To rapidly isolate epithelial cells from urine, this invention provides a method for extracting tumor cells from urine. By using specific antibody magnetic beads to selectively identify and bind to epithelial cells in urine, and then separating the magnetic beads, tumor cells can be isolated from urine. The tumor cells can then be identified by fluorescence in situ hybridization, and immunofluorescence staining can guide immunotherapy medication.

[0005] Specifically, the present invention adopts the following technical solution:

[0006] A method for extracting tumor cells from urine includes the following steps:

[0007] S1. Place a urine sample in a centrifuge tube and centrifuge at 2-8℃ and 1500-1900 rpm for 10-15 minutes. Discard the supernatant to obtain cell pellet.

[0008] S2. Resuspend the cell pellet in cell preservation solution, transfer the resulting cell suspension to an anticoagulant tube, add EpCAM antibody magnetic beads, mix well, and incubate at room temperature at 7-10 rpm for 30-60 min.

[0009] S3. After the cell sample incubated in step S2 is centrifuged briefly, it is transferred to a cell culture plate. The cell culture plate is placed under a magnetic field and stabilized for 20-30 minutes. The liquid is discarded, leaving the EpCAM antibody magnetic beads that have bound the tumor cells.

[0010] S4. Add cell fixation solution to the cell culture plate and fix for 10-15 minutes;

[0011] The method for extracting tumor cells from urine also includes the following steps:

[0012] S5-1. Discard the cell fixative, add the primary antibody mixture, mix well, and incubate at room temperature at 50-60 rpm for 30-60 min.

[0013] S6-1. Discard the liquid, add the secondary antibody mixture, mix well, and incubate at room temperature at 50-60 rpm for 30-60 min.

[0014] S7-1. Add staining reagent, mix well, and incubate at room temperature at 50-60 rpm for 3-5 min.

[0015] S8-1. After incubation, aspirate the staining solution, wash the magnetic beads with the first buffer solution, aspirate the liquid, and add the first buffer solution again to obtain the urine sample to be tested.

[0016] Alternatively, the method for extracting tumor cells from urine may further include the following steps:

[0017] S5-2. Discard the cell fixative; add the second buffer and soak at room temperature for 10-15 minutes; preferably, the second buffer is 2×SSC buffer.

[0018] S6-2. Remove the liquid and soak in water for 1-2 minutes; remove the water and soak in ethanol of 70%-100% gradient concentration for 1-2 minutes in sequence; remove the ethanol and air dry naturally.

[0019] S7-2. Add chromosome 7 probe reagent, place the cell culture plate at 82-83℃ for denaturation for 10-15 min, and then incubate at 50-52℃ for 2-4 h.

[0020] S8-2. Add staining reagent to the cell culture plate, let stand for 3-5 minutes, aspirate the staining solution, add the first buffer solution, and obtain the urine sample to be tested.

[0021] Alternatively, the method for extracting tumor cells from urine may further include the following steps:

[0022] S5-3. Discard the cell fixative, add the primary antibody mixture, mix well, and incubate at room temperature at 50-60 rpm for 30-60 min.

[0023] S6-3. Discard the liquid, add the secondary antibody mixture, mix well, and incubate at room temperature at 50-60 rpm for 30-60 min.

[0024] S7-3, Discard the liquid, add the second buffer solution and soak at room temperature for 10-15 minutes;

[0025] S8-3. Discard the liquid and soak in water for 1-2 minutes; discard the water and soak in ethanol of 70%-100% gradient concentration for 1-2 minutes in sequence; discard the ethanol and air dry naturally.

[0026] S9-3. Add chromosome 7 probe reagent, place the cell culture plate at 82-83℃ for denaturation for 10-15 min, and then incubate at 50-52℃ for 2-4 h.

[0027] S10-3. Add staining reagent to the cell culture plate, let stand for 3-5 minutes, aspirate the staining solution, add the first buffer solution, and obtain the urine sample to be tested.

[0028] Preferably, the first buffer is a 0.01 mol / L PBS buffer with pH = 7.4.

[0029] Preferably, the second buffer solution is a 2×SSC buffer solution.

[0030] Preferably, the staining reagent is DAPI.

[0031] The method for extracting tumor cells from urine in this invention utilizes EpCAM antibody magnetic beads to selectively recognize and bind to epithelial cells in urine. The cells maintain their intact morphology under liquid-phase conditions, and the magnetic beads binding the tumor cells are stabilized in the cell culture plate under a magnetic field, preventing loss. Immunofluorescence staining reagents and fluorescent probe reagents are then used to stain the cells with markers, allowing for observation of immunofluorescence and probe fluorescence under a fluorescence microscope to identify the cells and determine the purpose of immunotherapy.

[0032] The EpCAM antibody magnetic beads can be purchased commercially, for example, the EpCAM antibody magnetic beads are CD326 (EpCAM) magnetic beads, with specifications of EpCAM micron magnetic beads (1 mL, 4 × 10⁻⁶). 8 EpCAM nanobeads (1 mL, 1 mg / mL) or EpCAM nanobeads (1 mL, 1 mg / mL), preferably EpCAM microbeads.

[0033] In step S3, instantaneous centrifugation refers to centrifugation at a speed of 1000-1500 rpm for 5-10 seconds.

[0034] Preferably, in step S1, the cell pellet is prepared by centrifugation at 1900 rpm for 10 min.

[0035] Preferably, the method for discarding the supernatant in step S1 is to aspirate most of the supernatant with a Pasteur pipette and then aspirate the remaining supernatant with a pipette. When using a pipette to aspirate the supernatant, avoid touching the cell pellet with the pipette tip.

[0036] Preferably, the cell preservation solution in step S2 is 1×PBS buffer containing 0.1% to 0.5% BSA.

[0037] Preferably, in step S2, the amount of cell preservation solution used is 1 mL of the cell preservation solution per milligram of the cell precipitate, and if the cell precipitate is less than 1 mg, it is calculated as 1 mg.

[0038] Preferably, in step S2, the amount of EpCAM antibody magnetic beads used is 1 μL of the EpCAM antibody magnetic beads per milliliter of cell suspension.

[0039] Preferably, in step S2, the incubation is carried out at room temperature at a speed of 7 rpm for 30 min.

[0040] Preferably, the volume ratio of the cell fixative to the EpCAM antibody magnetic beads is 25:1.

[0041] Preferably, the cell fixative is 4% paraformaldehyde, and the fixation time is 10 min.

[0042] Preferably, the primary antibody mixture contains 20 v / v% cytokeratin antibody, 2 v / v% leukocyte common antigen antibody, and 2 v / v% programmed death ligand 1 antibody; preferably, the cytokeratin antibody is Pan-CK antibody, the leukocyte common antigen antibody is CD45 Ab-1 (Bra55 / 2), and the programmed death ligand 1 antibody is PD-L1 (Extracellular Domain Specific) (D8T4X) Rabbit mAB.

[0043] Preferably, after adding the primary antibody mixture in step S5-1 or S5-3, the mixture is incubated at 50 rpm for 60 min at room temperature.

[0044] Preferably, the secondary antibody mixture contains 0.25 v / v% red fluorescently labeled goat anti-mouse antibody, 0.25 v / v% green fluorescently labeled goat anti-mouse antibody, and 0.25 v / v% far-infrared fluorescently labeled goat anti-mouse antibody; preferably, the red fluorescently labeled goat anti-mouse antibody is Alexa. 568 goat anti-mouse IgG1, the green fluorescently labeled goat anti-mouse antibody is Alexa 488 anti-mouse IgG2a, the far-infrared fluorescently labeled goat anti-mouse antibody is Alexa 647-Goat Anti-Rabbit IgG.

[0045] Preferably, after adding the secondary antibody mixture in step S6-1 or S6-3, the mixture is incubated at 50 rpm for 30 min at room temperature.

[0046] Preferably, after adding the staining reagent in step S7-1, the mixture is incubated at 50 rpm for 5 min at room temperature.

[0047] The method for extracting tumor cells in urine provided by this invention can prepare circulating tumor cell test samples without invasiveness to the human body or relying on tissue biopsy samples, thereby characterizing tumor cells in urine. Therefore, it can be used for early screening of high-risk groups of cancer, efficacy evaluation of cancer patients, and recurrence monitoring after treatment.

[0048] Compared with the prior art, the beneficial effects of the present invention are:

[0049] (1) Compared with direct analysis of urine sediment, the method of the present invention is not affected by factors such as red blood cells, white blood cells, dead cell fragments, casts and crystals in urine. It uses EpCAM antibody magnetic beads to specifically identify and capture epithelial cells in urine, which is convenient to separate and highly purified, making the cell observation field clearer. Normal epithelial cells and epithelial tumor cells can be distinguished by fluorescent staining, without the need for judgment based on cell morphology recognition experience. For batch samples, it can greatly reduce the workload of detection and improve detection efficiency. Moreover, since the method of the present invention analyzes all epithelial cells in urine samples, it can avoid false positives or false negatives caused by missed cell detection, which is beneficial to the accuracy of the analysis results.

[0050] (2) Compared with the technique of staining and analyzing urine cells on a glass slide, the method of the present invention eliminates the steps of preparing cell smears and staining with organic solvents. After the instrument automatically captures epithelial cells and releases them into the cell culture plate, immunofluorescence staining with trace amounts of reagents can be performed directly on the cell culture plate.

[0051] (3) Compared with traditional urine exfoliative cytology analysis techniques, the method of the present invention has a high degree of automation, is easy to operate, and provides clear results analysis. It also has low requirements for the operating skills and cell analysis experience of laboratory physicians.

[0052] (4) Urine exfoliated cytology analysis is difficult to analyze all urinary epithelial cells due to interference from impurities. However, the method in this invention can fully enrich the epithelial cells in the urine sample and perform unified analysis.

[0053] (5) Compared with urine exfoliative cytology, the urine tumor cells in the method of the present invention are still in a single suspended state in the liquid after identification, which is easy to remove. The cells are intact and highly pure, and can be directly used for gene amplification and analysis, thereby determining the accurate source of solid tumors. It is especially suitable for early urinary tract tumors whose tumor source cannot be determined by imaging and other detection techniques. Attached Figure Description

[0054] Figure 1 This is a diagram showing the results of immunofluorescence staining analysis of epithelial cells isolated from the hematuria of a bladder cancer patient in Example 1 under liquid phase.

[0055] Figure 2 This is a diagram showing the results of immunofluorescence staining analysis of epithelial cells isolated from the urine of healthy individuals in a control experiment under liquid phase.

[0056] Figure 3 This is a graph showing the fluorescence in situ hybridization analysis results of epithelial cells isolated from the blood and urine of a bladder cancer patient in Example 2;

[0057] Figure 4This is a diagram showing the results of fluorescence in situ hybridization analysis of epithelial cells isolated from the urine of healthy individuals in Control Experiment 2;

[0058] Figure 5 This image shows the results of immunofluorescence staining and fluorescence in situ hybridization analysis of epithelial cells isolated from the blood and urine of a bladder cancer patient in Example 3. Detailed Implementation

[0059] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Any equivalent modifications or substitutions made by those skilled in the art based on the following embodiments are within the scope of protection of the present invention.

[0060] The patient in the following example is a low-grade urothelial carcinoma who presented with gross hematuria for one year. Color Doppler ultrasound revealed a slightly hyperechoic lesion in the bladder (possibly a space-occupying lesion, including one lesion measuring approximately 3.4 × 2.3 cm at the 8 o'clock position). 2 The object at the 6 o'clock position measures approximately 1.2 x 0.6 cm. 2 The patient was scheduled for transurethral resection of bladder tumor (TURP), followed by regular intravesical chemotherapy.

[0061] The main reagents or instruments used in the examples and control experiments are as follows:

[0062] Cell preservation solution: 1×PBS buffer containing 0.1%–0.5% BSA, wherein the percentage of BSA (bovine serum albumin) is by weight and volume.

[0063] EpCAM antibody magnetic bead solution: Place the EpCAM antibody magnetic bead reagent in a vortex mixer and mix thoroughly. Transfer 4 μL of this reagent to an EP tube and bring the volume to 100 μL with PBS buffer (pH 7.4). The EpCAM antibody magnetic bead reagent is CD326 (EpCAM) magnetic beads (micron-sized, 1 mL, 4 × 10⁻⁶). 8 (particles / mL), purchased from Yichang Meiguang Silicon Valley Life Science Co., Ltd.;

[0064] Circulating tumor cell separator: purchased from Yichang Meiguang Silicon Valley Life Science Co., Ltd.;

[0065] Cell fixative: 4% paraformaldehyde, purchased from Solarbio Science & Technology Co., Ltd.

[0066] Primary antibody mixture: Take 20 μL of Pan-CK antibody, 2 μL of CD45 Ab-1 (Bra55 / 2), and 2 μL of PD-L1 (Extracellular Domain Specific) (D8T4X) Rabbit mAB and add them to a 1.5 mL EP tube. Add 1×PBS buffer to bring the volume to 100 μL.

[0067] Secondary antibody mixture: Take 0.25 μL of each Alexa. 568 goat anti-mouse IgG1, 0.25μL Alexa 488 anti-mouse IgG2a, 0.25μL Alexa Add 647-Goat Anti-Rabbit IgG to a 1.5 mL EP tube and bring the volume to 100 μL with 1×PBS buffer;

[0068] Chromosome 7 probe reagent (CSP7): purchased from Xiamen Longjin Biotechnology Co., Ltd., specification: 50μL / vial.

[0069] Example 1

[0070] This embodiment provides a method for extracting urine epithelial cells, specifically including the following steps:

[0071] S1. With the patient's informed consent and the consent of the hospital's ethics committee, collect at least 30 mL of the patient's morning urine (hematuria) (patients with difficulty urinating may receive less than 30 mL; as long as there is urine sediment after centrifugation, it can be tested) and place it in a 50 mL centrifuge tube. Store it at 2–8°C and test it within 4 hours. Place the urine sample in a benchtop centrifuge and centrifuge at 1900 rpm for 10 minutes to precipitate blood cells. After centrifugation, carefully remove the centrifuge tube. Use a Pasteur pipette to aspirate most of the supernatant, and then use a pipette to carefully aspirate the supernatant until it is close to the cell layer to obtain blood cell sediment. Avoid touching the cell layer with the pipette tip when aspirating the liquid.

[0072] S2. Add cell preservation solution to the centrifuge tube to a final volume of 3 mL to resuspend the blood cell pellet, and transfer the resulting cell suspension (i.e., blood cell sample) to a 5 mL EDTA anticoagulant tube; wash the centrifuge tube with 1 mL of cell preservation solution, and transfer the washing solution to the EDTA anticoagulant tube; add 4 μL of LEpCAM antibody magnetic bead solution to the EDTA anticoagulant tube, tighten the stopper, gently invert 4-5 times, place on a 3D shaker and mix slowly at 7 rpm, incubate at room temperature for 30 min;

[0073] S3. After the blood cell sample incubated in step S2 is briefly centrifuged (e.g., at 1000 rpm for 5 seconds), immediately transfer it to a six-well cell culture plate. Then, wash the EDTA anticoagulant tube twice with PBS buffer (pH 7.4), and transfer the washing solution into the six-well cell culture plate. Place the six-well cell culture plate smoothly into the circulating tumor cell separator. Turn on the power and open the working chamber of the circulating tumor cell separator, add the magnetic head cap, and run the "capture" program (e.g., run for 20–30 minutes). After the instrument finishes running, carefully aspirate the PBS buffer from the six-well cell culture plate, leaving the EpCAM antibody beads that have bound tumor cells.

[0074] S4. Take out the six-well cell culture plate from the circulating tumor cell separator, aspirate 100 μL of cell fixation solution into the six-well cell culture plate, and fix at room temperature for 10-15 min;

[0075] The extracted tumor cells were subjected to immunofluorescence staining analysis, which specifically included the following steps:

[0076] S5-1. Discard the fixative, add 100 μL of primary antibody mixture to a six-well cell culture plate, mix slowly at 50 rpm on a horizontal shaker, and incubate at room temperature for 60 min; discard the primary antibody mixture, slowly add 1 mL of 1×PBS buffer (pH 7.4), and soak for 1 min;

[0077] S6-1. Discard the liquid, add 100 μL of secondary antibody mixture to a six-well cell culture plate, mix slowly at 50 rpm on a horizontal shaker, and incubate at room temperature for 30 min.

[0078] S7-1. Add 100 μL of DAPI (4',6-diamidinyl-2-phenylindole) to a six-well cell culture plate, mix slowly at 50 rpm on a horizontal shaker, and incubate at room temperature for 5 min.

[0079] After incubation in steps S8-1 and S7-1, aspirate the staining solution and slowly add 1 mL of PBS buffer (pH=7.4) along the well wall of the six-well cell culture plate, and soak for 1 min; aspirate the liquid and slowly add 1 mL of PBS buffer (pH=7.4) along the well wall of the six-well cell culture plate to obtain the urine sample to be tested.

[0080] S9-1. Immediately transfer the six-well cell culture plate to a fluorescence microscope for identification. Analyze the results by combining the fluorescence signals of the cell membrane and nucleus with the bright-field cell morphology (i.e., cell integrity and magnetic bead binding status). The analysis results are as follows: Figure 1 As shown.

[0081] Figure 1This image shows the immunofluorescence staining analysis of epithelial cells isolated from the hematuria of bladder cancer patients according to the method of the present invention in liquid phase. Thirty epithelial cells were detected from 30 mL of urine (9 cells were PD-L1 positive and 21 cells were PD-L1 negative). Figure 1 In the image, EpCAM represents the detection results of EpCAM antibody magnetic beads bound to tumor cells under a fluorescence microscope; DAPI represents the detection results of DAPI-stained cell nuclei under a fluorescence microscope (cell nuclei appear blue in the image); CK represents the detection results of CK-stained keratin under a fluorescence microscope (cells appear green in the image); CD45 represents the detection results of extracted tumor cells as white blood cells under a fluorescence microscope; PD-L1 represents the detection results of extracted tumor cells as PD-L1 under a fluorescence microscope (cells appear magenta in the image); and Merge represents the superimposed image of four images labeled DAPI, CK, CD45, and PD-L1. Image A shows the results of PD-L1 positive expression in urinary epithelial cells, and image B shows the results of PD-L1 negative expression in urinary epithelial cells. Figure 1 As shown in Figure A, after immunofluorescence staining under bright field, magnetic beads specifically adhered to the cell surface, exhibiting blue fluorescence (DAPI-stained cell nuclei), green fluorescence (CK-stained keratin), and magenta fluorescence (PD-L1, a tumor immunotherapy marker), but no red fluorescence (CD45 recognizing leukocytes). This indicates that the extracted cells were specifically labeled with magnetic beads, possessed nuclei, and were intact cells; they expressed tumor immunotherapy markers but not leukocyte markers. Specifically, under bright field light, EpCAM antibody magnetic beads recognizing epithelial cells were visible adhering to the cell surface of urinary epithelial cells. Under DAPI fluorescence, cell nuclei were visible, CK keratin staining was positive, CD45 was negative, and PD-L1 was positive, indicating that the extracted cells contained PD-L1-expressing epithelial cells.

[0082] Control Experiment 1

[0083] This experiment followed the method described in Example 1 to extract epithelial cells from the urine of healthy individuals, specifically including the following steps:

[0084] S1. Collect at least 30 mL of morning urine from healthy subjects and place it in a 50 mL centrifuge tube. Store the urine at 2–8°C and test within 4 hours. Place the urine sample in a benchtop centrifuge and centrifuge at 1900 rpm for 10 min to precipitate and remove cells. After centrifugation, carefully remove the centrifuge tube. Use a Pasteur pipette to aspirate most of the supernatant, and then use a pipette to carefully aspirate the supernatant until it is close to the cell layer to obtain cell precipitate. Avoid touching the cell layer with the pipette tip when aspirating the liquid.

[0085] S2. Add 3 mL of cell preservation solution to the centrifuge tube to resuspend the cell pellet. Transfer the resulting cell suspension to a 5 mL EDTA anticoagulant tube. Wash the centrifuge tube with 1 mL of cell preservation solution and transfer the washing solution to the EDTA anticoagulant tube. Add 4 μL of LEpCAM antibody magnetic bead solution to the cell suspension, cap the tube, gently invert it 4-5 times, place it on a 3D shaker and mix slowly at 7 rpm. Incubate at room temperature for 30 min.

[0086] S3. After briefly centrifuging the cell suspension from step S2 (e.g., at 1000 rpm for 5 seconds), immediately transfer it to a six-well cell culture plate. Then, wash the EP tubes twice with PBS buffer (pH 7.4), transferring the washing solution into the six-well cell culture plate. Place the six-well cell culture plate smoothly into the circulating tumor cell separator. Turn on the power and open the working chamber of the circulating tumor cell separator, add the magnetic head cap, and run the "capture" program. After the instrument finishes running, carefully aspirate the PBS buffer from the six-well cell culture plate, leaving the EpCAM antibody beads bound to epithelial cells.

[0087] S4. Take out the six-well cell culture plate from the circulating tumor cell separator, aspirate 100 μL of cell fixation solution into the six-well cell culture plate, and fix at room temperature for 10-15 min;

[0088] The obtained epithelial cells were subjected to immunofluorescence staining analysis, specifically including the following steps:

[0089] S5-1. Discard the cell fixative, add 100 μL of primary antibody mixture to a six-well cell culture plate, mix slowly at 50 rpm on a horizontal shaker, and incubate at room temperature for 60 min; discard the primary antibody mixture, slowly add 1 mL of PBS buffer (pH 7.4), and soak for 1 min;

[0090] S6-1. Discard the liquid, add 100 μL of secondary antibody mixture to a six-well cell culture plate, mix slowly at 50 rpm on a horizontal shaker, and incubate at room temperature for 30 min.

[0091] S7-1. Add 100 μL of DAPI (4',6-diamidinyl-2-phenylindole) to a six-well cell culture plate, mix slowly at 50 rpm on a horizontal shaker, and incubate at room temperature for 5 min.

[0092] After incubation in steps S8-1 and S7-1, aspirate the staining solution and slowly add 1 mL of PBS buffer (pH=7.4) along the well wall of the six-well cell culture plate, and soak for 1 min; aspirate the liquid and then slowly add 1 mL of PBS buffer (pH=7.4) along the well wall of the six-well cell culture plate.

[0093] S9-1. Immediately transfer the six-well cell culture plate to a fluorescence microscope for identification. Analyze the results by combining the fluorescence signals of the cell membrane and nucleus with the bright-field cell morphology (i.e., cell integrity and magnetic bead binding status). The analysis results are as follows: Figure 2 As shown.

[0094] Figure 2 This image shows the immunofluorescence analysis of epithelial cells isolated from the urine of healthy individuals in a control experiment under liquid phase. Figure 2 In the image, EpCAM represents the detection results of EpCAM antibody magnetic beads bound to epithelial cells under a fluorescence microscope; DAPI represents the detection results of DAPI-stained cell nuclei under a fluorescence microscope (cell nuclei are shown in blue in the image); CK represents the detection results of CK-stained keratin under a fluorescence microscope (cells are shown in green in the image); CD45 represents the results of leukocyte detection in extracted cells under a fluorescence microscope; PD-L1 represents the results of PD-L1 detection in extracted cells under a fluorescence microscope; and Merge represents the superimposed image of the four images labeled DAPI, CK, CD45, and PD-L1. Figure 2 As can be seen from the immunofluorescence staining, under bright field, the magnetic beads specifically adhered to the cell surface, exhibiting blue fluorescence (DAPI-stained cell nuclei), green fluorescence (CK-stained keratin), no magenta fluorescence (PD-L1 is a tumor immunotherapy marker), and no red fluorescence (CD45 recognizes leukocytes). This indicates that the extracted cells were specifically labeled with magnetic beads, had cell nuclei, and were intact cells; and there was no expression of tumor cell immunotherapy markers or leukocyte markers.

[0095] Example 2

[0096] This embodiment provides a method for extracting urine epithelial cells, specifically including the following steps:

[0097] Steps S1 to S4 are the same as in Example 1. The difference is that the rotation speed and centrifugation time in step S1 are adjusted appropriately according to the actual experimental conditions, with centrifugation time of 10 to 1500 rpm for 15 min. In step S2, the shaking speed and incubation time are adjusted appropriately according to the actual experimental conditions, with incubation time of 30 to 60 min at 7 to 10 rpm for 30 min.

[0098] S5-2. Discard the cell fixative, add 2 mL of 2×SSC (sodium citrate) solution and soak at room temperature for 10 min; discard the liquid, add 2 mL of pure water and soak for 2 min;

[0099] S6-2. Discard the pure water, add 2 mL of 70% ethanol solution, and soak for 2 min; discard the liquid, add 2 mL of 90% ethanol solution, and soak for 2 min; discard the liquid, add anhydrous ethanol, and soak for 2 min; discard the anhydrous ethanol, and air dry naturally.

[0100] S7-2, Add 10 μL of chromosome probe reagent No. 7 (CSP7), cover with a coverslip; seal the six-well cell culture plate in a waterproof self-sealing bag, immerse it in a water bath, denature it in an 82-83℃ water bath for 10-15 min, and then transfer it to a 50-52℃ water bath for immersion and incubation for 2-4 h.

[0101] S8-2. Add pure water to a six-well cell culture plate and soak for 1 minute to allow the coverslip to float. Remove the coverslip. Discard the pure water, add DAPI staining solution, let stand for 3-5 minutes, discard the staining solution, add 1×PBS buffer, and obtain the urine sample to be tested.

[0102] S9-2. Immediately transfer the six-well cell culture plate to a fluorescence microscope and analyze the results by combining nuclear fluorescence signals and bright-field cell morphology (i.e., cell integrity and magnetic bead binding status). The analysis results are as follows: Figure 3 As shown.

[0103] Figure 3 This image shows the results of fluorescence in situ hybridization analysis of epithelial cells isolated from the blood and urine of a bladder cancer patient according to the method described in this embodiment. Twenty-two tumor cells were detected from 30 mL of urine. Figure 3 In the image, EpCAM represents the detection results of EpCAM antibody magnetic beads bound to tumor cells under a fluorescence microscope; DAPI represents the detection results of cell nuclei stained with DAPI under a fluorescence microscope (cell nuclei are shown in blue in the image); CSP7 represents the detection results of chromosome 7 number under a fluorescence microscope (chromosomes in cell nuclei are shown in green in the image); and Merge represents the image obtained by overlaying two images labeled with DAPI and CSP7. Figure 3 As shown, after fluorescence in situ hybridization staining, under bright field light, magnetic beads specifically adhered to the cell surface, exhibiting blue fluorescence (DAPI-stained cell nuclei) and green fluorescent dots (more than 2) or clusters (CSP7-stained chromosomes). This indicates that the extracted cells were specifically labeled with EpCAM antibody magnetic beads, possessed nuclei, and were intact cells; furthermore, they showed abnormal polyploidy of chromosome 7. In other words, under bright field light, EpCAM antibody magnetic beads recognizing epithelial cells were visible adhering to the cell surface of urinary epithelial cells, and the cell nuclei were visible under DAPI fluorescence. The abnormal polyploidy of chromosome CSP7 indicated that the extracted cells were tumor cells.

[0104] Control Experiment 2

[0105] This control experiment followed the methods in steps S1-S4 of Control Experiment 1 and steps S5-2-S9-2 of Example 2, performing fluorescence in situ hybridization analysis on epithelial cells extracted from the urine of healthy individuals. The analysis results are as follows: Figure 4 As shown.

[0106] Figure 4 This image shows the results of fluorescence in situ hybridization analysis of epithelial cells isolated and extracted from the urine of healthy individuals according to the method of this invention. Figure 4 In the image, EpCAM represents the detection results of EpCAM antibody magnetic beads bound to epithelial cells under a fluorescence microscope; DAPI represents the detection results of cell nuclei stained with DAPI under a fluorescence microscope (cell nuclei are shown in blue in the image); CSP7 represents the detection results of chromosome 7 number under a fluorescence microscope (chromosomes in cell nuclei are shown in green in the image); and Merge represents the image obtained by overlaying two images labeled with DAPI and CSP7. Figure 4 As can be seen from the fluorescence in situ hybridization staining analysis, under bright field, magnetic beads specifically adhered to the cell surface, exhibiting blue fluorescence (DAPI-stained cell nuclei) and two green fluorescent dots (CSP7-stained chromosomes), indicating that the extracted cells were specifically labeled by magnetic beads, had cell nuclei, and were intact cells; moreover, chromosome 7 was a normal diploid, indicating that the extracted cells were normal healthy cells.

[0107] Example 3

[0108] This embodiment provides a method for extracting urine epithelial cells, specifically including the following steps:

[0109] Steps S1 to S4 are the same as in Example 1;

[0110] S5-3. Discard the fixative, add 100 μL of primary antibody mixture to a six-well cell culture plate, mix slowly at 50 rpm on a horizontal shaker, and incubate at room temperature for 60 min; discard the primary antibody mixture, slowly add 1 mL of PBS buffer (pH 7.4), and soak for 1 min;

[0111] S6-3. Discard the liquid, add 100μL of secondary antibody mixture to a six-well cell culture plate, mix slowly at 50rpm on a horizontal shaker, and incubate at room temperature for 30min.

[0112] S7-3. Discard the liquid, add 2 mL of 2×SSC (sodium citrate) solution and soak at room temperature for 10 min; discard the liquid, add 2 mL of pure water and soak for 2 min;

[0113] S8-3. Discard the pure water, add 2 mL of 70% ethanol solution, and soak for 2 min; discard the liquid, add 2 mL of 90% ethanol solution, and soak for 2 min; discard the liquid, add anhydrous ethanol, and soak for 2 min; discard the anhydrous ethanol, and air dry naturally.

[0114] S9-3. Add 10 μL of chromosome probe reagent No. 7 (CSP7), cover with a coverslip; seal the six-well cell culture plate in a waterproof self-sealing bag, immerse it in a water bath, denature it in an 82℃ water bath for 10 min, and then transfer it to a 51℃ water bath for 2 h of immersion and incubation.

[0115] S10-3. Add pure water to a six-well cell culture plate and soak for 1 minute to allow the coverslip to float. Remove the coverslip. Discard the pure water, add DAPI staining solution, let stand for 3-5 minutes, discard the staining solution, add 1×PBS buffer, and obtain the urine sample to be tested.

[0116] S11-3. Immediately transfer the six-well cell culture plate to a fluorescence microscope and analyze the results by combining nuclear fluorescence signals and bright-field cell morphology (i.e., cell integrity and magnetic bead binding status). The analysis results are as follows: Figure 5 As shown.

[0117] Figure 5 This image shows the results of immunofluorescence staining and fluorescence in situ hybridization analysis of epithelial cells isolated from the blood and urine of bladder cancer patients according to the method described in this embodiment. Twenty-two tumor cells were detected from 30 mL of urine, of which 9 cells were PD-L1 positive and 13 cells were PD-L1 negative. Figure 5 In the image, EpCAM represents the detection results of EpCAM antibody magnetic beads bound to tumor cells under a fluorescence microscope; DAPI represents the detection results of cell nuclei stained with DAPI under a fluorescence microscope (cell nuclei appear blue in the image); CSP7 represents the detection results of chromosome 7 number under a fluorescence microscope (chromosomes in cell nuclei appear green in the image); PD-L1 represents the detection results of PD-L1 in extracted epithelial cells under a fluorescence microscope (cells appear magenta in the image); and Merge represents the superimposed image of the three images labeled DAPI, CSP7, and PD-L1. Figure 5 As shown in Figure A, after immunofluorescence staining and fluorescence in situ hybridization, magnetic beads specifically adhered to the cell surface were visible under bright field. These beads exhibited blue fluorescence (DAPI-stained cell nuclei) and green fluorescent dots (more than two) or clusters (CSP7-stained chromosomes), and magenta fluorescence (PD-L1 is a biomarker for tumor immunotherapy). This indicates that the extracted cells were specifically labeled with EpCAM antibody magnetic beads, possessed nuclei, were intact cells, exhibited abnormal polyploidy of chromosome 7, and were positive for PD-L1 expression. Figure 5As shown in Figure B, after immunofluorescence staining and fluorescence in situ hybridization, specific magnetic beads adhered to the cell surface under bright field light. These beads exhibit blue fluorescence (DAPI-stained cell nuclei) and green fluorescent dots (more than two) or clusters (CSP7-stained chromosomes), but no magenta fluorescence (PD-L1 is a biomarker for tumor immunotherapy). This indicates that the extracted cells were specifically labeled with EpCAM antibody beads, possessed nuclei, were intact cells, exhibited abnormal polyploidy of chromosome 7, and were PD-L1 negative. In other words, under bright field light, EpCAM antibody beads recognizing epithelial cells were visible adhering to the cell surface. Under DAPI fluorescence, cell nuclei were visible, the CSP7 chromosome showed abnormal polyploidy, and PD-L1 expression may be positive or negative, indicating that the extracted epithelial cells may or may not be sensitive to immunosuppressants.

[0118] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Various modifications and variations can be made to the present invention by any person skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for extracting tumor cells from urine, characterized in that, Includes the following steps: S1. Place a urine sample in a centrifuge tube and centrifuge at 2-8℃ and 1500-1900rpm for 10-15 minutes. Discard the supernatant to obtain cell pellet. S2. Resuspend the cell pellet in cell preservation solution, transfer the resulting cell suspension to an anticoagulant tube, add EpCAM antibody magnetic beads, mix well, and incubate at room temperature at 7-10 rpm for 30-60 min. S3. After the cell sample incubated in step S2 is centrifuged briefly, it is transferred to a cell culture plate. The cell culture plate is placed under a magnetic field and stabilized for 20-30 minutes. The liquid is then discarded. S4. Add cell fixation solution to the cell culture plate and fix for 10-15 minutes; S5-1. Discard the cell fixative, add the primary antibody mixture, mix well, and incubate at room temperature at 50-60 rpm for 30-60 min; the primary antibody mixture contains 20 v / v% cytokeratin antibody, 2 v / v% leukocyte common antigen antibody, and 2 v / v% programmed death ligand 1 antibody. S6-1. Discard the liquid, add the secondary antibody mixture, mix well, and incubate at 50-60 rpm for 30-60 min at room temperature; the secondary antibody mixture contains 0.25 v / v% red fluorescently labeled goat anti-mouse antibody, 0.25 v / v% green fluorescently labeled goat anti-mouse antibody, and 0.25 v / v% far-infrared fluorescently labeled goat anti-mouse antibody. S7-1. Add staining reagent, mix well, and incubate at room temperature at 50-60 rpm for 3-5 min. S8-1. After incubation, aspirate the staining solution, wash the magnetic beads with the first buffer solution, aspirate the liquid, and add the first buffer solution again to obtain the urine sample to be tested.

2. The method for extracting tumor cells from urine according to claim 1, characterized in that, In step S2, the cell preservation solution is a 1×PBS buffer containing 0.1%~0.5% BSA.

3. The method for extracting tumor cells from urine according to claim 1, characterized in that, In step S2, the amount of cell preservation solution used is 1 mL of cell preservation solution per milligram of cell precipitate, and if the cell precipitate is less than 1 mg, it is calculated as 1 mg.

4. The method for extracting tumor cells from urine according to claim 1, characterized in that, In step S2, the amount of EpCAM antibody magnetic beads used is 1 μL of EpCAM antibody magnetic beads per milliliter of cell suspension.

5. The method for extracting tumor cells from urine according to claim 1, characterized in that, The volume ratio of the cell fixative to the EpCAM antibody magnetic beads is 25:1.

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