CN113916754B - Cell surface marker for detecting circulating tumor cells of breast cancer patient and application thereof - Google Patents

Cell surface marker for detecting circulating tumor cells of breast cancer patient and application thereof Download PDF

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CN113916754B
CN113916754B CN202111185982.2A CN202111185982A CN113916754B CN 113916754 B CN113916754 B CN 113916754B CN 202111185982 A CN202111185982 A CN 202111185982A CN 113916754 B CN113916754 B CN 113916754B
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何秀静
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West China Hospital of Sichuan University
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Abstract

The invention discloses a cell surface marker for detecting circulating tumor cells of a breast cancer patient and application thereof, wherein the cell surface marker comprises TSPAN1, FAIM2, KCNJ4, GABRD, PDK1L2, OR1OK1, UPK1B and OR51M1; performing immunocapture on the breast cancer CTC by adopting an antibody corresponding to the cell surface marker; the invention utilizes the obtained surface markers of the circulating tumor cells of the breast cancer patients to obtain the optimal antibody combination for immune enrichment screening, improves the enrichment and capture efficiency of the breast cancer CTC, effectively improves the CTC capture specificity and sensitivity, and has the effect of efficiently capturing the breast cancer CTC. Overcomes the problems of insufficient types, low detection rate and low purity of the existing breast cancer CTC enrichment markers.

Description

Cell surface marker for detecting circulating tumor cells of breast cancer patient and application thereof
Technical Field
The invention relates to the technical field of tumor cell detection, in particular to a cell surface marker for detecting CTC of a breast cancer patient and application thereof.
Background
The circulating tumor cells (circulating tumor cells, CTC) are tumor cells which are separated from tumor tissues and released into a blood circulation system, can truly represent tumor loads and characteristics, dynamically reflect tumor genomic information and gene expression profile changes, have close relationship with distant metastasis of tumors of patients, and have important significance for malignant tumor diagnosis and clinical stage judgment. At present, the research on the aspects of early tumor screening, dynamic monitoring, immunotherapy selection and the like of CTC (tumor cell control) has achieved remarkable results, and CTC detection has become one of the most potential noninvasive tumor diagnosis and treatment means. The field of liquid biopsy to which it belongs is rated MIT technology review as one of ten future medical techniques. Compared with the common tumor diagnosis methods such as imaging evidence and tissue biopsy, the CTC detection has the advantages of early stage, real-time high efficiency, non-invasiveness, convenient material taking, repeated monitoring and the like. Compared with the conventional tumor marker detection, the CTC detection can generally reflect the change of the disease earlier, can monitor the dynamic change of the tumor more sensitively, so as to evaluate the curative effect of the treatment, monitor the recurrence and metastasis and carry out the diagnosis along with the targeted drug in the treatment process, and has important guiding significance in the establishment of clinical treatment schemes. CTCs have now been formally listed in the tumor staging system and have become a further post-breast cancer assessment tool following four biological indicators of ER/PR, her-2, ki-67 and tumor histological grading. In 2019, CTCs are written into the diagnosis and treatment guide of breast cancer for the first time, and the CTCs can reflect the condition of tumor tissues, so that the CTCs can be used for pathological diagnosis, disease monitoring, molecular sequencing and the like by replacing tissue samples in a noninvasive mode, and not only can be dynamically monitored, but also can be used for judging after healing.
However, CTC detection presents a number of problems and challenges in clinical applications due to the specific nature of CTCs. Currently, CTC detection means are mainly based on physical and biological properties of CTCs. Including cell filtration, density gradient centrifugation, dielectrophoresis separation, etc., CTC detection is mainly based on biophysical characteristics such as cell size, density, deformability, charge characteristics, etc. However, CTCs have characteristics of rarity and heterogeneity, and the cell size is not uniform, and the cell itself has a certain flexibility, so that the above method lacks specificity when CTC separation is performed, resulting in problems of low purity, low efficiency, and the like.
The most widely used is the immunoaffinity-based CTC detection technique at present, wherein antibodies, nucleic acid aptamers or polypeptides combined with a proper matrix (such as magnetic beads) are combined with a CTC surface target antigen, and positive or negative selection is performed through magnetic force or other acting force to realize enrichment of CTCs. The negative selection method is to remove the white blood cells in the blood by utilizing the white blood cell surface specific antigen (mainly CD 45) so as to enrich the CTC, and has the characteristics of mature antibody, easy acquisition and high efficiency of capturing the white blood cells, but the method has certain limitations, red blood cells need to be cracked, the antibody is easy to carry out nonspecific binding on the CTC with poor state, the CTC detection purity is low, and false positive results are easy to cause. The positive selection method, i.e., the immunoenrichment method, captures CTCs directly by enriching the corresponding antibodies for certain specific biomarkers, such as the epithelial cell adhesion molecule (EpCAM), on the CTC surface. The method is insensitive to the state of CTC, has high detection purity, and has the characteristics of simpler operation, automation realization and durability. Currently, the CellSearch detection system approved by the United states Food and Drug Administration (FDA) and the CellCollector detection system approved by CFDA in China are all immune enrichment methods. It should be noted that CTCs have a high degree of heterogeneity and that there is also some difference in the markers on the surface of different types of tumor CTCs. Conventional detection systems for capturing CTCs do not adequately take into account the heterogeneity of CTCs and the specificity of tumors, and enrich and screen CTCs with relatively single markers or combinations of markers. For example, currently, the markers on CTC surfaces used in immune enrichment methods are mainly the adhesion molecule EpCAM and cytokeratin, etc. This immobilized antibody combination is clearly not suitable for all types of tumors, especially for capturing CTCs lacking the above surface markers, resulting in missed detection.
Disclosure of Invention
The invention provides a cell surface marker for detecting CTC of a breast cancer patient and application thereof aiming at the problems existing in the prior art.
The technical scheme adopted by the invention is as follows:
cell surface markers for detecting circulating tumor cells in breast cancer patients include TSPAN1, FAIM2, KCNJ4, GABRD, PDK1L2, OR1OK1, UPK1B and OR51M1.
The application of the cell surface markers for detecting the CTC of the breast cancer patient adopts the antibody corresponding to the cell surface markers to perform immunocapture of the breast cancer CTC.
Further, a HER2 antibody, epCam antibody, FAIM2 antibody, and GABRD antibody are used in combination, or a HER2 antibody, epCam antibody, KCNJ4 antibody, and GABRD antibody are used in combination.
Further, the application of the antibody corresponding to the cell surface marker in preparing reagents and/or medicines for detecting and/or treating breast cancer.
Further, the process of immunocapture of breast cancer CTC cells is as follows:
step 1: coating a micro-fluidic chip with a cell surface marker antibody;
step 2: the cultured breast cancer cell suspension or a blood sample of a breast cancer patient flows through the chip at a certain speed;
step 3: collecting cells captured by the chip with a collection solution and counting; counts represent the ability of the antibody to capture breast cancer cells.
A screening method for detecting cell surface markers of CTCs in breast cancer patients comprising the steps of:
analyzing CTC specific expression profile information and heterogeneity characteristics of the breast cancer patients according to the RNA-seq and scRNA-seq sequencing data of the circulating tumor cells CTC samples of the breast cancer patients in the public database to obtain CTC high expression genes;
filtering according to the gene expression abundance information of other cell types in the blood sample, and combining the surface protein expression characteristic information to obtain a CTC specific surface marker;
comparing the obtained CTC specific surface marker with the tumor tissue sample expression profile of the breast cancer patient in the public database, and determining the cell surface marker of the CTC.
The beneficial effects of the invention are as follows:
(1) The marker in the invention is a novel breast cancer CTC surface marker with enrichment capability;
(2) The invention utilizes the surface markers of the breast cancer CTC obtained by screening to obtain the optimal antibody combination for immune enrichment screening, improves the enrichment and capture efficiency of the breast cancer CTC, effectively improves the CTC capture specificity and sensitivity, and has the effect of efficiently capturing the breast cancer CTC;
(3) The invention solves the problems of insufficient types, low detection rate and low purity of the existing breast cancer CTC enrichment markers, realizes the efficient capture of breast cancer CTC, and promotes the application of CTC detection in auxiliary diagnosis, monitoring tumor dynamics, clinical treatment and post-healing evaluation of breast cancer; the method provides more accurate information and powerful support for realizing real-time accurate individual treatment, and has wide application prospect and market value.
Drawings
FIG. 1 is a schematic diagram of screening surface markers of CTC of breast cancer, a is the distribution of expression abundance of CTC specific expression markers of breast cancer in a CTC RNA-seq sample; b is the expression level of a common CTC specific expression marker in a CTC RNA-seq and a scRNA-seq data set of the breast cancer in a CTC RNA-seq sample; c is the expression level of a common CTC specific expression marker in a CTC scRNA-seq sample in a breast cancer CTC RNA-seq and scRNA-seq data set; d is the expression specificity of the expression marker protein level of the breast cancer CTC.
FIG. 2 is a schematic diagram showing the protein sequence analysis of the markers obtained by the screening of the present invention.
Fig. 3 is a schematic diagram of capturing CTCs of a blood sample using a microfluidic chip according to the present invention.
FIG. 4 is a graph showing the efficiency of screening breast cancer cells SKBR3 and MCF-7 by enrichment of the marker antibodies obtained by the screening in the present invention.
FIG. 5 is a graph showing the efficiency of screening of breast cancer cells SKBR3 and MCF-7 by combining the marker antibody and EpCAM obtained by screening in the present invention.
FIG. 6 is a graph showing the efficiency of screening breast cancer cells SKBR3 and MCF-7 by enrichment of the marker antibody and HER2 obtained by screening in the present invention.
FIG. 7 is a graph showing the efficiency of screening for breast cancer cells SKBR3 and MCF-7 by enrichment of the marker antibody and EpCAM+HER2 combination screened in the present invention.
FIG. 8 is a graph showing the efficiency of screening for breast cancer cells SKBR3 and MCF-7 by combining two by two and combining EpCAM+HER2 with four antibodies (FAIM 2, KCNJ4, GABRD, UPK 1B) screened in accordance with the present invention.
FIG. 9 is a graph showing the efficiency of the screening of four breast cancer cell enrichment by the selected combination antibodies of the present invention. (HER2+EpCAM, epCAM+HER2+FAIM2+GABRD, HER2+EpCAM+KCNJ4+GABRD)
FIG. 10 is a graph showing the efficiency of the screening of four breast cancer cell enrichment by the selected combination antibodies of the present invention. (HER2+EpCAM, epCAM+HER2+FAIM 2+GABRD)
FIG. 11 is a graph showing the capture capacity of the screened combination antibody (EpCAM+HER2+FAIM 2+GABRD) of the present invention for CTC in the blood of breast cancer patients.
Detailed Description
The invention will be further described with reference to the drawings and specific examples.
A cell surface marker for detecting circulating tumor cells of breast cancer patients comprises one OR two OR more of TSPAN1, FAIM2, KCNJ4, GABRD, PDK1L2, OR1OK1, UPK1B and OR51M1 mixed at any ratio.
A screening method comprising the steps of:
analyzing CTC specific expression profile information and heterogeneity characteristics of the breast cancer patients according to the RNA-seq and scRNA-seq sequencing data of the circulating tumor cells CTC samples of the breast cancer patients in the public database to obtain CTC high expression genes;
filtering according to the gene expression abundance information of other cell types in the blood sample, and combining the surface protein expression characteristic information to obtain a CTC specific surface marker;
comparing the obtained CTC specific surface markers with tumor tissue sample expression profiles of breast cancer patients in a public database, and determining the cell surface markers of the CTC, wherein the cell surface markers are shown in figure 1.
The surface markers unique to candidate CTCs were initially selected by the above screening, and further protein sequence analysis was performed on the genes obtained as described above, as shown in fig. 2. Markers having a transmembrane segment and an extracellular segment and having a relatively high expression level were selected from these markers for testing. The genes selected for FIG. 2 include: TSPAN1, FAIM2, KCNJ4, GABRD, PDK1L2, OR1OK1, UPK1B, and OR51M1.
And (3) carrying out relevant tumor cell enrichment screening experiments by adopting antibodies corresponding to the surface markers obtained through screening as shown in fig. 2, wherein the relevant tumor cell enrichment screening experiments comprise breast cancer cell lines and blood samples of breast cancer tumor patients. Experiments prove that the surface markers can enrich and screen the CTC in breast cancer tumor cells and blood.
The process of immunocapture of breast cancer CTC cells is as follows: as shown in fig. 3:
step 1: coating a micro-fluidic chip with a cell surface marker antibody;
step 2: the cultured breast cancer cell suspension or a blood sample of a breast cancer patient flows through the chip at a certain speed; wherein the cell suspension is formed by adding cells into 7.5mL of healthy human whole blood, uniformly mixing, treating by lymphocyte separating liquid, and adding 7.5mL of protective liquid into the collected cell precipitate.
Step 3: collecting cells captured by the chip with a collection solution and counting; counts represent the ability of the antibody to capture breast cancer cells.
Example 1
TSPAN1, FAIM2, KCNJ4, GABRD, PDK1L2, OR1OK1, UPK1B, and OR51M1 antibodies were prepared OR purchased, and the chips were individually coated with these antibodies, and each antibody was tested for screening ability for enrichment of breast cancer cells (the method is shown in fig. 3, excluding blood samples). In this example, classical breast cancer cells SKBR3 and MCF-7 were selected as experimental cells. After coating the chip, the cultured SKBR3 and MCF-7 cells were counted, 1000 cells per group flowed through the chip at a constant rate, and the cells captured by the chip were collected with a collection solution and counted. Capture ability of different antibodies on SKBR3 and MCF-7 breast cancer cells was counted. The results are shown in fig. 4, with the y-axis representing capture efficiency, i.e., the percentage of single antibody enriched captured cells. The x-axis represents newly developed breast cancer CTCs specifically express surface marker antibodies.
From the figure, the markers obtained by screening have a certain enrichment and capture capacity on SKBR3 cells, and the enrichment and capture efficiency is sequentially TSPAN1, FAIM2, KCNJ4, GABRD, PDK1L2, OR10K1, UPK1B and OR51M1 from high to low.
Example 2
The ability of the overlaid antibodies to enrich and capture breast cancer tumor cells was tested using the surface marker antibodies obtained by screening in combination with EpCam (the method is shown in fig. 3, excluding blood samples). The results are shown in fig. 5, with the y-axis representing capture efficiency, i.e., the percentage of antibody enriched captured cells. The x-axis represents EpCam and combinations with other newly developed surface marker antibodies. The mass ratio of Epcam to the corresponding antibody was 1:1.
Classical breast cancer cells SKBR3 and MCF-7 were selected as experimental cells. After coating the chip, the cultured SKBR3 and MCF-7 cells were counted, 1000 cells per group flowed through the chip at a constant rate, and the cells captured by the chip were collected with a collection solution and counted. Capture ability of different antibodies on SKBR3 and MCF-7 breast cancer cells was counted. From the figure, the combined antibody has better capturing capacity on breast cancer cells SKBR3 and MCF-7, and the capturing capacity is larger than that of a single EpCam antibody.
Example 3
The ability of the overlaid antibodies to enrich and capture breast cancer tumor cells was tested using the surface marker antibodies obtained by screening in combination with HER2 (the method is shown in fig. 3, excluding blood samples). The results are shown in fig. 6, with the y-axis representing capture efficiency, i.e., the percentage of antibody enriched captured cells. The x-axis represents HER2 and combinations with other newly developed surface marker antibodies. The mass ratio of HER2 and the corresponding antibody was 1:1.
Classical breast cancer cells SKBR3 and MCF-7 were selected as experimental cells. After coating the chip, the cultured SKBR3 and MCF-7 cells were counted, 1000 cells per group flowed through the chip at a constant rate, and the cells captured by the chip were collected with a collection solution and counted. Capture ability of different antibodies on SKBR3 and MCF-7 breast cancer cells was counted. From the figure, the combined antibodies have better capturing capacity on SKBR 3. The combined antibodies all have greater capture capacity for MCF-7 than the HER2 antibody alone.
Example 4
The ability of the superimposed antibodies to enrich and capture breast cancer tumor cells was tested using the surface marker antibodies obtained by screening in combination with EpCam and HER2 (the method is shown in fig. 3, excluding blood samples). The results are shown in fig. 7, with the y-axis representing capture efficiency, i.e., the percentage of antibody enriched captured cells. The x-axis represents EpCam and HER2 in combination with other surface marker antibodies. The mass ratio of the corresponding antibodies is 1:1:1.
Classical breast cancer cells SKBR3 and MCF-7 were selected as experimental cells. After coating the chip, the cultured SKBR3 and MCF-7 cells were counted, 1000 cells per group flowed through the chip at a constant rate, and the cells captured by the chip were collected with a collection solution and counted. Capture ability of different antibodies on SKBR3 and MCF-7 breast cancer cells was counted.
Through cell line experiments, the graph shows that the combined antibody has better capture capability on SKBR3 with higher selection efficiency. The combined antibodies all have greater capture capacity for MCF-7 than EpCam and HER2 combination.
Example 5
The ability of the overlaid antibodies to enrich and capture breast cancer tumor cells was tested (the method is shown in fig. 3, excluding blood samples) with the screened surface marker antibodies (FAIM 2, KCNJ4, GABRD, UPK 1B) in combination with EpCam and HER2 in pairwise. The results are shown in fig. 8, with the y-axis representing capture efficiency, i.e., the percentage of antibody enriched captured cells. The x-axis represents EpCam and HER2 in combination with other surface marker antibodies (FAIM 2, KCNJ4, GABRD, UPK 1B). The mass ratio of the corresponding antibodies is 1:1:1:1.
Classical breast cancer cells SKBR3 and MCF-7 were selected as experimental cells. After coating the chip, the cultured SKBR3 and MCF-7 cells were counted, 1000 cells per group flowed through the chip at a constant rate, and the cells captured by the chip were collected with a collection solution and counted. Capture ability of different antibodies on SKBR3 and MCF-7 breast cancer cells was counted.
Through cell line experiments, we selected the capture antibodies with higher efficiency as: FAIM2, KCNJ4, GABRD, UPK1B, combined two by two with EpCam and HER 2. As can be seen from the figure, the capture efficiency is significantly higher than the existing EpCam and HER2 capture schemes alone or in combination. Further optimization found that two highly efficient combinatorial approaches were HER2, epCam, FAIM2 and GABRD and HER2, epCam, KCNJ4 and GABRD. The capturing efficiency can reach 95% and 93% respectively.
Example 6
The chip is coated with HER2, epcam, FAIM2 and GABRD and HER2, epcam, KCNJ4 and GABRD combined antibodies, four breast cancer cells SKBR3, BT474, MCF-7 and MDA-MB-231 are cultured, 1000 of each cell is counted, the 1000 four breast cancer cells are respectively flowed through the chip at a certain flow rate, the cells captured by the chip are collected by a collecting liquid, and the capturing capacity of the combined antibodies on the four breast cancer cells is counted and calculated. As shown in fig. 9, the y-axis represents capture efficiency, i.e., the percentage of antibody enriched captured cells. The x-axis represents breast cancer cell lines, the antibody combinations were: HER2+EpCal antibody combination, antibody mass ratio of 1:1.HER2+Epcam+FAIM2+GABRD and HER2+Epcam+KCNJ4+GABRD, wherein the mass ratio of the antibodies is 1:1:1:1.
As can be seen from the figure, the capture efficiency of the combined antibodies on four breast cancer cells was significantly higher than for the her2+epcam antibody combination. And the enrichment and capture capacity of the HER2+Epcam+FAIM2+GABRD combined antibody on breast cancer cells is better than that of the HER2+Epcam+KCNJ4+GABRD combined antibody.
Example 7
HER2+ EpCam + faim2+ GABRD combination antibody coated chips were selected. Four breast cancer cells SKBR3, BT474, MCF-7 and MDA-MB-231 are cultured, 1000 cells are counted, the 1000 four breast cancer cells are added into 7.5ml of healthy human whole blood respectively, the four breast cancer cells are treated by lymphocyte separation liquid after being uniformly mixed, 7.5ml of protective liquid is added into collected cell sediment to form cell suspension, the cell suspension flows through a chip at a certain flow rate, the cells captured by the collecting liquid collecting chip are subjected to fluorescent staining by a fluorescent Marker. The capture capacity of the chip for four breast cancer cells in whole blood was calculated after scanning and counting. The results are shown in fig. 10, with the y-axis representing capture efficiency, i.e., the percentage of antibody enriched captured cells. The x-axis represents breast cancer cell lines, the antibody combinations are: HER2+EpCal antibody combination, antibody mass ratio of 1:1.HER2+Epcam+FAIM2+GABRD, the mass ratio of the antibodies is 1:1:1:1.
As can be seen from the figure, the capture efficiency of the combined antibodies on four breast cancer cells was significantly higher than for the her2+epcam antibody combination.
Example 8
HER2+ EpCam + faim2+ GABRD combination antibody coated chips were selected. And (3) extracting 7.5ml of whole blood of a breast cancer patient, treating the whole blood by using a red blood cell lysate, adding 7.5ml of protective solution into the collected cell precipitate to form a cell suspension, flowing the cell suspension through the chip at a certain flow rate, collecting cells captured by the chip by using the collecting solution, and performing fluorescent staining on the collected cells by using a fluorescent Marker. And (3) carrying out scanning counting analysis on the sample by using a fluorescent scanning counter, and recording the capturing quantity of the combined antibody coating chip on CTC in the blood of the breast cancer patient. The results are shown in fig. 11, with the y-axis representing capture efficiency, i.e., the percentage of antibody enriched captured cells. The x-axis represents breast cancer cell lines, the antibody combinations are: HER2+EpCal antibody combination, antibody mass ratio of 1:1.HER2+Epcam+FAIM2+GABRD, the mass ratio of the antibodies is 1:1:1:1.
As can be seen from the figure, the capture capacity of the combined antibodies for four breast cancer cells was significantly higher than for the her2+epcam antibody combination.
The invention accurately characterizes the expression of the breast cancer CTC cell gene by utilizing the sequencing data of the breast cancer patient CTC single cell, screens the breast cancer CTC specific expression gene by comparing with the expression abundance of the normal human blood single cell gene, integrates CTC RNA-seq data and protein expression profile, and further screens a series of potential surface markers (FAIM 2, KCNJ4, GABRD and UPK 1B) of the CTC in the breast cancer blood. And detecting the authenticity and the effectiveness of the breast cancer CTC specific surface marker by using a microfluidic chip coated with the surface marker antibody. On this basis, the antibody combinations HER2, epcam, FAIM2 and GABRD and HER2, epcam, KCNJ4 and GABRD with higher efficiency of capturing CTC are further optimized and screened. And CTC capture test experiments are carried out on breast cancer cell lines and blood samples of breast cancer tumor patients, and the results show that the optimized antibody combination is obviously superior to the currently mainstream antibody capture scheme in terms of capture efficiency.
The breast cancer CTC specific antibody and the combination provided by the invention solve the problems of low detection rate and low purity of the blood CTC of a breast cancer patient. The novel antibody combination identified by the invention can remarkably improve the enrichment and capture efficiency of CTC of breast cancer patients, and is helpful for the dynamic real-time monitoring and treatment effect evaluation of tumors of breast cancer patients. Effectively promotes the realization of real-time accurate individual treatment, has wide application prospect, and can create economic benefit and improve social benefit.

Claims (3)

1. The application of an antibody corresponding to a cell surface marker in preparing a reagent for detecting breast cancer is characterized in that the antibody corresponding to the cell surface marker is adopted to perform immunocapture on breast cancer circulating tumor cells CTC; cell surface markers include TSPAN1, FAIM2, KCNJ4, GABRD, PKD1L2, OR10K1, UPK1B and OR51M1; HER2 antibody, epCam antibody, FAIM2 antibody and GABRD antibody, or HER2 antibody, epCam antibody, KCNJ4 antibody and GABRD antibody.
2. The use of an antibody corresponding to a cell surface marker according to claim 1 for the preparation of a reagent for detecting breast cancer, wherein the immunocapture of breast cancer circulating tumor cells CTCs is as follows:
step 1: coating a micro-fluidic chip with a cell surface marker antibody;
step 2: the cultured breast cancer cell suspension or a blood sample of a breast cancer patient flows through the chip at a certain speed;
step 3: collecting cells captured by the chip with a collection solution and counting; counts represent the ability of the antibody to capture breast cancer cells.
3. The use of an antibody corresponding to a cell surface marker according to claim 1 for the preparation of a reagent for detecting breast cancer, wherein the method for screening the cell surface marker comprises the steps of: analyzing CTC specific expression profile information and heterogeneity characteristics of the breast cancer patients according to the RNA-seq and scRNA-seq sequencing data of the circulating tumor cells CTC samples of the breast cancer patients in the public database to obtain CTC high expression genes; filtering according to the gene expression abundance information of other cell types in the blood sample, and combining the surface protein expression characteristic information to obtain a CTC specific surface marker; comparing the obtained CTC specific surface marker with the tumor tissue sample expression profile of the breast cancer patient in the public database, and determining the cell surface marker of the CTC.
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