A marker combination and kit for detecting tumor cells in body fluid samples

Abstract

The present invention discloses a marker combination and a kit for detecting tumor cells in body fluid samples. The marker combination includes TERT and CD45, and further includes CK and a nuclear dye. The marker combination can be used for detecting tumor cells in body fluid samples, and the kit provided by the present invention includes the target composition, an erythrocyte lysis reagent, and a target cell enrichment reagent. The present invention provides a set of new tumor cell recognition markers TERT+ / CK+ / CD45‑ / DAPI+, wherein TERT is a telomerase reverse transcriptase. The advantages of the present invention are: first, it can significantly improve the sensitivity and specificity of detection; second, it can detect tumor cells of epithelial origin. For patients with urothelial carcinoma, the traditional EpCAM+ / CK+ / CD45‑ / DAPI+ detection method cannot distinguish between tumor cells and normal cells, and the TERT+ / CK+ / CD45‑ / DAPI+ detection method used in the present invention can clearly distinguish between tumor cells and normal cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical detection, and in particular relates to a marker combination and a kit for detecting tumor cells in body fluid samples. Background Art

[0002] Cancer has a high incidence and mortality rate worldwide, and has become a serious threat to human health. Liquid biopsy is a new non-invasive tumor detection technology that has emerged in recent years. Due to tumor metastasis, its cells fall off from the original site and enter the circulatory system, and then spread to the blood, pleural effusion, cerebrospinal fluid and even urine. Detecting free rare tumor cells and their molecular characteristics from these liquid samples can replace the biopsy of the original tumor lesions, so it is called liquid biopsy. Since lesion biopsy is traumatic and it is difficult to perform a second biopsy on patients in most cases, liquid biopsy, especially liquid biopsy on blood, is very popular in clinical practice because it is non-invasive and easy to obtain samples.

[0003] Currently, there are many technologies and equipment for detecting circulating tumor cells in the blood in the market and academia, which mainly adopt a two-step detection mode of enrichment followed by identification. Although there are many methods for enriching tumor cells, the methods for ultimately identifying tumor cells are extremely limited. They are basically identified by combining epithelial markers and leukocyte markers used in the CellSearch system approved by Johnson & Johnson for the US FDA. The CellSearch system uses magnetic particles labeled with antibodies against EpCAM to capture tumor cells in 7.5 ml of blood from cancer patients, and then through cell fixation, membrane permeabilization, and triple immunofluorescence staining (DAPI, CK, CD45), DAPI+ / CK+ / CD45- cells that meet certain morphological standards are defined as tumor cells and counted.

[0004] According to the above method, the marker combination used by the CellSearch system to identify tumor cells is EpCAM+ / CK+ / CD45- / DAPI+, where EpCAM and CK (cytokeratin) are both epithelial markers, CD45 is a leukocyte marker, and DAPI is a nuclear marker. According to the above definition, the CellSearch system can actually only identify the target cells as being of epithelial origin, but cannot clearly identify whether the target cells are benign or malignant. Therefore, when benign epithelial cells are present in the blood, CellSearch is prone to false positives. For example, the latest studies have shown that circulating epithelial cells can be detected in the blood of patients with benign diseases and even healthy people. In addition, since EpCAM and CK (cytokeratin) are both epithelial markers, it is impossible to distinguish tumor cells of epithelial origin, such as urothelial cancers such as bladder cancer and kidney cancer. Summary of the invention

[0005] The purpose of the present invention is to provide a marker combination and a kit for detecting tumor cells in body fluid samples, mainly to solve the problem that the prior art CellSearch system is prone to false positives in identifying tumor cells.

[0006] The technical solution adopted by the present invention to solve the above technical problems is as follows:

[0007] A marker combination for detecting tumor cells in a body fluid sample, the marker combination comprising TERT and CD45, specifically TERT positive and CD45 negative.

[0008] As a preferred embodiment, the marker combination further includes CK.

[0009] As a preferred embodiment, the marker combination further includes a nuclear dye.

[0010] As a preferred embodiment, the cell nuclear dye is DAPI.

[0011] The present invention also provides a kit for detecting tumor cells in body fluid samples, wherein the kit comprises the marker combination.

[0012] As a preferred embodiment, the kit includes 0.1-5ug / ml of TERT antibody, 0.1-5ug / ml of CK antibody and 0.1-5ug / ml of CD45 antibody.

[0013] As a preferred embodiment, the detection kit comprises the following components:

[0014] Fixative: 0.2-10% paraformaldehyde;

[0015] Membrane permeabilization solution: 0.1-5% polyethylene glycol octylphenyl ether;

[0016] Immunofluorescence staining solution: 0.1-5ug / ml TERT antibody, 0.1-5ug / ml CK antibody, 0.1-5ug / ml CD45 antibody, all of which are labeled with fluorescent groups;

[0017] Nuclear dye: 0.5-5uM DAPI in phosphate buffer;

[0018] Buffer II: phosphate buffer with a pH of 7.2-7.6 and a concentration of 0.15M-0.2M.

[0019] As a preferred embodiment, the fluorescent group labeled with the antibody is FITC, PE or APC.

[0020] As a preferred embodiment, the detection kit further comprises a red blood cell lysis reagent, and the red blood cell lysis reagent comprises the following components:

[0021] Red blood cell lysis solution: 0.1-5.0g potassium bicarbonate, 1-10g ammonium chloride, 0.01-0.1g EDTA-Na2, add double distilled water to 1000ml;

[0022] Buffer I: Phosphate buffer containing 0.1-3% bovine serum albumin, pH 7.2-7.6.

[0023] As a preferred embodiment, the components of the red blood cell lysis solution are: 1.0g potassium bicarbonate, 8.3g ammonium chloride, 0.037g EDTA-Na2, and double distilled water is added to 1000ml.

[0024] As a preferred embodiment, the detection kit further comprises a target cell enrichment reagent, and the target cell enrichment reagent comprises the following components:

[0025] Magnetic bead suspension: 0.1-5 mg / mL CD45 antibody-coated carboxyl magnetic beads;

[0026] Buffer II: phosphate buffer with a pH of 7.2-7.6 and a concentration of 0.15M-0.2M.

[0027] The abbreviations involved in the present invention are as follows:

[0028] TERT: telomerase reverse transcriptase

[0029] CK: Cytokeratin

[0030] CD45: expressed on all white blood cells, called the common leukocyte antigen

[0031] DAPI: 4',6-diamidino-2-phenylindole

[0032] FITC: fluorescein isothiocyanate

[0033] PE: Phycoerythrin

[0034] APC: Allophycocyanin

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] 1. The present invention provides a new tumor cell identification marker combination of TERT+ / CK+ / CD45- / DAPI+, wherein TERT is telomerase reverse transcriptase. Its advantages are: first, it can significantly improve the sensitivity and specificity of detection. Second, it can detect epithelial tumor cells.

[0037] 2. The monitoring results of the present invention show that for patients with urothelial carcinoma, the traditional EpCAM+ / CK+ / CD45- / DAPI+ detection method cannot distinguish between tumor cells and normal cells, while the TERT+ / CK+ / CD45- / DAPI+ detection method used in the present invention can clearly distinguish between tumor cells and normal cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is a cell image with different fluorescence characteristics obtained by scanning a glass slide using a high-speed fluorescence imaging device in Example 1 of the present invention; wherein BF is the cell field of view under white light, the detection of TERT protein is displayed in green, and DAPI is the staining of the cell nucleus displayed in blue.

[0039] Figure 2 This is a fluorescent image of tumor cells identified in a pleural effusion sample of a lung cancer patient in Example 2 of the present invention, wherein the framed cells are TERT-positive, DAPI-positive, CK-negative, and CD45-negative.

[0040] Figure 3 It is the statistical result of CD45 expression and TERT expression of the detected cells in the pleural effusion sample of Example 2 of the present invention; wherein TERT+ / CD45- indicates TERT positive expression and CD45 negative expression, and TERT- / CD45+ indicates TERT negative expression and CD45 positive expression.

[0041] Figure 4A This is a fluorescent image of tumor cells identified in a urine sample of a bladder cancer patient according to Example 3 of the present invention, wherein the framed portion shows a cell that is CD45 negative, TERT positive, CK positive, and DAPI positive.

[0042] Figure 4B This is a fluorescent image of tumor cells identified in a urine sample of a bladder cancer patient according to Example 3 of the present invention, wherein the framed portion shows a normal epithelial cell that is CD45 negative, EpCAM positive, CK positive, and DAPI positive.

[0043] Figure 5 It is the statistical result of CD45 expression and TERT expression of the detected cells in the urine sample of Example 3 of the present invention; wherein TERT+ / CD45- indicates TERT positive expression and CD45 negative expression, and TERT- / CD45+ indicates TERT negative expression and CD45 positive expression. DETAILED DESCRIPTION

[0044] The technical solution of the present invention is described in detail below in conjunction with the embodiments. The reagents and biological materials used below are all commercial products unless otherwise specified.

[0045] Example 1: Verification of telomerase expression in tumor cell lines

[0046] (1) Centrifuge 1 mL of MCF-7 cell line (500 g, 5 min), discard the supernatant, and add 2 mL of HBSS buffer to resuspend the cells;

[0047] (2) After counting the cell count, take an appropriate amount of the above resuspension and drop it on a glass slide and let it stand for 10 minutes;

[0048] (3) Remove the solution on the slide surface, add 100 μl of TERT-FITC antibody at a concentration of 2 μg / ml, and incubate at room temperature for 1 h;

[0049] (4) After the incubation, wash the chip three times with PBS, add 100 μl of DAPI onto the slide, and incubate at room temperature for 10 min;

[0050] (5) After incubation, the chip was washed three times with PBS and imaged using a high-speed fluorescence imaging device.

[0051] The imaging results are as follows Figure 1 As shown in the figure, a cell image with different fluorescence characteristics obtained by scanning a slide with a high-speed fluorescence imaging device is shown, where BF is the cell field of view under white light, TERT protein detection shows green, and DAPI is the staining of the cell nucleus shows blue. Under BF conditions, there are obvious cells with blue fluorescence and green fluorescence at the corresponding positions. This feature indicates that the tumor cell line MCF7 has high expression of telomerase.

[0052] Example 2: Pleural effusion sample testing in lung cancer patients

[0053] The testing method for pleural effusion samples from lung cancer patients includes the following steps:

[0054] (1) 20 mL of pleural effusion from lung cancer patients was centrifuged (500 g, 5 min) to separate cells, and 5 mL of red blood cell lysis buffer (BD Company) was added to lyse for 5 min in the dark, and centrifuged again (500 g, 5 min). The supernatant was discarded, and the cells were resuspended and washed with Hank's balanced salt solution (HBSS), centrifuged (500 g, 5 min), the supernatant was discarded, and 2 mL of HBSS was added to resuspend the cells;

[0055] (2) After counting the cell count, take an appropriate amount of the above resuspension and drop it on a glass slide and let it stand for 10 minutes;

[0056] (3) Remove the solution on the surface of the slide and drop 100 ul of antibody mixture onto the slide, where the antibody mixture includes: 2 ug / ml FITC-labeled TERT antibody, 1 ug / ml PE-labeled CK antibody, and 2 ug / ml APC-labeled CD45 antibody, and incubate at room temperature for 1 h;

[0057] (4) After the incubation, wash the chip three times with PBS, add 100 μl of DAPI onto the slide, and incubate at room temperature for 10 min;

[0058] (5) After incubation, the chip was washed three times with PBS and imaged using a high-speed fluorescence imaging device.

[0059] Test results such as Figure 2 As shown in the figure, the selected cells are TERT positive, DAPI positive, CK negative and CD45 negative, and it can be determined that the cells are tumor cells. Figure 2 The other cells shown in the figure do not have this fluorescent feature.

[0060] The corresponding TERT telomerase fluorescence signal intensity was further counted for CD45-positive cells and CD45-negative cells in some areas of the slide. Figure 3 , is the statistical result of CD45 expression and TERT expression of cells detected in pleural effusion samples; TERT+ / CD45- indicates TERT positive expression and CD45 negative expression, and TERT- / CD45+ indicates TERT negative expression and CD45 positive expression. Figure 3 The results show that for CD45-positive cells, TERT presents a low level of background signal value in the range of 1000-2000, while for CD45-negative, TERT-positive cells, the TERT signal value is above 5000. TERT-positive cells can be clearly distinguished from negative cells, indicating that the detection system of the present invention has high sensitivity.

[0061] Example 3: Urine testing of bladder cancer patients

[0062] The approach to urine testing for bladder cancer patients involves the following steps:

[0063] (1) Separate cells from 20 mL of urine from bladder cancer patients (500 g, 5 min), add 5 mL of red blood cell lysis buffer (BD Company) and lyse for 5 min in the dark, centrifuge again (500 g, 5 min), discard the supernatant, resuspend and wash the cells with Hank's balanced salt solution (HBSS), centrifuge (500 g, 5 min), discard the supernatant, and add 2 mL of HBSS to resuspend the cells;

[0064] (2) After counting the cell count, take an appropriate amount of the above resuspension and drop it on a glass slide and let it stand for 10 minutes;

[0065] (3A) Remove the solution on the slide and drop 100ul of antibody mixture onto the slide, where the antibody mixture includes: 2ug / ml FITC-labeled TERT antibody, 1ug / ml PE

[0066] The labeled CK antibody and 2ug / ml APC-labeled CD45 antibody were incubated at room temperature for 1h;

[0067] (3B) As a control group, add another 100ul antibody mixture onto the slide, where the antibody mixture includes: 2ug / ml FITC-labeled EpCAM antibody, 1ug / ml PE-labeled CK antibody and 2ug / ml APC-labeled CD45 antibody, and incubate at room temperature for 1h;

[0068] (4) After the incubation, wash the chip three times with PBS, add 100 μl of DAPI onto the slide, and incubate at room temperature for 10 min;

[0069] (5) After incubation, the chip was washed three times with PBS and imaged using a high-speed fluorescence imaging device.

[0070] Test results such as Figure 4A and 4B As shown, the boxed portion 4A shows a CD45-negative, TERT-positive, CK-positive and DAPI-positive cell, which can be determined to be a tumor cell. Figure 4A The remaining CD45-negative cells also have obvious CK positivity, but these cells have no obvious TERT positivity, and are epithelial cells but not tumor cells. Among them, 4B shows a normal epithelial cell that is CD45-negative, EpCAM-positive, CK-positive, and DAPI-positive. For normal urothelial cells, EpCAM is also highly expressed. Therefore, for patients with urothelial cancer, the traditional EpCAM+ / CK+ / CD45- / DAPI+ detection method cannot distinguish between tumor cells and normal cells, and the detection method of the present invention has more obvious advantages.

[0071] The corresponding TERT telomerase fluorescence signal intensities of CD45-positive cells and CD45-negative cells in some areas on the slide were further statistically analyzed. Figure 5 As shown, the results show that for CD45-positive cells, TERT presents a low level of background signal value in the range of 500-2000, while for CD45-negative, TERT-positive cells, the TERT signal value is above 5000. TERT-positive cells can be clearly distinguished from negative cells, indicating that the detection system of the present invention has high sensitivity and specificity.

[0072] The above are only some preferred embodiments of the present invention, and the present invention is not limited to the contents of the embodiments. For those skilled in the art, various changes and modifications can be made within the scope of the technical solution of the present invention, and any changes and modifications made are within the protection scope of the present invention.

Claims

1. Use of the marker combination TERT, CK, CD45, and DAPI in the preparation of a detection kit, characterized in that: The detection kit is used for detecting bladder cancer tumor cells in urine samples, and the tumor cells are epithelial-derived tumor cells.

2. Use of the marker combination TERT, CK, CD45, and DAPI according to claim 1 in preparing a detection kit, characterized in that: The detection kit includes 0.1-5ug / ml of TERT antibody, 0.1-5ug / ml of CK antibody and 0.1-5ug / ml of CD45 antibody.

3. Use of the marker combination TERT, CK, CD45, and DAPI in preparing a detection kit according to claim 1, characterized in that: The detection kit comprises the following components: Fixative: 0.2-10% paraformaldehyde; Membrane permeabilization solution: 0.1-5% polyethylene glycol octylphenyl ether; Immunofluorescence staining solution: 0.1-5ug / ml TERT antibody, 0.1-5ug / ml CK antibody, 0.1-5ug / ml CD45 antibody, all of which are labeled with fluorescent groups; Nuclear dye: 0.5-5uM DAPI in phosphate buffer; Buffer II: phosphate buffer with a pH of 7.2-7.6 and a concentration of 0.15M-0.2M.

4. Use of the marker combination TERT, CK, CD45, and DAPI in preparing a detection kit according to claim 3, characterized in that: The fluorescent group labeled with the antibody is FITC, PE or APC.

5. Use of the marker combination TERT, CK, CD45, and DAPI in preparing a detection kit according to claim 3, characterized in that: The detection kit also includes a red blood cell lysis reagent, which includes the following components: Red blood cell lysis solution: 0.1-5.0g potassium bicarbonate, 1-10g ammonium chloride, 0.01-0.1g EDTA-Na2, add double distilled water to 1000ml; Buffer I: Phosphate buffer containing 0.1-3% bovine serum albumin, pH 7.2-7.

6.

6. Use of the marker combination TERT, CK, CD45, and DAPI in preparing a detection kit according to claim 5, characterized in that: The components of the red blood cell lysis solution are: 1.0g potassium bicarbonate, 8.3g ammonium chloride, 0.037g EDTA-Na2, and double distilled water is added to 1000ml.

7. Use of the marker combination TERT, CK, CD45, and DAPI in preparing a detection kit according to claim 3, characterized in that: The detection kit also includes a target cell enrichment reagent, which includes the following components: Magnetic bead suspension: 0.1-5 mg / mL of carboxyl magnetic beads coated with CD45 antibody; Buffer II: phosphate buffer with a pH of 7.2-7.6 and a concentration of 0.15M-0.2M.

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