CN115197914A - Method for separating and detecting circulating tumor cells - Google Patents
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Abstract
The invention belongs to the field of cell detection, and particularly relates to a method for separating and detecting circulating tumor cells. The invention relates to a method for separating and detecting circulating tumor cells, which comprises the following steps: centrifuging a blood sample by adopting a Ficoll density gradient centrifugation method, absorbing a mononuclear cell layer, washing, adding erythrocyte lysate, centrifuging and washing to obtain peripheral blood mononuclear cells; dividing the cell into a first part of cells and a second part of cells; performing positive sorting enrichment on the first part of cells by using an immunomagnetic bead sorting (MACS), and then performing flow cell sorting detection after staining the enriched cells by using a fluorescence labeling antibody; and detecting the expression of the following markers by applying a real-time fluorescent quantitative PCR method to the second part of cells: ep-CAM, CK8, CK18, CK19, CD44v6, CD45, CXCR4, SDF-1; and D, combining the detection results of the step C and the step D, and determining the separation result of the circulating tumor cells of the blood sample.
Description
Technical Field
The invention belongs to the field of cell detection, and particularly relates to a method for separating and detecting circulating tumor cells.
Background
The concept of circulating tumor cells was first proposed in 1896 by Ashworth, an australian scholarian, and cancer cells gradually differentiate and shed from primary tumors or metastatic tumors to enter the blood circulation. Specifically, the primary tumor grows in blood vessels to a certain stage, and directly invades the peripheral blood vessels, and tumor cells firstly grow by the integrin attached to the basement membrane of the blood vessels; when the number of tumor cells is gradually increased, the matrix metalloproteinase secreted by the tumor cells is gradually increased, and the IV type collagen is gradually digested and removed, so that the barrier of a basement membrane is broken through, and the IV type collagen enters blood, namely the tumor cells are called as circulating tumor cells; after entering human blood, circulating tumor cells can migrate to the whole body along with the blood circulation of the tumor to form recurrent metastasis. Therefore, there is a need to identify an effective method for isolating and detecting circulating tumor cells.
In recent years, more and more researchers have identified and evaluated circulating tumor cells by a "liquid biopsy" method, and the analysis of circulating tumor cells is helpful for patients with a high risk of recurrence, in terms of stratification for specific adjuvant medical treatment and monitoring of treatment response. The technology can discover high risk group of recurrence as soon as possible, reduce the risk of tumor recurrence and obviously improve survival rate. Compared with the traditional imaging diagnosis, endoscopy and pathological diagnosis, the cell count of the circulating tumor is earlier than the tumor discovery or tumor recurrence discovery, the high-frequency monitoring can be realized, the purposes of monitoring the disease progression and changing the treatment scheme in real time can be achieved, and only a small amount of peripheral blood of a patient needs to be extracted, so that the method has no side effect on the patient and is easy to accept.
In the prior art, methods for directly separating and identifying circulating tumor cells are divided into two main types: one is a crude separation based on the physical size of circulating tumor cells, i.e., circulating tumor cells are screened by filtration methods. Filters with a pore size of 8 μm were used, but many leukocytes had a particle size greater than 8 μm, resulting in clogging of the filter with leukocytes; the circulating tumor cells in different periods have different sizes, and if the circulating tumor cells in the dormant period can easily pass through the filter membrane with the aperture of 8 mu m, the circulating tumor cells can be missed for detection. The other type is based on the separation of cell surface antigen markers, the most representative product of the method is a CellSearch full-automatic circulating tumor cell detector of Qiangsheng company, and the method is the only technology approved by FDA and CFDA for clinically detecting circulating tumor cells, but the method only uses immunomagnetic beads, and the separated circulating tumor cells have poor activity, poor detection sensitivity, omission and other problems.
Disclosure of Invention
In order to solve the problems of the prior art, the present invention aims to provide a method for separating and detecting circulating tumor cells, which can effectively separate the circulating tumor cells from a blood sample, improve the sensitivity and specificity of circulating tumor cell detection, and effectively reduce the omission ratio.
The invention relates to a method for separating and detecting circulating tumor cells, which comprises the following steps:
A. blood sample treatment: centrifuging the blood sample by a Ficoll density gradient centrifugation method, wherein the centrifuged system is at least divided into four layers, namely a plasma platelet layer, a mononuclear cell layer, a layered liquid layer, polymorphonuclear leukocytes and a red blood cell layer; sucking a mononuclear cell layer, washing, adding erythrocyte lysate, centrifuging and washing to obtain peripheral blood mononuclear cells;
B. dividing the peripheral blood mononuclear cells obtained in the step A into a first part of cells and a second part of cells;
C. performing positive sorting enrichment on the first part of cells by using an immunomagnetic bead sorting technology (MACS), and then performing flow cell sorting detection after staining the enriched cells by using a fluorescence labeling antibody;
D. and (3) detecting the expression of the following markers by applying a real-time fluorescent quantitative PCR method to the second part of cells: ep-CAM, CK8, CK18, CK19, CD44v6, CD45, CXCR4, SDF-1;
E. and D, integrating the detection results of the step C and the step D, and determining the separation result of the circulating tumor cells of the blood sample.
According to a further feature of the method for isolating and detecting circulating tumor cells of the present invention, in step a, the blood sample is diluted with HBSS buffer at 1.
According to a further feature of the method for separating and detecting circulating tumor cells of the present invention, in step a, the centrifugal liquid used in the Ficoll density gradient centrifugation method is lymphocyte separation liquid, and the volume of the centrifugal liquid is 2 times of that of the blood sample.
According to a further feature of the method for separating and detecting circulating tumor cells of the present invention, in the step C, the positive cell sorting magnetic beads are CD326 (Ep-CAM) magnetic beads, and the amount of the CD326 (Ep-CAM) magnetic beads is 20ul/10 7 And (4) cells.
According to still further features in the described methods of isolating and detecting circulating tumor cells of the invention, in step C, the fluorescently labeled antibody is selected from the group consisting of: the antibody is characterized by comprising a leukocyte surface common antigen CD45 antibody marked by a fluorescent dye PE, a human cytokeratin CK (8/18) antibody marked by a fluorescent dye FITC, and an epithelial cell adhesion molecule CD326/Ep-CAM antibody marked by a fluorescent dye APC.
The method for separating and detecting the circulating tumor cells has the following advantages:
(1) Peripheral blood circulating tumor cell counts were measured by a combination of immunomagnetic activated cell sorting (MACS) and flow cytometric sorting (FACS). The method relies primarily on the expression of specific markers for epithelial cells, such as Cytokeratin (CK) in epithelial cells, which are expressed predominantly on epithelial cells but not on leukocytes. Cytokeratins are proteins formed by the passage of an intermediate filament of keratin through the cytosol interior and the cytoskeleton; the expression of which is mainly dependent on the type of epithelial cells, the terminal differentiation processTime point and growth phase. Epithelial cell adhesion molecule (Ep-CAM), also known as CD326, is a common surface marker for positive selection of cell populations, while a specific leukocyte surface marker (CD 45) is used for negative selection. Thus, circulating tumor cells are defined as CD45 - CK + CD326 + A cell. Generally, the number of circulating tumor cells in a normal human is 0 to 1 per 7.5mL of peripheral blood; a low risk of recurrence or metastasis is defined as containing 1-5 circulating tumor cells; if the number of circulating tumor cells is more than 5, the patient has high risk of relapse or metastasis, and the prognosis is poor.
(2) The real-time fluorescent quantitative PCR method is used for detecting the expression of circulating tumor cell related surface markers, including Ep-CAM, CK8, CK18, CK19, CD44v6, CD45, CXCR4 and SDF-1.
(3) Can improve the sensitivity and specificity of circulating tumor cell detection, effectively reduce the omission factor and facilitate clinical popularization and application.
Drawings
FIG. 1 is a graph of the results of testing circulating tumor cells isolated from a 58 year old male patient with advanced lung adenocarcinoma.
FIG. 2 shows the real-time fluorescent quantitative PCR expression of circulating tumor cell surface markers in the same 58-year-old male patient with advanced lung adenocarcinoma.
Detailed description of the preferred embodiment
The invention is further illustrated with reference to the following specific examples.
Example 1: detection of circulating tumor cell number of clinical middle-late stage non-small cell lung cancer patient
(1) Sample preparation
30 cases of the middle-stage and late-stage non-small cell lung cancer of the group of clinical diagnosis are collected, and 30 peripheral blood samples are collected.
(2) Sample detection
1) Gently inverting 7.5mL of the above peripheral blood sample for several times and mixing;
2) Mixing the above blood with 7.5mL HBSS, slowly adding to the surface of a 50mL centrifuge tube containing 15mL of lymphocyte separation medium, centrifuging at 1,200rpm for 20min at room temperature, aspirating the atomized mononuclear cell layer with a 5mL pipette, and dividing it into two parts (one part for example 1 and the other part for example 2);
3) Adding 10mL of HBSS into the solution, centrifuging at 1,200rpm for 5min, removing the supernatant again, washing twice, adding 5mL of erythrocyte lysate (1 = 10 concentrate: sterile water), standing the solution at room temperature in a dark place for 10min, centrifuging at 1,200rpm for 5min, and removing the supernatant;
4) Adding 10mL of HBSS for resuspending cells, then centrifuging at 1,200rpm for 5min, removing supernatant, adding 1mL of HBSS again, mixing uniformly and counting; adding 5mL of magnetic bead sorting buffer (buffer) to resuspend the cells, centrifuging again at 1,200rpm for 5min, and removing the supernatant;
5) Resuspending cells with a residual solution, adding CD326 magnetic beads, incubating for 30min in shade at 4 ℃, washing the cells with a MACS buffer solution, centrifuging for 5min at 1,200rpm, and removing a supernatant;
6) Taking LS type magnetic bead separation tube, lubricating and cleaning magnetic bead separation column with 3mL MACS buffer solution, and using 500uLMACS buffer solution (less than or equal to 50 × 10) 6 ) After the cells in the liquid of the rinsing separation column are resuspended, adding the separation column, and waiting until the separation liquid completely and uniformly flows out;
7) The tube wall was washed with 3mL of MACS buffer and then re-applied to one column (1 time), and 3mL of MACS buffer r was applied to one column (2 times);
8) Adding 5mL of MACS buffer solution into the tube, quickly taking out the separation column from the tube, placing the separation column on a flow tube, pushing all the separation liquid in the separation tube into the flow tube by an electric pusher, centrifuging at 1,200rpm for 5min, and removing the supernatant;
9) Adding 1mL of HBSS, then resuspending the supernatant cells, centrifuging at 1,200rpm for 5min, and removing the supernatant;
10 Sequentially adding 10. Mu.L each of the antibody, CD326, CK, CD45 (10. Mu.L/10) 6 ) Mixing, incubating for 12min at room temperature in dark place, centrifuging and washing with 4mL PBS for 1 time, removing supernatant, adding 500 μ LPBS, and loading to flow cytometer;
11 Collecting circulating tumor cells (CD 45) - CK + CD326 + Cell) numberVolume, turn off flow cytometer.
(3) Analysis of detection results
The detection rate of circulating tumor cells in peripheral blood was 46.67% (14 cases), and the number of circulating tumor cells detected was 0 to 28, with an average of 7.97/mL. FIG. 1 is a graph of the results of testing circulating tumor cells isolated from a 58 year old male patient with advanced lung adenocarcinoma, showing that the number of circulating tumor cells was 21.
Example 2: detection of circulating tumor cell related gene expression of patient with middle and late stage clinical non-small cell lung cancer
(1) Sample detection
1) Counting 10 6 Diluting LC-5 cells (a lung squamous carcinoma cell line) by 10 times step by step to obtain 10 5 、10 4 、10 3 、10 2 10, 1 LC-5 cells;
2) RNA extracted from peripheral blood mononuclear cells in blood samples of healthy volunteers is respectively compared with RNA extracted from 1, 10 2 、10 3 、10 4 、10 5 RNA mixing of individual LC-5 cells;
3) Carrying out real-time fluorescence quantitative PCR detection on the mixed sample, wherein the amplification efficiency is 96-104%, the slope is set to-3.1-3.5 and-3.113 2 Not less than 0.99. The detection threshold is set as the Ct value when the circulating tumor cell related genes Ep-CAM, CK8, CK18, CK19, CD44v6, CD45, CXCR4 and SDF-1 have amplification signals at the same time under the condition of the lowest sample concentration, and the positive result is obtained when the amplification Ct value of any one gene is larger than the threshold.
4) The atomized mononuclear cell layer obtained in example 1 was centrifuged at 5,000rpm for 5min, and the supernatant was removed;
5) Adding 1mL Trizol, incubating at 4 deg.C for 10min, and slowly blowing to blow cells to lyse the cells;
6) Adding 200 mu L chloroform, and mixing by vortex;
7) Incubating at 4 ℃ for 10min, then centrifuging at 13,500rpm at 4 ℃ for 15min, and taking 300-500 mu L of upper-layer water phase to a new EP tube;
8) Adding 300-500 μ L isopropanol (volume ratio to water phase is 1);
9) Adding 1mL of precooled 75% ethanol (DEPC water), lightly blowing, centrifuging at 4 ℃ and 13,500rpm for 5min, and removing the supernatant;
10 30-50 μ L DEPC water is added, and the mixture is placed for 10min at room temperature, thus obtaining the total RNA reaction solution.
11 Prepare the following reaction solution (one reaction amount): total RNA reaction 2. Mu.L, 2 Xone Step
RT-PCRBuffer 410. Mu.L, 50 XROX Reference Dye or Dye II 0.4. Mu.L, primeScript 1Step Enzyme Mix 2. Mu.L, 10. Mu. Mol/L upstream and downstream primers 0.8. Mu.L each, RNase Free dH 2 O5.2. Mu.L, 20. Mu.L in total.
12 On a fluorescent quantitative PCR instrument (ABI 7500) to detect the expression of Ep-CAM, CK8, CK18, CK19, CD44v6, CD45, CXCR4, SDF-1, GAPDH genes. The amplification conditions were: 5min at 42 ℃,10 s at 95 ℃, 5s at 95 ℃, 34s at 60 ℃, 15s at 95 ℃, 1min at 60 ℃, 15s at 95 ℃, 59s at 4 ℃ and 59s at 40 cycles.
(2) Analysis of detection results
The detection rate of the circulating tumor cell-associated gene in peripheral blood was 43.33% (13 cases). FIG. 2 shows real-time fluorescent quantitative PCR expression of cell markers associated with circulating tumor cells in the same 58 year old male patient with advanced lung adenocarcinoma.
Example 3: separation and detection of circulating tumor cells by combination of MACS, FACS and real-time fluorescence quantitative PCR method
30 patients with advanced lung adenocarcinoma were selected, and the data obtained by combining MACS, FACS (see example 1) and real-time fluorescent quantitative PCR (see example 2) were analyzed in combination to find a detection rate of 63.33% of circulating tumor cells (19 cases). The attached Table 1 shows the detection of circulating tumor cells in 30 patients.
Attached table 1
Note: n.s was not detected.
After the MACS technology is applied to positive sorting and enrichment, circulating tumor cells are gathered, the concentration degree is provided, detection is convenient, and the possibility of missed detection can be effectively reduced by combining FACS.
The circulating tumor cells can be detected on the genome level by using real-time fluorescent quantitative PCR, and the specificity is high.
As can be seen from the table above, the combination of MACS, FACS and real-time fluorescence quantitative PCR method can improve the sensitivity and specificity of circulating tumor cell detection more efficiently and effectively reduce the omission factor.
Claims (5)
1. A method for isolating and detecting circulating tumor cells, comprising the steps of:
A. blood sample treatment: centrifuging the blood sample by adopting a Ficoll density gradient centrifugation method, wherein a centrifuged system is at least divided into four layers, namely a plasma platelet layer, a mononuclear cell layer, a layered liquid layer, polymorphonuclear leukocytes and a red blood cell layer; sucking a mononuclear cell layer, washing, adding erythrocyte lysate, centrifuging and washing to obtain peripheral blood mononuclear cells;
B. dividing the peripheral blood mononuclear cells obtained in the step A into a first part of cells and a second part of cells;
C. performing positive sorting enrichment on the first part of cells by using an immunomagnetic bead sorting (MACS), and then performing flow cell sorting detection after staining the enriched cells by using a fluorescence labeling antibody;
D. and (3) detecting the expression of the following markers by applying a real-time fluorescent quantitative PCR method to the second part of cells: ep-CAM, CK8, CK18, CK19, CD44v6, CD45, CXCR4, SDF-1;
E. and D, integrating the detection results of the step C and the step D, and determining the separation result of the circulating tumor cells of the blood sample.
2. The method of isolating and detecting circulating tumor cells of claim 1, wherein: in step a, the blood sample is diluted with HBSS buffer at 1.
3. The method of isolating and detecting circulating tumor cells of claim 1, wherein: in the step A, the centrifugal liquid adopted by the Ficoll density gradient centrifugation method is lymphocyte separation liquid, and the volume of the centrifugal liquid is 2 times that of a blood sample.
4. The method of isolating and detecting circulating tumor cells of claim 1, wherein: in the step C, the positive cell sorting magnetic beads are CD326 (Ep-CAM) magnetic beads, and the dosage is 20ul/10 7 And (4) one cell.
5. The method for isolating and detecting circulating tumor cells of claim 1, wherein: in step C, the fluorescently labeled antibody is selected from the group consisting of: the antibody is characterized by comprising a leukocyte surface common antigen CD45 antibody marked by a fluorescent dye PE, a human cytokeratin CK (8/18) antibody marked by a fluorescent dye FITC, and an epithelial cell adhesion molecule CD326/Ep-CAM antibody marked by a fluorescent dye APC.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103866016A (en) * | 2014-03-07 | 2014-06-18 | 复旦大学附属中山医院 | Circulating tumor cell detection kit and application thereof |
| CN105675378A (en) * | 2014-11-18 | 2016-06-15 | 上海张江转化医学研发中心有限公司 | Simple single circulating tumor cell separation method and apparatus |
| CN107656044A (en) * | 2017-09-25 | 2018-02-02 | 亚能生物技术（深圳）有限公司 | The detection kit and detection method of a kind of circulating tumor cell |
| CN108982839A (en) * | 2018-06-12 | 2018-12-11 | 广州四叶草健康科技有限公司 | Circulating tumor cell detection method based on immunomagnetic beads and flow cytometer |
| CN110456034A (en) * | 2018-05-07 | 2019-11-15 | 上海市第十人民医院 | A kind of detection method of circulating tumor cell |
-
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103866016A (en) * | 2014-03-07 | 2014-06-18 | 复旦大学附属中山医院 | Circulating tumor cell detection kit and application thereof |
| CN105675378A (en) * | 2014-11-18 | 2016-06-15 | 上海张江转化医学研发中心有限公司 | Simple single circulating tumor cell separation method and apparatus |
| CN107656044A (en) * | 2017-09-25 | 2018-02-02 | 亚能生物技术（深圳）有限公司 | The detection kit and detection method of a kind of circulating tumor cell |
| CN110456034A (en) * | 2018-05-07 | 2019-11-15 | 上海市第十人民医院 | A kind of detection method of circulating tumor cell |
| CN108982839A (en) * | 2018-06-12 | 2018-12-11 | 广州四叶草健康科技有限公司 | Circulating tumor cell detection method based on immunomagnetic beads and flow cytometer |
Non-Patent Citations (1)
| Title |
|---|
| 中日联谊医院普外科会议筹备组编辑: "《普外进展论坛 孟宪民基金会首届学术讨论会暨 第十二届全国普外基础与临床进展学术大会》", vol. 2018, 上海交通大学出版社, pages: 565 - 566 * |
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