A method and kit for improving the sensitivity and specificity of circulating tumor cell detection

Abstract

This invention belongs to the field of biomedical technology, specifically relating to a method and kit for improving the sensitivity and specificity of circulating tumor cell (CTC) detection. The method first treats anticoagulated blood with erythrocyte lysis buffer, centrifuges to obtain a cell suspension (including leukocytes and circulating tumor cells); then, a radiosensitizer is added and the suspension is irradiated; next, single-cell gel electrophoresis is used to detect the degree of DNA damage in the test cells; finally, leukocytes and tumor cells are distinguished based on cell morphology using a fluorescence microscope or fluorescence scanner. This invention is the first to detect the number and type of circulating tumor cells (CTCs) in peripheral blood based on ionizing radiation and radiosensitization effects, according to the degree of DNA damage in tumor cells. Compared with existing technologies, this detection method and kit are not only simple and rapid, but also significantly improve detection sensitivity and specificity. Furthermore, it is independent of the physical properties of cells and tumor cell surface markers, requires a small sample volume, is highly versatile, and can detect CTCs in different types of solid tumors.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically a method and kit for improving the sensitivity and specificity of circulating tumor cell detection. Background Technology

[0002] Circulating tumor cells (CTCs) are tumor cells that detach from the primary tumor tissue or metastatic lesions and enter the peripheral blood circulation. CTCs have two main characteristics: first, they are rare; the number of CTCs in peripheral blood is extremely small, with a background blood cell count of up to 10⁻⁶ cells per mL of blood. 9 Firstly, there are only 1 to 10 CTCs; secondly, there is heterogeneity, with differences existing between different tumors, different locations within the same tumor, and different tumor cells within the same location. Due to the rarity and heterogeneity of CTCs, achieving accurate counting and type detection of CTCs presents a significant challenge.

[0003] Currently, the detection strategy for CTCs consists of two steps: (1) CTC enrichment, which involves enriching rare CTCs from a large number of blood cells; and (2) CTC identification, which involves identifying the number and type of tumor cells in the enriched cells. CTC enrichment mainly utilizes the physical characteristics (size, density, charge properties, etc.) and biological characteristics (surface-specific antigens) of CTCs; CTC identification involves recognizing tumor cell-specific markers, with immunofluorescence being the most commonly used technique. Based on the above strategy, many methods have been established for CTC detection.

[0004] The most classic method for isolating CTCs based on physical properties is membrane filtration separation enrichment technology (isolation by size of epithelial tumor cell, ISET). This method utilizes the difference in cell size, allowing smaller white blood cells to pass through the membrane while retaining larger tumor cells. Based on this, methods such as Rarecells@Diagnostic (RareCell, France), ScreenCell (ScreenCell, France), and CanPatrol (Guangzhou Yishan) in my country are relatively simple to operate, preserve the original cell morphology well, and have good sensitivity, especially for larger CTC clusters. However, the enrichment purity is relatively low, often containing a large number of normal blood cells, and smaller CTCs may be filtered directly, leading to a certain degree of missed detection. In recent years, companies such as Parsortix have introduced microfluidic technology based on cell size for CTC detection, but these methods also suffer from insufficient specificity and low purity. Density gradient centrifugation is a method for obtaining whole blood cells (CTCs) by high-speed centrifugation using a separation medium, such as OncoQuick (Germany's Greiner Bio-one). It utilizes a 50mL centrifuge tube with a built-in porous barrier to prevent contamination of CTCs with whole blood cells after gradient separation, thus improving enrichment efficiency. However, it is costly, requires a large blood volume, and is susceptible to the effects of centrifugation time and environmental factors. In summary, methods for separating CTCs based on physical properties rarely achieve both high efficiency and high purity simultaneously. This is mainly due to the overlap in physical properties between CTCs and leukocytes, making it difficult to effectively remove leukocytes from the separated CTCs.

[0005] Biologically based separation methods utilize the differences in specific cell membrane antigen expression between CTCs and blood cells, employing antibodies or aptamers bound to immunomagnetic beads for separation. The most classic method is the CellSearch system from Johnson & Johnson. Its basic principle is to use the CTC-specific antigen epithelial cell adhesion molecule (EpCAM) for positive enrichment and the leukocyte-specific antigen CD45 for negative enrichment. Simultaneously, magnetic beads are used to sort the enriched CTCs, and finally, the enriched cells are identified by immunofluorescence (anti-CD45, anti-CK8 / 18 / 19, and nuclear DAPI). However, studies have found that CTCs undergo an epithelial-mesenchymal transition (EMT) process after entering the circulatory system, resulting in low or no EpCAM expression, leading to a high false negative rate. Furthermore, the blood collection process can damage the body's epithelial tissue, causing blood to mix with normal epithelial cells, producing false positive results. Therefore, detecting CTCs using cell surface markers is prone to false positives or false negatives.

[0006] In addition, in recent years, CTC detection technologies that combine the physical and biological characteristics of CTCs have emerged, such as CTC-chip, HB-Chip, CTC-iChip and CytoSorter in my country. Although the development of microfluidic chip technology has greatly improved the detection / capture capability of CTCs, the sensitivity and specificity of CTC enrichment and capture still need to be improved because microfluidic chip technology is still based on the physical or biological characteristics of CTCs, or a combination of both.

[0007] Due to low detection sensitivity, existing CTC detection methods require at least 7.5 mL or 10 mL of blood to complete the test. Summary of the Invention

[0008] Radiation therapy is a common treatment for tumors, using high-energy radiation beams to kill tumor cells. Because tumor cells proliferate rapidly and have weak repair capabilities, their sensitivity to radiation is far greater than that of healthy cells. Radiosensitizers are chemical substances or drugs that enhance the tumor-killing effect by accelerating DNA damage and indirectly generating free radicals. To address the shortcomings of existing technologies, this invention proposes a novel method for directly detecting tumor cells (CTCs) based on the ionizing radiation sensitization mechanism, utilizing differences in the degree of DNA damage in cells, and improving detection sensitivity and specificity. This method uses the radiosensitizer CMNa to enhance the sensitivity of tumor cells to X-ray radiation, followed by single-cell gel electrophoresis to detect the degree of DNA double-strand breaks. Due to their high radiosensitivity, tumor cells exhibit significant DNA damage, displaying a comet-like cell morphology, which can be used to distinguish tumor cells from white blood cells.

[0009] The principle of the detection method of this invention is that, compared with normal blood cells, tumor cells often exhibit higher sensitivity to ionizing radiation, and the use of radiosensitizers can further enhance the radiosensitivity of tumor cells. Single-cell gel electrophoresis (SCGE) is a method that can detect the degree of DNA damage in cells at the single-cell level and is currently widely used in environmental monitoring, occupational exposure, and food safety. The biggest advantage of SCGE is that it can intuitively provide an image of each cell when DNA damage occurs, and the degree of DNA damage in the cell can be directly determined through the image. Therefore, by using the SCGE experiment to detect DNA damage in tumor cells after radiation sensitization and comparing it with background blood cells, a method for detecting CTCs based on the degree of DNA damage in cells can be established. That is, cells with a high degree of DNA damage are tumor cells, and cells with a low degree of DNA damage or no damage are normal blood cells. The method provided by this invention is called radiosensitizing-single-cell gel electrophoresis (rsSCGE).

[0010] Therefore, the primary objective of this invention is to provide a method for detecting CTCs, the technical solution of which is as follows:

[0011] 1) Red blood cells (RBCs) in peripheral anticoagulated blood were removed using red blood cell lysis buffer, and the cells were centrifuged to obtain a cell suspension (including white blood cells (WBCs) and circulating tumor cells (CTCs)).

[0012] 2) Add a radiosensitizer to the cell suspension and subject it to ionizing radiation;

[0013] 3) Single-cell gel electrophoresis was used to analyze the degree of DNA damage in cells;

[0014] 4) Counting and classifying tumor cells based on cell comet morphology: Tumor cells and white blood cells exhibit different cell morphologies. Tumor cells are counted and classified using a fluorescence microscope or fluorescence scanner.

[0015] Furthermore,

[0016] Step 1) Remove RBCs from peripheral anticoagulated blood using red blood cell lysis buffer.

[0017] Step 2) describes ionizing radiation including X-rays; the radiosensitizer is a reagent that enhances radiation-induced DNA damage, including sodium glycinate. Further, the ionizing radiation intensity is 2-6 Gy, and the sodium glycinate concentration is 10-30 mg / mL.

[0018] Step 3) The degree of cellular DNA damage was analyzed using single-cell gel electrophoresis, including neutral single-cell gel electrophoresis.

[0019] The cell morphology in step 4) is recorded using a fluorescence microscope or fluorescence scanner, which serves as a basis for determining the number and type of tumor cells.

[0020] Specifically: Four types of cellular comet morphologies were identified using a fluorescence microscope or fluorescence scanner, and the results were interpreted according to the following rules:

[0021] The first type is the leukocyte comet morphology: round cell shape, with no or few comet tails; the second type is the apoptotic leukocyte comet morphology: the cell comet head is smaller than the leukocyte, and the comet tail is completely separated from the head; the third type is the CTC comet morphology: the comet head is larger than the leukocyte, and the comet tail is completely connected to the head; the fourth type is the apoptotic CTC comet morphology: the comet head is larger than the leukocyte, and the comet tail is completely separated from the head.

[0022] The tumor cell types detected include different cell lines of solid tumors such as breast cancer, nasopharyngeal carcinoma, colorectal cancer, lung cancer, prostate cancer, gastric cancer, thyroid cancer, esophageal cancer, liver cancer, and lymphoma.

[0023] This invention is used for detection purposes other than diagnosis or treatment.

[0024] A second objective of this invention is to provide a circulating tumor cell detection kit that complements the method described above, comprising: erythrocyte lysis buffer, radiosensitizer, and single-cell gel electrophoresis reagent.

[0025] A third objective of this invention is to provide the application of erythrocyte lysis buffer, radiosensitizer, and single-cell gel electrophoresis reagent in the preparation of the circulating tumor cell detection kit.

[0026] Compared with existing CTC detection technologies, this invention has the following advantages:

[0027] 1. Not dependent on tumor cell surface antigens: It avoids the problem of heterogeneity in marker expression caused by CTC epithelial-mesenchymal transition, as well as the interference of normal epithelial cells on the results.

[0028] 2. Not dependent on the size difference between tumor cells and white blood cells: Tumor cells are on average larger than white blood cells, but monocytes and some lymphocytes are also relatively large, and tumor cells with high degree of malignancy will be smaller; in addition, tumor cell lines are larger than tumor cells in the human body, which leads to a theoretically high recovery rate of CTC detection, but the effect is not ideal in the real world.

[0029] 3. High specificity and sensitivity: After radiation sensitization, due to differences in DNA damage, different comet cell morphologies are observed after single-cell gel electrophoresis. The differences in cell morphology can be used to distinguish tumor cells from blood cells; even a single tumor cell in 1 mL of blood can be detected.

[0030] 4. Small sample size: 0.5 mL of blood can detect 1 to 10 tumor cells.

[0031] 5. Simple and fast method: Traditional methods require tumor cell enrichment and identification, which involves many steps and takes up to 10 hours to complete the test; this method only requires lysing red blood cells, and the obtained cell suspension can be directly tested, and the test can be completed within 4 hours.

[0032] 6. Low cost and wide applicability: No complex equipment or operating procedures are required; it is applicable to the detection of various types of CTCs and has wide versatility.

[0033] 7. This invention can be used for scientific research on CTC detection. Attached Figure Description

[0034] Figure 1 : A schematic diagram of the process for detecting circulating tumor cells (CTC) in this invention.

[0035] Figure 2 Results of single-cell gel electrophoresis analysis of breast cancer cell lines and peripheral blood mononuclear cells (PBMCs) treated with CMNa combined with X-rays;

[0036] in: Figure 2 A shows single-cell gel electrophoresis images of breast cancer cell lines MCF7, SKBR3, MDA-MB-231 and normal blood cells PBMC after radiation sensitization. Figure 2 B is a bar chart showing the statistical results of DNA damage detection after cell radiation sensitization. An asterisk indicates that the difference between tumor cells and PBMCs is statistically significant. P < 0.05, P < 0.01, P < 0.001, P < 0.0001.

[0037] Figure 3 Results of single-cell gel electrophoresis analysis of nasopharyngeal carcinoma cell lines and PBMCs treated with CMNa combined with X-rays;

[0038] in: Figure 3 A shows single-cell gel electrophoresis images of nasopharyngeal carcinoma cell lines CNE2, CNE1, 5-8F and normal blood cell PBMCs after radiation sensitization. Figure 3B is a bar chart showing the statistical results of DNA damage detection after cell radiation sensitization. An asterisk indicates that the difference between tumor cells and PBMCs is statistically significant. P < 0.05, P < 0.01, P < 0.001, P < 0.0001.

[0039] Figure 4 Results of single-cell gel electrophoresis analysis of colorectal cancer cell lines and PBMCs after treatment with CMNa combined with X-rays;

[0040] in: Figure 4 A shows single-cell gel electrophoresis images of colorectal cancer cell lines HT-29, HCT-116, SW620, and normal blood cells PBMC after radiation sensitization. Figure 4 B is a bar chart showing the statistical results of DNA damage detection after cell radiation sensitization. An asterisk indicates that the difference between tumor cells and PBMCs is statistically significant. P < 0.05, P < 0.01, P < 0.001, P < 0.0001.

[0041] Figure 5 Results of single-cell gel electrophoresis analysis of lung cancer cell lines and PBMCs treated with CMNa combined with X-rays;

[0042] in: Figure 5 A shows single-cell gel electrophoresis images of lung cancer cell lines A549 and PC-9 and normal blood cells PBMC after radiation sensitization. Figure 5 B is a bar chart showing the statistical results of DNA damage detection after cell radiation sensitization. An asterisk indicates that the difference between tumor cells and PBMCs is statistically significant. P < 0.05, P < 0.01, P < 0.001, P < 0.0001.

[0043] Figure 6 Results of single-cell gel electrophoresis analysis of prostate cancer cell lines and PBMCs treated with CMNa combined with X-rays;

[0044] in: Figure 6 A shows single-cell gel electrophoresis images of prostate cancer cell lines LNCaP and PC-3 and normal blood cells PBMC after radiation sensitization. Figure 6B is a bar chart showing the statistical results of DNA damage detection after cell radiation sensitization. An asterisk indicates that the difference between tumor cells and PBMCs is statistically significant. P < 0.05, P < 0.01, P < 0.001, P < 0.0001.

[0045] Figure 7 Results of single-cell gel electrophoresis analysis of gastric cancer cell lines and PBMCs treated with CMNa combined with X-rays;

[0046] in: Figure 7 A shows single-cell gel electrophoresis images of gastric cancer cell lines MGC-803 and HGC-27 and normal blood cell PBMCs after radiation sensitization. Figure 7 B is a bar chart showing the statistical results of DNA damage detection after cell radiation sensitization. An asterisk indicates that the difference between tumor cells and PBMCs is statistically significant. P < 0.05, P < 0.01, P < 0.001, P < 0.0001.

[0047] Figure 8 Results of single-cell gel electrophoresis analysis of thyroid cancer cell lines and PBMCs treated with CMNa combined with X-rays;

[0048] in: Figure 8 A shows single-cell gel electrophoresis images of thyroid cancer cell lines BCPAP and KTC-1 and normal blood cell PBMCs after radiation sensitization. Figure 8 B is a bar chart showing the statistical results of DNA damage detection after cell radiation sensitization. An asterisk indicates that the difference between tumor cells and PBMCs is statistically significant. P < 0.05, P < 0.01, P < 0.001, P < 0.0001.

[0049] Figure 9 Results of single-cell gel electrophoresis analysis of esophageal cancer cell lines and PBMCs after treatment with CMNa combined with X-rays;

[0050] in: Figure 9 A shows single-cell gel electrophoresis images of esophageal cancer cell lines KYSE150 and TE-10 and normal blood cell PBMCs after radiation sensitization. Figure 9B is a bar chart showing the statistical results of DNA damage detection after cell radiation sensitization. An asterisk indicates that the difference between tumor cells and PBMCs is statistically significant. P < 0.05, P < 0.01, P < 0.001, P < 0.0001.

[0051] Figure 10 Results of single-cell gel electrophoresis analysis of hepatocellular carcinoma cell lines and PBMCs after treatment with CMNa combined with X-rays;

[0052] in: Figure 10 A shows single-cell gel electrophoresis images of liver cancer cell lines HepG2 and Hep3B2.1-7 and normal blood cells PBMC after radiation sensitization; Figure 10 B is a bar chart showing the statistical results of DNA damage detection after cell radiation sensitization. An asterisk indicates that the difference between tumor cells and PBMCs is statistically significant. P < 0.05, P < 0.01, P < 0.001, P < 0.0001.

[0053] Figure 11 Results of single-cell gel electrophoresis analysis of lymphoma cell lines and PBMCs treated with CMNa combined with X-rays;

[0054] in: Figure 11 A shows single-cell gel electrophoresis images of the Raji lymphoma cell line and normal blood cell PBMCs after radiation sensitization. Figure 11 B is a bar chart showing the statistical results of DNA damage detection after cell radiation sensitization. An asterisk indicates that the difference between tumor cells and PBMCs is statistically significant. P < 0.05, P < 0.01, P < 0.001, P < 0.0001.

[0055] Figure 12 A fluorescence scanner was used to scan the results of single-cell gel electrophoresis and obtain images of cell types.

[0056] Figure 13 Immunofluorescence assay for identifying tumor cells and leukocytes;

[0057] in: Figure 13 A is the identification of different tumor cell lines and leukocytes using cell immunofluorescence assay; Figure 13B represents the identification of tumor cells and white blood cells in different tumor patients using cellular immunofluorescence assay.

[0058] Figure 14 Immunofluorescence assay revealed three cell types in single-cell gel electrophoresis results;

[0059] The cell morphologies were as follows: apoptotic leukocytes: small head, long, discontinuous comet tail; CTCs: long, continuous comet tail; apoptotic CTCs: large head, long, discontinuous comet tail.

[0060] Figure 15: Identification of CTCs by fluorescence in situ hybridization (FISH);

[0061] in: Figure 15 A is the breast cancer cell line AU565, which was identified as a CTC using this invention and was also confirmed as a CTC by FISH method; Figure 15 B represents CTCs identified in the peripheral blood of HER2-positive breast cancer patients by this invention, and also confirmed as CTCs by FISH method; White arrow: Breast cancer cells with high degree of cell DNA damage and positive HER2 amplification; Yellow arrow: Normal blood cells with low degree of cell DNA damage and no HER2 amplification.

[0062] Figure 16: Mass spectrometry proteomics comparison of CTCs and apoptotic CTCs with leukocytes;

[0063] in: Figure 16 A is a comparison of CTCs and differentially expressed proteins in leukocytes based on mass spectrometry proteomics; Figure 16 B. Comparison of differentially expressed proteins between apoptotic CTCs and leukocytes based on mass spectrometry proteomics. The upregulated proteins marked in the figure are tumor-derived proteins, while the downregulated proteins are leukocyte-specific proteins. The results in the figure confirm that the cells detected in this invention are tumor cells.

[0064] Figure 17 Mass spectrometry proteomics comparisons were performed between apoptotic CTCs and between apoptotic leukocytes and leukocytes, respectively.

[0065] in: Figure 17 A represents a comparison of differentially expressed proteins between apoptotic CTCs and CTCs based on mass spectrometry proteomics. Figure 17 B represents a comparison of differentially expressed proteins between apoptotic WBCs and WBCs based on mass spectrometry proteomics.

[0066] Figure 18 shows the analysis of the degree of DNA damage in leukocytes of tumor patients and healthy controls after ionizing radiation sensitization. Detailed Implementation

[0067] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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.

[0068] Unless otherwise specified, the raw materials used in the embodiments of this invention, such as various tumor cells, can be obtained commercially (all from ATCC). Clinical samples were obtained from Hunan Cancer Hospital and used for research with the consent of the patients or their families.

[0069] Example 1. Single-cell gel electrophoresis detection of DNA damage during the ionization radiation sensitization process of different tumor cell lines and normal blood cell PBMCs.

[0070] This embodiment aims to evaluate the differences in DNA damage caused by radiation sensitization treatment to different tumor cell lines and normal blood cells (PBMCs), thereby verifying the theoretical basis and feasibility of the present invention for detecting circulating tumor cells (CTCs).

[0071] First, the following single-cell suspensions were prepared: breast cancer cell lines (MCF7, SKBR-3, MDA-MB-231), nasopharyngeal carcinoma cells (CNE2, CNE1, 5-8F), colorectal carcinoma cells (HT-29, HCT-116, SW620), lung cancer cells (A549, PC-9), prostate cancer cells (LNCaP, PC-3), gastric cancer cells (MGC-803, HGC-27), thyroid cancer cells (BCPAP, KTC-1), esophageal cancer cells (KYSE150, TE-10), liver cancer cells (HepG2, Hep3B2.1-7), and Raji lymphoma cells as CTC models; peripheral blood mononuclear cells (PBMCs) from healthy individuals were obtained by gradient centrifugation using Ficoll separation medium as normal blood cell models. Tumor cell lines were cultured according to the ATCC recommended medium type, with the addition of 10% fetal bovine serum to prepare complete medium, and cultured in a 37℃ / 5% CO2 incubator. Cells were collected when they reached the logarithmic growth phase. Then, the prepared single-cell suspensions were treated with 4 Gy X-rays, 20 mg / mL CMNa, and 4 Gy X-rays combined with 20 mg / mL CMNa for about 10 minutes respectively. Finally, the degree of DNA damage in the cells was detected by neutral single-cell gel electrophoresis (neutral SCGE), and the cells were photographed under a fluorescence microscope. The degree of DNA damage in the cells was quantitatively analyzed using CASP comet analysis software. Figures 2-11The results of single-cell gel electrophoresis analysis of different cell types after X-ray combined with CMNa treatment are presented. Table 1 lists the percentage of cell tail DNA (%Tail DNA) obtained by CASP Comet analysis software for each group of cells under different treatment methods, and bar charts are drawn accordingly to visually display the comparison results. The charts show that the degree of DNA damage in various tumor cells treated with X-ray + CMNa was significantly higher than that in the X-ray or CMNa treatment groups alone, and the differences were statistically significant (P < 0.001). In contrast, the degree of DNA damage in normal blood cells (PBMCs) remained low under all three treatment conditions. This method can significantly enhance the radiosensitivity of tumor cells, but has little effect on normal PBMCs. The strategy of identifying tumor cells and normal blood cells based on the degree of DNA damage is highly feasible and specific, providing theoretical and experimental basis for subsequent CTC detection.

[0072]

[0073] Example 2. A method for detecting circulating tumor cells in peripheral anticoagulated blood based on ionizing radiation-sensitized single-cell gel electrophoresis.

[0074] Figure 1 This is a flowchart of the present invention for detecting circulating tumor cells in peripheral blood based on ionizing radiation-sensitized single-cell gel electrophoresis; Figure 12 The results and cell types were obtained using a fluorescence scanner.

[0075] For detailed operating procedures, please refer to this embodiment.

[0076] 1. Preparation of cell suspension:

[0077] (1) 0.5 mL of fresh anticoagulated blood from cancer patients (breast cancer and nasopharyngeal carcinoma) was added to 1.5 mL of red blood cell lysis buffer (ammonium chloride 150 mmol / L, potassium bicarbonate 10 mmol / L, Na2EDTA 0.1 mmol / L, pH adjusted to 7.2), and the mixture was gently blown to mix. The mixture was lysed at 4°C for 15 min.

[0078] (2) Centrifuge at 800~1000 rpm for 5 min and discard the upper red clear liquid;

[0079] (3) Collect the precipitate, wash with PBS 1-2 times, and centrifuge to collect the cells.

[0080] 2. Treatment of test cells with the radiation sensitizer CMNa and ionizing X-ray radiation:

[0081] (1) Adjust the volume of the cell suspension and add 40 mg / mL CMNa at a 1:1 volume ratio to make the final concentration 20 mg / mL. After mixing the cells by pipetting, transfer them to 12-well plates and incubate at room temperature in the dark for 1 h.

[0082] (2) The X-ray irradiator irradiates the well plate containing the test cells with a total X-ray amount of 4 Gy for about 10 minutes.

[0083] 3. Neutral single-cell gel electrophoresis to detect the degree of DNA damage in the test cells:

[0084] (1) Preparation of cell suspension: After irradiation, transfer the cell suspension in the well plate to a 1.5 mL conical centrifuge tube, centrifuge at 500 g for 5 min, and discard the supernatant. Adjust the volume of cells to 1000 μL with PBS for later use;

[0085] (2) Gel preparation: The normal melting point agarose gel and the low melting point agarose gel were preheated and melted. The concentration of the normal melting point agarose gel was 0.75%, and the concentration of the low melting point agarose gel was 1%.

[0086] (3) Laying the bottom layer gel: Use a clean glass slide of 18cm×15cm as the electrophoresis plate, and evenly spread 3000 μL of normal melting point agarose gel on the electrophoresis plate. Let it stand in a refrigerator at 4℃ for 2min. To avoid gel detachment during electrophoresis, the electrophoresis plate after gel laying can be placed in an oven at 50℃ for 15min to dry;

[0087] (4) Spread the top layer of gel: Add the cell suspension to be tested to 3000 μL of low melting point agarose gel (ensure that the volume ratio of cell suspension to low melting point agarose gel is 1:3), mix well, spread the gel evenly on the bottom layer of gel, and let it stand in a refrigerator at 4°C for 2 min.

[0088] (5) Cell lysis: Prepare neutral cell lysis buffer in advance, place the gel electrophoresis plate in the cell lysis buffer, and freeze at 4°C for at least 2 hours; after lysis, wash the gel electrophoresis plate in double-distilled water 3 times, 5 minutes each time;

[0089] (The neutral lysis buffer formula is as follows: Weigh 58.44g NaCl, 5.584g EDTA Na2∙2H2O, and 0.6057g Tris, add 445mL of double-distilled water, and heat to dissolve. Before use, add 10% DMSO and 1% Triton X-100, mix thoroughly, and adjust the pH to 8.0.)

[0090] (6) Unwinding: After lysis, the gel electrophoresis plate was transferred to the pre-cooled electrophoresis buffer (1×TBE solution) at 4℃ and unwound at 4℃ for 40 min to unwind the cell DNA into single strands;

[0091] (7) Electrophoresis: After unwinding, place the gel electrophoresis plate on a horizontal electrophoresis tank. The surface of the electrophoresis buffer (1×TBE solution) should be about 0.2 cm above the gel surface. Electrophoresis should be performed at a constant voltage of 20V for 20 min. After electrophoresis, the gel should be dehydrated in sequence with 70%, 85%, and 100% ethanol and then air-dried.

[0092] (8) Results observation and data analysis: After adding nucleic acid fluorescent dye to stain the cell DNA in the gel in the dark for 5 min, wash away the excess dye with double distilled water; use green as the excitation light source and perform image analysis with a fluorescence microscope or fluorescence scanner.

[0093] Figure 12 The four types of leukocyte comet morphology are as follows: the first type is the leukocyte comet morphology, characterized by round cells with little or no comet tail; the second type is the apoptotic leukocyte comet morphology, characterized by a comet head significantly smaller than the leukocyte and a tail completely separated from the head; the third type is the CTC comet morphology, characterized by a comet head larger than or slightly larger than the leukocyte and a distinct comet tail, with the tail completely connected to the head; and the fourth type is the apoptotic CTC comet morphology, characterized by a comet head larger than or slightly larger than the leukocyte and a tail completely separated from the head.

[0094] Example 3. Cell immunofluorescence technique was used to identify the CTCs and their types detected in this invention.

[0095] The study used breast cancer cell lines MCF7, nasopharyngeal carcinoma cell line CNE-2, and colorectal carcinoma cell line HT-29. Clinical samples were obtained from peripheral blood of patients with the corresponding cancer types. Given that most solid tumors originate from epithelial tissue, cytokeratin (CK) was considered a specific marker for CTC identification; CK-positive cells were tumor cells, while CK-negative cells were normal blood cells.

[0096] refer to Figure 13 A. Identification using tumor cell lines. Cultured tumor cell lines were infused into the peripheral blood of healthy volunteers and detected using the method provided in this invention. Identification was then performed using a cell immunofluorescence assay, as follows: MCF7 breast cancer cells, CNE-2 nasopharyngeal carcinoma cells, and HT-29 colorectal carcinoma cells (1×10⁻⁶) were counted. 6 (Preferably a few cells) was mixed into 1 mL of peripheral blood from a healthy volunteer and analyzed according to the method described in Example 2. Subsequently, the cells in the gel after electrophoresis were analyzed by immunofluorescence according to the standard cellular immunofluorescence procedure. The results showed that cells with high DNA damage (showing tails) were CK positive, while cells with low or no DNA damage were CK negative, thus confirming that the cells with high DNA damage were indeed tumor cells.

[0097] refer to Figure 13B describes the use of immunofluorescence staining to identify tumor cells from peripheral blood of patients with breast cancer, nasopharyngeal carcinoma, and colorectal cancer. The procedure is as follows: 2 mL of peripheral blood was collected from patients with breast cancer, colorectal cancer, and nasopharyngeal carcinoma. The cells were analyzed according to the method described in Example 2. Subsequently, immunofluorescence detection was performed on the cells in the gel after electrophoresis following standard immunofluorescence procedures. The results showed that cells with high DNA damage (exhibiting tailing) were CK-positive, while cells with low or no DNA damage were CK-negative.

[0098] refer to Figure 14 To identify different cell types in single-cell gel electrophoresis results, three types of cells with high DNA damage were found in clinical specimens from tumor patients, compared to leukocytes. The first type, "tightly connected," had a comet head larger than or slightly larger than leukocytes, with the tail completely connected to the head. The second type, "large-headed breakage," had a comet head larger than or slightly larger than leukocytes, but the tail was completely disconnected from the head. The third type, "small-headed breakage," had a comet head smaller than leukocytes, and the tail was completely disconnected from the head. Immunofluorescence results confirmed that the first two types of cells were closed-cell tumor cells (CTCs), termed "typical CTCs" and "apoptotic CTCs," respectively; the third type of cells were leukocytes (apoptotic WBCs).

[0099] Example 4. The CTCs detected by this invention and their types were identified using fluorescence in situ hybridization (FISH).

[0100] In this embodiment, tumor cell lines and clinical blood samples were used to confirm the CTCs and their types detected by this invention. HER2 positivity is a special molecular pathological type of breast cancer, specifically characterized by an increased copy number of the HER2 gene, which can be detected by HER2 amplification fluorescence in situ hybridization, and is currently an important clinical method for determining the molecular pathological type of breast cancer. Based on the characteristics of HER2-positive breast cancer cells, this embodiment selected the HER2 gene-amplified breast cancer cell line AU565, and clinically diagnosed HER2-positive breast cancer patients. (Reference) Figure 15 A, Counting breast cancer cells AU565 (1×10⁻⁶) 6 (Preferably 100 cells), mixed into 1 mL of peripheral blood from a healthy volunteer, and tested according to the method provided in Example 2. Subsequently, fluorescence in situ hybridization was performed on the cells in the gel after electrophoresis using a HER2 gene amplification detection kit (Vysis, Abbott). The results showed that cells with high DNA damage (exhibiting tails) showed HER2 gene amplification, while cells with low or no DNA damage did not show HER2 gene amplification.

[0101] refer to Figure 15B. Two mL of peripheral blood was collected from patients with pathologically confirmed HER2-positive breast cancer and tested according to the method provided in Example 2. Subsequently, fluorescence in situ hybridization (FISH) was performed on the cells in the electrophoresis gel using a HER2 gene amplification detection kit (Vysis, Abbott). The results showed that in clinical blood samples, cells with high DNA damage (tailing) exhibited HER2 gene amplification, indicating tumor cells; while cells with low or no DNA damage did not amplify HER2. These results demonstrate that the established CTC detection method has good specificity.

[0102] Example 5. Identification of the CTCs and their types detected by this invention using proteomics mass spectrometry.

[0103] In this embodiment, clinical blood samples were used to confirm the CTCs and their types detected by this invention. Taking breast cancer as an example, peripheral blood was collected from one breast cancer patient, and CTCs were detected using this invention. Subsequently, CTCs, apoptotic CTCs, apoptotic WBCs, and WBCs were picked under a microscope and subjected to proteomics mass spectrometry analysis. The results are shown in Tables 2 and 3. Compared with the WBC group, both CTCs and apoptotic CTCs showed significantly increased expression of tumor-derived proteins, indicating that these cells were indeed CTCs. Simultaneously, both apoptotic CTCs and apoptotic WBCs showed significantly increased expression of apoptosis-related proteins. (Reference) Figure 16 The expression levels of tumor-derived proteins in the CTC group and the apoptotic CTC group were significantly higher than those in the WBC group, while the expression levels of leukocyte-specific proteins were significantly lower than those in the WBC group; Reference Figure 17 Compared with the general CTC group, the apoptotic CTC group showed a significant increase in the expression of apoptosis-related proteins, and the apoptotic WBC group also showed a significant increase in the expression of apoptosis-related proteins compared with the general WBC group. In summary, all the above results indicate that the established CTC detection method has good specificity and can accurately identify CTCs.

[0104]

[0105] Note: nd indicates that the protein was not detected; — indicates that it is not applicable.

[0106]

[0107] Note: nd indicates that the protein was not detected; — indicates that it is not applicable.

[0108] Example 6. The Spiking assay was used to study the high sensitivity of the present invention in detecting CTCs.

[0109] To investigate the sensitivity of this invention for detecting CTCs, a peripheral blood model of cancer patients was constructed by adding cultured tumor cell lines to the peripheral blood of healthy volunteers. Taking breast cancer, nasopharyngeal carcinoma, and colorectal cancer as examples, the procedure is as follows: 1 mL of peripheral blood from healthy volunteers was taken, and 1, 5, 10, and 100 tumor cells were added, respectively. The experiment was then conducted according to the steps in Example 2. The results are shown in Tables 4 and 5. The overall recovery rate of this method ranged from 82.2% to 93.1%. The coefficient of variation increased as the number of added tumor cells decreased, mainly because a small number of cells easily affects the results. Even when the number of added cells was as low as one, a good recovery rate was still observed. The results of this example demonstrate that the CTC detection method established in this invention has excellent sensitivity and stability, and can effectively detect extremely low numbers of tumor cells.

[0110]

[0111]

[0112] Example 7: CTC detection in newly diagnosed patients with breast cancer and nasopharyngeal carcinoma

[0113] This invention detected an average of 3.9 CTCs / mL in 100 newly diagnosed breast cancer patients and an average of 8.22 CTCs / mL in 134 newly diagnosed nasopharyngeal carcinoma patients, significantly higher than the classic CTC detection method (CellSearch's method). The degree of DNA damage in tumor cells of cancer patients is significantly higher than that in healthy cells. Figure 18 ).

[0114] The embodiments described above are not related to diagnosis or treatment, but are merely for the purpose of verifying and highlighting the advantages of the detection method of the present invention compared with other existing detection methods, and are still used for scientific research purposes.

Claims

1. A method for improving the sensitivity and specificity of circulating tumor cell detection, characterized in that, Includes the following steps: 1) Red blood cells in peripheral anticoagulated blood were removed using red blood cell lysis buffer, and centrifugation was used to obtain a cell suspension containing white blood cells and circulating tumor cells; 2) Add a radiosensitizer to the cell suspension and subject it to ionizing radiation; 3) Single-cell gel electrophoresis was used to analyze the degree of DNA damage in cells; 4) Counting and classifying tumor cells based on cell comet morphology: Tumor cells and white blood cells exhibit different cell morphologies. Tumor cells are counted and their types are identified using a fluorescence microscope or fluorescence scanner.

2. The method according to claim 1, characterized in that, The radiosensitizer mentioned in step 2) is a reagent that enhances radiation-induced DNA damage.

3. The method according to claim 1, characterized in that, Step 2) The ionizing radiation sensitizer includes sodium glycinate; the ionizing radiation includes X-rays; preferably, the ionizing radiation intensity is 2-6 Gy and the sodium glycinate concentration is 10-30 mg / mL.

4. The method according to claim 1, characterized in that, Step 3) The degree of cellular DNA damage was analyzed using single-cell gel electrophoresis, including neutral single-cell gel electrophoresis.

5. The method according to claim 1, characterized in that, The cell morphology in step 4) is recorded using a fluorescence microscope or fluorescence scanner, which serves as a basis for determining the number and type of tumor cells.

6. The method according to claim 5, characterized in that, Four types of cellular comet morphologies were identified using fluorescence microscopy or a fluorescence scanner: the first type is the leukocyte comet morphology: round cells with little or no comet tail; the second type is the apoptotic leukocyte comet morphology: the comet head is smaller than the leukocyte, and the tail is completely separated from the head; the third type is the CTC comet morphology: the comet head is larger than the leukocyte, and the tail is completely connected to the head; the fourth type is the apoptotic CTC comet morphology: the comet head is larger than the leukocyte, and the tail is completely separated from the head.

7. The method according to claim 1, characterized in that, Tumor cell types include different cell lines of solid tumors such as breast cancer, nasopharyngeal carcinoma, colorectal cancer, lung cancer, prostate cancer, gastric cancer, thyroid cancer, esophageal cancer, liver cancer, and lymphoma.

8. The method according to claim 1, characterized in that, For testing purposes other than diagnosis or treatment.

9. A circulating tumor cell detection kit compatible with the method according to any one of claims 1-8, characterized in that, include: Red blood cell lysis buffer, radiosensitizer, single-cell gel electrophoresis reagent.

10. The use of erythrocyte lysis buffer, radiosensitizer, and single-cell gel electrophoresis reagent in the preparation of the circulating tumor cell detection kit according to claim 9.

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