A marker group, a kit and a device for capturing circulating tumor cells

Abstract

The present invention belongs to the field of tumor diagnosis, and specifically relates to a marker group, a kit and a device for capturing circulating tumor cells. The marker group includes a first marker and a second marker; wherein: the first marker is EPCAM; the second marker is one or a combination of two or more of CD9, CD41, THBS1, RGS18, and RGS10; the circulating tumor cells are pancreatic cancer circulating tumor cells and / or breast cancer circulating tumor cells. The present invention significantly improves the enrichment and detection rate of CTC in the patient's blood by using antibodies against new surface markers of pancreatic cancer or breast cancer circulating tumor cells (CTC), effectively reducing the possibility of missed detection; at the same time, it enhances the capture and enrichment efficiency of CTC, providing strong support for dynamic tumor monitoring, prognosis evaluation and personalized precision treatment of cancer patients, and has broad market value and application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of tumor diagnosis, and in particular relates to a marker group, a kit and a device for capturing circulating tumor cells. Background Art

[0002] Circulating tumor cells (CTCs) are cancer cells released into the blood circulation by primary tumors or metastatic lesions and are considered to be the "seeds" of cancer metastasis. The presence and number of CTCs are closely related to the patient's risk of metastasis, treatment response and survival rate. Therefore, capturing and analyzing CTCs can provide key information for early diagnosis, prognosis prediction and personalized treatment of cancer. However, since the content of CTCs in peripheral blood is extremely rare, with only 1 to 10 CTCs per 1 billion blood cells, their detection and separation have always been technical challenges in tumor research and clinical applications. Existing CTC detection technologies mainly include immunological methods, separation methods based on physical properties (such as density gradient centrifugation) and microfluidics. Current microfluidics technology has shown great potential in CTC capture. Microfluidic chips can achieve efficient enrichment and capture of CTCs by precisely controlling fluid dynamics conditions and utilizing cell size differences. However, due to the uneven size of circulating tumor cells and the extremely low proportion of cells, the separation method of microfluidics lacks specificity. With the development of CTC detection technology, the advantages of immune screening are gradually being reflected. Immunoassay detection of CTCs mainly relies on the high affinity binding between specific markers on the surface of tumor cells and antibodies. By combining tumor-related antibodies (such as antibodies against EPCAM, CK, HER2, etc.) with detection equipment, CTCs can be specifically captured or labeled. Immunoassays are divided into two categories: immunoenrichment and immunostaining. Immunoenrichment is to enrich CTCs in the blood by combining antibodies with specific markers on the surface of CTCs, while immunostaining is to use antibodies to label CTCs and then detect them using a flow cytometer or fluorescence microscope.

[0003] At present, the mainstream method of CTC capture relies on tumor cell-specific surface markers, such as epithelial cell adhesion molecule (EPCAM). Many commercial devices (such as the CellSearch system) enrich CTCs based on EPCAM antibodies and have been widely used in CTC detection of various cancers. However, EPCAM expression is not high or heterogeneous in some tumor types, resulting in the failure of some CTCs to be effectively captured. In addition, cancer cells may undergo epithelial-mesenchymal transition (EMT) during metastasis, during which the expression of EPCAM may decrease, further reducing the effectiveness of EPCAM-based capture methods. Using this single marker to enrich and screen CTCs will inevitably lead to a certain degree of missed detection. Therefore, identifying new tumor cell surface markers and developing new circulating tumor cell CTC capture devices will greatly promote the development of CTC detection technology.

[0004] Therefore, based on this, the technical solution of the present invention is proposed. Summary of the invention

[0005] In view of the shortcomings of existing CTC detection technology, the present invention performs single-cell sequencing of CTCs and WBCs of pancreatic cancer and breast cancer patients, and uses bioinformatics technology to perform big data analysis on the sequencing results, and summarizes a series of new markers unique to the surface of CTCs.

[0006] Furthermore, the results of protein expression profiles and CTC expression profile group sequencing of a large number of pancreatic cancer or breast cancer patients are compared to find common CTC surface-specific markers. On this basis, new markers are used to perform enrichment (capture) tests on pancreatic cancer cell lines and pancreatic cancer patient blood, select surface markers with enrichment capabilities, optimize the best combination of new capture antibodies, and perform immune enrichment screening. The antibodies or antibody combinations of new surface markers screened by the above method greatly improve the capture and enrichment efficiency of CTCs, which can provide support for clinical evaluation of treatment effects and realization of individual precision treatment, and have huge market value and application prospects.

[0007] The relevant product is used to detect the level of cell surface marker gene expression products in the sample. If the detected level of the cell surface marker gene expression product is higher than the reference level, the presence of pancreatic cancer or breast cancer circulating tumor cells CTC is determined.

[0008] In order to solve the problems existing in the prior art, the present invention provides a marker group for capturing circulating tumor cells, wherein the marker group includes a first marker and a second marker; wherein:

[0009] The first marker is EPCAM;

[0010] The second marker is one or a combination of two or more of CD9, CD41, THBS1, RGS18, and RGS10;

[0011] The circulating tumor cells are pancreatic cancer circulating tumor cells;

[0012] And / or, the circulating tumor cells are breast cancer circulating tumor cells.

[0013] Based on the same technical concept, another embodiment of the present invention is to provide a use of a marker group in preparing a kit for capturing circulating tumor cells.

[0014] Based on the same technical concept, another embodiment of the present invention is to provide a kit, which includes a detection reagent for detecting a marker group.

[0015] Preferably, the kit further comprises a detection antibody corresponding to the marker, a microfluidic chip, a buffer and a flushing solution; wherein:

[0016] The detection antibodies are antibodies against CD9, CD41, THBS1, RGS18, RGS10, and EPCAM;

[0017] The microfluidic chip is composed of a substrate layer containing multiple channels and a cover layer;

[0018] The buffer solution is a balanced salt solution containing 0.1wt.% BSA and 5wt.% glucose components;

[0019] The flushing solution is a balanced salt solution containing 0.1wt.% BSA, 0.2wt.% pancreatin and 0.5mg / mL papain.

[0020] Based on the same technical concept, another embodiment of the present invention is to provide a use of a marker group in preparing a device for capturing circulating tumor cells.

[0021] Based on the same technical concept, another solution of the present invention is to provide a device, which includes a microfluidic chip, a syringe pump, a connecting tube, a pre-cooling ice table, and a sample collection tube; wherein:

[0022] The microfluidic chip is used to detect a marker group on the surface of circulating tumor cells in a sample and capture the marker group;

[0023] The injection pump is used to inject the sample into the microfluidic chip;

[0024] The connecting tube is used to connect the injection pump, the microfluidic chip and the sample collection tube;

[0025] The pre-cooling ice table is used to keep the microfluidic chip in a low temperature state;

[0026] The sample collection tube is used to collect the captured CTCs.

[0027] Preferably, the sample is a human blood sample.

[0028] The present invention also provides a method for detecting or capturing circulating tumor cells (CTCs) in the blood of pancreatic cancer and / or breast cancer patients in a sample, the method comprising: contacting the sample with a product targeting cell surface or intracellular markers on circulating tumor cells (CTCs) of pancreatic cancer and / or breast cancer to capture circulating tumor cells, wherein the product is immobilized on a solid support surface.

[0029] The beneficial effects of the present invention are:

[0030] The present invention aims at the limitations of existing CTC capture products. By performing single-cell sequencing on CTC samples isolated from the blood of pancreatic cancer and breast cancer patients, and comparing and analyzing the pancreatic cancer and breast cancer data in the public database, potential CTC surface markers in the blood of pancreatic cancer and breast cancer patients are screened. Through further bioinformatics analysis, a series of candidate CTC surface markers (CD9, CD41, THBS1, RGS18, RGS10) for pancreatic cancer and breast cancer are determined. These markers are used alone or in combination with existing markers (EPCAM), and CTC capture experiments are performed using microfluidic chips coated with corresponding antibodies. CTCs in pancreatic cancer and breast cancer cell lines and patient blood samples are enriched and screened in vitro, and the reliability and effectiveness of the markers are verified. The present invention significantly improves the enrichment number and detection rate of CTCs in the blood of pancreatic cancer and breast cancer patients by using new surface markers and antibodies combined with them, effectively reduces missed detection, and overcomes the shortcomings of low CTC detection rate and small detection amount in traditional technologies. This technology greatly improves the efficiency of CTC capture and enrichment, and provides strong support for dynamic tumor monitoring, efficacy evaluation, and personalized precision treatment of pancreatic cancer and breast cancer patients. It has broad market application potential and will benefit more cancer patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0032] Figure 1 It is the gene expression abundance distribution map of circulating tumor cell surface markers.

[0033] Figure 2It is a schematic diagram of the process of sample capture and collection, fluorescence observation, and single-cell transcriptome sequencing.

[0034] Figure 3 Figure 2 is a diagram of a CTC capture and collection device.

[0035] Figure 4 The captured CTC images are shown in Figure 2 (the left image is a view with a scale of 100 μm, and the right image is an enlarged view with a scale of 50 μm).

[0036] Figure 5 The whole blood from the portal vein of pancreatic cancer patients flows through a microfluidic chip on which EPCAM antibodies combined with one or more antibodies of RGS18, RSG10, CD41, CD9, and THBS1 are fixed, and the data graph shows the capture and enrichment efficiency of CTCs for each group of chips.

[0037] Figure 6 The whole blood from the peripheral vein of breast cancer patients flows through a microfluidic chip that is immobilized with EPCAM antibodies combined with one or more antibodies of RGS18, RSG10, CD41, CD9, and THBS1. The data graph shows the capture and enrichment efficiency of CTCs for each group of chips.

[0038] Figure 7 The human pancreatic cancer cell lines SU86.86, CFPAC-1, PANC-1, ASPC-1, and MIA PACA-2 cells flowed through a microfluidic chip fixed with EPCAM antibodies combined with one or more antibodies of RGS18, RSG10, CD41, CD9, and THBS1. The data graph shows the capture and enrichment efficiency of each group of chips for the five types of human pancreatic cancer cells.

[0039] Figure 8 The human breast cancer cell lines MCF-7, SKBR-3, MCF-12A, MCF-10A, and MDA-MB-231 cells flowed through a microfluidic chip fixed with EPCAM antibodies combined with one or more antibodies of RGS18, RSG10, CD41, CD9, and THBS1. The capture and enrichment efficiency data of each group of chips for the five types of human breast cancer cells are shown in the figure. DETAILED DESCRIPTION

[0040] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology and laboratory operation procedures used herein are terms and routine procedures widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0041] As used herein, the terms "individual," "patient," or "subject" are used interchangeably and refer to any single animal, more preferably a mammal (including non-human animals, such as, for example, cats, dogs, horses, rabbits, zoo animals, cattle, pigs, sheep, and non-human primates) for which treatment is desired. In certain embodiments, the patient herein is a human. The patient may be a "cancer patient," i.e., a patient who has cancer, or is at risk of developing cancer, or who has one or more symptoms of cancer.

[0042] As used herein, the term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues.

[0043] As used herein, the terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.

[0044] As used herein, the terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" are not mutually exclusive when referred to herein.

[0045] As used herein, the term "disorder" is any condition that would benefit from treatment, including, but not limited to, chronic and acute disorders or diseases, including those pathological conditions that predispose a mammal to the disorder in question.

[0046] As used herein, the term "tumor cell" refers to any tumor cell present in a tumor or a sample thereof. Tumor cells can be distinguished from other cells that may be present in a tumor sample, such as stromal cells and tumor-infiltrating immune cells, using methods known in the art and / or described herein.

[0047] As used herein, the term "circulating tumor cells" or "CTCs" refers to tumor cells that are shed from a tumor and are present in the blood (ie, in the circulation).

[0048] Herein, the term "tumor cell surface marker" refers to biomolecules such as proteins, carbohydrates, glycoproteins, etc. The biomolecules are specifically or preferentially or differentially expressed on tumor cells, and / or are found to be associated with tumor cells, and therefore can be used as preferred or specific targets for tumors. In specific embodiments, the dominant expression can be the dominant expression compared to other cells in the organism, or the dominant expression in a specific region of the organism (e.g., in a specific organ or tissue).

[0049] Herein, the term "sample" or "test sample" refers to a sample obtained or separated from a biological organism, such as a tumor sample from a subject. Exemplary biological samples include, but are not limited to, biological fluid samples, serum, plasma, urine, saliva, tumor samples, tumor biopsies, and / or tissue samples, etc. The term also includes a mixture of the above samples. The term "test sample" also includes untreated or pretreated (or preprocessed) biological samples. In some embodiments, the test sample may include cells from a subject. In some embodiments, the test sample may be a tumor cell test sample, such as a sample may include cancer cells, cells from a tumor, and / or a tumor biopsy. In some embodiments, the test sample may be a blood sample. The test sample may be obtained by removing a cell sample from a subject, but may also be accomplished by using previously separated cells (e.g., separated at a previous time point and separated by the same person or another person). In addition, the test sample may be a freshly collected or previously collected sample.

[0050] As used herein, the term "detecting" includes any means of detection, including direct and indirect detection.

[0051] Example 1: Screening and determination of circulating tumor cell surface markers

[0052] 1. Single-cell transcriptome sequencing of circulating tumor cells from clinical samples

[0053] 1. Clinical sample collection and CTC isolation

[0054] In order to find and screen circulating tumor cell surface markers, blood was collected from cancer patients, CTCs were extracted, and CTCs in portal vein blood were captured using a microfluidic chip for single-cell transcriptome sequencing. The capture process includes the following steps: (1) Before CTCs are captured, anti-EPCAM capture antibodies are injected into the lanes of the microfluidic chip and coated overnight at 4°C to prepare a microfluidic capture system that can be used to capture pancreatic cancer CTCs; (2) On the day of capture, blood samples from patients were collected and red blood cells were removed using red blood cell lysis buffer; (3) The cells were resuspended in HBSS to a density of 2×10 7 / mL, and injected into the microfluidic chip at a flow rate of 5mL / h; (4) rinsed three times with 10mL of HBSS at a flow rate of 30mL / h to remove non-specifically bound cells; (5) rinsed the chip with 15mL of elution buffer at a flow rate of 50mL / h to release and collect CTCs. The collected CTCs can be used for single-cell transcriptome sequencing.

[0055] 2.10×Genomics single-cell library construction and sequencing

[0056] In the present invention, the 10×Genomics Chromium 3' Gene Expression Kit V3 kit is used to prepare a single-cell transcriptome library for sequencing, and the detailed steps strictly follow the 10×Genomics single-cell operation specifications. Specifically, after obtaining a single-cell suspension through the above steps, the target cells and the corresponding 10×Genomics reagents are added to the Chromium chip to generate a latex droplet (Gel Bead in Emulsion, GEM) containing a single cell and a single gel bead, and subsequent reverse transcription, double-stranded DNA generation, library construction and sequencing are performed. In the laboratory, we set the target capture cells for each sample to 6000~8000. The final constructed library was sequenced on the Illumina HiSeq 4000 platform, and the target sequencing depth of each cell was 100,000 reads.

[0057] 2. Identification of circulating tumor cell (CTC) cell surface markers

[0058] 1. Single-cell transcriptome data preprocessing and quality control

[0059] First, the raw sequencing data images obtained from the above sequencing were converted into Fastq format sequence information with double-end 150 base pairs using Illumina's bcl2fastq software. Then, the obtained sequences were compared with the GRCh38 version of the human reference genome using Cell Ranger (v.3.0.0, 10×Genomics) software to obtain a gene expression matrix. The UMI tags of the sequences that can be specifically aligned to the exon regions of the transcriptome genes will be used for subsequent statistical counting.

[0060] 2. Copy number variation analysis and cell type identification and single-cell transcriptome data dimensionality reduction and clustering analysis

[0061] We first globally normalized the gene expression matrix of the single-cell data using the “LogNormalize” method. The specific operation is to divide the expression value of each gene by the total expression of the corresponding cell, multiply it by the default parameter factor 10000, and then take the logarithm. Next, the Find Variable Features function was used to screen out 2000 genes with high variability, and the expression data of these genes were scaled to z-scores using the ScaleData function. Subsequently, the RunPCA function was used to perform principal component analysis (PCA) on these highly variable genes to achieve data dimensionality reduction. After the dimensionality reduction was completed, the Find Neighbors function was used to construct a KNN graph to determine the weight relationship between cells. The Find Clusters function was combined with the Louvain algorithm to perform cell clustering, and the resolution parameter was set to 1. Finally, the t-SNE method was used to visualize the clustering results.

[0062] 3. Differential Gene Expression Analysis

[0063] In order to find and identify differentially expressed genes in CTCs, the present invention uses R studio to perform differential expression analysis through the Find Markers function, and evaluates the significance of genes based on the non-parametric Wilcoxon rank sum test. The min.pct parameter is set to 0.25, and the logfc threshold parameter is set to 0.25. The final circulating tumor cell surface markers identified are CD9, CD41 (ITGA2B), THBS1, RGS18, RGS10 (such as Figure 1 As shown), T cells (CD3D+, CD3E+, CD3G+), natural killer cells (KLRD1+, KLRF1+, GNLY+), B cells (CD79A+, CD79B+, MS4A1+), monocytes (CD14+, FCN1+, S100A12+), neutrophils (FCGR3B+, FPR1+), and dendritic cells (CD1C+, FCER1A+, CLEC10A+) were identified through analysis.

[0064] The relevant information of the markers is shown in Tables 1 to 5.

[0065] Table 1

[0066]

[0067] Table 2

[0068]

[0069] Table 3

[0070]

[0071] Table 4

[0072]

[0073] Table 5

[0074]

[0075] Example 2: Capture ability of combined antibodies on CTCs in the blood of pancreatic cancer patients

[0076] The screened cell surface markers are used to capture and enrich CTCs in the blood of pancreatic cancer patients. The specific process is as follows Figure 2 As shown: (1) CTC surface marker combination antibodies (EPCAM, EPCAM and RGS18, EPCAM and CD41, EPCAM and RGS10, EPCAM and CD9, EPCAM and THBS1, EPCAM and RGS18, CD41, RGS10, CD9, THBS1) were injected into the lanes of the microfluidic chip and coated overnight at 4°C to prepare a microfluidic capture system for capturing pancreatic cancer CTCs. (2) Fresh blood samples were collected from patients and red blood cells were removed using red blood cell lysis buffer. (3) The obtained cell samples were used Figure 3 The CTC capture device is used to capture and collect CTCs. (4) The collected cells are stained with a fluorescent staining reagent, and finally scanned and counted with a fluorescent counter to count the number of captured CTCs (e.g. Figure 5 as shown).

[0077] The results showed that the ability of the chip coated with the combination of EPCAM and RGS18, EPCAM and CD41, EPCAM and RGS10, EPCAM and CD9, and EPCAM and THBS1 antibodies to enrich and screen CTCs in blood samples of pancreatic cancer patients was significantly higher than that of the chip coated with EPCAM alone (e.g. Figure 5 The enrichment and screening ability of the chip coated with the combination of EPCAM and RGS18, CD41, RGS10, CD9, and THBS1 antibodies in the blood samples of pancreatic cancer patients was significantly higher than that of the chip coated with the combination of EPCAM and RGS18, EPCAM and CD41, EPCAM and RGS10, EPCAM and CD9, and EPCAM and THBS1 antibodies (as shown in Figure 5 As shown). Product device Figure 3 As shown in FIG. 1 , the system mainly includes a capture and collection system, which includes a microfluidic injection pump, a chip coated with a combination of different surface marker antibodies, and a collection device. Figure 4 shown.

[0078] The specific operation flow chart is as follows Figure 2The figure shows the process of extracting cells from the patient's peripheral venous whole blood, flowing through a microfluidic chip with EPCAM and new marker antibodies fixed on it, and enriching tumor cells after being washed with buffer and eluted with washing solution. The enriched cells are then fluorescently stained, observed, and subjected to single-cell transcriptome sequencing.

[0079] Example 3: Capture ability of combined antibodies on CTCs in the blood of breast cancer patients

[0080] The screened cell surface markers are used to capture and enrich CTCs in the blood of breast cancer patients. The specific process is as follows Figure 2 As shown: (1) CTC surface marker combination antibodies (EPCAM, EPCAM and RGS18, EPCAM and CD41, EPCAM and RGS10, EPCAM and CD9, EPCAM and THBS1, EPCAM and RGS18, CD41, RGS10, CD9, THBS1) were injected into the lanes of the microfluidic chip and coated overnight at 4°C to prepare a microfluidic capture system for capturing breast cancer CTCs. (2) Fresh blood samples from patients were collected and red blood cells were removed using red blood cell lysis buffer. (3) The obtained cell samples were used Figure 3 The CTC capture device is used to capture and collect CTCs. (4) The collected cells are stained with a fluorescent staining reagent, and finally scanned and counted with a fluorescent counter to count the number of captured CTCs (e.g. Figure 6 as shown).

[0081] The results showed that the ability of the chip coated with the combination of EPCAM and RGS18, EPCAM and CD41, EPCAM and RGS10, EPCAM and CD9, and EPCAM and THBS1 antibodies to enrich and screen CTCs in blood samples of breast cancer patients was significantly higher than that of the chip coated with EPCAM alone (e.g. Figure 6 The enrichment and screening ability of the chip coated with the combination of EPCAM and RGS18, CD41, RGS10, CD9, and THBS1 antibodies in the blood samples of breast cancer patients was significantly higher than that of the chip coated with the combination of EPCAM and RGS18, EPCAM and CD41, EPCAM and RGS10, EPCAM and CD9, and EPCAM and THBS1 antibodies (as shown in Figure 6 As shown). Product device Figure 3 As shown, it mainly includes a capture and collection system, which includes a microfluidic injection pump, a chip coated with a combination of different surface marker antibodies, and a collection device.

[0082] The specific operation flow chart is as follows Figure 2The figure shows the process of extracting cells from the patient's peripheral venous whole blood, flowing through a microfluidic chip with EPCAM and new marker antibodies fixed on it, and enriching tumor cells after being washed with buffer and eluted with washing solution. The enriched cells are then fluorescently stained, observed, and subjected to single-cell transcriptome sequencing.

[0083] Example 4: Capture efficiency of combined antibodies on different pancreatic cancer cells

[0084] Human pancreatic cancer cell lines (SU86.86, CFPAC-1, PANC-1, ASPC-1, MIA PACA-2) were flowed through a microfluidic chip fixed with EPCAM antibody alone or in combination with one or more antibodies such as RGS18, RSG10, CD41, CD9, and THBS1. 1000 cells in each group flowed through the chip at a certain speed. The cells captured by the chip were collected with a collection fluid and counted. The capture capacity of different antibodies for the five types of cells, SU86.86, CFPAC-1, PANC-1, ASPC-1, and MIA PACA-2, was calculated (e.g. Figure 7 as shown).

[0085] EPCAM antibody was coated alone or in combination with one or more antibodies such as RGS18, RSG10, CD41, CD9, THBS1 to capture human pancreatic cancer cells SU86.86, CFPAC-1, PANC-1, ASPC-1, MIA PACA-2, and the percentage of captured enriched cells (total number of input cells) was tested. Figure 7 It can be seen that the capture efficiency of EPCAM combined with RGS18, RSG10, CD41, CD9, and THBS1 antibodies is significantly higher than that of EPCAM alone, and the capture ability of the chip coated with EPCAM and RGS18, CD41, RGS10, CD9, and THBS1 combined antibodies to human pancreatic cancer cells is significantly higher than that of the chip coated with EPCAM and RGS18, EPCAM and CD41, EPCAM and RGS10, EPCAM and CD9, and EPCAM and THBS1 combined antibodies (e.g. Figure 7 as shown).

[0086] Example 5: Capture efficiency of combined antibodies on different breast cancer cells in blood

[0087] Human breast cancer cell lines (MCF-7, SKBR-3, MCF-12A, MCF-10A, MDA-MB-231) were flowed through a microfluidic chip immobilized with EPCAM antibody alone or in combination with one or more antibodies such as RGS18, RSG10, CD41, CD9, and THBS1. 1000 cells in each group flowed through the chip at a certain speed. The cells captured by the chip were collected with a collection fluid and counted. The capture capacity of different antibodies for the five cell types MCF-7, SKBR-3, MDA-MB-231, MCF-12A, and MCF-10A was calculated (e.g. Figure 8 as shown).

[0088] EPCAM antibody was coated alone or in combination with one or more antibodies such as RGS18, RSG10, CD41, CD9, THBS1 to capture human breast cancer cells MCF-7, SKBR-3, MDA-MB-231, MCF-12A, and MCF-10A, and the percentage of captured enriched cells (total number of input cells) was tested. Figure 8 It can be seen that the capture efficiency of EPCAM combined with RGS18, RSG10, CD41, CD9, and THBS1 antibodies is significantly higher than that of EPCAM alone, and the capture ability of the chip coated with EPCAM and RGS18, CD41, RGS10, CD9, and THBS1 combined antibodies to human breast cancer cells is significantly higher than that of the chip coated with EPCAM and RGS18, EPCAM and CD41, EPCAM and RGS10, EPCAM and CD9, and EPCAM and THBS1 combined antibodies (e.g. Figure 8 as shown).

[0089] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. Use of a marker group in preparing a kit for capturing circulating tumor cells, characterized in that: The marker panel includes EPCAM, CD9, CD41, THBS1, RGS18 and RGS10; The circulating tumor cells are pancreatic cancer circulating tumor cells, And / or, the circulating tumor cells are breast cancer circulating tumor cells.

2. The use according to claim 1, characterized in that The kit includes a detection reagent for detecting a marker panel including EPCAM, CD9, CD41, THBS1, RGS18 and RGS10.

3. The use according to claim 1, characterized in that The kit also includes detection antibodies corresponding to the markers, a microfluidic chip, a buffer and a flushing solution; wherein: The detection antibodies are antibodies against CD9, CD41, THBS1, RGS18, RGS10 and EPCAM; The microfluidic chip is composed of a substrate layer containing multiple channels and a cover layer; The buffer solution is a balanced salt solution containing 0.1wt% BSA and 5wt% glucose components; The flushing solution is a balanced salt solution containing 0.1wt% BSA, 0.2wt% pancreatin and 0.5mg / mL papain.

4. Use of a marker panel in preparing a device for capturing circulating tumor cells, characterized in that: The marker panel includes EPCAM, CD9, CD41, THBS1, RGS18 and RGS10.

5. The use according to claim 4, characterized in that The device comprises a microfluidic chip, a syringe pump, a connecting tube, a precooling ice table and a sample collection tube; wherein: The microfluidic chip is used to detect a marker group on the surface of circulating tumor cells in a sample and capture the marker group; the marker group includes EPCAM, CD9, CD41, THBS1, RGS18 and RGS10; The injection pump is used to inject the sample into the microfluidic chip; The connecting tube is used to connect the injection pump, the microfluidic chip and the sample collection tube; The pre-cooling ice table is used to keep the microfluidic chip in a low temperature state; The sample collection tube is used to collect the captured CTCs.

6. The use according to claim 5, characterized in that The sample is a human blood sample.

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