Prostate cancer AR-V7 positive circulating tumor cell detection method

Abstract

The invention provides a prostate cancer AR-V7 positive circulating tumor cell detection method, and relates to the technical field of medical examination, the detection method can detect AR-V7 positive circulating tumor cells in the early stage of prostate cancer, breaks through the limitation that tumor is difficult to find in the early stage in a traditional diagnosis method, and improves the detection accuracy of the prostate cancer AR-V7 positive circulating tumor cells. Prostate cancer cells and normal cells can be effectively distinguished through joint detection and accurate interpretation of multiple markers, the risk of misdiagnosis is reduced, a more reliable basis is provided for clinical diagnosis, the expression condition of AR-V7 is accurately detected, and the kit has the advantages of being high in sensitivity, high in accuracy and high in reliability. According to the prostate cancer treatment system, medication guidance is provided for the treatment process of a prostate cancer patient, a doctor is helped to formulate a personalized treatment scheme, the treatment effect is improved, only a blood sample of the patient needs to be collected for detection, pain and risks caused by invasive operation such as traditional biopsy are avoided, and the patient acceptability is higher.

Description

Technical Field

[0001] The present invention belongs to the field of medical testing technology, and more specifically, particularly relates to a method for detecting AR-V7 positive circulating tumor cells of prostate cancer. Background Art

[0002] Prostate cancer (PCa) is common in middle-aged and elderly men and is currently one of the most common malignant tumors of the urinary system. According to the 2020 World Cancer Report, prostate cancer ranks sixth in the incidence of male malignant tumors. With the aging of the population, changes in living habits, and the increase in the detection rate of prostate cancer, the incidence of prostate cancer in my country is increasing year by year. In 2022, the number of new cases of prostate cancer in my country was 134,200, and the number of deaths was 47,500. Studies have found that most patients have already spread at the time of initial diagnosis, and the prognosis is extremely poor. About 79% of prostate malignancies are diagnosed at the local stage, 12% of patients are diagnosed after regional lymph node invasion, and another 5% are diagnosed after local metastasis. In patients with local or local tumors, the probability of five-year survival is about 100%. However, distant metastasis can lead to a significant drop in the patient's five-year survival rate to about 30%. Risk factors for prostate cancer include genetic susceptibility, family history of prostate or breast malignancies, and age. The median age of patients at diagnosis is 72 years old. Therefore, early diagnosis of prostate cancer and timely intervention and treatment play an important role in preventing its occurrence and development. For early screening and diagnosis of prostate cancer, blood biomarker detection, digital rectal examination (DRE) and prostate biopsy are the main detection methods. At present, prostate-specific antigen (PSA) detection occupies a dominant position in prostate cancer screening due to its high sensitivity and low-cost non-invasive means, but its specificity still needs to be improved. The popularity of rectal ultrasound-guided prostate puncture biopsy is an important reason for improving the early diagnosis rate of prostate cancer. In terms of prostate cancer treatment, especially for patients with localized prostate cancer, hormone therapy remains the cornerstone of prostate cancer treatment. For patients with castration-resistant prostate cancer (CRPC), the use of new drugs such as PARP inhibitors and AR antagonists has become the key to treatment. With the development of medical technology, radiotherapy equipment also plays an important role in the treatment of prostate cancer, such as intensity modulated radiation therapy (IMRT) and proton therapy, which can more accurately target tumors and reduce damage to surrounding healthy tissues.

[0003] Circulating tumor cells (CTCs) are cells released from primary tumors and carry tumor-specific biological information. Due to the convenience and timeliness of peripheral blood testing, they can be used as a technical means for auxiliary diagnosis and screening of early prostate cancer, and can also be used as a reference indicator for medication guidance and prognosis. Androgen receptor (AR) plays an important role in the occurrence and development of prostate cancer and can promote the progression and metastasis of prostate cancer. Studies have found that AR-V7 is one of the most frequently detected splice variants in CRPC, which can be used as a molecular marker to guide drug selection for CRPC patients. AR-V7 is currently the only protein variant that can be detected in clinical samples. By detecting the expression of AR-V7 in CTCs, patients can be guided in medication. For example, patients with AR-V7 positive may have better efficacy with paclitaxel drugs, and AR-V7 negative patients can be treated with androgen receptor signal inhibitors.

[0004] In order to provide more technical means for early screening and treatment prognosis of prostate cancer, the present invention develops a method for detecting circulating tumor cells that are positive for AR-V7 in prostate cancer. This method can quickly and accurately detect whether there are tumor cells in the blood of prostate cancer patients and whether the tumor cells express AR-V7, which is of great significance for early screening of prostate cancer and medication guidance. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a method for detecting circulating tumor cells that are positive for AR-V7 in prostate cancer to solve the above problems.

[0006] A method for detecting AR-V7-positive circulating tumor cells in prostate cancer, comprising the following steps:

[0007] S1: Prepare capture chip, select stainless steel mesh with pore size of 18×18 μm, perform gold plating, cut, wash with ethanol, wash with specific mixed solution, wash with deionized water and ethanol, incubate with thiolated sodium hyaluronate, wash with PBS solution, activate with EDC and Sulfo-NHS, wash with PBS solution, incubate with EpCAM antibody, wash with PBS solution, block with BSA, assemble, add lyophilization protectant, freeze-dry and store for later use;

[0008] S2: Prepare fixative, membrane permeabilization agent, blocking solution, staining solution and cleaning solution;

[0009] S3: Perform cell line verification, including the preparation of Jurkat cells and 22RV1 cells, and place the cell suspension in the chip loading wells, use an automatic sampler for sample loading and staining, and remove the reagent strip for slide reading after the instrument runs.

[0010] S4: Process clinical samples, including blood dilution, centrifugation, supernatant removal, washing, etc.

[0011] S5: Detect and interpret the samples. Place the samples in the sample wells of the chip and use an automatic sample injector to add samples and stain them. After the instrument is finished running, take out the reagent strips for reading, and interpret and compile data according to specific standards.

[0012] Preferably, during the preparation of the capture chip, the diameter of the chip cut from the stainless steel mesh is 8 mm.

[0013] Preferably, during the preparation of the capture chip, when the thiolated sodium hyaluronate is incubated, the oscillation speed of the oscillator is 200 rpm / min and the incubation time is 2 h.

[0014] Preferably, during the preparation of the capture chip, EDC and Sulfo-NHS are activated and incubated at room temperature in the dark for 30 minutes.

[0015] Preferably, during the preparation of the capture chip, EpCAM antibody incubation is performed at 37° C. for 2 hours.

[0016] Preferably, the fixing solution is 250 mL 4% paraformaldehyde.

[0017] Preferably, the membrane permeabilization agent is 250 mL 0.1% Triton TM X-100.

[0018] Preferably, the blocking solution is 250 mL (1.5 mg / mL glycine + 50 mg / mL BSA + 3% goat serum + 2% Tween 20 dissolved in PBS).

[0019] Preferably, the dye solution is 250 mL (1 μg 647 fluorescent AR-V7 antibody + 1μg 488 fluorescent anti-panCytokeratin antibody + 5μg PE anti-human CD45 antibody dissolved in PBS).

[0020] Preferably, the cleaning solution is PBS buffer.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1. The detection method of the present invention can detect AR-V7-positive circulating tumor cells in the early stage of prostate cancer, breaking through the limitation of traditional diagnostic methods that are difficult to detect tumors in the early stages, providing patients with the possibility of receiving timely treatment in the early stages of the disease, and significantly improving the accuracy of early diagnosis.

[0023] 2. Through the combined detection and precise interpretation of multiple markers, it is possible to effectively distinguish prostate cancer cells from normal cells, reduce the risk of misdiagnosis, and provide a more reliable basis for clinical diagnosis.

[0024] 3. Accurately detect the expression of AR-V7 and provide medication guidance for the treatment of prostate cancer patients, which will help doctors develop personalized treatment plans and improve treatment outcomes.

[0025] 4. Only blood samples from patients need to be collected for testing, which avoids the pain and risks caused by invasive operations such as traditional biopsies, and is more acceptable to patients.

[0026] 5. The entire testing process is relatively simple and quick, and the test results can be obtained in a relatively short time, which helps to formulate treatment strategies for patients in a timely manner.

[0027] 6. The detection sensitivity of CTC in prostate cancer patients is as high as 100%, and the specificity of detection in healthy control samples is also 100%, which can effectively identify patients and healthy people and reduce missed diagnoses and misdiagnoses.

[0028] 7. It provides a powerful technical means for the study of the pathogenesis, disease progression and treatment effect evaluation of prostate cancer, which helps to promote the in-depth development of prostate cancer research.

[0029] 8. Early screening and accurate medication guidance can help improve treatment outcomes, prolong patient survival, and improve patient prognosis and quality of life.

[0030] 9. Through early diagnosis and precise treatment, unnecessary treatment and examination can be avoided, and the medical expenses of patients and social medical costs can be reduced.

[0031] 10. The reagents and equipment used in the test are common and easy to obtain, and the test method is easy to operate and master, which is conducive to promotion and application in medical institutions at all levels and improving the overall diagnosis and treatment level of prostate cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is a representative picture of the cell line validation results in the present invention;

[0033] Figure 2 This is a representative picture of AR-V7 positive CTC in the present invention;

[0034] Figure 3 This is a representative picture of AR-V7 negative CTC in the present invention;

[0035] Figure 4 It is a representative picture of the healthy control sample in the present invention;

[0036] Figure 5It is a schematic diagram of the process of the present invention. DETAILED DESCRIPTION

[0037] The following embodiments of the present invention are described in further detail in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0038] Prostate cancer is one of the most common malignant tumors in men, and its early diagnosis and effective treatment are crucial to the survival rate and quality of life of patients. The present invention provides a method for detecting circulating tumor cells positive for AR-V7 in prostate cancer, aiming to provide a more accurate and convenient technical means for the screening, diagnosis and treatment of prostate cancer.

[0039] Experimental Materials:

[0040] Stainless steel screen: pore size 18×18 microns;

[0041] Thiolated sodium hyaluronate;

[0042] EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride);

[0043] Sulfo-NHS (N-hydroxysulfosuccinimide);

[0044] EpCAM antibody;

[0045] 4% paraformaldehyde (fixative solution);

[0046] 0.1% Triton TM X-100 (membrane rupture agent);

[0047] 1.5 mg / mL glycine, 50 mg / mL BSA, 3% goat serum, 2% Tween20 (blocking solution components);

[0048] 647 fluorescent AR-V7 antibody, 488 fluorescent Anti-panCytokeratin antibody, PEanti-humanCD45 antibody (staining solution components);

[0049] Jurkat cells and 22RV1 cells;

[0050] Blood samples from prostate cancer patients and healthy controls.

[0051] Experimental equipment: gold plating equipment, oil bath, oscillator, pipette, cell counter, centrifuge, automatic pipettor, fluorescence microscope, freeze dryer, 24-well plate, lens cleaning paper, high-purity nitrogen bottle.

[0052] Capture chip preparation:

[0053] Select a stainless steel mesh with an aperture of 18×18 microns and place it in a gold plating device for gold plating. The purpose of gold plating is to increase the chemical stability and biocompatibility of the mesh, which is conducive to the subsequent combination with biological molecules. During the gold plating process, the concentration, temperature and time of the gold plating solution are strictly controlled to ensure that the uniformity and thickness of the gold plating layer meet the requirements;

[0054] After the gold plating is completed, use a precision cutting tool to cut the screen into a circular chip with a diameter of 8 mm. The cut chips are first washed with ethanol for 2 minutes. Ethanol washing helps to remove grease and impurities on the surface of the chip. Transfer the chip after ethanol washing to a clean beaker, and then add a mixed solution of water, 25% ammonia water, and 30% hydrogen peroxide in a volume ratio of 5:1:1 to the beaker. Seal the beaker with plastic wrap to prevent the solution from volatilizing and being contaminated during heating. Then put the beaker into an oil bath that has been preheated to 75°C and heat it for 5 minutes at a fixed time.

[0055] After the heating and cleaning is completed, the chip is placed in deionized water, ethanol, and ethanol for washing in turn. Each washing time is about 1 minute to completely remove the residual washing liquid. After the washing is completed, the liquid on the surface of the chip is blown dry with high-purity nitrogen, or the chip is placed in a well-ventilated place to dry naturally. 3mL of thiolated sodium hyaluronate is placed in an EP tube, and then 15-18 washed chips are carefully added to the EP tube. The EP tube is placed on an oscillator, the shaking speed is set to 200rpm / min, and the shaking is incubated for 2 hours. Shaking incubation helps ensure that the chip and the thiolated sodium hyaluronate are fully and evenly contacted and reacted;

[0056] After the incubation, the chip was washed with PBS solution for 2 times, each time for 3 minutes. After the washing was completed, 0.609 mg of EDC was weighed and dissolved in 35 μL of MES buffer (0.05 M, pH = 6), and 0.348 mg of Sulfo-NHS was weighed and dissolved in 20 μL of MES solution. After the two were dissolved separately, they were mixed to obtain 55 μL of solution. 55 μL of activator solution was evenly transferred to the chip surface using a pipette, and then the chip was placed in a dark environment at room temperature for 30 minutes;

[0057] After activation, the chip was washed again with PBS solution, and the washing was repeated twice to remove the unreacted activator. After washing, 1.75 μg of EpCAM antibody was dissolved in MES buffer (0.05 M, pH = 6), and the total volume after mixing was 53.5 mL. The antibody solution was completely absorbed and evenly placed on the chip surface, and then the chip was placed in a constant temperature incubator at 37°C for 2 hours. After incubation, 1 mL of PBS solution was taken in the well plate to wash the chip, and 500 μL of 1% BSA was added to the well plate with the chip, and it was blocked at room temperature for 1 hour. After blocking, 1 mL of PBS solution was taken in the well plate to wash the chip again, and the chip was assembled, and 300 μL of freeze-drying protective agent was added to the sample well, and it was precooled at -80°C for 2 hours. After precooling, the assembled chip was placed in a freeze dryer for freeze drying. After freeze drying, the chip was placed in a 4°C refrigerator for storage.

[0058] Reagent preparation:

[0059] Weigh an appropriate amount of paraformaldehyde powder and dissolve it in deionized water to prepare 250 mL of 4% paraformaldehyde solution. During the preparation process, use a magnetic stirrer to stir continuously to accelerate the dissolution of paraformaldehyde. Stir for about 30 minutes until the solution becomes clear and transparent.

[0060] Accurately measure 25mL of Triton TM Add the X-100 stock solution to an appropriate amount of deionized water and dilute to 250 mL to prepare 0.1% Triton TM After the X-100 membrane rupturing agent solution is prepared, mix the solution thoroughly and store it in a brown bottle in a refrigerator at 4°C for later use;

[0061] Weigh 375 mg of glycine and dissolve it in an appropriate amount of PBS solution. Weigh 12.5 g of BSA and add it to the above solution. Stir to fully dissolve it. Measure 7.5 mL of goat serum and 5 mL of 2% Tween20 and add them to the solution. Then dilute to 250 mL with PBS solution. After fully mixing, store in a 4°C refrigerator for later use.

[0062] Accurately weigh 1 μg 647 fluorescent AR-V7 antibody, 1 μg 488 fluorescent anti-panCytokeratin antibody and 5μg PE anti-human CD45 antibody, dissolve the above antibodies in an appropriate amount of PBS solution, then mix the three antibody solutions together, and dilute to 250mL with PBS solution. After fully mixing, store in a 4℃ refrigerator and keep away from light;

[0063] Directly use PBS buffer as the cleaning solution. Prepare an appropriate amount of PBS buffer in advance and store it at room temperature for later use.

[0064] Jurkat cell preparation:

[0065] Take out the Jurkat cell culture flask that has been cultured to the logarithmic growth phase from the incubator, operate in the clean bench, gently shake the cell suspension in the culture flask, then use a pipette to aspirate an appropriate amount of cell suspension, transfer it to a 15mL centrifuge tube, put the centrifuge tube in the centrifuge, set the speed to 1300rpm, and centrifuge at room temperature for 3 minutes. After centrifugation, carefully use a pipette to aspirate the supernatant, be careful not to aspirate the cell pellet, add 1mL PBS solution to the centrifuge tube, gently blow to resuspend the cell pellet, and then centrifuge again. The centrifugation conditions are the same as above. Repeat the above PBS resuspension and washing steps once to ensure that the residual culture medium components are fully removed. Finally, resuspend and wash the cells with 1mL PBS, and use a cell counter to count the cells. After counting, according to the counting results, use PBS solution to dilute the Jurkat cell concentration to 9×106 / mL for standby use;

[0066] 22RV1 cell preparation:

[0067] As in the preparation of Jurkat cells, cultured 22RV1 cells were collected, centrifuged and resuspended in PBS for washing. The 22RV1 cells were counted using a cell counter, and then the cells were graded diluted. 100 22RV1 cells were accurately pipetted and placed in 300 μl of the prepared Jurkat cell suspension. The cells were gently pipetted to mix thoroughly and set aside.

[0068] On-machine testing:

[0069] Take out the prepared staining reagent and chip from the 4℃ refrigerator, let it balance at room temperature for a while, use a pipette to accurately absorb 300μL of the cell suspension prepared in step (ii), slowly and evenly place it in the sample loading hole of the chip to avoid bubbles, start the automatic sample loading instrument produced by Huixian Pharmaceutical Technology Co., Ltd., and perform sample loading and staining operations according to the preset procedures and parameters. After the instrument is finished running, carefully take out the reagent strip and place it under a fluorescence microscope for reading. First, use the green light channel (AF488) to preliminarily observe and determine the green bright spot, and then switch to the blue light channel (DAPI), orange light channel (PE) and red light channel (AF647) in turn to take pictures and record the staining and morphological characteristics of the cells.

[0070] Clinical sample processing steps:

[0071] Confirmed prostate cancer patients and healthy controls were selected, and peripheral blood was collected using disposable blood collection needles and vacuum blood collection tubes. 8 mL of blood was collected from each patient. The collected blood samples were immediately transferred to the laboratory and operated in an ultra-clean workbench. 4 mL of PBS was taken into a 15 mL centrifuge tube, and then 4 mL of whole blood was slowly and dropwise added using a pipette. At the same time, the centrifuge tube was gently shaken to fully mix the blood and PBS to complete a 1:1 dilution.

[0072] Take 4mL of sample density separation liquid and put it into another 15mL centrifuge tube. Slowly add 8mL of diluted blood into the centrifuge tube. During the operation, keep the tip of the pipette close to the inner wall of the centrifuge tube to allow the blood to flow down slowly to form a clear layered interface. Avoid violent shaking and mixing. After the addition is completed, the total volume is about 12mL. Gently cover the centrifuge tube lid.

[0073] The centrifuge tubes containing blood and separation solution were placed symmetrically in a centrifuge, and the centrifugal speed was set to 2500 rpm / min and the centrifugal time was set to 20 minutes. After the centrifugation was completed, the centrifuge tubes were carefully taken out. At this time, it can be observed that the liquid in the tube is divided into three obvious layers: the top layer is the plasma layer, the middle layer is the buffy coat layer (PBMC layer), and the bottom layer is the red blood cell and granulocyte layer;

[0074] Use a pipette to carefully discard the plasma layer (the top layer), taking care to avoid the middle white film layer. Then, pipette the PBMC layer (white film layer) into another 15 mL centrifuge tube, pipette about 2 mL, add 10 mL of PBS solution to the centrifuge tube, gently invert the centrifuge tube to fully suspend the PBMC for washing;

[0075] Put the washed centrifuge tube back into the centrifuge, set the centrifugal speed to 1500rpm / min, and the centrifugal time to 5 minutes. After the centrifugation is completed, take out the centrifuge tube and carefully discard the supernatant with a pipette;

[0076] Add 5 mL of PBS solution to the centrifuge tube again, gently invert the centrifuge tube to resuspend the precipitate, wash it for the second time, and repeat the above centrifugation operation under the same centrifugation conditions;

[0077] After centrifugation, remove the centrifuge tube, discard the supernatant, add 300 μL PBS solution to resuspend the precipitate, and use a pipette to gently blow to mix the cells thoroughly for later use.

[0078] Sample testing and interpretation:

[0079] Take out the staining reagent and the prepared chip from the 4°C refrigerator, let them balance at room temperature for a while, turn on the automatic sampler and fluorescence microscope, preheat to the working state, use a pipette to accurately absorb 300 μL of the processed clinical sample, slowly and evenly load it into the sample loading hole of the chip to avoid bubbles and overflow, put the chip loaded with the sample into the automatic sampler, and perform the sample loading and staining operations according to the preset program. After the instrument is finished running, carefully take out the reagent strip and place it under a fluorescence microscope for reading. First, use the green light channel (AF488) to preliminarily observe and determine the green bright spot, and then take pictures in turn according to the green light channel (AF488), blue light channel (DAPI), orange light channel (PE), and red light channel (AF647). According to the photo results, analyze and interpret the staining of the cells; DAPI+, PanCK+, CD45-, and AR-V7+ are interpreted as AR-V7-positive circulating tumor cells, and DAPI+, PanCK+, CD45-, and AR-V7- are interpreted as AR-V7-negative circulating tumor cells.

[0080] Representative images of cell line validation results are shown in Figure 1 , Representative images of AR-V7 positive CTCs are shown in Figure 2 , Representative images of AR-V7 negative CTCs are shown in Figure 3 , representative pictures of healthy control samples are shown in Figure 4 , the clinical sample test results are statistically as follows:

[0081]

[0082] Jurkat and 22RV1 cells were used to verify the detection method. Jurkat cells simulated leukocytes in human whole blood, and the staining results were DAPI+, PanCK-, CD45+, and AR-V7-, indicating that the staining was normal and no markers related to prostate cancer were detected. 22RV1 cells simulated prostate cancer CTCs in whole blood, and the staining results were DAPI+, PanCK+, CD45-, and AR-V7+. The staining results were in line with expectations, indicating that the detection method can accurately identify markers related to prostate cancer. Combined with the cell line verification results, it was proved that the detection method of the present invention is technically feasible, can effectively distinguish leukocytes and prostate cancer CTCs, and accurately detect the expression of AR-V7.

[0083] The detection method of the present invention was used to detect AR-V7-positive circulating tumor cells in 6 prostate cancer patients and 6 healthy controls. The results showed that CTCs were detected in the samples of prostate cancer patients, and the CTCs of some patients were AR-V7 positive. No CTCs or AR-V7-positive CTCs were detected in the healthy control samples, indicating that the detection method has good specificity for healthy people.

[0084] The embodiments of the present invention are given for the purpose of illustration and description, and are not intended to be exhaustive or to limit the invention to the disclosed forms. Many modifications and variations will be apparent to those of ordinary skill in the art. The embodiments are selected and described in order to better illustrate the principles and practical applications of the present invention and to enable those of ordinary skill in the art to understand the present invention and thereby design various embodiments with various modifications suitable for specific uses.

Claims

1. A method for detecting circulating tumor cells positive for AR-V7 in prostate cancer, characterized in that: The following steps are involved: S1: Prepare capture chip, select stainless steel mesh with pore size of 18×18 μm, perform gold plating, cut, wash with ethanol, wash with specific mixed solution, wash with deionized water and ethanol, incubate with thiolated sodium hyaluronate, wash with PBS solution, activate with EDC and Sulfo-NHS, wash with PBS solution, incubate with EpCAM antibody, wash with PBS solution, block with BSA, assemble, add lyophilization protectant, freeze-dry and store for later use; S2: Prepare fixative, membrane permeabilization agent, blocking solution, staining solution and cleaning solution; S3: Perform cell line verification, including the preparation of Jurkat cells and 22RV1 cells, and place the cell suspension in the chip loading wells, use an automatic sampler for sample loading and staining, and remove the reagent strip for slide reading after the instrument runs. S4: Process clinical samples; S5: Detect and interpret the sample. Place the sample in the sample well of the chip and use an automatic sample injector to add the sample and stain it. After the instrument is finished running, take out the reagent strip for reading.

2. A method for detecting circulating tumor cells positive for AR-V7 in prostate cancer as claimed in claim 1, characterized in that: During the preparation of the capture chip, the diameter of the chip cut from the stainless steel mesh is 8 mm.

3. A method for detecting circulating tumor cells positive for AR-V7 in prostate cancer as claimed in claim 1, characterized in that: During the preparation of the capture chip, during the incubation of the thiolated sodium hyaluronate, the oscillation speed of the oscillator was 200 rpm / min, and the incubation time was 2 h.

4. A method for detecting circulating tumor cells positive for AR-V7 in prostate cancer as claimed in claim 1, characterized in that: During the preparation of the capture chip, EDC and Sulfo-NHS were activated and incubated at room temperature in the dark for 30 minutes.

5. A method for detecting circulating tumor cells positive for AR-V7 in prostate cancer as claimed in claim 1, characterized in that: During the preparation of the capture chip, EpCAM antibody incubation was performed at 37° C. for 2 h.

6. A method for detecting circulating tumor cells positive for AR-V7 in prostate cancer as claimed in claim 1, characterized in that: The fixing solution is 250 mL 4% paraformaldehyde.

7. A method for detecting AR-V7 positive circulating tumor cells in prostate cancer as claimed in claim 1, characterized in that: The membrane permeabilization agent is 250mL 0.1% Triton TM X-100.

8. A method for detecting AR-V7 positive circulating tumor cells in prostate cancer as claimed in claim 1, characterized in that: The blocking solution is 250 mL.

9. A method for detecting AR-V7 positive circulating tumor cells in prostate cancer as claimed in claim 1, characterized in that: The dyeing solution is 250 mL.

10. A method for detecting AR-V7 positive circulating tumor cells in prostate cancer as claimed in claim 1, characterized in that: The cleaning solution is PBS buffer.

Images